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Sample records for 5-ht3a receptor channel

  1. Pathways and Barriers for Ion Translocation through the 5-HT3A Receptor Channel

    PubMed Central

    Di Maio, Danilo; Chandramouli, Balasubramanian; Brancato, Giuseppe

    2015-01-01

    Pentameric ligand gated ion channels (pLGICs) are ionotropic receptors that mediate fast intercellular communications at synaptic level and include either cation selective (e.g., nAChR and 5-HT3) or anion selective (e.g., GlyR, GABAA and GluCl) membrane channels. Among others, 5-HT3 is one of the most studied members, since its first cloning back in 1991, and a large number of studies have successfully pinpointed protein residues critical for its activation and channel gating. In addition, 5-HT3 is also the target of a few pharmacological treatments due to the demonstrated benefits of its modulation in clinical trials. Nonetheless, a detailed molecular analysis of important protein features, such as the origin of its ion selectivity and the rather low conductance as compared to other channel homologues, has been unfeasible until the recent crystallization of the mouse 5-HT3A receptor. Here, we present extended molecular dynamics simulations and free energy calculations of the whole 5-HT3A protein with the aim of better understanding its ion transport properties, such as the pathways for ion permeation into the receptor body and the complex nature of the selectivity filter. Our investigation unravels previously unpredicted structural features of the 5-HT3A receptor, such as the existence of alternative intersubunit pathways for ion translocation at the interface between the extracellular and the transmembrane domains, in addition to the one along the channel main axis. Moreover, our study offers a molecular interpretation of the role played by an arginine triplet located in the intracellular domain on determining the characteristic low conductance of the 5-HT3A receptor, as evidenced in previous experiments. In view of these results, possible implications on other members of the superfamily are suggested. PMID:26465896

  2. Pathways and Barriers for Ion Translocation through the 5-HT3A Receptor Channel.

    PubMed

    Di Maio, Danilo; Chandramouli, Balasubramanian; Brancato, Giuseppe

    2015-01-01

    Pentameric ligand gated ion channels (pLGICs) are ionotropic receptors that mediate fast intercellular communications at synaptic level and include either cation selective (e.g., nAChR and 5-HT3) or anion selective (e.g., GlyR, GABAA and GluCl) membrane channels. Among others, 5-HT3 is one of the most studied members, since its first cloning back in 1991, and a large number of studies have successfully pinpointed protein residues critical for its activation and channel gating. In addition, 5-HT3 is also the target of a few pharmacological treatments due to the demonstrated benefits of its modulation in clinical trials. Nonetheless, a detailed molecular analysis of important protein features, such as the origin of its ion selectivity and the rather low conductance as compared to other channel homologues, has been unfeasible until the recent crystallization of the mouse 5-HT3A receptor. Here, we present extended molecular dynamics simulations and free energy calculations of the whole 5-HT3A protein with the aim of better understanding its ion transport properties, such as the pathways for ion permeation into the receptor body and the complex nature of the selectivity filter. Our investigation unravels previously unpredicted structural features of the 5-HT3A receptor, such as the existence of alternative intersubunit pathways for ion translocation at the interface between the extracellular and the transmembrane domains, in addition to the one along the channel main axis. Moreover, our study offers a molecular interpretation of the role played by an arginine triplet located in the intracellular domain on determining the characteristic low conductance of the 5-HT3A receptor, as evidenced in previous experiments. In view of these results, possible implications on other members of the superfamily are suggested. PMID:26465896

  3. Length and Amino Acid Sequence of Peptides Substituted for the 5-HT3A Receptor M3M4 Loop May Affect Channel Expression and Desensitization

    PubMed Central

    McKinnon, Nicole K.; Bali, Moez; Akabas, Myles H.

    2012-01-01

    5-HT3A receptors are pentameric neurotransmitter-gated ion channels in the Cys-loop receptor family. Each subunit contains an extracellular domain, four transmembrane segments (M1, M2, M3, M4) and a 115 residue intracellular loop between M3 and M4. In contrast, the M3M4 loop in prokaryotic homologues is <15 residues. To investigate the limits of M3M4 loop length and composition on channel function we replaced the 5-HT3A M3M4 loop with two to seven alanine residues (5-HT3A-An = 2–7). Mutants were expressed in Xenopus laevis oocytes and characterized using two electrode voltage clamp recording. All mutants were functional. The 5-HT EC50's were at most 5-fold greater than wild-type (WT). The desensitization rate differed significantly among the mutants. Desensitization rates for 5-HT3A-A2, 5-HT3A-A4, 5-HT3A-A6, and 5-HT3A-A7 were similar to WT. In contrast, 5-HT3A-A3 and 5-HT3A-A5 had desensitization rates at least an order of magnitude faster than WT. The one Ala loop construct, 5-HT3A-A1, entered a non-functional state from which it did not recover after the first 5-HT application. These results suggest that the large M3M4 loop of eukaryotic Cys-loop channels is not required for receptor assembly or function. However, loop length and amino acid composition can effect channel expression and desensitization. We infer that the cytoplasmic ends of the M3 and M4 segments may undergo conformational changes during channel gating and desensitization and/or the loop may influence the position and mobility of these segments as they undergo gating-induced conformational changes. Altering structure or conformational mobility of the cytoplasmic ends of M3 and M4 may be the basis by which phosphorylation or protein binding to the cytoplasmic loop alters channel function. PMID:22539982

  4. Agonist- and antagonist-induced up-regulation of surface 5-HT3A receptors

    PubMed Central

    Morton, Russell A; Baptista-Hon, Daniel T; Hales, Tim G; Lovinger, David M

    2015-01-01

    Background and Purpose The 5-HT3 receptor is a member of the pentameric ligand-gated ion channel family and is pharmacologically targeted to treat irritable bowel syndrome and nausea/emesis. Furthermore, many antidepressants elevate extracellular concentrations of 5-HT. This study investigates the functional consequences of exposure of recombinant 5-HT3A receptors to agonists and antagonists. Experimental Approach We used HEK cells stably expressing recombinant 5-HT3A receptors and the ND7/23 (mouse neuroblastoma/dorsal root ganglion hybrid) cell line, which expresses endogenous 5-HT3 receptors. Surface expression of recombinant 5-HT3A receptors, modified to contain the bungarotoxin (BTX) binding sequence, was quantified using fluorescence microscopy to image BTX-conjugated fluorophores. Whole cell voltage-clamp electrophysiology was used to measure the density of current mediated by 5-HT3A receptors. Key Results 5-HT3A receptors were up-regulated by the prolonged presence of agonists (5-HT and m-chlorophenylbiguanide) and antagonists (MDL-72222 and morphine). The up-regulation of 5-HT3A receptors by 5-HT and MDL-72222 was time- and concentration-dependent but was independent of newly translated receptors. The phenomenon was observed for recombinant rodent and human 5-HT3A receptors and for endogenous 5-HT3 receptors in neuronal ND7/23 cells. Conclusions and Implications Up-regulation of 5-HT3A receptors, following exposure to either agonists or antagonists suggests that this phenomenon may occur in response to different therapeutic agents. Medications that elevate 5-HT levels, such as the antidepressant inhibitors of 5-HT reuptake and antiemetic inhibitors of 5-HT3 receptor function, may both raise receptor expression. However, this will require further investigation in vivo. PMID:25989383

  5. Fluorophore assisted light inactivation (FALI) of recombinant 5-HT3A receptor constitutive internalization and function

    PubMed Central

    Morton, Russell A.; Luo, Guoxiang; Davis, Margaret I.; Hales, Tim G.; Lovinger, David M.

    2011-01-01

    Fluorescent proteins and molecules are now widely used to tag and visualize proteins resulting in an improved understanding of protein trafficking, localization, and function. In addition, fluorescent tags have also been used to inactivate protein function in a spatially and temporally-defined manner, using a technique known as fluorophore-assisted light inactivation (FALI) or chromophore-assisted light inactivation (CALI). In this study we tagged the serotonin3 A subunit with the α-bungarotoxin binding sequence (BBS) and subsequently labeled 5-HT3A/BBS receptors with fluorescently conjugated α-bungarotoxin in live cells. We show that 5-HT3A/BBS receptors are constitutively internalized in the absence of an agonist and internalization as well as receptor function are inhibited by fluorescence. The fluorescence-induced disruption of function and internalization was reduced with oxygen radical scavengers suggesting the involvement of reactive oxygen species, implicating the FALI process. Furthermore, these data suggest that intense illumination during live-cell microscopy may result in inadvertent FALI and inhibition of protein trafficking. PMID:21338684

  6. Influence of the 5-HT3A Receptor Gene Polymorphism and Childhood Sexual Trauma on Central Serotonin Activity

    PubMed Central

    Huh, Hyu Jung; Chae, Jeong-Ho

    2015-01-01

    Background Gene-environment interactions are important for understanding alterations in human brain function. The loudness dependence of auditory evoked potential (LDAEP) is known to reflect central serotonergic activity. Single nucleotide polymorphisms (SNPs) in the 5-HT3A serotonin receptor gene are associated with psychiatric disorders. This study aimed to investigate the effect between 5-HT3A receptor gene polymorphisms and childhood sexual trauma on the LDAEP as an electrophysiological marker in healthy subjects. Methods A total of 206 healthy subjects were recruited and evaluated using the childhood trauma questionnaire (CTQ) and hospital anxiety and depression scale (HADS). Peak-to-peak N1/P2 was measured at five stimulus intensities, and the LDAEP was calculated as the linear-regression slope. In addition, the rs1062613 SNPs of 5-HT3A (CC, CT, and TT) were analyzed in healthy subjects. Results There was a significant interaction between scores on the CTQ-sexual abuse subscale and 5-HT3A genotype on the LDAEP. Subjects with the CC polymorphism had a significantly higher LDEAP than T carriers in the sexually abused group. In addition, CC genotype subjects in the sexually abused group showed a significantly higher LDAEP compared with CC genotype subjects in the non-sexually abused group. Conclusions Our findings suggest that people with the CC polymorphism of the 5-HT3A gene have a greater risk of developing mental health problems if they have experienced childhood sexual abuse, possibly due to low central serotonin activity. Conversely, the T polymorphism may be protective against any central serotonergic changes following childhood sexual trauma. PMID:26701104

  7. Kampo Medicine: Evaluation of the Pharmacological Activity of 121 Herbal Drugs on GABAA and 5-HT3A Receptors.

    PubMed

    Hoffmann, Katrin M; Herbrechter, Robin; Ziemba, Paul M; Lepke, Peter; Beltrán, Leopoldo; Hatt, Hanns; Werner, Markus; Gisselmann, Günter

    2016-01-01

    Kampo medicine is a form of Japanese phytotherapy originating from traditional Chinese medicine (TCM). During the last several decades, much attention has been paid to the pharmacological effects of these medical plants and their constituents. However, in many cases, a systematic screening of Kampo remedies to determine pharmacologically relevant targets is still lacking. In this study, a broad screening of Kampo remedies was performed to look for pharmacologically relevant 5-HT3A and GABAA receptor ligands. Several of the Kampo remedies are currently used for symptoms such as nausea, emesis, gastrointestinal motility disorders, anxiety, restlessness, or insomnia. Therefore, the pharmacological effects of 121 herbal drugs from Kampo medicine were analyzed as ethanol tinctures on heterologously expressed 5-HT3A and GABAA receptors, due to the involvement of these receptors in such pathophysiological processes. The tinctures of Lindera aggregata (radix) and Leonurus japonicus (herba) were the most effective inhibitory compounds on the 5-HT3A receptor. Further investigation of known ingredients in these compounds led to the identification of leonurine from Leonurus as a new natural 5-HT3A receptor antagonist. Several potentiating herbs (e.g., Magnolia officinalis (cortex), Syzygium aromaticum (flos), and Panax ginseng (radix)) were also identified for the GABAA receptor, which are all traditionally used for their sedative or anxiolytic effects. A variety of tinctures with antagonistic effects Salvia miltiorrhiza (radix) were also detected. Therefore, this study reveals new insights into the pharmacological action of a broad spectrum of herbal drugs from Kampo, allowing for a better understanding of their physiological effects and clinical applications. PMID:27524967

  8. Kampo Medicine: Evaluation of the Pharmacological Activity of 121 Herbal Drugs on GABAA and 5-HT3A Receptors

    PubMed Central

    Hoffmann, Katrin M.; Herbrechter, Robin; Ziemba, Paul M.; Lepke, Peter; Beltrán, Leopoldo; Hatt, Hanns; Werner, Markus; Gisselmann, Günter

    2016-01-01

    Kampo medicine is a form of Japanese phytotherapy originating from traditional Chinese medicine (TCM). During the last several decades, much attention has been paid to the pharmacological effects of these medical plants and their constituents. However, in many cases, a systematic screening of Kampo remedies to determine pharmacologically relevant targets is still lacking. In this study, a broad screening of Kampo remedies was performed to look for pharmacologically relevant 5-HT3A and GABAA receptor ligands. Several of the Kampo remedies are currently used for symptoms such as nausea, emesis, gastrointestinal motility disorders, anxiety, restlessness, or insomnia. Therefore, the pharmacological effects of 121 herbal drugs from Kampo medicine were analyzed as ethanol tinctures on heterologously expressed 5-HT3A and GABAA receptors, due to the involvement of these receptors in such pathophysiological processes. The tinctures of Lindera aggregata (radix) and Leonurus japonicus (herba) were the most effective inhibitory compounds on the 5-HT3A receptor. Further investigation of known ingredients in these compounds led to the identification of leonurine from Leonurus as a new natural 5-HT3A receptor antagonist. Several potentiating herbs (e.g., Magnolia officinalis (cortex), Syzygium aromaticum (flos), and Panax ginseng (radix)) were also identified for the GABAA receptor, which are all traditionally used for their sedative or anxiolytic effects. A variety of tinctures with antagonistic effects Salvia miltiorrhiza (radix) were also detected. Therefore, this study reveals new insights into the pharmacological action of a broad spectrum of herbal drugs from Kampo, allowing for a better understanding of their physiological effects and clinical applications. PMID:27524967

  9. Investigation of 5-HT3A receptor gene expression in peripheral blood mononuclear cells of individuals who had been exposed to air pollution.

    PubMed

    Ahangari, Ghasem; Amirabad, Leila Mohammadi; Mozafari, Sona; Majeidi, Ali; Deilami, Gholamreza Derkhshan

    2013-12-01

    The role of air pollution in exacerbation of allergic symptoms is well known. Several studies have shown the effect of air pollution on serotonergic system. The changes in serotonergic system could trigger several allergic symptoms. 5-HT(3A) is among serotonin receptors on the peripheral Blood Mononuclear Cells (PBMCs) as well as other cells. In the present study we compared the 5-HT(3A) gene expression in PBMCs of the asthmatic patients as well as individuals who had been exposed to the air pollution. Normal individuals were also included in the study as control for comparison of 5-HT(3A) gene expression. Following the synthesis of the cDNA using mRNA extracted from PBMCs the level of 5- HT(3A) gene expression was measured using real-time PCR. The results showed t a significant increase in the relative expression level of 5-HT(3A) receptor in PBMCs from asthmatic patients and individuals exposed to the air pollutants compared to normal controls. Our result indicates that significant increase in 5-HT(3A) receptor may contribute to the pathogenesis as well as allergic symptoms which resulted from air pollution.

  10. Regulation of the 5-HT3A receptor-mediated current by alkyl 4-hydroxybenzoates isolated from the seeds of Nelumbo nucifera.

    PubMed

    Youn, Ui Joung; Lee, Jun-Ho; Lee, Yoo Jin; Nam, Joo Won; Bae, Hyunsu; Seo, Eun-Kyoung

    2010-09-01

    Four known alkyl 4-hydroxybenzoates, i.e., methyl 4-hydroxybenzoate (1), ethyl 4-hydroxybenzoate (2), propyl 4-hydroxybenzoate (3), and butyl 4-hydroxybenzoate (4), were isolated from the seeds of Nelumbo nucifera Gaertner (Nymphaeaceae) for the first time. The structures of the isolates were identified by 1D- and 2D-NMR spectroscopy and comparison with published values. The compounds were evaluated for their effects on the 5-HT-stimulated inward current (I(5-HT)) mediated by the human 5-HT(3)A receptors expressed in Xenopus oocytes. Compounds 1 and 2 enhanced the I(5-HT), but 4 reduced it. These results indicate that 4 is an inhibitor of the 5-HT(3)A receptors expressed in Xenopus oocytes.

  11. Regulation of the 5-HT3A receptor-mediated current by alkyl 4-hydroxybenzoates isolated from the seeds of Nelumbo nucifera.

    PubMed

    Youn, Ui Joung; Lee, Jun-Ho; Lee, Yoo Jin; Nam, Joo Won; Bae, Hyunsu; Seo, Eun-Kyoung

    2010-09-01

    Four known alkyl 4-hydroxybenzoates, i.e., methyl 4-hydroxybenzoate (1), ethyl 4-hydroxybenzoate (2), propyl 4-hydroxybenzoate (3), and butyl 4-hydroxybenzoate (4), were isolated from the seeds of Nelumbo nucifera Gaertner (Nymphaeaceae) for the first time. The structures of the isolates were identified by 1D- and 2D-NMR spectroscopy and comparison with published values. The compounds were evaluated for their effects on the 5-HT-stimulated inward current (I(5-HT)) mediated by the human 5-HT(3)A receptors expressed in Xenopus oocytes. Compounds 1 and 2 enhanced the I(5-HT), but 4 reduced it. These results indicate that 4 is an inhibitor of the 5-HT(3)A receptors expressed in Xenopus oocytes. PMID:20860031

  12. Serotonin homeostasis and serotonin receptors as actors of cortical construction: special attention to the 5-HT3A and 5-HT6 receptor subtypes

    PubMed Central

    Vitalis, Tania; Ansorge, Mark S.; Dayer, Alexandre G.

    2013-01-01

    Cortical circuits control higher-order cognitive processes and their function is highly dependent on their structure that emerges during development. The construction of cortical circuits involves the coordinated interplay between different types of cellular processes such as proliferation, migration, and differentiation of neural and glial cell subtypes. Among the multiple factors that regulate the assembly of cortical circuits, 5-HT is an important developmental signal that impacts on a broad diversity of cellular processes. 5-HT is detected at the onset of embryonic telencephalic formation and a variety of serotonergic receptors are dynamically expressed in the embryonic developing cortex in a region and cell-type specific manner. Among these receptors, the ionotropic 5-HT3A receptor and the metabotropic 5-HT6 receptor have recently been identified as novel serotonergic targets regulating different aspects of cortical construction including neuronal migration and dendritic differentiation. In this review, we focus on the developmental impact of serotonergic systems on the construction of cortical circuits and discuss their potential role in programming risk for human psychiatric disorders. PMID:23801939

  13. Design and validation of a homogeneous time-resolved fluorescence cell-based assay targeting the ligand-gated ion channel 5-HT3A.

    PubMed

    Blanc, Emilie; Wagner, Patrick; Plaisier, Fabrice; Schmitt, Martine; Durroux, Thierry; Bourguignon, Jean-Jacques; Partiseti, Michel; Dupuis, Elodie; Bihel, Frederic

    2015-09-01

    Ligand-gated ion channels (LGICs) are considered as attractive protein targets in the search for new therapeutic agents. Nowadays, this strategy involves the capability to screen large chemical libraries. We present a new Tag-lite ligand binding assay targeting LGICs on living cells. This technology combines the use of suicide enzyme tags fused to channels of interest with homogeneous time-resolved fluorescence (HTRF) as the detection readout. Using the 5-HT3 receptor as system model, we showed that the pharmacology of the HALO-5HT3 receptor was identical to that of the native receptor. After validation of the assay by using 5-HT3 agonists and antagonists of reference, a pilot screen enabled us to identify azelastine, a well-known histamine H1 antagonist, as a potent 5-HT3 antagonist. This interesting result was confirmed with electrophysiological experiments. The method described here is easy to implement and could be applicable for other LGICs, opening new ways for the screening of chemical libraries. PMID:25998104

  14. Electrical interfacing of neurotransmitter receptor and field effect transistor

    NASA Astrophysics Data System (ADS)

    Peitz, I.; Fromherz, P.

    2009-10-01

    The interfacing of a ligand-gated ion channel to a transistor is studied. It relies on the transduction of ion current to a voltage in a cell-transistor junction. For the first time, a genetically modified cell is used without external driving voltage as applied by a patch-pipette. Using a core-coat conductor model, we show that an autonomous dynamics gives rise to a signal if a driving voltage is provided by potassium channels, and if current compensation is avoided by an inhomogeneous activation of channels. In a proof-of-principle experiment, we transfect HEK293 cells with the serotonin receptor 5-HT3A and the potassium channel Kv1.3. The interfacing is characterized under voltage-clamp with a negative transistor signal for activated 5-HT3A and a positive signal for activated Kv1.3. Without patch-pipette, a biphasic transient is induced by serotonin. The positive wave is assigned to 5-HT3A receptors in the free membrane that drive a potassium outward current through the adherent membrane. The negative wave is attributed to 5-HT3A receptors in the adherent membrane that are activated with a delay due to serotonin diffusion. The implementation of a receptor-cell-transistor device is a fundamental step in the development of biosensors that combine high specificity and universal microelectronic readout.

  15. Expression of 5-HT3 receptors and TTX resistant sodium channels (NaV1.8) on muscle nerve fibers in pain-free humans and patients with chronic myofascial temporomandibular disorders

    PubMed Central

    2014-01-01

    Background Previous studies have shown that 5-HT3-antagonists reduce muscle pain, but there are no studies that have investigated the expression of 5-HT3-receptors in human muscles. Also, tetrodotoxin resistant voltage gated sodium-channels (NaV) are involved in peripheral sensitization and found in trigeminal ganglion neurons innervating the rat masseter muscle. This study aimed to investigate the frequency of nerve fibers that express 5-HT3A-receptors alone and in combination with NaV1.8 sodium-channels in human muscles and to compare it between healthy pain-free men and women, the pain-free masseter and tibialis anterior muscles, and patients with myofascial temporomandibular disorders (TMD) and pain-free controls. Methods Three microbiopsies were obtained from the most bulky part of the tibialis and masseter muscles of seven and six healthy men and seven and six age-matched healthy women, respectively, while traditional open biopsies were obtained from the most painful spot of the masseter of five female patients and from a similar region of the masseter muscle of five healthy, age-matched women. The biopsies were processed by routine immunohistochemical methods. The biopsy sections were incubated with monoclonal antibodies against the specific axonal marker PGP 9.5, and polyclonal antibodies against the 5-HT3A-receptors and NaV1.8 sodium-channels. Results A similar percentage of nerve fibers in the healthy masseter (85.2%) and tibialis (88.7%) muscles expressed 5-HT3A-receptors. The expression of NaV1.8 by 5-HT3A positive nerve fibers associated with connective tissue was significantly higher than nerve fibers associated with myocytes (P < .001). In the patients, significantly more fibers per section were found with an average of 3.8 ± 3 fibers per section in the masseter muscle compared to 2.7 ± 0.2 in the healthy controls (P = .024). Further, the frequency of nerve fibers that co-expressed NaV1.8 and 5-HT3A receptors was significantly

  16. Activation and modulation of recombinantly expressed serotonin receptor type 3A by terpenes and pungent substances.

    PubMed

    Ziemba, Paul M; Schreiner, Benjamin S P; Flegel, Caroline; Herbrechter, Robin; Stark, Timo D; Hofmann, Thomas; Hatt, Hanns; Werner, Markus; Gisselmann, Günter

    2015-11-27

    Serotonin receptor type 3 (5-HT3 receptor) is a ligand-gated ion channel that is expressed in the central nervous system (CNS) as well as in the peripheral nervous system (PNS). The receptor plays an important role in regulating peristalsis of the gastrointestinal tract and in functions such as emesis, cognition and anxiety. Therefore, a variety of pharmacologically active substances target the 5-HT3 receptor to treat chemotherapy-induced nausea and vomiting. The 5-HT3 receptors are activated, antagonized, or modulated by a wide range of chemically different substances, such as 2-methyl-serotonin, phenylbiguanide, setrones, or cannabinoids. Whereas the action of all of these substances is well described, less is known about the effect of terpenoids or fragrances on 5-HT3A receptors. In this study, we screened a large number of natural odorous and pungent substances for their pharmacological action on recombinantly expressed human 5-HT3A receptors. The receptors were functionally expressed in Xenopus oocytes and characterized by electrophysiological recordings using the two-electrode voltage-clamp technique. A screening of two odorous mixes containing a total of 200 substances revealed that the monoterpenes, thymol and carvacrol, act as both weak partial agonists and positive modulators on the 5-HT3A receptor. In contrast, the most effective blockers were the terpenes, citronellol and geraniol, as well as the pungent substances gingerol, capsaicin and polygodial. In our study, we identified new modulators of 5-HT3A receptors out of the classes of monoterpenes and vanilloid substances that frequently occur in various plants. PMID:26456648

  17. Cloning and expression of ligand-gated ion-channel receptor L2 in central nervous system

    SciTech Connect

    Houtani, Takeshi; Munemoto, Yumi; Kase, Masahiko; Sakuma, Satoru; Tsutsumi, Toshiyuki; Sugimoto, Tetsuo . E-mail: sugimoto@takii.kmu.ac.jp

    2005-09-23

    An orphan receptor of ligand-gated ion-channel type (L2, also termed ZAC according to the presence of zinc ion for channel activation) was identified by computer-assisted search programs on human genome database. The L2 protein shares partial homology with serotonin receptors 5HT3A and 5HT3B. We have cloned L2 cDNA derived from human caudate nucleus and characterized the exon-intron structure as follows: (1) The L2 protein has four transmembrane regions (M1-M4) and a long cytoplasmic loop between M3 and M4. (2) The sequence is conserved in species including chimpanzee, dog, cow, and opossum. (3) Nine exons form its protein-coding region and especially exon 5 corresponds to a disulfide bond region on the amino-terminal side. Our analysis using multiple tissue cDNA panels revealed that at least two splicing variants of L2 mRNA are present. The cDNA PCR amplification study revealed that L2 mRNA is expressed in tissues including brain, pancreas, liver, lung, heart, kidney, and skeletal muscle while 5HT3A mRNA could be detected in brain, heart, placenta, lung, kidney, pancreas, and skeletal muscle, and 5HT3B mRNA in brain, kidney, and skeletal muscle, suggesting different significance in tissue expression of these receptors. Regional expression of L2 mRNA and protein was examined in brain. The RT-PCR studies confirmed L2 mRNA expression in hippocampus, striatum, amygdala, and thalamus in adult brain. The L2 protein was immunolocalized by using antipeptide antibodies. Immunostained tissue sections revealed that L2-like immunoreactivity was dominantly expressed in the hippocampal CA3 pyramidal cells and in the polymorphic layer of the dentate gyrus. We analyzed the expression of L2 protein in HEK293 cells using GFP fusion protein reporter system. Western blots revealed that L2 protein confers sugar chains on the extracellular side. In transfected HEK293 cells, cellular membranes and intracellular puncta were densely labeled with GFP, suggesting selective dispatch to the

  18. Pentameric quaternary structure of the intracellular domain of serotonin type 3A receptors

    PubMed Central

    Pandhare, Akash; Grozdanov, Petar N.; Jansen, Michaela

    2016-01-01

    In spite of extensive efforts over decades an experimentally-derived structure of full-length eukaryotic pentameric ligand-gated ion channels (pLGICs) is still lacking. These pharmaceutically highly-relevant channels contain structurally well-conserved and characterized extracellular and transmembrane domains. The intracellular domain (ICD), however, has been orphaned in structural studies based on the consensus assumption of being largely disordered. In the present study, we demonstrate for the first time that the serotonin type 3A (5-HT3A) ICD assembles into stable pentamers in solution in the absence of the other two domains, thought to be the drivers for oligomerization. Additionally, the soluble 5-HT3A-ICD construct interacted with the protein RIC-3 (resistance to inhibitors of cholinesterase). The interaction provides evidence that the 5-HT3A-ICD is not only required but also sufficient for interaction with RIC-3. Our results suggest the ICD constitutes an oligomerization domain. This novel role significantly adds to its known contributions in receptor trafficking, targeting, and functional fine-tuning. The innate diversity of the ICDs with sizes ranging from 50 to 280 amino acids indicates new methodologies need to be developed to determine the structures of these domains. The use of soluble ICD proteins that we report in the present study constitutes a useful approach to address this gap. PMID:27045630

  19. Calcium Channel Signaling Complexes with Receptors and Channels.

    PubMed

    Zamponi, Gerald W

    2015-01-01

    Voltage-gated calcium channels are not only mediators of cell signalling events, but also are recipients of signalling inputs from G protein coupled receptors (GPCRs) and their associated second messenger pathways. The coupling of GPCRs to calcium channels is optimized through the formation of receptor-channel complexes. In addition, this provides a mechanism for receptorchannel co-trafficking to and from the plasma membrane. On the other hand, voltage-gated calcium channel activity affects other types of ion channels such as voltage-and calcium-activated potassium channels. Coupling efficiency between these two families of channels is also enhanced through the formation of channel-channel complexes. This review provides a concise overview of the current state of knowledge on the physical interactions between voltage-gated calcium channels and members of the GPCR family, and with other types of ion channels.

  20. Impact of intracellular domain flexibility upon properties of activated human 5-HT3 receptors*

    PubMed Central

    Kozuska, J L; Paulsen, I M; Belfield, W J; Martin, I L; Cole, D J; Holt, A; Dunn, S M J

    2014-01-01

    Background and Purpose It has been proposed that arginine residues lining the intracellular portals of the homomeric 5-HT3A receptor cause electrostatic repulsion of cation flow, accounting for a single-channel conductance substantially lower than that of the 5-HT3AB heteromer. However, comparison of receptor homology models for wild-type pentamers suggests that salt bridges in the intracellular domain of the homomer may impart structural rigidity, and we hypothesized that this rigidity could account for the low conductance. Experimental Approach Mutations were introduced into the portal region of the human 5-HT3A homopentamer, such that putative salt bridges were broken by neutralizing anionic partners. Single-channel and whole cell currents were measured in transfected tsA201 cells and in Xenopus oocytes respectively. Computational simulations of protein flexibility facilitated comparison of wild-type and mutant receptors. Key Results Single-channel conductance was increased substantially, often to wild-type heteromeric receptor values, in most 5-HT3A mutants. Conversely, introduction of arginine residues to the portal region of the heteromer, conjecturally creating salt bridges, decreased conductance. Gating kinetics varied significantly between different mutant receptors. EC50 values for whole-cell responses to 5-HT remained largely unchanged, but Hill coefficients for responses to 5-HT were usually significantly smaller in mutants. Computational simulations suggested increased flexibility throughout the protein structure as a consequence of mutations in the intracellular domain. Conclusions and Implications These data support a role for intracellular salt bridges in maintaining the quaternary structure of the 5-HT3 receptor and suggest a role for the intracellular domain in allosteric modulation of cooperativity and agonist efficacy. Linked Article This article is commented on by Vardy and Kenakin, pp. 1614–1616 of volume 171 issue 7. To view this commentary

  1. Dipicrylamine Modulates GABAρ1 Receptors through Interactions with Residues in the TM4 and Cys-Loop Domains.

    PubMed

    Limon, Agenor; Estrada-Mondragón, Argel; Ruiz, Jorge M Reyes; Miledi, Ricardo

    2016-04-01

    Dipicrylamine (DPA) is a commonly used acceptor agent in Förster resonance energy transfer experiments that allows the study of high-frequency neuronal activity in the optical monitoring of voltage in living cells. However, DPA potently antagonizes GABAA receptors that contain α1 and β2 subunits by a mechanism which is not clearly understood. In this work, we aimed to determine whether DPA modulation is a general phenomenon of Cys-loop ligand-gated ion channels (LGICs), and whether this modulation depends on particular amino acid residues. For this, we studied the effects of DPA on human homomeric GABAρ1, α7 nicotinic, and 5-HT3A serotonin receptors expressed in Xenopus oocytes. Our results indicate that DPA is an allosteric modulator of GABAρ1 receptors with an IC50 of 1.6 µM, an enhancer of α7 nicotinic receptors at relatively high concentrations of DPA, and has little, if any, effect on 5-HT3A receptors. DPA antagonism of GABAρ1 was strongly enhanced by preincubation, was slightly voltage-dependent, and its washout was accelerated by bovine serum albumin. These results indicate that DPA modulation is not a general phenomenon of LGICs, and structural differences between receptors may account for disparities in DPA effects. In silico modeling of DPA docking to GABAρ1, α7 nicotinic, and 5-HT3A receptors suggests that a hydrophobic pocket within the Cys-loop and the M4 segment in GABAρ1, located at the extracellular/membrane interface, facilitates the interaction with DPA that leads to inhibition of the receptor. Functional examinations of mutant receptors support the involvement of the M4 segment in the allosteric modulation of GABAρ1 by DPA. PMID:26869399

  2. Endogenous ion channel complexes: the NMDA receptor.

    PubMed

    Frank, René A W

    2011-06-01

    Ionotropic receptors, including the NMDAR (N-methyl-D-aspartate receptor) mediate fast neurotransmission, neurodevelopment, neuronal excitability and learning. In the present article, the structure and function of the NMDAR is reviewed with the aim to condense our current understanding and highlight frontiers where important questions regarding the biology of this receptor remain unanswered. In the second part of the present review, new biochemical and genetic approaches for the investigation of ion channel receptor complexes will be discussed.

  3. Transient Receptor Potential Channels in the Vasculature

    PubMed Central

    Earley, Scott; Brayden, Joseph E.

    2015-01-01

    The mammalian genome encodes 28 distinct members of the transient receptor potential (TRP) superfamily of cation channels, which exhibit varying degrees of selectivity for different ionic species. Multiple TRP channels are present in all cells and are involved in diverse aspects of cellular function, including sensory perception and signal transduction. Notably, TRP channels are involved in regulating vascular function and pathophysiology, the focus of this review. TRP channels in vascular smooth muscle cells participate in regulating contractility and proliferation, whereas endothelial TRP channel activity is an important contributor to endothelium-dependent vasodilation, vascular wall permeability, and angiogenesis. TRP channels are also present in perivascular sensory neurons and astrocytic endfeet proximal to cerebral arterioles, where they participate in the regulation of vascular tone. Almost all of these functions are mediated by changes in global intracellular Ca2+ levels or subcellular Ca2+ signaling events. In addition to directly mediating Ca2+ entry, TRP channels influence intracellular Ca2+ dynamics through membrane depolarization associated with the influx of cations or through receptor- or store-operated mechanisms. Dysregulation of TRP channels is associated with vascular-related pathologies, including hypertension, neointimal injury, ischemia-reperfusion injury, pulmonary edema, and neurogenic inflammation. In this review, we briefly consider general aspects of TRP channel biology and provide an in-depth discussion of the functions of TRP channels in vascular smooth muscle cells, endothelial cells, and perivascular cells under normal and pathophysiological conditions. PMID:25834234

  4. A cluster of novel serotonin receptor 3-like genes on human chromosome 3.

    PubMed

    Karnovsky, Alla M; Gotow, Lisa F; McKinley, Denise D; Piechan, Julie L; Ruble, Cara L; Mills, Cynthia J; Schellin, Kathleen A B; Slightom, Jerry L; Fitzgerald, Laura R; Benjamin, Christopher W; Roberds, Steven L

    2003-11-13

    The ligand-gated ion channel family includes receptors for serotonin (5-hydroxytryptamine, 5-HT), acetylcholine, GABA, and glutamate. Drugs targeting subtypes of these receptors have proven useful for the treatment of various neuropsychiatric and neurological disorders. To identify new ligand-gated ion channels as potential therapeutic targets, drafts of human genome sequence were interrogated. Portions of four novel genes homologous to 5-HT(3A) and 5-HT(3B) receptors were identified within human sequence databases. We named the genes 5-HT(3C1)-5-HT(3C4). Radiation hybrid (RH) mapping localized these genes to chromosome 3q27-28. All four genes shared similar intron-exon organizations and predicted protein secondary structure with 5-HT(3A) and 5-HT(3B). Orthologous genes were detected by Southern blotting in several species including dog, cow, and chicken, but not in rodents, suggesting that these novel genes are not present in rodents or are very poorly conserved. Two of the novel genes are predicted to be pseudogenes, but two other genes are transcribed and spliced to form appropriate open reading frames. The 5-HT(3C1) transcript is expressed almost exclusively in small intestine and colon, suggesting a possible role in the serotonin-responsiveness of the gut.

  5. Integrin receptors and ligand-gated channels.

    PubMed

    Morini, Raffaella; Becchetti, Andrea

    2010-01-01

    Plastic expression of different integrin subunits controls the different stages of neural development, whereas in the adult integrins regulate synaptic stability. Evidence of integrin-channel crosstalk exists for ionotropic glutamate receptors. As is often the case in other tissues, integrin engagement regulates channel activity through complex signaling pathways that often include tyrosine phosphorylation cascades. The specific pathways recruited by integrin activation depend on cerebral region and cell type. In turn, ion channels control integrin expression onto the plasma membrane and their ligand binding affinity. The most extensive studies concern the hippocampus and suggest implications for neuronal circuit plasticity. The physiological relevance of these findings depends on whether adhesion molecules, aside from determining tissue stability, contribute to synaptogenesis and the responsiveness of mature synapses, thus contributing to long-term circuit consolidation. Little evidence is available for other ligand-gated channels, with the exception of nicotinic receptors. These exert a variety of functions in neurons and non neural tissue, both in development and in the adult, by regulating cell cycle, synaptogenesis and synaptic circuit refinement. Detailed studies in epidermal keratinocytes have shed some light on the possible mechanisms through which ACh can regulate cell motility, which may be of general relevance for morphogenetic processes. As to the control of mature synapses, most results concern the integrinic control of nicotinic receptors in the neuromuscular junction. Following this lead, a few studies have addressed similar topics in adult cerebral synapses. However, pursuing and interpreting these results in the brain is especially difficult because of the complexity of the nicotinic roles and the widespread contribution of nonsynaptic, paracrine transmission. From a pathological point of view, considering the well-known contribution of both

  6. Effect of homologous serotonin receptor loop substitutions on the heterologous expression in Pichia of a chimeric acetylcholine-binding protein with alpha-bungarotoxin-binding activity.

    PubMed

    Paulo, Joao A; Hawrot, Edward

    2009-10-01

    The molluscan acetylcholine-binding protein (AChBP) is a soluble homopentameric homolog of the extracellular domain of various ligand-gated ion channels. Previous studies have reported that AChBP, when fused to the ion pore domain of the serotonin receptor (5HT(3A)R), can form a functional ligand-gated chimeric channel only if the AChBP loop regions between beta-strands beta1 and beta2 (beta1-beta2), beta6 and beta7 (beta6-beta7), and beta8 and beta9 (beta8-beta9) are replaced with those of the 5HT(3A)R. To investigate further the potential interactions among these three important loop regions in a membrane- and detergent-free system, we designed AChBP constructs in which loops beta1-beta2, beta6-beta7, and beta8-beta9 of the AChBP were individually and combinatorially substituted in all permutations with the analogous loops of the 5HT(3A)R. These chimeras were expressed as secreted proteins using the Pichia pastoris yeast expression system. [(125)I]-alpha-Bungarotoxin-binding was detected in the culture media obtained from homologous recombinant clones expressing the wild-type AChBP, the beta1-beta2 loop-only chimera, and the chimera containing all three 5HT(3A)R loop substitutions. The remaining chimeras failed to show [(125)I]-alpha-bungarotoxin binding, and further analysis of cellular extracts allowed us to determine that these binding-negative chimeric constructs accumulated intracellularly and were not secreted into the culture medium. Our results demonstrate that coordinated interactions among loops beta1-beta2, beta6-beta7, and beta8-beta9 are essential for the formation of a functional ligand-binding site, as evidenced by [(125)I]-alpha-bungarotoxin-binding, and for efficient protein secretion. In addition, the constructs described here demonstrate the feasibility of utilizing soluble scaffolds to explore functionally important interactions within the extracellular domain of membrane-bound proteins. PMID:19427904

  7. Inositol trisphosphate receptor and ion channel models based on single-channel data

    NASA Astrophysics Data System (ADS)

    Gin, Elan; Wagner, Larry E.; Yule, David I.; Sneyd, James

    2009-09-01

    The inositol trisphosphate receptor (IPR) plays an important role in controlling the dynamics of intracellular Ca2+. Single-channel patch-clamp recordings are a typical way to study these receptors as well as other ion channels. Methods for analyzing and using this type of data have been developed to fit Markov models of the receptor. The usual method of parameter fitting is based on maximum-likelihood techniques. However, Bayesian inference and Markov chain Monte Carlo techniques are becoming more popular. We describe the application of the Bayesian methods to real experimental single-channel data in three ion channels: the ryanodine receptor, the K+ channel, and the IPR. One of the main aims of all three studies was that of model selection with different approaches taken. We also discuss the modeling implications for single-channel data that display different levels of channel activity within one recording.

  8. Crystal structure of a heterotetrameric NMDA receptor ion channel.

    PubMed

    Karakas, Erkan; Furukawa, Hiro

    2014-05-30

    N-Methyl-D-aspartate (NMDA) receptors belong to the family of ionotropic glutamate receptors, which mediate most excitatory synaptic transmission in mammalian brains. Calcium permeation triggered by activation of NMDA receptors is the pivotal event for initiation of neuronal plasticity. Here, we show the crystal structure of the intact heterotetrameric GluN1-GluN2B NMDA receptor ion channel at 4 angstroms. The NMDA receptors are arranged as a dimer of GluN1-GluN2B heterodimers with the twofold symmetry axis running through the entire molecule composed of an amino terminal domain (ATD), a ligand-binding domain (LBD), and a transmembrane domain (TMD). The ATD and LBD are much more highly packed in the NMDA receptors than non-NMDA receptors, which may explain why ATD regulates ion channel activity in NMDA receptors but not in non-NMDA receptors.

  9. Allosterism and Structure in Thermally Activated Transient Receptor Potential Channels.

    PubMed

    Diaz-Franulic, Ignacio; Poblete, Horacio; Miño-Galaz, Germán; González, Carlos; Latorre, Ramón

    2016-07-01

    The molecular sensors that mediate temperature changes in living organisms are a large family of proteins known as thermosensitive transient receptor potential (TRP) ion channels. These membrane proteins are polymodal receptors that can be activated by cold or hot temperatures, depending on the channel subtype, voltage, and ligands. The stimuli sensors are allosterically coupled to a pore domain, increasing the probability of finding the channel in its ion conductive conformation. In this review we first discuss the allosteric coupling between the temperature and voltage sensor modules and the pore domain, and then discuss the thermodynamic foundations of thermo-TRP channel activation. We provide a structural overview of the molecular determinants of temperature sensing. We also posit an anisotropic thermal diffusion model that may explain the large temperature sensitivity of TRP channels. Additionally, we examine the effect of several ligands on TRP channel function and the evidence regarding their mechanisms of action. PMID:27297398

  10. Zinc as Allosteric Ion Channel Modulator: Ionotropic Receptors as Metalloproteins.

    PubMed

    Peralta, Francisco Andrés; Huidobro-Toro, Juan Pablo

    2016-01-01

    Zinc is an essential metal to life. This transition metal is a structural component of many proteins and is actively involved in the catalytic activity of cell enzymes. In either case, these zinc-containing proteins are metalloproteins. However, the amino acid residues that serve as ligands for metal coordination are not necessarily the same in structural proteins compared to enzymes. While crystals of structural proteins that bind zinc reveal a higher preference for cysteine sulfhydryls rather than histidine imidazole rings, catalytic enzymes reveal the opposite, i.e., a greater preference for the histidines over cysteines for catalysis, plus the influence of carboxylic acids. Based on this paradigm, we reviewed the putative ligands of zinc in ionotropic receptors, where zinc has been described as an allosteric modulator of channel receptors. Although these receptors do not strictly qualify as metalloproteins since they do not normally bind zinc in structural domains, they do transitorily bind zinc at allosteric sites, modifying transiently the receptor channel's ion permeability. The present contribution summarizes current information showing that zinc allosteric modulation of receptor channels occurs by the preferential metal coordination to imidazole rings as well as to the sulfhydryl groups of cysteine in addition to the carboxyl group of acid residues, as with enzymes and catalysis. It is remarkable that most channels, either voltage-sensitive or transmitter-gated receptor channels, are susceptible to zinc modulation either as positive or negative regulators. PMID:27384555

  11. New insights into TRP channels: Interaction with pattern recognition receptors.

    PubMed

    Han, Huirong; Yi, Fan

    2014-01-01

    An increasing number of studies have implicated that the activation of innate immune system and inflammatory mechanisms are of importance in the pathogenesis of numerous diseases. The innate immune system is present in almost all multicellular organisms in response to pathogens or tissue injury, which is performed via germ-line encoded pattern-recognition receptors (PRRs) to recognize pathogen-associated molecular patterns (PAMPs) or dangers-associated molecular patterns (DAMPs). Intracellular pathways linking immune and inflammatory response to ion channel expression and function have been recently identified. Among ion channels, transient receptor potential (TRP) channels are a major family of non-selective cation-permeable channels that function as polymodal cellular sensors involved in many physiological and pathological processes. In this review, we summarize current knowledge about classifications, functions, and interactions of TRP channels and PRRs, which may provide new insights into their roles in the pathogenesis of inflammatory diseases.

  12. Calcium Channels and Associated Receptors in Malignant Brain Tumor Therapy.

    PubMed

    Morrone, Fernanda B; Gehring, Marina P; Nicoletti, Natália F

    2016-09-01

    Malignant brain tumors are highly lethal and aggressive. Despite recent advances in the current therapies, which include the combination of surgery and radio/chemotherapy, the average survival rate remains poor. Altered regulation of ion channels is part of the neoplastic transformation, which suggests that ion channels are involved in cancer. Distinct classes of calcium-permeable channels are abnormally expressed in cancer and are likely involved in the alterations underlying malignant growth. Specifically, cytosolic Ca(2+) activity plays an important role in the regulation of cell proliferation, and Ca(2+) signaling is altered in proliferating tumor cells. A series of previous studies emphasized the importance of the T-type low-voltage-gated calcium channels (VGCC) in different cancer types, including gliomas, and remarkably, pharmacologic inhibition of T-type VGCC caused antiproliferative effects and triggered apoptosis of human glioma cells. Other calcium permeable channels, such as transient receptor potential (TRP) channels, contribute to changes in Ca(2+) by modulating the driving force for Ca(2+) entry, and some TRP channels are required for proliferation and migration in gliomas. Furthermore, recent evidence shows that TRP channels contribute to the progression and survival of the glioblastoma patients. Likewise, the purinergic P2X7 receptor acts as a direct conduit for Ca(2+)-influx and an indirect activator of voltage-gated Ca(2+)-channel. Evidence also shows that P2X7 receptor activation is linked to elevated expression of inflammation promoting factors, tumor cell migration, an increase in intracellular mobilization of Ca(2+), and membrane depolarization in gliomas. Therefore, this review summarizes the recent findings on calcium channels and associated receptors as potential targets to treat malignant gliomas. PMID:27418672

  13. Zinc as Allosteric Ion Channel Modulator: Ionotropic Receptors as Metalloproteins

    PubMed Central

    Peralta, Francisco Andrés; Huidobro-Toro, Juan Pablo

    2016-01-01

    Zinc is an essential metal to life. This transition metal is a structural component of many proteins and is actively involved in the catalytic activity of cell enzymes. In either case, these zinc-containing proteins are metalloproteins. However, the amino acid residues that serve as ligands for metal coordination are not necessarily the same in structural proteins compared to enzymes. While crystals of structural proteins that bind zinc reveal a higher preference for cysteine sulfhydryls rather than histidine imidazole rings, catalytic enzymes reveal the opposite, i.e., a greater preference for the histidines over cysteines for catalysis, plus the influence of carboxylic acids. Based on this paradigm, we reviewed the putative ligands of zinc in ionotropic receptors, where zinc has been described as an allosteric modulator of channel receptors. Although these receptors do not strictly qualify as metalloproteins since they do not normally bind zinc in structural domains, they do transitorily bind zinc at allosteric sites, modifying transiently the receptor channel’s ion permeability. The present contribution summarizes current information showing that zinc allosteric modulation of receptor channels occurs by the preferential metal coordination to imidazole rings as well as to the sulfhydryl groups of cysteine in addition to the carboxyl group of acid residues, as with enzymes and catalysis. It is remarkable that most channels, either voltage-sensitive or transmitter-gated receptor channels, are susceptible to zinc modulation either as positive or negative regulators. PMID:27384555

  14. Modification of Glutamate Receptor Channels: Molecular Mechanisms and Functional Consequences

    NASA Astrophysics Data System (ADS)

    Hatt, Hanns

    Of the many possible mechanisms for modulating the efficiency of ion channels, the phosphorylation of receptor channel proteins may be the primary one. Changes in the set of molecular subunits of which the channels are composed are also important, especially for long-term regulation. In the central nervous system synaptic plasticity may be altered by modulating the ligand-activated neuronal ion channels involved in synaptic transmission; among them are channels gated directly by glutamate, the regulation of which we are only beginning to understand. This paper focuses on modulation of these channels [α-amino-3-hydroxy-5-methyl-4-isoxazoleprionic acid (AMPA), kainate, and N-methyl-d-aspartate (NMDA) types] by phosphorylation and changes in subunit composition. AMPA- and kainate-activated receptors are modulated by adenosine 3, 5-monophosphate (cAMP) dependent protein kinase A (PKA) coupled via D1 dopamine receptors. An increase in the intracellular concentration of cAMP and protein kinase A potentiates kainate-activated currents in α-motoneurons of the spinal cord by increasing the affinity of the ligand (glutamate) for the phosphorylated receptor protein (GluR6 and 7). The rapid desensitization of AMPA-evoked currents normally observed in horizontal cells of the retina is completely blocked by increasing the intracellular concentration of cAMP. The effects of changes in subunit composition were examined in rat hippocampal neurons. The subunit composition of the NMDA receptor determines the kinetic properties of synaptic currents and can be regulated by the type of innervating neuron. Similar changes also occur during development. An important determinant here is the activity of the system. Dynamic regulation of excitatory receptors by both mechanisms may well be associated with some forms of learning and memory in the mammalian brain.

  15. Coupled gating between cardiac calcium release channels (ryanodine receptors).

    PubMed

    Marx, S O; Gaburjakova, J; Gaburjakova, M; Henrikson, C; Ondrias, K; Marks, A R

    2001-06-01

    Excitation-contraction coupling in heart muscle requires the activation of Ca(2+)-release channels/type 2 ryanodine receptors (RyR2s) by Ca(2+) influx. RyR2s are arranged on the sarcoplasmic reticular membrane in closely packed arrays such that their large cytoplasmic domains contact one another. We now show that multiple RyR2s can be isolated under conditions such that they remain physically coupled to one another. When these coupled channels are examined in planar lipid bilayers, multiple channels exhibit simultaneous gating, termed "coupled gating." Removal of the regulatory subunit, the FK506 binding protein (FKBP12.6), functionally but not physically uncouples multiple RyR2 channels. Coupled gating between RyR2 channels may be an important regulatory mechanism in excitation-contraction coupling as well as in other signaling pathways involving intracellular Ca(2+) release. PMID:11397781

  16. Transient Receptor Potential (TRP) channels in T cells.

    PubMed

    Bertin, Samuel; Raz, Eyal

    2016-05-01

    The transient receptor potential (TRP) family of ion channels is widely expressed in many cell types and plays various physiological roles. Growing evidence suggests that certain TRP channels are functionally expressed in the immune system. Indeed, an increasing number of reports have demonstrated the functional expression of several TRP channels in innate and adaptive immune cells and have highlighted their critical role in the activation and function of these cells. However, very few reviews have been entirely dedicated to this subject. Here, we will summarize the recent findings with regards to TRP channel expression in T cells and discuss their emerging role as regulators of T cell activation and functions. Moreover, these studies suggest that beyond their pharmaceutical interest in pain management, certain TRP channels may represent potential novel therapeutic targets for various immune-related diseases.

  17. Glutamate Receptor Ion Channels: Structure, Regulation, and Function

    PubMed Central

    Wollmuth, Lonnie P.; McBain, Chris J.; Menniti, Frank S.; Vance, Katie M.; Ogden, Kevin K.; Hansen, Kasper B.; Yuan, Hongjie; Myers, Scott J.; Dingledine, Ray

    2010-01-01

    The mammalian ionotropic glutamate receptor family encodes 18 gene products that coassemble to form ligand-gated ion channels containing an agonist recognition site, a transmembrane ion permeation pathway, and gating elements that couple agonist-induced conformational changes to the opening or closing of the permeation pore. Glutamate receptors mediate fast excitatory synaptic transmission in the central nervous system and are localized on neuronal and non-neuronal cells. These receptors regulate a broad spectrum of processes in the brain, spinal cord, retina, and peripheral nervous system. Glutamate receptors are postulated to play important roles in numerous neurological diseases and have attracted intense scrutiny. The description of glutamate receptor structure, including its transmembrane elements, reveals a complex assembly of multiple semiautonomous extracellular domains linked to a pore-forming element with striking resemblance to an inverted potassium channel. In this review we discuss International Union of Basic and Clinical Pharmacology glutamate receptor nomenclature, structure, assembly, accessory subunits, interacting proteins, gene expression and translation, post-translational modifications, agonist and antagonist pharmacology, allosteric modulation, mechanisms of gating and permeation, roles in normal physiological function, as well as the potential therapeutic use of pharmacological agents acting at glutamate receptors. PMID:20716669

  18. Ion selectivity in the ryanodine receptor and other calcium channels.

    NASA Astrophysics Data System (ADS)

    Gillespie, Dirk

    2006-03-01

    Biological ion channels passively conduct ions across cell membranes, some with great specificity. Calcium channels are selective channels that range in their Ca^2+ affinity depending on the channel's physiological role. For example, the L-type calcium channel has micromolar affinity while the ryanodine receptor (RyR) has millimolar affinity. On the other hand, both of these channels have the chemically-similar EEEE and DDDD amino acid motifs in their selectivity filters. An electrodiffusion model of RyR that reproduces and predicts >50 data curves will be presented. In this model, ions are charged, hard spheres and the chemical potential is computed using density functional theory of fluids. Ion selectivity arises from a competition between the need for cations to screen the negative charges of the channel and the crowding of ions in the tiny space of the channel. Charge/space competition implies that selectivity increases as the channel volume decreases (thereby increasing the protein charge density), something that has recently been experimentally confirmed in mutant channels. Dielectric properties can also increase selectivity. In Monte Carlo simulations, Ca^2+ affinity is much higher when the channel protein has a low dielectric constant. This counterintuitive result occurs because calcium channel selectivity filters are lined with negatively-charged (acidic) amino acids (EEEE or DDDD). These permanent negative charges induce negative polarization charge at the protein/lumen interface. The total negative charge of the protein (polarization plus permanent) is increased, resulting in increased ion densities, increased charge/space competition, and there in increased Ca^2+ affinity. If no negative protein charges were present, cations would induce enough positive polarization charge to prevent flux.

  19. Kir3 channel signaling complexes: focus on opioid receptor signaling

    PubMed Central

    Nagi, Karim; Pineyro, Graciela

    2014-01-01

    Opioids are among the most effective drugs to treat severe pain. They produce their analgesic actions by specifically activating opioid receptors located along the pain perception pathway where they inhibit the flow of nociceptive information. This inhibition is partly accomplished by activation of hyperpolarizing G protein-coupled inwardly-rectifying potassium (GIRK or Kir3) channels. Kir3 channels control cellular excitability in the central nervous system and in the heart and, because of their ubiquitous distribution, they mediate the effects of a large range of hormones and neurotransmitters which, upon activation of corresponding G protein-coupled receptors (GPCRs) lead to channel opening. Here we analyze GPCR signaling via these effectors in reference to precoupling and collision models. Existing knowledge on signaling bias is discussed in relation to these models as a means of developing strategies to produce novel opioid analgesics with an improved side effects profile. PMID:25071446

  20. Prolactin receptor in regulation of neuronal excitability and channels.

    PubMed

    Patil, Mayur J; Henry, Michael A; Akopian, Armen N

    2014-01-01

    Prolactin (PRL) activates PRL receptor isoforms to exert regulation of specific neuronal circuitries, and to control numerous physiological and clinically-relevant functions including; maternal behavior, energy balance and food intake, stress and trauma responses, anxiety, neurogenesis, migraine and pain. PRL controls these critical functions by regulating receptor potential thresholds, neuronal excitability and/or neurotransmission efficiency. PRL also influences neuronal functions via activation of certain neurons, resulting in Ca(2+) influx and/or electrical firing with subsequent release of neurotransmitters. Although PRL was identified almost a century ago, very little specific information is known about how PRL regulates neuronal functions. Nevertheless, important initial steps have recently been made including the identification of PRL-induced transient signaling pathways in neurons and the modulation of neuronal transient receptor potential (TRP) and Ca(2+) -dependent K(+) channels by PRL. In this review, we summarize current knowledge and recent progress in understanding the regulation of neuronal excitability and channels by PRL.

  1. Lipid modulation of thermal transient receptor potential channels.

    PubMed

    Hernández-García, Enrique; Rosenbaum, Tamara

    2014-01-01

    There is a subgroup of transient receptor potential (TRP) ion channels that are responsive to temperature (thermo-TRP channels). These are important to a variety of sensory and physiological phenomena such as pain and taste perception. All thermo-TRP channels known to date are subject to modulation by lipidic molecules of many kinds, from the ubiquitous cholesterol to more specialized molecules such as prostaglandins. Although the mechanisms and sites of binding of lipids on thermo-TRPs are largely unknown, the explosion on research of lipids and ion channels has revealed previously unsuspected roles for them. Diacyl glycerol is a lipid produced by phospholipase C (PLC) and it was discovered to modulate TRP channels in the eye of the fly, and many mammal TRP channels have been found to interact with lipids. While most of the lipids acting on thermo-TRP channels have been found to activate them, there are a few capable of inhibition. Phosphatidylinositol 4,5-bisphosphate is even capable of both inhibition and activation on a couple of thermo-TRPs, depending on the cellular context. More data is required to assess the mechanism through which lipids affect thermo-TRP channel activity and the physiological importance of this interaction.

  2. Lipid modulation of thermal transient receptor potential channels.

    PubMed

    Hernández-García, Enrique; Rosenbaum, Tamara

    2014-01-01

    There is a subgroup of transient receptor potential (TRP) ion channels that are responsive to temperature (thermo-TRP channels). These are important to a variety of sensory and physiological phenomena such as pain and taste perception. All thermo-TRP channels known to date are subject to modulation by lipidic molecules of many kinds, from the ubiquitous cholesterol to more specialized molecules such as prostaglandins. Although the mechanisms and sites of binding of lipids on thermo-TRPs are largely unknown, the explosion on research of lipids and ion channels has revealed previously unsuspected roles for them. Diacyl glycerol is a lipid produced by phospholipase C (PLC) and it was discovered to modulate TRP channels in the eye of the fly, and many mammal TRP channels have been found to interact with lipids. While most of the lipids acting on thermo-TRP channels have been found to activate them, there are a few capable of inhibition. Phosphatidylinositol 4,5-bisphosphate is even capable of both inhibition and activation on a couple of thermo-TRPs, depending on the cellular context. More data is required to assess the mechanism through which lipids affect thermo-TRP channel activity and the physiological importance of this interaction. PMID:25366236

  3. Transient receptor potential channels and regulation of lung endothelial permeability

    PubMed Central

    2013-01-01

    Abstract This review highlights our current knowledge regarding expression of transient receptor potential (TRP) cation channels in lung endothelium and evidence for their involvement in regulation of lung endothelial permeability. Six mammalian TRP families have been identified and organized on the basis of sequence homology: TRPC (canonical), TRPV (vanilloid), TRPM (melastatin), TRPML (mucolipin), TRPP (polycystin), and TRPA (ankyrin). To date, only TRPC1/4, TRPC6, TRPV4, and TRPM2 have been extensively studied in lung endothelium. Calcium influx through each of these channels has been documented to increase lung endothelial permeability, although their channel-gating mechanisms, downstream signaling mechanisms, and impact on endothelial structure and barrier integrity differ. While other members of the TRPC, TRPV, and TRPM families may be expressed in lung endothelium, we have little or no evidence linking these to regulation of lung endothelial permeability. Further, neither the expression nor functional role(s) of any TRPML, TRPP, and TRPA family members has been studied in lung endothelium. In addition to this assessment organized by TRP channel family, we also discuss TRP channels and lung endothelial permeability from the perspective of lung endothelial heterogeneity, using outcomes of studies focused on TRPC1/4 and TRPV4 channels. The diversity within the TRP channel family and the relative paucity of information regarding roles of a number of these channels in lung endothelium make this field ripe for continued investigation. PMID:25006396

  4. Functional expression of purinergic P2 receptors and transient receptor potential channels by the human urothelium

    PubMed Central

    Shabir, Saqib; Cross, William; Kirkwood, Lisa A.; Pearson, Joanna F.; Appleby, Peter A.; Walker, Dawn; Eardley, Ian

    2013-01-01

    In addition to its role as a physical barrier, the urothelium is considered to play an active role in mechanosensation. A key mechanism is the release of transient mediators that activate purinergic P2 receptors and transient receptor potential (TRP) channels to effect changes in intracellular Ca2+. Despite the implied importance of these receptors and channels in urothelial tissue homeostasis and dysfunctional bladder disease, little is known about their functional expression by the human urothelium. To evaluate the expression and function of P2X and P2Y receptors and TRP channels, the human ureter and bladder were used to separate urothelial and stromal tissues for RNA isolation and cell culture. RT-PCR using stringently designed primer sets was used to establish which P2 and TRP species were expressed at the transcript level, and selective agonists/antagonists were used to confirm functional expression by monitoring changes in intracellular Ca2+ and in a scratch repair assay. The results confirmed the functional expression of P2Y4 receptors and excluded nonexpressed receptors/channels (P2X1, P2X3, P2X6, P2Y6, P2Y11, TRPV5, and TRPM8), while a dearth of specific agonists confounded the functional validation of expressed P2X2, P2X4, P2Y1, P2Y2, TRPV2, TRPV3, TRPV6 and TRPM7 receptors/channels. Although a conventional response was elicited in control stromal-derived cells, the urothelial cell response to well-characterized TRPV1 and TRPV4 agonists/antagonists revealed unexpected anomalies. In addition, agonists that invoked an increase in intracellular Ca2+ promoted urothelial scratch repair, presumably through the release of ATP. The study raises important questions about the ligand selectivity of receptor/channel targets expressed by the urothelium. These pathways are important in urothelial tissue homeostasis, and this opens the possibility of selective drug targeting. PMID:23720349

  5. Receptor for protons: First observations on Acid Sensing Ion Channels.

    PubMed

    Krishtal, Oleg

    2015-07-01

    The history of ASICs began in 1980 with unexpected observation. The concept of highly selective Na(+) current gated by specific receptors for protons was not easily accepted. It took 16 years to get these receptor/channels cloned and start a new stage in their investigation. "The receptor for protons" became ASIC comprising under this name a family of receptor/channels ubiquitous for mammalian nervous system, both peripheral and central. The role of ASICs as putative nociceptors was suggested almost immediately after their discovery. This role subsequently was proven in many forms of pain-related phenomena. Many other functions of ASICs have been also found or primed for speculations both in physiology and in disease. Despite the width of field and strength of efforts, numerous basic questions are to be answered before we understand how the local changes in pH in the nervous tissue transform into electric and messenger signaling via ASICs as transducers. This article is part of the Special Issue entitled 'Acid-Sensing Ion Channels in the Nervous System'.

  6. Transient receptor potential (TRP) channels: a clinical perspective

    PubMed Central

    Kaneko, Yosuke; Szallasi, Arpad

    2014-01-01

    Transient receptor potential (TRP) channels are important mediators of sensory signals with marked effects on cellular functions and signalling pathways. Indeed, mutations in genes encoding TRP channels are the cause of several inherited diseases in humans (the so-called ‘TRP channelopathies’) that affect the cardiovascular, renal, skeletal and nervous systems. TRP channels are also promising targets for drug discovery. The initial focus of research was on TRP channels that are expressed on nociceptive neurons. Indeed, a number of potent, small-molecule TRPV1, TRPV3 and TRPA1 antagonists have already entered clinical trials as novel analgesic agents. There has been a recent upsurge in the amount of work that expands TRP channel drug discovery efforts into new disease areas such as asthma, cancer, anxiety, cardiac hypertrophy, as well as obesity and metabolic disorders. A better understanding of TRP channel functions in health and disease should lead to the discovery of first-in-class drugs for these intractable diseases. With this review, we hope to capture the current state of this rapidly expanding and changing field. LINKED ARTICLES This article is part of a themed section on the pharmacology of TRP channels. To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2014.171.issue-10 PMID:24102319

  7. Transient Receptor Potential Channels as Targets for Phytochemicals

    PubMed Central

    2015-01-01

    To date, 28 mammalian transient receptor potential (TRP) channels have been cloned and characterized. They are grouped into six subfamilies on the basis of their amino acid sequence homology: TRP Ankyrin (TRPA), TRP Canonical (TRPC), TRP Melastatin (TRPM), TRP Mucolipin (TRPML), TRP Polycystin (TRPP), and TRP Vanilloid (TRPV). Most of the TRP channels are nonselective cation channels expressed on the cell membrane and exhibit variable permeability ratios for Ca2+ versus Na+. They mediate sensory functions (such as vision, nociception, taste transduction, temperature sensation, and pheromone signaling) and homeostatic functions (such as divalent cation flux, hormone release, and osmoregulation). Significant progress has been made in our understanding of the specific roles of these TRP channels and their activation mechanisms. In this Review, the emphasis will be on the activation of TRP channels by phytochemicals that are claimed to exert health benefits. Recent findings complement the anecdotal evidence that some of these phytochemicals have specific receptors and the activation of which is responsible for the physiological effects. Now, the targets for these phytochemicals are being unveiled; a specific hypothesis can be proposed and tested experimentally to infer a scientific validity of the claims of the health benefits. The broader and pressing issues that have to be addressed are related to the quantities of the active ingredients in a given preparation, their bioavailability, metabolism, adverse effects, excretion, and systemic versus local effects. PMID:24926802

  8. Acetylcholine receptor channel imaged in the open state

    NASA Astrophysics Data System (ADS)

    Unwin, Nigel

    1995-01-01

    The structure of the open-channel form of the acetylcholine receptor has been determined from electron images of Torpedo ray postsynaptic membranes activated by brief (<5ms) mixing with droplets containing acetylcholine. Comparison with the closed-channel form shows that acetylcholine initiates small rotations of the subunits in the extracellular domain, which trigger a change in configuration of α-helices lining the membrane-spanning pore. The open pore tapers towards the intracellular membrane face, where it is shaped by a 'barrel' of α-helices having a pronounced right-handed twist.

  9. Hot channels in airways: pharmacology of the vanilloid receptor.

    PubMed

    Hwang, Sun Wook; Oh, Uhtaek

    2002-06-01

    Airway hyperresponsiveness of the tracheobronchial path is recognized as the critical feature of bronchial asthma. Sensory nerves in the airway are implicated strongly in this hyperresponsiveness. The vanilloid VR1 receptor, a cloned capsaicin receptor and a nociceptor-specific cation channel, is known to detect and transduce various harmful stimuli to electrical signals. Recent findings suggest that bradykinin can activate VR1 through generation of lipoxygenase products and that protein kinase C and phospholipase C mediate the sensitization of VR1 by many key inflammatory mediators. Such findings will lead to a better understanding of the enigmatic etiology of asthma.

  10. Ion channels gated by acetylcholine and serotonin: structures, biology, and drug discovery

    PubMed Central

    Wu, Zhong-shan; Cheng, Hao; Jiang, Yi; Melcher, Karsten; Xu, H Eric

    2015-01-01

    The nicotinic acetylcholine receptors (nAChRs) and the 5-HT3 receptors (5-HT3Rs) are cation-selective members of the pentameric ligand-gated ion channels (pLGICs), which are oligomeric protein assemblies that convert a chemical signal into an ion flux through postsynaptic membrane. They are critical components for synaptic transmission in the nervous system, and their dysfunction contributes to many neurological disorders. The diverse subunit compositions of pLGICs give rise to complex mechanisms of ligand recognition, channel gating, and ion-selective permeability, which have been demonstrated in numerous electrophysiological and molecular biological studies, and unraveled by progress in studying the structural biology of this protein family. In this review, we discuss recent insights into the structural and functional basis of two cation-selective pLGICs, the nAChR and the 5-HT3R, including their subunit compositions, ligand binding, and channel gating mechanisms. We also discuss their relevant pharmacology and drug discovery for treating various neurological disorders. Finally, we review a model of two alternative ion conducting pathways based on the latest 5-HT3A crystal structure. PMID:26238288

  11. Functional ryanodine receptor channels in flatworm muscle fibres.

    PubMed

    Day, T A; Haithcock, J; Kimber, M; Maule, A G

    2000-04-01

    Caffeine, which stimulates intracellular Ca2+ release channels known as ryanodine receptor (RyR) channels, induces contraction of individual muscle fibres dissociated from the trematode Schistosoma mansoni, and the turbellarians Dugesia tigrina and Procerodes littoralis. Caffeine is much more potent on S. mansoni fibres (EC50 0.7 mM) than those from D. tigrina or P. littoralis (3.2 mM and 4.6 mM, respectively). These caffeine-induced contractions are blocked by ryanodine, confirming the presence of functional RyR channels in these flatworm muscles. However, the contractions are not blocked by typical RyR channel blockers ruthenium red or neomycin, indicating that there may be important pharmacological differences between the RyR channels in this early-diverging phylum and those of later animals. These studies demonstrate that RyR channels are present in the muscle of these flatworms, and that the sarcoplasmic reticulum stores sufficient Ca2+ to support contraction.

  12. Transient receptor potential channel C5 in cancer chemoresistance

    PubMed Central

    He, Dong-xu; Ma, Xin

    2016-01-01

    The transient receptor potential (TRP) superfamily contains at least 28 homologs in mammalian. These proteins form TRP channels are permeable to monovalent and divalent cations and participate in a variety of physiological functions. Dysregulation of TRP channels is responsible for numerous diseases. This review provides a brief short overview of mammalian TRP channels with a focus on TRPC5 and its role in cancers. Dysregulation of TRPC5 interrupts Ca2+ homeostasis in cancer cells, which activates signaling pathways that are highly associated with cancer progression, especially cancer chemoresistance. Based on the important role of TRPC5, we also discuss the potential of TRPC5 as a target for therapeutic intervention. Either direct targeting of TRPC5 or indirect interruption of TRPC5-related signaling pathways may effectively overcome cancer chemoresistance. PMID:26657058

  13. Post-transcriptional regulation of GABAB receptor and GIRK1 channels by Nogo receptor 1

    PubMed Central

    2013-01-01

    Background Type B GABA receptors (GABA Rs) play a critical role in synaptic transmission. We carried out studies to determine whether neuronal cell surface expression of GABAB-Rs might be regulated by the Nogo receptor 1 (NgR1). Results siRNA knock-down of NgR1 resulted in a selective increase of GABAB R1 and GABAB R2 protein without altering the expression of GABAA receptor or GAD65. The increase in GABAB receptor subunits was unaccompanied by a change in mRNA, but inhibition of mTOR by rapamycin blocked the increase in GABAB protein. NgR1 siRNA also caused an increase in G protein coupled inwardly rectifying potassium channel (GIRK1). The increase in GABAB receptor and GIRK1 channel proteins was in the plasma membrane, determined by cell surface biotinylation. In NgR1 knockout mice, the amount of GABAB R2 and GIRK1 in hippocampus-derived synaptosomes was increased. Conclusions Together these findings suggest that NgR1 mediated modulation of synaptic transmission may be accomplished, at least in part, through modulation of G protein coupled receptors and channels. PMID:23829864

  14. Physiology and pathophysiology of canonical transient receptor potential channels

    PubMed Central

    Abramowitz, Joel; Birnbaumer, Lutz

    2009-01-01

    The existence of a mammalian family of TRPC ion channels, direct homologues of TRP, the visual transduction channel of flies, was discovered during 1995–1996 as a consequence of research into the mechanism by which the stimulation of the receptor-Gq-phospholipase Cβ signaling pathway leads to sustained increases in intracellular calcium. Mammalian TRPs, TRPCs, turned out to be nonselective, calcium-permeable cation channels, which cause both a collapse of the cell’s membrane potential and entry of calcium. The family comprises 7 members and is widely expressed. Many cells and tissues express between 3 and 4 of the 7 TRPCs. Despite their recent discovery, a wealth of information has accumulated, showing that TRPCs have widespread roles in almost all cells studied, including cells from excitable and nonexcitable tissues, such as the nervous and cardiovascular systems, the kidney and the liver, and cells from endothelia, epithelia, and the bone marrow compartment. Disruption of TRPC function is at the root of some familial diseases. More often, TRPCs are contributing risk factors in complex diseases. The present article reviews what has been uncovered about physiological roles of mammalian TRPC channels since the time of their discovery. This analysis reveals TRPCs as major and unsuspected gates of Ca2+ entry that contribute, depending on context, to activation of transcription factors, apoptosis, vascular contractility, platelet activation, and cardiac hypertrophy, as well as to normal and abnormal cell proliferation. TRPCs emerge as targets for a thus far nonexistent field of pharmacological intervention that may ameliorate complex diseases.—Abramowitz, J., Birnbaumer, L. Physiology and pathophysiology of canonical transient receptor potential channels. PMID:18940894

  15. Acid-sensing ion channels and transient-receptor potential ion channels in zebrafish taste buds.

    PubMed

    Levanti, M; Randazzo, B; Viña, E; Montalbano, G; Garcia-Suarez, O; Germanà, A; Vega, J A; Abbate, F

    2016-09-01

    Sensory information from the environment is required for life and survival, and it is detected by specialized cells which together make up the sensory system. The fish sensory system includes specialized organs that are able to detect mechanical and chemical stimuli. In particular, taste buds are small organs located on the tongue in terrestrial vertebrates that function in the perception of taste. In fish, taste buds occur on the lips, the flanks, and the caudal (tail) fins of some species and on the barbels of others. In fish taste receptor cells, different classes of ion channels have been detected which, like in mammals, presumably participate in the detection and/or transduction of chemical gustatory signals. However, since some of these ion channels are involved in the detection of additional sensory modalities, it can be hypothesized that taste cells sense stimuli other than those specific for taste. This mini-review summarizes current knowledge on the presence of transient-receptor potential (TRP) and acid-sensing (ASIC) ion channels in the taste buds of teleosts, especially adult zebrafish. Up to now ASIC4, TRPC2, TRPA1, TRPV1 and TRPV4 ion channels have been found in the sensory cells, while ASIC2 was detected in the nerves supplying the taste buds. PMID:27513962

  16. Activation and Regulation of Purinergic P2X Receptor Channels

    PubMed Central

    Coddou, Claudio; Yan, Zonghe; Obsil, Tomas; Huidobro-Toro, J. Pablo

    2011-01-01

    Mammalian ATP-gated nonselective cation channels (P2XRs) can be composed of seven possible subunits, denoted P2X1 to P2X7. Each subunit contains a large ectodomain, two transmembrane domains, and intracellular N and C termini. Functional P2XRs are organized as homomeric and heteromeric trimers. This review focuses on the binding sites involved in the activation (orthosteric) and regulation (allosteric) of P2XRs. The ectodomains contain three ATP binding sites, presumably located between neighboring subunits and formed by highly conserved residues. The detection and coordination of three ATP phosphate residues by positively charged amino acids are likely to play a dominant role in determining agonist potency, whereas an AsnPheArg motif may contribute to binding by coordinating the adenine ring. Nonconserved ectodomain histidines provide the binding sites for trace metals, divalent cations, and protons. The transmembrane domains account not only for the formation of the channel pore but also for the binding of ivermectin (a specific P2X4R allosteric regulator) and alcohols. The N- and C- domains provide the structures that determine the kinetics of receptor desensitization and/or pore dilation and are critical for the regulation of receptor functions by intracellular messengers, kinases, reactive oxygen species and mercury. The recent publication of the crystal structure of the zebrafish P2X4.1R in a closed state provides a major advance in the understanding of this family of receptor channels. We will discuss data obtained from numerous site-directed mutagenesis experiments accumulated during the last 15 years with reference to the crystal structure, allowing a structural interpretation of the molecular basis of orthosteric and allosteric ligand actions. PMID:21737531

  17. Structure and function of glutamate receptor ion channels.

    PubMed

    Mayer, Mark L; Armstrong, Neali

    2004-01-01

    A vast number of proteins are involved in synaptic function. Many have been cloned and their functional role defined with varying degrees of success, but their number and complexity currently defy any molecular understanding of the physiology of synapses. A beacon of success in this medieval era of synaptic biology is an emerging understanding of the mechanisms underlying the activity of the neurotransmitter receptors for glutamate. Largely as a result of structural studies performed in the past three years we now have a mechanistic explanation for the activation of channel gating by agonists and partial agonists; the process of desensitization, and its block by allosteric modulators, is also mostly explained; and the basis of receptor subtype selectivity is emerging with clarity as more and more structures are solved. In the space of months we have gone from cartoons of postulated mechanisms to hard fact. It is anticipated that this level of understanding will emerge for other synaptic proteins in the coming decade.

  18. Receptors, Ion Channels, and Signaling Mechanisms Underlying Microglial Dynamics*

    PubMed Central

    Madry, Christian; Attwell, David

    2015-01-01

    Microglia, the innate immune cells of the CNS, play a pivotal role in brain injury and disease. Microglia are extremely motile; their highly ramified processes constantly survey the brain parenchyma, and they respond promptly to brain damage with targeted process movement toward the injury site. Microglia play a key role in brain development and function by pruning synapses during development, phagocytosing apoptotic newborn neurons, and regulating neuronal activity by direct microglia-neuron or indirect microglia-astrocyte-neuron interactions, which all depend on their process motility. This review highlights recent discoveries about microglial dynamics, focusing on the receptors, ion channels, and signaling pathways involved. PMID:25855789

  19. Membrane coordination of receptors and channels mediating the inhibition of neuronal ion currents by ADP.

    PubMed

    Gafar, Hend; Dominguez Rodriguez, Manuel; Chandaka, Giri K; Salzer, Isabella; Boehm, Stefan; Schicker, Klaus

    2016-09-01

    ADP and other nucleotides control ion currents in the nervous system via various P2Y receptors. In this respect, Cav2 and Kv7 channels have been investigated most frequently. The fine tuning of neuronal ion channel gating via G protein coupled receptors frequently relies on the formation of higher order protein complexes that are organized by scaffolding proteins and harbor receptors and channels together with interposed signaling components. However, ion channel complexes containing P2Y receptors have not been described. Therefore, the regulation of Cav2.2 and Kv7.2/7.3 channels via P2Y1 and P2Y12 receptors and the coordination of these ion channels and receptors in the plasma membranes of tsA 201 cells have been investigated here. ADP inhibited currents through Cav2.2 channels via both P2Y1 and P2Y12 receptors with phospholipase C and pertussis toxin-sensitive G proteins being involved, respectively. The nucleotide controlled the gating of Kv7 channels only via P2Y1 and phospholipase C. In fluorescence energy transfer assays using conventional as well as total internal reflection (TIRF) microscopy, both P2Y1 and P2Y12 receptors were found juxtaposed to Cav2.2 channels, but only P2Y1, and not P2Y12, was in close proximity to Kv7 channels. Using fluorescence recovery after photobleaching in TIRF microscopy, evidence for a physical interaction was obtained for the pair P2Y12/Cav2.2, but not for any other receptor/channel combination. These results reveal a membrane juxtaposition of P2Y receptors and ion channels in parallel with the control of neuronal ion currents by ADP. This juxtaposition may even result in apparent physical interactions between receptors and channels.

  20. Modulation of cardiac ryanodine receptor channels by alkaline earth cations.

    PubMed

    Diaz-Sylvester, Paula L; Porta, Maura; Copello, Julio A

    2011-01-01

    Cardiac ryanodine receptor (RyR2) function is modulated by Ca(2+) and Mg(2+). To better characterize Ca(2+) and Mg(2+) binding sites involved in RyR2 regulation, the effects of cytosolic and luminal earth alkaline divalent cations (M(2+): Mg(2+), Ca(2+), Sr(2+), Ba(2+)) were studied on RyR2 from pig ventricle reconstituted in bilayers. RyR2 were activated by M(2+) binding to high affinity activating sites at the cytosolic channel surface, specific for Ca(2+) or Sr(2+). This activation was interfered by Mg(2+) and Ba(2+) acting at low affinity M(2+)-unspecific binding sites. When testing the effects of luminal M(2+) as current carriers, all M(2+) increased maximal RyR2 open probability (compared to Cs(+)), suggesting the existence of low affinity activating M(2+)-unspecific sites at the luminal surface. Responses to M(2+) vary from channel to channel (heterogeneity). However, with luminal Ba(2+)or Mg(2+), RyR2 were less sensitive to cytosolic Ca(2+) and caffeine-mediated activation, openings were shorter and voltage-dependence was more marked (compared to RyR2 with luminal Ca(2+)or Sr(2+)). Kinetics of RyR2 with mixtures of luminal Ba(2+)/Ca(2+) and additive action of luminal plus cytosolic Ba(2+) or Mg(2+) suggest luminal M(2+) differentially act on luminal sites rather than accessing cytosolic sites through the pore. This suggests the presence of additional luminal activating Ca(2+)/Sr(2+)-specific sites, which stabilize high P(o) mode (less voltage-dependent) and increase RyR2 sensitivity to cytosolic Ca(2+) activation. In summary, RyR2 luminal and cytosolic surfaces have at least two sets of M(2+) binding sites (specific for Ca(2+) and unspecific for Ca(2+)/Mg(2+)) that dynamically modulate channel activity and gating status, depending on SR voltage. PMID:22039534

  1. Mutations of L293 in transmembrane two of the mouse 5-hydroxytryptamine3A receptor alter gating and alcohol modulatory actions.

    PubMed

    Hu, Xiang-Qun; Hayrapetyan, Volodya; Gadhiya, Jay J; Rhubottom, Heather E; Lovinger, David M; Machu, Tina K

    2006-05-01

    1 The goal of this study was to determine whether mutations of L293 at the 15' position of TM2 in the 5-HT(3A) receptor alter macroscopic current kinetics, and if these changes could account for alterations in alcohol modulation. Receptor function was assessed in Xenopus oocytes under voltage-clamp and in HEK293 cells with whole-cell patch-clamp recording and rapid drug application. 2 Examination of responses of L293C and L293S receptors to agonist alone revealed enhanced activation, deactivation, and desensitization rates relative to the wild-type receptor. The L293G mutation produced marked slowing of deactivation and desensitization rates. Increased potency of 5-HT and increased efficacy of the partial agonist, DA, was also observed in these mutant receptors. 3 Ethanol and trichloroethanol (TCEt) enhancement of receptor function was reduced or eliminated in receptors containing L293 mutations to C, G, or S. The L293I mutant receptor retained ethanol and TCEt sensitivity. Ethanol and TCEt enhanced activation rate in the wild-type, but not the L293G and L293S receptors. No relationship was observed between any physicochemical property of the substituted amino acids and the change in alcohol potentiation of function. 4 The changes in receptor-channel properties in the mutant receptors support the idea that the L293 residue has important roles in channel gating. Our findings indicate that loss of allosteric modulation by alcohols is not related in any simple way to changes in channel kinetic properties brought about by L293 mutants. We did not observe any evidence that L293 is part of an alcohol binding site. PMID:16520747

  2. The sigma receptor as a ligand-regulated auxiliary potassium channel subunit.

    PubMed

    Aydar, Ebru; Palmer, Christopher P; Klyachko, Vitaly A; Jackson, Meyer B

    2002-04-25

    The sigma receptor is a novel protein that mediates the modulation of ion channels by psychotropic drugs through a unique transduction mechanism depending neither on G proteins nor protein phosphorylation. The present study investigated sigma receptor signal transduction by reconstituting responses in Xenopus oocytes. Sigma receptors modulated voltage-gated K+ channels (Kv1.4 or Kv1.5) in different ways in the presence and absence of ligands. Association between Kv1.4 channels and sigma receptors was demonstrated by coimmunoprecipitation. These results indicate a novel mechanism of signal transduction dependent on protein-protein interactions. Domain accessibility experiments suggested a structure for the sigma receptor with two cytoplasmic termini and two membrane-spanning segments. The ligand-independent effects on channels suggest that sigma receptors serve as auxiliary subunits to voltage-gated K+ channels with distinct functional interactions, depending on the presence or absence of ligand.

  3. A ligand channel through the G protein coupled receptor opsin.

    PubMed

    Hildebrand, Peter W; Scheerer, Patrick; Park, Jung Hee; Choe, Hui-Woog; Piechnick, Ronny; Ernst, Oliver P; Hofmann, Klaus Peter; Heck, Martin

    2009-01-01

    The G protein coupled receptor rhodopsin contains a pocket within its seven-transmembrane helix (TM) structure, which bears the inactivating 11-cis-retinal bound by a protonated Schiff-base to Lys296 in TM7. Light-induced 11-cis-/all-trans-isomerization leads to the Schiff-base deprotonated active Meta II intermediate. With Meta II decay, the Schiff-base bond is hydrolyzed, all-trans-retinal is released from the pocket, and the apoprotein opsin reloaded with new 11-cis-retinal. The crystal structure of opsin in its active Ops* conformation provides the basis for computational modeling of retinal release and uptake. The ligand-free 7TM bundle of opsin opens into the hydrophobic membrane layer through openings A (between TM1 and 7), and B (between TM5 and 6), respectively. Using skeleton search and molecular docking, we find a continuous channel through the protein that connects these two openings and comprises in its central part the retinal binding pocket. The channel traverses the receptor over a distance of ca. 70 A and is between 11.6 and 3.2 A wide. Both openings are lined with aromatic residues, while the central part is highly polar. Four constrictions within the channel are so narrow that they must stretch to allow passage of the retinal beta-ionone-ring. Constrictions are at openings A and B, respectively, and at Trp265 and Lys296 within the retinal pocket. The lysine enforces a 90 degrees elbow-like kink in the channel which limits retinal passage. With a favorable Lys side chain conformation, 11-cis-retinal can take the turn, whereas passage of the all-trans isomer would require more global conformational changes. We discuss possible scenarios for the uptake of 11-cis- and release of all-trans-retinal. If the uptake gate of 11-cis-retinal is assigned to opening B, all-trans is likely to leave through the same gate. The unidirectional passage proposed previously requires uptake of 11-cis-retinal through A and release of photolyzed all-trans-retinal through

  4. AMPA receptors undergo channel arrest in the anoxic turtle cortex.

    PubMed

    Pamenter, Matthew Edward; Shin, Damian Seung-Ho; Buck, Leslie Thomas

    2008-02-01

    Without oxygen, all mammals suffer neuronal injury and excitotoxic cell death mediated by overactivation of the glutamatergic N-methyl-D-aspartate receptor (NMDAR). The western painted turtle can survive anoxia for months, and downregulation of NMDAR activity is thought to be neuroprotective during anoxia. NMDAR activity is related to the activity of another glutamate receptor, the alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid receptor (AMPAR). AMPAR blockade is neuroprotective against anoxic insult in mammals, but the role of AMPARs in the turtle's anoxia tolerance has not been investigated. To determine whether AMPAR activity changes during hypoxia or anoxia in the turtle cortex, whole cell AMPAR currents, AMPAR-mediated excitatory postsynaptic potentials (EPSPs), and excitatory postsynaptic currents (EPSCs) were measured. The effect of AMPAR blockade on normoxic and anoxic NMDAR currents was also examined. During 60 min of normoxia, evoked peak AMPAR currents and the frequencies and amplitudes of EPSPs and EPSCs did not change. During anoxic perfusion, evoked AMPAR peak currents decreased 59.2 +/- 5.5 and 60.2 +/- 3.5% at 20 and 40 min, respectively. EPSP frequency (EPSP(f)) and amplitude decreased 28.7 +/- 6.4% and 13.2 +/- 1.7%, respectively, and EPSC(f) and amplitude decreased 50.7 +/- 5.1% and 51.3 +/- 4.7%, respectively. In contrast, hypoxic (Po(2) = 5%) AMPAR peak currents were potentiated 56.6 +/- 20.5 and 54.6 +/- 15.8% at 20 and 40 min, respectively. All changes were reversed by reoxygenation. AMPAR currents and EPSPs were abolished by 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX). In neurons pretreated with CNQX, anoxic NMDAR currents were reversibly depressed by 49.8 +/- 7.9%. These data suggest that AMPARs may undergo channel arrest in the anoxic turtle cortex. PMID:18056983

  5. Electrochemical evaluation of chemical selectivity of glutamate receptor ion channel proteins with a multi-channel sensor.

    PubMed

    Sugawara, M; Hirano, A; Rehák, M; Nakanishi, J; Kawai, K; Sato, H; Umezawa, Y

    1997-01-01

    A new method for evaluating chemical selectivity of agonists towards receptor ion channel proteins is proposed by using glutamate receptor (GluR) ion channel proteins and their agonists N-methyl-D-aspartic acid (NMDA), L-glutamate, and (2S, 3R, 4S) isomer of 2-(carboxycyclopropyl)glycine (L-CCG-IV). Integrated multi-channel currents, corresponding to the sum of total amount of ions passed through the multiple open channels, were used as a measure of agonists' selectivity to recognize ion channel proteins and induce channel currents. GluRs isolated from rat synaptic plasma membranes were incorporated into planar bilayer lipid membranes (BLMs) formed by the folding method. The empirical factors that affect the selectivity were demonstrated: (i) the number of GluRs incorporated into BLMs varied from one membrane to another; (ii) each BLM contained different subtypes of GluRs (NMDA and/or non-NMDA subtypes); and (iii) the magnitude of multi-channel responses induced by L-glutamate at negative applied potentials was larger than at positive potentials, while those by NMDA and L-CCG-IV were linearly related to applied potentials. The chemical selectivity among NMDA, L-glutamate and L-CCG-IV for NMDA subtype of GluRs was determined with each single BLM in which only NMDA subtype of GluRs was designed to be active by inhibiting the non-NMDA subtypes using a specific antagonist DNQX. The order of selectivity among the relevant agonists for the NMDA receptor subtype was found to be L-CCG-IV > L-glutamate > NMDA, which is consistent with the order of binding affinity of these agonists towards the same NMDA subtypes. The potential use of this approach for evaluating chemical selectivity towards non-NMDA receptor subtypes of GluRs and other receptor ion channel proteins is discussed.

  6. Combined single channel and single molecule detection identifies subunit composition of STIM1-activated transient receptor potential canonical (TRPC) channels.

    PubMed

    Asanov, Alexander; Sampieri, Alicia; Moreno, Claudia; Pacheco, Jonathan; Salgado, Alfonso; Sherry, Ryan; Vaca, Luis

    2015-01-01

    Depletion of intracellular calcium ion stores initiates a rapid cascade of events culminating with the activation of the so-called Store-Operated Channels (SOC) at the plasma membrane. Calcium influx via SOC is essential in the initiation of calcium-dependent intracellular signaling and for the refilling of internal calcium stores, ensuring the regeneration of the signaling cascade. In spite of the significance of this evolutionary conserved mechanism, the molecular identity of SOC has been the center of a heated controversy spanning over the last 20 years. Initial studies positioned some members of the transient receptor potential canonical (TRPC) channel superfamily of channels (with the more robust evidence pointing to TRPC1) as a putative SOC. Recent evidence indicates that Stromal Interacting Molecule 1 (STIM1) activates some members from the TRPC family of channels. However, the exact subunit composition of TRPC channels remains undetermined to this date. To identify the subunit composition of STIM1-activated TRPC channels, we developed novel method, which combines single channel electrophysiological measurements based on the patch clamp technique with single molecule fluorescence imaging. We termed this method Single ion Channel Single Molecule Detection technique (SC-SMD). Using SC-SMD method, we have obtained direct evidence of the subunit composition of TRPC channels activated by STIM1. Furthermore, our electrophysiological-imaging SC-SMD method provides evidence at the molecular level of the mechanism by which STIM1 and calmodulin antagonize to modulate TRPC channel activity.

  7. Functional Coupling of Ca2+ Channels and Ryanodine Receptors in Cardiac Myocytes

    NASA Astrophysics Data System (ADS)

    Sham, James S. K.; Cleemann, Lars; Morad, Martin

    1995-01-01

    In skeletal muscle, dihydropyridine receptors are functionally coupled to ryanodine receptors of the sarcoplasmic reticulum in triadic or diadic junctional complexes. In cardiac muscle direct physical or functional couplings have not been demonstrated. We have tested the hypothesis of functional coupling of L-type Ca2+ channels and ryanodine receptors in rat cardiac myocytes by comparing the efficacies of Ca2+ in triggering Ca2+ release when the ion enters the cell via the Ca2+ channels or the Na^+/Ca2+ exchanger. Ca2+ transported through the Ca2+ channels was 20-160 times more effective than Ca2+ influx via the Na^+/Ca2+ exchanger in gating Ca2+ release from the sarcoplasmic reticulum, suggesting privileged communication between Ca2+ channels and ryanodine receptors. In support of this hypothesis we found that Ca2+ channels were inactivated by Ca2+ release from the sarcoplasmic reticulum, even though the myoplasmic Ca2+ concentrations were buffered with 10 mM EGTA. The data thus suggest privileged cross signaling between the dihydropyridine and ryanodine receptors such that Ca2+ flux through either the Ca2+ channel or the ryanodine receptor alters the gating kinetics of the other channel.

  8. NR2 subunit-dependence of NMDA receptor channel block by external Mg2+

    PubMed Central

    Qian, Anqi; Buller, Amy L; Johnson, Jon W

    2005-01-01

    The vital roles played by NMDA receptors in CNS physiology depend critically on powerful voltage-dependent channel block by external Mg2+ (Mg2+o). NMDA receptor channel block by Mg2+o depends on receptor subunit composition: NR1/2A receptors (receptors composed of NR1 and NR2A subunits) and NR1/2B receptors are more strongly inhibited by Mg2+o than are NR1/2C or NR1/2D receptors. We investigated the effects of Mg2+o on single-channel and whole-cell currents recorded from recombinant NR1/2D and NR1/2A receptors expressed in HEK293 and 293T cells. The main conclusions are as follows: (1) Voltage-dependent inhibition by Mg2+o of whole-cell NR1/2D receptor responses was at least 4-fold weaker than inhibition of NR1/2A receptor responses at all voltages tested. (2) Channel block by Mg2+o reduced the duration of NR1/2D receptor single-channel openings; this reduction was used to estimate the apparent blocking rate of Mg2+o (k+,app). The k+,app for NR1/2D receptors was similar to but moderately slower than the k+,app obtained from cortical NMDA receptors composed of NR1, NR2A and NR2B subunits at all voltages tested. (3) Mg2+o blocking events induced an additional component in the closed-duration distribution; this component was used to estimate the apparent unblocking rate of Mg2+o (k−,app). The k−,app for NR1/2D receptors was much faster than the k−,app for cortical receptors at all voltages tested. The voltage-dependence of the k−,app of NR1/2D and cortical receptors differed in a manner that suggested that Mg2+o may permeate NR1/2D receptors more easily than cortical receptors. (4) Mg2+o inhibits NR1/2D receptors less effectively than cortical receptors chiefly because Mg2+o unbinds much more rapidly from NR1/2D receptors. PMID:15513936

  9. Domain-based identification and analysis of glutamate receptor ion channels and their relatives in prokaryotes.

    PubMed

    Ger, Mao-Feng; Rendon, Gloria; Tilson, Jeffrey L; Jakobsson, Eric

    2010-10-06

    Voltage-gated and ligand-gated ion channels are used in eukaryotic organisms for the purpose of electrochemical signaling. There are prokaryotic homologues to major eukaryotic channels of these sorts, including voltage-gated sodium, potassium, and calcium channels, Ach-receptor and glutamate-receptor channels. The prokaryotic homologues have been less well characterized functionally than their eukaryotic counterparts. In this study we identify likely prokaryotic functional counterparts of eukaryotic glutamate receptor channels by comprehensive analysis of the prokaryotic sequences in the context of known functional domains present in the eukaryotic members of this family. In particular, we searched the nonredundant protein database for all proteins containing the following motif: the two sections of the extracellular glutamate binding domain flanking two transmembrane helices. We discovered 100 prokaryotic sequences containing this motif, with a wide variety of functional annotations. Two groups within this family have the same topology as eukaryotic glutamate receptor channels. Group 1 has a potassium-like selectivity filter. Group 2 is most closely related to eukaryotic glutamate receptor channels. We present analysis of the functional domain architecture for the group of 100, a putative phylogenetic tree, comparison of the protein phylogeny with the corresponding species phylogeny, consideration of the distribution of these proteins among classes of prokaryotes, and orthologous relationships between prokaryotic and human glutamate receptor channels. We introduce a construct called the Evolutionary Domain Network, which represents a putative pathway of domain rearrangements underlying the domain composition of present channels. We believe that scientists interested in ion channels in general, and ligand-gated ion channels in particular, will be interested in this work. The work should also be of interest to bioinformatics researchers who are interested in the use

  10. The N-terminal domain of GluR6-subtype glutamate receptor ion channels

    SciTech Connect

    Kumar, Janesh; Schuck, Peter; Jin, Rongsheng; Mayer, Mark L.

    2009-09-25

    The amino-terminal domain (ATD) of glutamate receptor ion channels, which controls their selective assembly into AMPA, kainate and NMDA receptor subtypes, is also the site of action of NMDA receptor allosteric modulators. Here we report the crystal structure of the ATD from the kainate receptor GluR6. The ATD forms dimers in solution at micromolar protein concentrations and crystallizes as a dimer. Unexpectedly, each subunit adopts an intermediate extent of domain closure compared to the apo and ligand-bound complexes of LIVBP and G protein-coupled glutamate receptors (mGluRs), and the dimer assembly has a markedly different conformation from that found in mGluRs. This conformation is stabilized by contacts between large hydrophobic patches in the R2 domain that are absent in NMDA receptors, suggesting that the ATDs of individual glutamate receptor ion channels have evolved into functionally distinct families.

  11. A Transient Receptor Potential Ion Channel in Chlamydomonas Shares Key Features with Sensory Transduction-Associated TRP Channels in Mammals

    PubMed Central

    Arias-Darraz, Luis; Cabezas, Deny; Colenso, Charlotte K.; Alegría-Arcos, Melissa; Bravo-Moraga, Felipe; Varas-Concha, Ignacio; Almonacid, Daniel E.; Madrid, Rodolfo; Brauchi, Sebastian

    2015-01-01

    Sensory modalities are essential for navigating through an ever-changing environment. From insects to mammals, transient receptor potential (TRP) channels are known mediators for cellular sensing. Chlamydomonas reinhardtii is a motile single-celled freshwater green alga that is guided by photosensory, mechanosensory, and chemosensory cues. In this type of alga, sensory input is first detected by membrane receptors located in the cell body and then transduced to the beating cilia by membrane depolarization. Although TRP channels seem to be absent in plants, C. reinhardtii possesses genomic sequences encoding TRP proteins. Here, we describe the cloning and characterization of a C. reinhardtii version of a TRP channel sharing key features present in mammalian TRP channels associated with sensory transduction. In silico sequence-structure analysis unveiled the modular design of TRP channels, and electrophysiological experiments conducted on Human Embryonic Kidney-293T cells expressing the Cr-TRP1 clone showed that many of the core functional features of metazoan TRP channels are present in Cr-TRP1, suggesting that basic TRP channel gating characteristics evolved early in the history of eukaryotes. PMID:25595824

  12. Transient Receptor Potential Ion Channels Control Thermoregulatory Behaviour in Reptiles

    PubMed Central

    Seebacher, Frank; Murray, Shauna A.

    2007-01-01

    Biological functions are governed by thermodynamics, and animals regulate their body temperature to optimise cellular performance and to avoid harmful extremes. The capacity to sense environmental and internal temperatures is a prerequisite for the evolution of thermoregulation. However, the mechanisms that enable ectothermic vertebrates to sense heat remain unknown. The recently discovered thermal characteristics of transient receptor potential ion channels (TRP) render these proteins suitable to act as temperature sensors. Here we test the hypothesis that TRPs are present in reptiles and function to control thermoregulatory behaviour. We show that the hot-sensing TRPV1 is expressed in a crocodile (Crocodylus porosus), an agamid (Amphibolurus muricatus) and a scincid (Pseudemoia entrecasteauxii) lizard, as well as in the quail and zebrafinch (Coturnix chinensis and Poephila guttata). The TRPV1 genes from all reptiles form a unique clade that is delineated from the mammalian and the ancestral Xenopus sequences by an insertion of two amino acids. TRPV1 and the cool-sensing TRPM8 are expressed in liver, muscle (transversospinalis complex), and heart tissues of the crocodile, and have the potential to act as internal thermometer and as external temperatures sensors. Inhibition of TRPV1 and TRPM8 in C. porosus abolishes the typically reptilian shuttling behaviour between cooling and heating environments, and leads to significantly altered body temperature patterns. Our results provide the proximate mechanism of thermal selection in terrestrial ectotherms, which heralds a fundamental change in interpretation, because TRPs provide the mechanism for a tissue-specific input into the animals' thermoregulatory response. PMID:17356692

  13. From The Cover: Microtransplantation of functional receptors and channels from the Alzheimer's brain to frog oocytes

    NASA Astrophysics Data System (ADS)

    Miledi, R.; Dueñas, Z.; Martinez-Torres, A.; Kawas, C. H.; Eusebi, F.

    2004-02-01

    About a decade ago, cell membranes from the electric organ of Torpedo and from the rat brain were transplanted to frog oocytes, which thus acquired functional Torpedo and rat neurotransmitter receptors. Nevertheless, the great potential that this method has for studying human diseases has remained virtually untapped. Here, we show that cell membranes from the postmortem brains of humans that suffered Alzheimer's disease can be microtransplanted to the plasma membrane of Xenopus oocytes. We show also that these postmortem membranes carry neurotransmitter receptors and voltage-operated channels that are still functional, even after they have been kept frozen for many years. This method provides a new and powerful approach to study directly the functional characteristics and structure of receptors, channels, and other membrane proteins of the Alzheimer's brain. This knowledge may help in understanding the basis of Alzheimer's disease and also help in developing new treatments. -aminobutyric acid receptors | sodium channels | calcium channels | postmortem brain

  14. Heterologously expressed serotonin 1A receptors couple to muscarinic K+ channels in heart.

    PubMed Central

    Karschin, A; Ho, B Y; Labarca, C; Elroy-Stein, O; Moss, B; Davidson, N; Lester, H A

    1991-01-01

    In cardiac atrial cells, muscarinic acetylcholine receptors activate a K+ current directly via a guanine nucleotide-binding protein (G protein). Serotonin type 1A receptors may activate a similar pathway in hippocampal neurons. To develop a system in which receptor/G protein/K+ channel coupling can be experimentally manipulated, we have used a highly efficient recombinant vaccinia virus vector system to express human serotonin 1A receptors in primary cultures of rat atrial myocytes. The expressed 1A receptors activated the inwardly rectifying K+ conductance that is normally activated by the endogenous muscarinic acetylcholine receptors. Maximal responses to either agonist occluded further activation by the other agonist. The average activation time constants for serotonin were about 5 times slower than for acetylcholine. The data support suggestions that the intracellular signaling pathway from seven-helix receptors to G proteins and directly to ion channels is widespread in excitable cells. After a fraction of the G proteins are activated irreversibly by guanosine 5'-[gamma-thio]triphosphate, subsequent transduction proceeds more efficiently. One possible interpretation is that multiple G-protein molecules are required to activate each channel. Vaccinia virus expression vectors are thus useful for expressing seven-helix receptors in primary cultures of postmitotic cells and have provided a heterologous expression system for the signaling pathway from seven-helix receptors to G proteins and directly to ion channels. Images PMID:1905814

  15. Structure-Driven Pharmacology of Transient Receptor Potential Channel Vanilloid 1.

    PubMed

    Díaz-Franulic, Ignacio; Caceres-Molina, Javier; Sepulveda, Romina V; Gonzalez-Nilo, Fernando; Latorre, Ramon

    2016-09-01

    The transient receptor potential vanilloid 1 (TRPV1) ion channel is a polymodal receptor that mediates the flux of cations across the membrane in response to several stimuli, including heat, voltage, and ligands. The best known agonist of TRPV1 channels is capsaicin, the pungent component of "hot" chili peppers. In addition, peptides found in the venom of poisonous animals, along with the lipids phosphatidylinositol 4,5-biphosphate, lysophosphatidic acid, and cholesterol, bind to TRPV1 with high affinity to modulate channel gating. Here, we discuss the functional evidence regarding ligand-dependent activation of TRPV1 channels in light of structural data recently obtained by cryoelectron microscopy. This review focuses on the mechanistic insights into ligand binding and allosteric gating of TRPV1 channels and the relevance of accurate polymodal receptor biophysical characterization for drug design in novel pain therapies. PMID:27335334

  16. Thinking in cycles: MWC is a good model for acetylcholine receptor-channels

    PubMed Central

    Auerbach, Anthony

    2012-01-01

    Abstract Neuromuscular acetylcholine receptors have long been a model system for understanding the mechanisms of operation of ligand-gated ion channels and fast chemical synapses. These five subunit membrane proteins have two allosteric (transmitter) binding sites and a distant ion channel domain. Occupation of the binding sites by agonist molecules transiently increases the probability that the channel is ion-permeable. Recent experiments show that the Monod, Wyman and Changeux formalism for allosteric proteins, originally developed for haemoglobin, is an excellent model for acetylcholine receptors. By using mutations and single-channel electrophysiology, the gating equilibrium constants for receptors with zero, one or two bound agonist molecules, and the agonist association and dissociation rate constants from both the closed- and open-channel conformations, have been estimated experimentally. The change in affinity for each transmitter molecule between closed and open conformations provides ∼–5.1 kcal mol−1 towards the global gating isomerization of the protein. PMID:21807612

  17. Structure-Driven Pharmacology of Transient Receptor Potential Channel Vanilloid 1.

    PubMed

    Díaz-Franulic, Ignacio; Caceres-Molina, Javier; Sepulveda, Romina V; Gonzalez-Nilo, Fernando; Latorre, Ramon

    2016-09-01

    The transient receptor potential vanilloid 1 (TRPV1) ion channel is a polymodal receptor that mediates the flux of cations across the membrane in response to several stimuli, including heat, voltage, and ligands. The best known agonist of TRPV1 channels is capsaicin, the pungent component of "hot" chili peppers. In addition, peptides found in the venom of poisonous animals, along with the lipids phosphatidylinositol 4,5-biphosphate, lysophosphatidic acid, and cholesterol, bind to TRPV1 with high affinity to modulate channel gating. Here, we discuss the functional evidence regarding ligand-dependent activation of TRPV1 channels in light of structural data recently obtained by cryoelectron microscopy. This review focuses on the mechanistic insights into ligand binding and allosteric gating of TRPV1 channels and the relevance of accurate polymodal receptor biophysical characterization for drug design in novel pain therapies.

  18. Molecular mechanism of ATP binding and ion channel activation in P2X receptors

    SciTech Connect

    Hattori, Motoyuki; Gouaux, Eric

    2012-10-24

    P2X receptors are trimeric ATP-activated ion channels permeable to Na{sup +}, K{sup +} and Ca{sup 2+}. The seven P2X receptor subtypes are implicated in physiological processes that include modulation of synaptic transmission, contraction of smooth muscle, secretion of chemical transmitters and regulation of immune responses. Despite the importance of P2X receptors in cellular physiology, the three-dimensional composition of the ATP-binding site, the structural mechanism of ATP-dependent ion channel gating and the architecture of the open ion channel pore are unknown. Here we report the crystal structure of the zebrafish P2X4 receptor in complex with ATP and a new structure of the apo receptor. The agonist-bound structure reveals a previously unseen ATP-binding motif and an open ion channel pore. ATP binding induces cleft closure of the nucleotide-binding pocket, flexing of the lower body {beta}-sheet and a radial expansion of the extracellular vestibule. The structural widening of the extracellular vestibule is directly coupled to the opening of the ion channel pore by way of an iris-like expansion of the transmembrane helices. The structural delineation of the ATP-binding site and the ion channel pore, together with the conformational changes associated with ion channel gating, will stimulate development of new pharmacological agents.

  19. Principal pathway coupling agonist binding to channel gating in nicotinic receptors

    NASA Astrophysics Data System (ADS)

    Lee, Won Yong; Sine, Steven M.

    2005-11-01

    Synaptic receptors respond to neurotransmitters by opening an intrinsic ion channel in the final step in synaptic transmission. How binding of the neurotransmitter is conveyed over the long distance to the channel remains a central question in neurobiology. Here we delineate a principal pathway that links neurotransmitter binding to channel gating by using a structural model of the Torpedo acetylcholine receptor at 4-Å resolution, recordings of currents through single receptor channels and determinations of energetic coupling between pairs of residues. We show that a pair of invariant arginine and glutamate residues in each receptor α-subunit electrostatically links peripheral and inner β-sheets from the binding domain and positions them to engage with the channel. The key glutamate and flanking valine residues energetically couple to conserved proline and serine residues emerging from the top of the channel-forming α-helix, suggesting that this is the point at which the binding domain triggers opening of the channel. The series of interresidue couplings identified here constitutes a primary allosteric pathway that links neurotransmitter binding to channel gating.

  20. Channel catfish, Ictalurus punctatus, chemokine receptor CXCR4 cDNA

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Chemokine receptor CXCR4, a member of the G protein-coupled receptor superfamily, binds selectively CXCL12. This protein plays many important roles in immunological as well as pathophysiological functions. In this communication, we identified and characterized the channel catfish CXCR4 transcript....

  1. GIRK channel activation via adenosine or muscarinic receptors has similar effects on rat atrial electrophysiology.

    PubMed

    Wang, Xiaodong; Liang, Bo; Skibsbye, Lasse; Olesen, Søren-Peter; Grunnet, Morten; Jespersen, Thomas

    2013-08-01

    G protein-coupled inwardly rectifying K⁺ channels (GIRK) are important in the regulation of heart rate and atrial electrophysiology. GIRK channels are activated by G protein-coupled receptors, including muscarinic M₂ receptors and adenosine A₁ receptors. The aim of this study was to characterize and compare the electrophysiological effects of acetylcholine (ACh) and adenosine on GIRK channels in rat atria. Action potential duration at 90% repolarization (APD₉₀), effective refractory period (ERP), and resting membrane potential (RMP) were investigated in isolated rat atria by intracellular recordings. Both the adenosine analog N6-cyclopentyladenosine (CPA) and ACh profoundly shortened APD₉₀ and ERP and hyperpolarized the RMP. No additive or synergistic effect of CPA and ACh coapplication was observed. To antagonize GIRK channel activation, the specific inhibitor rTertiapin Q (TTQ) was applied. The coapplication of TTQ reversed the CPA and ACh-induced effects. When TTQ was applied without exogenous receptor activator, both APD₉₀ and ERP were prolonged and RMP was depolarized, confirming a basal activity of the GIRK current. The results reveal that activation of A₁ and M₂ receptors has a profound and equal effect on the electrophysiology in rat atrium. This effect is to a major extent mediated through GIRK channels. Furthermore, these results support the notion that atrial GIRK currents from healthy hearts have a basal component and additional activation can be mediated via at least 2 different receptor mechanisms. PMID:23609329

  2. The opioid peptide dynorphin directly blocks NMDA receptor channels in the rat.

    PubMed Central

    Chen, L; Gu, Y; Huang, L Y

    1995-01-01

    1. The actions of dynorphin on N-methyl-D-aspartate (NMDA) responses were examined in acutely dissociated trigeminal neurons in rat. Whole-cell and single-channel currents were recorded using the patch clamp technique. 2. Dynorphins reduced NMDA-activated currents (INMDA). The IC50 was 0.25 microM for dynorphin (1-32), 1.65 microM for dynorphin (1-17) and 1.8 microM for dynorphin (1-13). 3. The blocking action of dynorphin is voltage independent. 4. The inhibitory action of dynorphin cannot be blocked by high concentration of the non-selective opioid receptor antagonist naloxone, nor by the specific kappa-opioid receptor antagonist nor-Binaltorphimine (nor-BNI). 5. Single-channel analyses indicate that dynorphin reduces the fraction of time the channel is open without altering the channel conductance. 6. We propose that dynorphin acts directly on NMDA receptors. PMID:7537820

  3. Peptide fragments of the dihydropyridine receptor can modulate cardiac ryanodine receptor channel activity and sarcoplasmic reticulum Ca2+ release.

    PubMed Central

    Dulhunty, Angela F; Curtis, Suzanne M; Cengia, Louise; Sakowska, Magdalena; Casarotto, Marco G

    2004-01-01

    We show that peptide fragments of the dihydropyridine receptor II-III loop alter cardiac RyR (ryanodine receptor) channel activity in a cytoplasmic Ca2+-dependent manner. The peptides were AC (Thr-793-Ala-812 of the cardiac dihydropyridine receptor), AS (Thr-671-Leu-690 of the skeletal dihydropyridine receptor), and a modified AS peptide [AS(D-R18)], with an extended helical structure. The peptides added to the cytoplasmic side of channels in lipid bilayers at > or = 10 nM activated channels when the cytoplasmic [Ca2+] was 100 nM, but either inhibited or did not affect channel activity when the cytoplasmic [Ca2+] was 10 or 100 microM. Both activation and inhibition were independent of bilayer potential. Activation by AS, but not by AC or AS(D-R18), was reduced at peptide concentrations >1 mM in a voltage-dependent manner (at +40 mV). In control experiments, channels were not activated by the scrambled AS sequence (ASS) or skeletal II-III loop peptide (NB). Resting Ca2+ release from cardiac sarcoplasmic reticulum was not altered by peptide AC, but Ca2+-induced Ca2+ release was depressed. Resting and Ca2+-induced Ca2+ release were enhanced by both the native and modified AS peptides. NMR revealed (i) that the structure of peptide AS(D-R18) is not influenced by [Ca2+] and (ii) that peptide AC adopts a helical structure, particularly in the region containing positively charged residues. This is the first report of specific functional interactions between dihydropyridine receptor A region peptides and cardiac RyR ion channels in lipid bilayers. PMID:14678014

  4. Ion channel profile of TRPM8 cold receptors reveals a role of TASK-3 potassium channels in thermosensation.

    PubMed

    Morenilla-Palao, Cruz; Luis, Enoch; Fernández-Peña, Carlos; Quintero, Eva; Weaver, Janelle L; Bayliss, Douglas A; Viana, Félix

    2014-09-11

    Animals sense cold ambient temperatures through the activation of peripheral thermoreceptors that express TRPM8, a cold- and menthol-activated ion channel. These receptors can discriminate a very wide range of temperatures from innocuous to noxious. The molecular mechanism responsible for the variable sensitivity of individual cold receptors to temperature is unclear. To address this question, we performed a detailed ion channel expression analysis of cold-sensitive neurons, combining bacterial artificial chromosome (BAC) transgenesis with a molecular-profiling approach in fluorescence-activated cell sorting (FACS)-purified TRPM8 neurons. We found that TASK-3 leak potassium channels are highly enriched in a subpopulation of these sensory neurons. The thermal threshold of TRPM8 cold neurons is decreased during TASK-3 blockade and in mice lacking TASK-3, and, most importantly, these mice display hypersensitivity to cold. Our results demonstrate a role of TASK-3 channels in thermosensation, showing that a channel-based combinatorial strategy in TRPM8 cold thermoreceptors leads to molecular specialization and functional diversity. PMID:25199828

  5. Regulation of Transient Receptor Potential channels by the phospholipase C pathway

    PubMed Central

    Rohacs, Tibor

    2013-01-01

    Transient Receptor Potential (TRP) channels were discovered while analyzing visual mutants in drosophila. The protein encoded by the transient receptor potential (trp) gene is a Ca2+ permeable cation channel activated downstream of the phospholipase C (PLC) pathway. While searching for homologues in other organisms, a surprisingly large number of mammalian TRP channels were cloned. The regulation of TRP channels is quite diverse, but many of them are either activated downstream of the PLC pathway, or modulated by it. This review will summarize the current knowledge on regulation of TRP channels by the PLC pathway, with special focus on TRPC-s, which can be considered as effectors of the PLC pathway, and the heat and capsaicin sensitive TRPV1, which is modulated by the PLC pathway in a complex manner. PMID:23916247

  6. The human histamine H3 receptor couples to GIRK channels in Xenopus oocytes.

    PubMed

    Sahlholm, Kristoffer; Nilsson, Johanna; Marcellino, Daniel; Fuxe, Kjell; Arhem, Peter

    2007-07-19

    The histamine H(3) receptor mediates inhibitory responses in the nervous system. Here, we demonstrate the coupling of the human histamine H(3) receptor to G protein-coupled inward rectifier potassium (GIRK) channels in Xenopus oocytes, using voltage-clamp. The histamine H(3) receptor agonist (R)-alpha-methylhistamine increased GIRK currents with an EC(50) of 2.5 nM. The response to (R)-alpha-methylhistamine was inhibited by the specific antagonist/inverse agonist clobenpropit. GIRK channels represent a novel effector pathway for the histamine H(3) receptor, also suggesting the use of electrophysiology assays in histamine H(3) receptor drug screening, allowing for the resolution of G protein activation kinetics.

  7. Electron density topography based model to explore N-methyl-D-aspartate receptor channel blockers

    NASA Astrophysics Data System (ADS)

    Ingle, Snehal V.; Joshi, Kaustubh A.

    2016-03-01

    The dwell time of a molecule in a voltage dependent NMDA receptor channel is an important factor in defining its activity as channel blocker. A model has been designed, based on quantum chemical descriptors like geometrical parameters, charge distribution, electron density topography and global reactivity descriptors, to shed lights on the dwell time of a channel blocker. Structure and charge distribution studies indicate polarization of molecules with the electron density located at the core of the molecule. Electron density topography reveals ring critical point (ρrcp), emerging as a signature parameter to understand the dwell time of a channel blocker molecule.

  8. Block of NMDA receptor channels by endogenous neurosteroids: implications for the agonist induced conformational states of the channel vestibule

    PubMed Central

    Vyklicky, Vojtech; Krausova, Barbora; Cerny, Jiri; Balik, Ales; Zapotocky, Martin; Novotny, Marian; Lichnerova, Katarina; Smejkalova, Tereza; Kaniakova, Martina; Korinek, Miloslav; Petrovic, Milos; Kacer, Petr; Horak, Martin; Chodounska, Hana; Vyklicky, Ladislav

    2015-01-01

    N-methyl-D-aspartate receptors (NMDARs) mediate synaptic plasticity, and their dysfunction is implicated in multiple brain disorders. NMDARs can be allosterically modulated by numerous compounds, including endogenous neurosteroid pregnanolone sulfate. Here, we identify the molecular basis of the use-dependent and voltage-independent inhibitory effect of neurosteroids on NMDAR responses. The site of action is located at the extracellular vestibule of the receptor’s ion channel pore and is accessible after receptor activation. Mutations in the extracellular vestibule in the SYTANLAAF motif disrupt the inhibitory effect of negatively charged steroids. In contrast, positively charged steroids inhibit mutated NMDAR responses in a voltage-dependent manner. These results, in combination with molecular modeling, characterize structure details of the open configuration of the NMDAR channel. Our results provide a unique opportunity for the development of new therapeutic neurosteroid-based ligands to treat diseases associated with dysfunction of the glutamate system. PMID:26086919

  9. Liposome reconstitution and modulation of recombinant N-methyl-d-aspartate receptor channels by membrane stretch

    PubMed Central

    Kloda, Anna; Lua, Linda; Hall, Rhonda; Adams, David J.; Martinac, Boris

    2007-01-01

    In this study, the heteromeric N-methyl-d-aspartate (NMDA) receptor channels composed of NR1a and NR2A subunits were expressed, purified, reconstituted into liposomes, and characterized by using the patch clamp technique. The protein exhibited the expected electrophysiological profile of activation by glutamate and glycine and internal Mg2+ blockade. We demonstrated that the mechanical energy transmitted to membrane-bound NMDA receptor channels can be exerted directly by tension developed in the lipid bilayer. Membrane stretch and application of arachidonic acid potentiated currents through NMDA receptor channels in the presence of intracellular Mg2+. The correlation of membrane tension induced by either mechanical or chemical stimuli with the physiological Mg2+ block of the channel suggests that the synaptic transmission can be altered if NMDA receptor complexes experience local changes in bilayer thickness caused by dynamic targeting to lipid microdomains, electrocompression, or chemical modification of the cell membranes. The ability to study gating properties of NMDA receptor channels in artificial bilayers should prove useful in further study of structure–function relationships and facilitate discoveries of new therapeutic agents for treatment of glutamate-mediated excitotoxicity or analgesic therapies. PMID:17242368

  10. Temperature-sensitive transient receptor potential channels in corneal tissue layers and cells.

    PubMed

    Mergler, Stefan; Valtink, Monika; Takayoshi, Sumioka; Okada, Yuka; Miyajima, Masayasu; Saika, Shizuya; Reinach, Peter S

    2014-01-01

    We here provide a brief summary of the characteristics of transient receptor potential channels (TRPs) identified in corneal tissue layers and cells. In general, TRPs are nonselective cation channels which are Ca(2+) permeable. Most TRPs serve as thermosensitive molecular sensors (thermo-TRPs). Based on their functional importance, the possibilities are described for drug-targeting TRP activity in a clinical setting. TRPs are expressed in various tissues of the eye including both human corneal epithelial and endothelial layers as well as stromal fibroblasts and stromal nerve fibers. TRP vanilloid type 1 (TRPV1) heat receptor, also known as capsaicin receptor, along with TRP melastatin type 8 (TRPM8) cold receptor, which is also known as menthol receptor, are prototypes of the thermo-TRP family. The TRPV1 functional channel is the most investigated TRP channel in these tissues, owing to its contribution to maintaining tissue homeostasis as well as eliciting wound healing responses to injury. Other thermo-TRP family members identified in these tissues are TRPV2, 3 and 4. Finally, there is the TRP ankyrin type 1 (TRPA1) cold receptor. All of these thermo-TRPs can be activated within specific temperature ranges and transduce such inputs into chemical and electrical signals. Although several recent studies have begun to unravel complex roles for thermo-TRPs such as TRPV1 in corneal layers and resident cells, additional studies are needed to further elucidate their roles in health and disease.

  11. LE135, a retinoid acid receptor antagonist, produces pain through direct activation of TRP channels

    PubMed Central

    Yin, Shijin; Luo, Jialie; Qian, Aihua; Yu, Weihua; Hu, Hongzhen

    2014-01-01

    Background and PurposeRetinoids, through their activation of retinoic acid receptors (RARs) and retinoid X receptors, regulate diverse cellular processes, and pharmacological intervention in their actions has been successful in the treatment of skin disorders and cancers. Despite the many beneficial effects, administration of retinoids causes irritating side effects with unknown mechanisms. Here, we demonstrate that LE135 [4-(7,8,9,10-tetrahydro-5,7,7,10,10-pentamethyl-5H-benzo[e]naphtho[2,3-b][1,4]diazepin-13-yl)benzoic acid], a selective antagonist of RARβ, is a potent activator of the capsaicin (TRPV1) and wasabi (TRPA1) receptors, two critical pain-initiating cation channels. Experimental ApproachWe performed to investigate the excitatory effects of LE135 on TRPV1 and TRPA1 channels expressed in HEK293T cells and in dorsal root ganglia neurons with calcium imaging and patch-clamp recordings. We also used site-directed mutagenesis of the channels to determine the structural basis of LE135-induced activation of TRPV1 and TRPA1 channels and behavioural testing to examine if pharmacological inhibition and genetic deletion of the channels affected LE135-evoked pain-related behaviours. Key ResultsLE135 activated both the capsaicin receptor (TRPV1) and the allyl isothiocyanate receptor (TRPA1) heterologously expressed in HEK293T cells and endogenously expressed by sensory nociceptors. Mutations disrupting the capsaicin-binding site attenuated LE135 activation of TRPV1 channels and a single mutation (K170R) eliminated TRPA1 activity evoked by LE135. Intraplantar injection of LE135 evoked pain-related behaviours. Both TRPV1 and TRPA1 channels were involved in LE135-elicited pain-related responses, as shown by pharmacological and genetic ablation studies. Conclusions and ImplicationsThis blocker of retinoid acid signalling also exerted non-genomic effects through activating the pain-initiating TRPV1 and TRPA1 channels. PMID:24308840

  12. Discovery of 2-substituted benzoxazole carboxamides as 5-HT3 receptor antagonists.

    PubMed

    Yang, Zhicai; Fairfax, David J; Maeng, Jun-Ho; Masih, Liaqat; Usyatinsky, Alexander; Hassler, Carla; Isaacson, Soshanna; Fitzpatrick, Kevin; DeOrazio, Russell J; Chen, Jianqing; Harding, James P; Isherwood, Matthew; Dobritsa, Svetlana; Christensen, Kevin L; Wierschke, Jonathan D; Bliss, Brian I; Peterson, Lisa H; Beer, Cathy M; Cioffi, Christopher; Lynch, Michael; Rennells, W Martin; Richards, Justin J; Rust, Timothy; Khmelnitsky, Yuri L; Cohen, Marlene L; Manning, David D

    2010-11-15

    A new class of 2-substituted benzoxazole carboxamides are presented as potent functional 5-HT(3) receptor antagonists. The chemical series possesses nanomolar in vitro activity against human 5-HT(3)A receptors. A chemistry optimization program was conducted and identified 2-aminobenzoxazoles as orally active 5-HT(3) receptor antagonists with good metabolic stability. These novel analogues possess drug-like characteristics and have potential utility for the treatment of diseases attributable to improper 5-HT(3) receptor function, especially diarrhea predominant irritable bowel syndrome (IBS-D).

  13. Vector-averaged gravity does not alter acetylcholine receptor single channel properties

    NASA Technical Reports Server (NTRS)

    Reitstetter, R.; Gruener, R.

    1994-01-01

    To examine the physiological sensitivity of membrane receptors to altered gravity, we examined the single channel properties of the acetylcholine receptor (AChR), in co-cultures of Xenopus myocytes and neurons, to vector-averaged gravity in the clinostat. This experimental paradigm produces an environment in which, from the cell's perspective, the gravitational vector is "nulled" by continuous averaging. In that respect, the clinostat simulates one aspect of space microgravity where the gravity force is greatly reduced. After clinorotation, the AChR channel mean open-time and conductance were statistically not different from control values but showed a rotation-dependent trend that suggests a process of cellular adaptation to clinorotation. These findings therefore suggest that the ACHR channel function may not be affected in the microgravity of space despite changes in the receptor's cellular organization.

  14. Transient Receptor Potential Channel Polymorphisms Are Associated with the Somatosensory Function in Neuropathic Pain Patients

    PubMed Central

    Baron, Ralf; Maier, Christoph; Tölle, Thomas R.; Treede, Rolf-Detlef; Berthele, Achim; Faltraco, Frank; Flor, Herta; Gierthmühlen, Janne; Haenisch, Sierk; Huge, Volker; Magerl, Walter; Maihöfner, Christian; Richter, Helmut; Rolke, Roman; Scherens, Andrea; Üçeyler, Nurcan; Ufer, Mike; Wasner, Gunnar; Zhu, Jihong; Cascorbi, Ingolf

    2011-01-01

    Transient receptor potential channels are important mediators of thermal and mechanical stimuli and play an important role in neuropathic pain. The contribution of hereditary variants in the genes of transient receptor potential channels to neuropathic pain is unknown. We investigated the frequency of transient receptor potential ankyrin 1, transient receptor potential melastin 8 and transient receptor potential vanilloid 1 single nucleotide polymorphisms and their impact on somatosensory abnormalities in neuropathic pain patients. Within the German Research Network on Neuropathic Pain (Deutscher Forscbungsverbund Neuropathischer Schmerz) 371 neuropathic pain patients were phenotypically characterized using standardized quantitative sensory testing. Pyrosequencing was employed to determine a total of eleven single nucleotide polymorphisms in transient receptor potential channel genes of the neuropathic pain patients and a cohort of 253 German healthy volunteers. Associations of quantitative sensory testing parameters and single nucleotide polymorphisms between and within groups and subgroups, based on sensory phenotypes, were analyzed. Single nucleotide polymorphisms frequencies did not differ between both the cohorts. However, in neuropathic pain patients transient receptor potential ankyrin 1 710G>A (rs920829, E179K) was associated with the presence of paradoxical heat sensation (p = 0.03), and transient receptor potential vanilloid 1 1911A>G (rs8065080, I585V) with cold hypoalgesia (p = 0.0035). Two main subgroups characterized by preserved (1) and impaired (2) sensory function were identified. In subgroup 1 transient receptor potential vanilloid 1 1911A>G led to significantly less heat hyperalgesia, pinprick hyperalgesia and mechanical hypaesthesia (p = 0.006, p = 0.005 and p<0.001) and transient receptor potential vanilloid 1 1103C>G (rs222747, M315I) to cold hypaesthesia (p = 0.002), but there was absence of associations in subgroup 2. In

  15. Structural Insights into Divalent Cation Modulations of ATP-Gated P2X Receptor Channels.

    PubMed

    Kasuya, Go; Fujiwara, Yuichiro; Takemoto, Mizuki; Dohmae, Naoshi; Nakada-Nakura, Yoshiko; Ishitani, Ryuichiro; Hattori, Motoyuki; Nureki, Osamu

    2016-02-01

    P2X receptors are trimeric ATP-gated cation channels involved in physiological processes ranging widely from neurotransmission to pain and taste signal transduction. The modulation of the channel gating, including that by divalent cations, contributes to these diverse physiological functions of P2X receptors. Here, we report the crystal structure of an invertebrate P2X receptor from the Gulf Coast tick Amblyomma maculatum in the presence of ATP and Zn(2+) ion, together with electrophysiological and computational analyses. The structure revealed two distinct metal binding sites, M1 and M2, in the extracellular region. The M1 site, located at the trimer interface, is responsible for Zn(2+) potentiation by facilitating the structural change of the extracellular domain for pore opening. In contrast, the M2 site, coupled with the ATP binding site, might contribute to regulation by Mg(2+). Overall, our work provides structural insights into the divalent cation modulations of P2X receptors. PMID:26804916

  16. Single Expressed Glycine Receptor Domains Reconstitute Functional Ion Channels without Subunit-specific Desensitization Behavior*

    PubMed Central

    Meiselbach, Heike; Vogel, Nico; Langlhofer, Georg; Stangl, Sabine; Schleyer, Barbara; Bahnassawy, Lamia'a; Sticht, Heinrich; Breitinger, Hans-Georg; Becker, Cord-Michael; Villmann, Carmen

    2014-01-01

    Cys loop receptors are pentameric arrangements of independent subunits that assemble into functional ion channels. Each subunit shows a domain architecture. Functional ion channels can be reconstituted even from independent, nonfunctional subunit domains, as shown previously for GlyRα1 receptors. Here, we demonstrate that this reconstitution is not restricted to α1 but can be transferred to other members of the Cys loop receptor family. A nonfunctional GlyR subunit, truncated at the intracellular TM3–4 loop by a premature stop codon, can be complemented by co-expression of the missing tail portion of the receptor. Compared with α1 subunits, rescue by domain complementation was less efficient when GlyRα3 or the GABAA/C subunit ρ1 was used. If truncation disrupted an alternative splicing cassette within the intracellular TM3–4 loop of α3 subunits, which also regulates receptor desensitization, functional rescue was not possible. When α3 receptors were restored by complementation using domains with and without the spliced insert, no difference in desensitization was found. In contrast, desensitization properties could even be transferred between α1/α3 receptor chimeras harboring or lacking the α3 splice cassette proving that functional rescue depends on the integrity of the alternative splicing cassette in α3. Thus, an intact α3 splicing cassette in the TM3–4 loop environment is indispensable for functional rescue, and the quality of receptor restoration can be assessed from desensitization properties. PMID:25143388

  17. Single-Particle Cryo-EM of the Ryanodine Receptor Channel in an Aqueous Environment.

    PubMed

    Baker, Mariah R; Fan, Guizhen; Serysheva, Irina I

    2015-01-01

    Ryanodine receptors (RyRs) are tetrameric ligand-gated Ca(2+) release channels that are responsible for the increase of cytosolic Ca(2+) concentration leading to muscle contraction. Our current understanding of RyR channel gating and regulation is greatly limited due to the lack of a high-resolution structure of the channel protein. The enormous size and unwieldy shape of Ca(2+) release channels make X-ray or NMR methods difficult to apply for high-resolution structural analysis of the full-length functional channel. Single-particle electron cryo-microscopy (cryo-EM) is one of the only effective techniques for the study of such a large integral membrane protein and its molecular interactions. Despite recent developments in cryo-EM technologies and break-through single-particle cryo-EM studies of ion channels, cryospecimen preparation, particularly the presence of detergent in the buffer, remains the main impediment to obtaining atomic-resolution structures of ion channels and a multitude of other integral membrane protein complexes. In this review we will discuss properties of several detergents that have been successfully utilized in cryo-EM studies of ion channels and the emergence of the detergent alternative amphipol to stabilize ion channels for structure-function characterization. Future structural studies of challenging specimen like ion channels are likely to be facilitated by cryo-EM amenable detergents or alternative surfactants.

  18. Insights into the channel gating of P2X receptors from structures, dynamics and small molecules

    PubMed Central

    Wang, Jin; Yu, Ye

    2016-01-01

    P2X receptors, as ATP-gated non-selective trimeric ion channels, are permeable to Na+, K+ and Ca2+. Comparing with other ligand-gated ion channel families, P2X receptors are distinct in their unique gating properties and pathophysiological roles, and have attracted attention as promising drug targets for a variety of diseases, such as neuropathic pain, multiple sclerosis, rheumatoid arthritis and thrombus. Several small molecule inhibitors for distinct P2X subtypes have entered into clinical trials. However, many questions regarding the gating mechanism of P2X remain unsolved. The structural determinations of P2X receptors at the resting and ATP-bound open states revealed that P2X receptor gating is a cooperative allosteric process involving multiple domains, which marks the beginning of the post-structure era of P2X research at atomic level. Here, we review the current knowledge on the structure-function relationship of P2X receptors, depict the whole picture of allosteric changes during the channel gating, and summarize the active sites that may contribute to new strategies for developing novel allosteric drugs targeting P2X receptors. PMID:26725734

  19. Functional Chimeras of GLIC Obtained by Adding the Intracellular Domain of Anion- and Cation-Conducting Cys-Loop Receptors.

    PubMed

    Mnatsakanyan, Nelli; Nishtala, Sita Nirupama; Pandhare, Akash; Fiori, Mariana C; Goyal, Raman; Pauwels, Jonathan E; Navetta, Andrew F; Ahrorov, Afzal; Jansen, Michaela

    2015-04-28

    Pentameric ligand-gated ion channels (pLGICs), also called Cys-loop receptors in eukaryotic superfamily members, play diverse roles in neurotransmission and serve as primary targets for many therapeutic drugs. Structural studies of full-length eukaryotic pLGICs have been challenging because of glycosylation, large size, pentameric assembly, and hydrophobicity. X-ray structures of prokaryotic pLGICs, including the Gloeobacter violaceus LGIC (GLIC) and the Erwinia chrysanthemi LGIC (ELIC), and truncated eukaryotic pLGICs have significantly improved and complemented the understanding of structural details previously obtained with acetylcholine-binding protein and Torpedo nicotinic acetylcholine receptors. Prokaryotic pLGICs share their overall structural features with eukaryotic pLGICs for the ligand-binding extracellular and channel-lining transmembrane domains. The large intracellular domain (ICD) is present only in eukaryotic members and is characterized by a low level of sequence conservation and significant variability in length (50-250 amino acids), making the ICD a potential target for the modulation of specific pLGIC subunits. None of the structures includes a complete ICD. Here, we created chimeras by adding the ICD of cation-conducting (nAChR-α7) and anion-conducting (GABAρ1, Glyα1) eukaryotic homopentamer-forming pLGICs to GLIC. GLIC-ICD chimeras assemble into pentamers to form proton-gated channels, as does the parent GLIC. Additionally, the sensitivity of the chimeras toward modulation of functional maturation by chaperone protein RIC-3 is preserved as in those of the parent eukaryotic channels. For a previously described GLIC-5HT3A-ICD chimera, we now provide evidence of its successful large-scale expression and purification to homogeneity. Overall, the chimeras provide valuable tools for functional and structural studies of eukaryotic pLGIC ICDs. PMID:25861708

  20. Biophysical analysis of thermosensitive TRP channels with a special focus on the cold receptor TRPM8.

    PubMed

    Carrasquel-Ursulaez, Willy; Moldenhauer, Hans; Castillo, Juan Pablo; Latorre, Ramón; Alvarez, Osvaldo

    2015-01-01

    Mammals maintain homeostatic control of their body temperature. Therefore, these organisms are expected to have adaptations that confer the ability to detect and react to both self and ambient temperature. Temperature-activated ion channels have been discovered to be the primary molecular determinants of thermosensation. The most representative group of these determinants constitutes members of the transient receptor potential superfamily, TRP, which are activated by either low or high temperatures covering the whole range of physiologically relevant temperatures. This review makes a critical assessment of existing analytical methods of temperature-activated TRP channel mechanisms using the cold-activated TRPM8 channel as a paradigm. PMID:27227023

  1. Biophysical analysis of thermosensitive TRP channels with a special focus on the cold receptor TRPM8

    PubMed Central

    Carrasquel-Ursulaez, Willy; Moldenhauer, Hans; Castillo, Juan Pablo; Latorre, Ramón; Alvarez, Osvaldo

    2015-01-01

    Mammals maintain homeostatic control of their body temperature. Therefore, these organisms are expected to have adaptations that confer the ability to detect and react to both self and ambient temperature. Temperature-activated ion channels have been discovered to be the primary molecular determinants of thermosensation. The most representative group of these determinants constitutes members of the transient receptor potential superfamily, TRP, which are activated by either low or high temperatures covering the whole range of physiologically relevant temperatures. This review makes a critical assessment of existing analytical methods of temperature-activated TRP channel mechanisms using the cold-activated TRPM8 channel as a paradigm. PMID:27227023

  2. A common mechanism underlies stretch activation and receptor activation of TRPC6 channels

    PubMed Central

    Spassova, Maria A.; Hewavitharana, Thamara; Xu, Wen; Soboloff, Jonathan; Gill, Donald L.

    2006-01-01

    The TRP family of ion channels transduce an extensive range of chemical and physical signals. TRPC6 is a receptor-activated nonselective cation channel expressed widely in vascular smooth muscle and other cell types. We report here that TRPC6 is also a sensor of mechanically and osmotically induced membrane stretch. Pressure-induced activation of TRPC6 was independent of phospholipase C. The stretch responses were blocked by the tarantula peptide, GsMTx-4, known to specifically inhibit mechanosensitive channels by modifying the external lipid-channel boundary. The GsMTx-4 peptide also blocked the activation of TRPC6 channels by either receptor-induced PLC activation or by direct application of diacylglycerol. The effects of the peptide on both stretch- and diacylglycerol-mediated TRPC6 activation indicate that the mechanical and chemical lipid sensing by the channel has a common molecular mechanism that may involve lateral-lipid tension. The mechanosensing properties of TRPC6 channels highly expressed in smooth muscle cells are likely to play a key role in regulating myogenic tone in vascular tissue. PMID:17056714

  3. Transient Receptor Potential Channels in Microglia: Roles in Physiology and Disease.

    PubMed

    Echeverry, Santiago; Rodriguez, María Juliana; Torres, Yolima P

    2016-10-01

    Microglia modulate the nervous system cellular environment and induce neuroprotective and neurotoxic effects. Various molecules are involved in these processes, including families of ion channels expressed in microglial cells, such as transient receptor potential (TRP) channels. TRP channels comprise a family of non-selective cation channels that can be activated by mechanical, thermal, and chemical stimuli, and which contribute to the regulation of intracellular calcium concentrations. TRP channels have been shown to be involved in cellular processes such as osmotic regulation, cytokine production, proliferation, activation, cell death, and oxidative stress responses. Given the significance of these processes in microglial activity, studies of TRP channels in microglia have focused on determining their roles in both neuroprotective and neurotoxic processes. TRP channel activity has been proposed to play an important function in neurodegenerative diseases, ischemia, inflammatory responses, and neuropathic pain. Modulation of TRP channel activity may thus be considered as a potential therapeutic strategy for the treatment of various diseases associated with alterations of the central nervous system (CNS). In this review, we describe the expression of different subfamilies of TRP channels in microglia, focusing on their physiological and pathophysiological roles, and consider their potential use as therapeutic targets in CNS diseases. PMID:27260222

  4. Transient Receptor Potential Channels in Microglia: Roles in Physiology and Disease.

    PubMed

    Echeverry, Santiago; Rodriguez, María Juliana; Torres, Yolima P

    2016-10-01

    Microglia modulate the nervous system cellular environment and induce neuroprotective and neurotoxic effects. Various molecules are involved in these processes, including families of ion channels expressed in microglial cells, such as transient receptor potential (TRP) channels. TRP channels comprise a family of non-selective cation channels that can be activated by mechanical, thermal, and chemical stimuli, and which contribute to the regulation of intracellular calcium concentrations. TRP channels have been shown to be involved in cellular processes such as osmotic regulation, cytokine production, proliferation, activation, cell death, and oxidative stress responses. Given the significance of these processes in microglial activity, studies of TRP channels in microglia have focused on determining their roles in both neuroprotective and neurotoxic processes. TRP channel activity has been proposed to play an important function in neurodegenerative diseases, ischemia, inflammatory responses, and neuropathic pain. Modulation of TRP channel activity may thus be considered as a potential therapeutic strategy for the treatment of various diseases associated with alterations of the central nervous system (CNS). In this review, we describe the expression of different subfamilies of TRP channels in microglia, focusing on their physiological and pathophysiological roles, and consider their potential use as therapeutic targets in CNS diseases.

  5. Characterization of additional novel immune type receptors in channel catfish, Ictalurus punctatus

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Mining of channel catfish (Ictalurus punctatus) expressed sequence tag databases identified seven new novel immune type receptors (IpNITRs). These differed in sequence, but not structure, from previously described IpNITR1-11. IpNITR12a, 12b, 13 and 14, encode proteins containing a single variable (V...

  6. Reconstitution of Purified Acetylcholine Receptors with Functional Ion Channels in Planar Lipid Bilayers

    NASA Astrophysics Data System (ADS)

    Nelson, N.; Anholt, R.; Lindstrom, J.; Montal, M.

    1980-05-01

    Acetylcholine receptor, solubilized and purified from Torpedo californica electric organ under conditions that preserve the activity of its ion channel, was reconstituted into vesicles of soybean lipid by the cholate-dialysis technique. The reconstituted vesicles were then spread into monolayers at an air-water interface and planar bilayers were subsequently formed by apposition of two monolayers. Addition of carbamoylcholine caused an increase in membrane conductance that was transient and relaxed spontaneously to the base level (i.e., became desensitized). The response to carbamoylcholine was dose dependent and competitively inhibited by curare. Fluctuations of membrane conductance corresponding to the opening and closing of receptor channels were observed. Fluctuation analysis indicated a single-channel conductance of 16± 3 pS (in 0.1 M NaCl) with a mean channel open time estimated to be 35± 5 ms. Thus, purified acetylcholine receptor reconstituted into lipid bilayers exhibited the pharmacological specificity, activation, and desensitization properties expected of this receptor in native membranes.

  7. Main ion channels and receptors associated with visceral hypersensitivity in irritable bowel syndrome

    PubMed Central

    de Carvalho Rocha, Heraldo Arcela; Dantas, Bruna Priscilla Vasconcelos; Rolim, Thaísa Leite; Costa, Bagnólia Araújo; de Medeiros, Arnaldo Correia

    2014-01-01

    Irritable bowel syndrome (IBS) is a very frequent functional gastrointestinal disorder characterized by recurrent abdominal pain or discomfort and alteration of bowel habits. The IBS physiopathology is extremely complex. Visceral hypersensitivity plays an important role in the pathogenesis of abdominal pain in both in vitro and in vivo models of this functional disorder. In order to obtain a general view of the participation of the main ion channels and receptors regarding the visceral hypersensitivity in the IBS and to describe their chemical structure, a literature review was carried out. A bibliographical research in the following electronic databases: Pubmed and Virtual Library in Health (BVS) was fulfilled by using the search terms “ion channels” “or” “receptors” “and” “visceral hypersensitivity” “or” “visceral nociception” “and” “irritable bowel syndrome”. Original and review articles were considered for data acquisition. The activation of the ATP ion-gated channels, voltage-gated sodium (Nav) and calcium (Cav) channels, as well as the activation of protease-activated receptors (PAR2), transient receptor potential vanilloide-1, serotonin, cannabinoids and cholecystokinin are involved in the genesis of visceral hypersensitivity in IBS. The involvement of ion channels and receptors concerning visceral hypersensitivity is noteworthy in IBS models. PMID:24976114

  8. Regulation of Calcium Channels and Exocytosis in Mouse Adrenal Chromaffin Cells by Prostaglandin EP3 Receptors

    PubMed Central

    Jewell, Mark L.; Breyer, Richard M.

    2011-01-01

    Prostaglandin (PG) E2 controls numerous physiological functions through a family of cognate G protein-coupled receptors (EP1–EP4). Targeting specific EP receptors might be therapeutically useful and reduce side effects associated with nonsteroidal anti-inflammatory drugs and selective cyclooxygenase-2 inhibitors that block prostanoid synthesis. Systemic immune challenge and inflammatory cytokines have been shown to increase expression of the synthetic enzymes for PGE2 in the adrenal gland. Catecholamines and other hormones, released from adrenal chromaffin cells in response to Ca2+ influx through voltage-gated Ca2+ channels, play central roles in homeostatic function and the coordinated stress response. However, long-term elevation of circulating catecholamines contributes to the pathogenesis of hypertension and heart failure. Here, we investigated the EP receptor(s) and cellular mechanisms by which PGE2 might modulate chromaffin cell function. PGE2 did not alter resting intracellular [Ca2+] or the peak amplitude of nicotinic acetylcholine receptor currents, but it did inhibit CaV2 voltage-gated Ca2+ channel currents (ICa). This inhibition was voltage-dependent and mediated by pertussis toxin-sensitive G proteins, consistent with a direct Gβγ subunit-mediated mechanism common to other Gi/o-coupled receptors. mRNA for all four EP receptors was detected, but using selective pharmacological tools and EP receptor knockout mice, we demonstrated that EP3 receptors mediate the inhibition of ICa. Finally, changes in membrane capacitance showed that Ca2+-dependent exocytosis was reduced in parallel with ICa. To our knowledge, this is the first study of EP receptor signaling in mouse chromaffin cells and identifies a molecular mechanism for paracrine regulation of neuroendocrine function by PGE2. PMID:21383044

  9. Purification and reconstitution of the calcium antagonist receptor of the voltage-sensitive calcium channel

    SciTech Connect

    Curtis, B.M.

    1986-01-01

    Treatment with digitonin solubilized the calcium antagonist receptor as a stable complex with (/sup 3/H)nitrendipine from rat brain membranes. The solubilized complex retains allosteric coupling to binding sites for diltiazem, verapamil, and inorganic calcium antagonist sites. The calcium antagonist receptor from cardiac sarcolemma and the transverse-tubule membrane of skeletal muscle is also efficiently solubilized with digitonin and the receptor in all three tissues is a large glycoprotein with a sedimentation coefficient of 20 S. The T-tubule calcium antagonist receptor complex was extensively purified by a combination of chromatography on WGA-Sepharose, ion exchange chromatography, and sedimentation on sucrose gradients to yield preparations estimated to be 41% homogeneous by specific activity and 63% homogeneous by SDS gel electrophoresis. Analysis of SDS gels detect three polypeptides termed ..cap alpha..(Mr 135,000), ..beta..(Mr 50,000), and ..gamma..(Mr 32,000) as noncovalently associated subunits of the calcium antagonist receptor. The ..cap alpha.. and ..gamma.. subunits are glycosylated polypeptides, and the molecular weight of the core polypeptides are 108,000 and 24,000 respectively. The calcium antagonist receptor was reconstituted into a phospholipid bilayer by adding CHAPS and exogeneous lipid to the purified receptor followed by rapid detergent removal. This procedure resulted in the incorporation of 45% of the calcium antagonist receptor into closed phospholipid vesicles. Data suggests that the ..cap alpha.., ..beta.., and ..gamma.. subunits of the T-tubule calcium antagonist receptor are sufficient to form a functional calcium channel.

  10. Receptor model for the molecular basis of tissue selectivity of 1, 4-dihydropyridine calcium channel drugs.

    PubMed

    Langs, D A; Strong, P D; Triggle, D J

    1990-09-01

    Our analysis of the solid state conformations of nifedipine [dimethyl 1,4-dihydro-2,6-dimethyl-4-(2-nitrophenyl)-3,5-pyridinecarboxylate ] and its 1,4-dihydropyridine (1,4-DHP) analogues produced a cartoon description of the important interactions between these drugs and their voltage-dependent calcium channel receptor. In the present study a molecular-level detailed model of the 1,4-DHP receptor binding site has been built from the published amino acid sequence of the alpha 1 subunit of the voltage-dependent calcium channel isolated from rabbit skeletal muscle transverse tubule membranes. The voltage-sensing component of the channel described in this work differs from other reported for the homologous sodium channel in that it incorporates a water structure and a staggered, rather than eclipsed, hydrogen bonded S4 helix conformation. The major recognition surfaces of the receptor lie in helical grooves on the S4 or voltage-sensing alpha-helix that is positioned in the center of the bundle of transmembrane helices that define each of the four calcium channel domains. Multiple binding clefts defined by Arg-X-X-Arg-P-X-X-S 'reading frames' exist on the S4 strand. The tissue selectivity of nifedipine and its analogues may arise, in part, from conservative changes in the amino acid residues at the P and S positions of the reading frame that define the ester-binding regions of receptors from different tissues. The crystal structures of two tissue-selective nifedipine analogues, nimodipine [isopropyl (2-methoxyethyl) 1,4-dihydro-2,6-dimethyl-4-(3-nitrophenyl)-3,5-pyridinecarboxylate ] and nitrendipine [ethyl methyl 1,4-dihydro-2,6-dimethyl-4-(3-nitrophenyl)-3, 5-pyridinecarboxylate] are reported. Nimodipine was observed to have an unusual ester side chain conformation that enhances the fit to the proposed ester-sensing region of the receptor.

  11. Molecular mechanism of the assembly of an acid-sensing receptor ion channel complex.

    PubMed

    Yu, Yong; Ulbrich, Maximilian H; Li, Ming-Hui; Dobbins, Scott; Zhang, Wei K; Tong, Liang; Isacoff, Ehud Y; Yang, Jian

    2012-01-01

    Polycystic kidney disease (PKD) family proteins associate with transient receptor potential (TRP) channel family proteins to form functionally important complexes. PKD proteins differ from known ion channel-forming proteins and are generally thought to act as membrane receptors. Here we find that PKD1L3, a PKD protein, functions as a channel-forming subunit in an acid-sensing heteromeric complex formed by PKD1L3 and TRPP3, a TRP channel protein. Single amino-acid mutations in the putative pore region of both proteins alter the channel's ion selectivity. The PKD1L3/TRPP3 complex in the plasma membrane of live cells contains one PKD1L3 and three TRPP3. A TRPP3 C-terminal coiled-coil domain forms a trimer in solution and in crystal, and has a crucial role in the assembly and surface expression of the PKD1L3/TRPP3 complex. These results demonstrate that PKD subunits constitute a new class of channel-forming proteins, enriching our understanding of the function of PKD proteins and PKD/TRPP complexes. PMID:23212381

  12. Kainate receptor pore‐forming and auxiliary subunits regulate channel block by a novel mechanism

    PubMed Central

    Brown, Patricia M. G. E.; Aurousseau, Mark R. P.; Musgaard, Maria; Biggin, Philip C.

    2016-01-01

    Key points Kainate receptor heteromerization and auxiliary subunits, Neto1 and Neto2, attenuate polyamine ion‐channel block by facilitating blocker permeation.Relief of polyamine block in GluK2/GluK5 heteromers results from a key proline residue that produces architectural changes in the channel pore α‐helical region.Auxiliary subunits exert an additive effect to heteromerization, and thus relief of polyamine block is due to a different mechanism.Our findings have broad implications for work on polyamine block of other cation‐selective ion channels. Abstract Channel block and permeation by cytoplasmic polyamines is a common feature of many cation‐selective ion channels. Although the channel block mechanism has been studied extensively, polyamine permeation has been considered less significant as it occurs at extreme positive membrane potentials. Here, we show that kainate receptor (KAR) heteromerization and association with auxiliary proteins, Neto1 and Neto2, attenuate polyamine block by enhancing blocker permeation. Consequently, polyamine permeation and unblock occur at more negative and physiologically relevant membrane potentials. In GluK2/GluK5 heteromers, enhanced permeation is due to a single proline residue in GluK5 that alters the dynamics of the α‐helical region of the selectivity filter. The effect of auxiliary proteins is additive, and therefore the structural basis of polyamine permeation and unblock is through a different mechanism. As native receptors are thought to assemble as heteromers in complex with auxiliary proteins, our data identify an unappreciated impact of polyamine permeation in shaping the signalling properties of neuronal KARs and point to a structural mechanism that may be shared amongst other cation‐selective ion channels. PMID:26682513

  13. Receptors, channels, and signalling in the urothelial sensory system in the bladder.

    PubMed

    Merrill, Liana; Gonzalez, Eric J; Girard, Beatrice M; Vizzard, Margaret A

    2016-04-01

    The storage and periodic elimination of urine, termed micturition, requires a complex neural control system to coordinate the activities of the urinary bladder, urethra, and urethral sphincters. At the level of the lumbosacral spinal cord, lower urinary tract reflex mechanisms are modulated by supraspinal controls with mechanosensory input from the urothelium, resulting in regulation of bladder contractile activity. The specific identity of the mechanical sensor is not yet known, but considerable interest exists in the contribution of transient receptor potential (TRP) channels to the mechanosensory functions of the urothelium. The sensory, transduction, and signalling properties of the urothelium can influence adjacent urinary bladder tissues including the suburothelial nerve plexus, interstitial cells of Cajal, and detrusor smooth muscle cells. Diverse stimuli, including those that activate TRP channels expressed by the urothelium, can influence urothelial release of chemical mediators (such as ATP). Changes to the urothelium are associated with a number of bladder pathologies that underlie urinary bladder dysfunction. Urothelial receptor and/or ion channel expression and the release of signalling molecules (such as ATP and nitric oxide) can be altered with bladder disease, neural injury, target organ inflammation, or psychogenic stress. Urothelial receptors and channels represent novel targets for potential therapies that are intended to modulate micturition function or bladder sensation. PMID:26926246

  14. Purification and characterization of an. alpha. -bungarotoxin receptor that forms a functional nicotinic channel

    SciTech Connect

    Gotti, C.; Ogando, A.E.; Moretti, M.; Clementi, F. ); Hanke, W.; Schlue, R. )

    1991-04-15

    Neither the structure nor the function of {alpha}-bungarotoxin ({alpha}Bgtx) binding molecules in the nervous system have yet been completely defined, although it is known that some of these molecules are related to cation channels and some are not. Using an improved method of affinity chromatography, the authors have isolated a toxin binding molecule from chicken optic lobe that contains at least three subunits with apparent M{sub r} values of 52,000, 57,000, and 67,000. The M{sub r} 57,000 subunit binds {alpha}Bgtx receptors of human neuroblastoma cells, fetal calf muscle, and chicken optic lobe but not by antibodies raised against Torpedo acetylcholine receptor, the serum of myasthenic patients, or monoclonal antibody 35. {sup 125}I-labeled {alpha}Bgtx binding to the isolated receptor is blocked, with the same potency, by nicotinic agonists and antagonists, such as nicotine, neuronal bungarotoxin and, d-tubocurarine. When reconstituted in a planar lipid bilayer, the purified {alpha}Bgtx receptor forms cationic channels with a conductance of 50 pS. These channels are activated in a dose-dependent manner by carbamylcholine and blocked by d-tubocurarine.

  15. Calcium entry through nicotinic receptor channels and calcium channels in cultured rat superior cervical ganglion cells.

    PubMed Central

    Trouslard, J; Marsh, S J; Brown, D A

    1993-01-01

    1. Patch-clamp techniques in conjunction with indo-1 fluorescent measurements were used to measure increases in intracellular free calcium concentration and membrane conductance induced by the activation of nicotinic and calcium channels in cultured rat sympathetic neurons. 2. Under voltage-clamp conditions, pressure application of the nicotinic agonist DMPP (1,1-dimethyl-4-phenylpiperazinium iodide, 100 microM, 100 ms) increased [Ca2+]i by 193 +/- 26 nM at a clamp potential of -60 mV. This was accompanied by an inward current of -4.53 +/- 0.89 nA, giving a mean ratio of the delta (Ca2+]i to the total inward charge transfer of 42.7 nmoles per litre of free calcium per nanocoulomb of charge (M/q ratio). 3. The DMPP-induced current and associated delta [Ca2+]i were reduced by mecamylamine (100 nM-10 microM) but were unaffected by alpha-bungarotoxin (100 nM) or cadmium (100 microM). 4. The M/q ratio was not affected by the holding potential (from -80 to -40 mV) but was a function of the external calcium concentration. 5. The M/q ratio was reduced by increasing the intracellular calcium buffering capacity and increased by heparin but not affected by ryanodine or by depletion of the caffeine-sensitive calcium store. 6. Under the same recording conditions, we quantified the increase in [Ca2+]i associated with activation of the voltage-dependent calcium current. On average at -60 mV, the M/q ratio of this highly calcium-selective permeability was 1961 mM nC-1, which is 46 times that obtained for the nicotinic channel. 7. Assuming constant-field theory, ion-substitution experiments suggest that in 2.5 mM external calcium, the permeability sequence for the nicotinic conductance was Cs+ < Li+ < Na+ < K+ < Ca2+. 8. We conclude that the nicotinic channels in rat sympathetic neurones are significantly permeant to Ca2+ and that the influx of Ca2+ through these channels is the principal cause of the rise in [Ca2+]i seen under voltage clamp. PMID:8254522

  16. The Role of 5-HT3 Receptors in Signaling from Taste Buds to Nerves.

    PubMed

    Larson, Eric D; Vandenbeuch, Aurelie; Voigt, Anja; Meyerhof, Wolfgang; Kinnamon, Sue C; Finger, Thomas E

    2015-12-01

    Activation of taste buds triggers the release of several neurotransmitters, including ATP and serotonin (5-hydroxytryptamine; 5-HT). Type III taste cells release 5-HT directly in response to acidic (sour) stimuli and indirectly in response to bitter and sweet tasting stimuli. Although ATP is necessary for activation of nerve fibers for all taste stimuli, the role of 5-HT is unclear. We investigated whether gustatory afferents express functional 5-HT3 receptors and, if so, whether these receptors play a role in transmission of taste information from taste buds to nerves. In mice expressing GFP under the control of the 5-HT(3A) promoter, a subset of cells in the geniculate ganglion and nerve fibers in taste buds are GFP-positive. RT-PCR and in situ hybridization confirmed the presence of 5-HT(3A) mRNA in the geniculate ganglion. Functional studies show that only those geniculate ganglion cells expressing 5-HT3A-driven GFP respond to 10 μM 5-HT and this response is blocked by 1 μM ondansetron, a 5-HT3 antagonist, and mimicked by application of 10 μM m-chlorophenylbiguanide, a 5-HT3 agonist. Pharmacological blockade of 5-HT3 receptors in vivo or genetic deletion of the 5-HT3 receptors reduces taste nerve responses to acids and other taste stimuli compared with controls, but only when urethane was used as the anesthetic. We find that anesthetic levels of pentobarbital reduce taste nerve responses apparently by blocking the 5-HT3 receptors. Our results suggest that 5-HT released from type III cells activates gustatory nerve fibers via 5-HT3 receptors, accounting for a significant proportion of the neural taste response.

  17. The Role of 5-HT3 Receptors in Signaling from Taste Buds to Nerves.

    PubMed

    Larson, Eric D; Vandenbeuch, Aurelie; Voigt, Anja; Meyerhof, Wolfgang; Kinnamon, Sue C; Finger, Thomas E

    2015-12-01

    Activation of taste buds triggers the release of several neurotransmitters, including ATP and serotonin (5-hydroxytryptamine; 5-HT). Type III taste cells release 5-HT directly in response to acidic (sour) stimuli and indirectly in response to bitter and sweet tasting stimuli. Although ATP is necessary for activation of nerve fibers for all taste stimuli, the role of 5-HT is unclear. We investigated whether gustatory afferents express functional 5-HT3 receptors and, if so, whether these receptors play a role in transmission of taste information from taste buds to nerves. In mice expressing GFP under the control of the 5-HT(3A) promoter, a subset of cells in the geniculate ganglion and nerve fibers in taste buds are GFP-positive. RT-PCR and in situ hybridization confirmed the presence of 5-HT(3A) mRNA in the geniculate ganglion. Functional studies show that only those geniculate ganglion cells expressing 5-HT3A-driven GFP respond to 10 μM 5-HT and this response is blocked by 1 μM ondansetron, a 5-HT3 antagonist, and mimicked by application of 10 μM m-chlorophenylbiguanide, a 5-HT3 agonist. Pharmacological blockade of 5-HT3 receptors in vivo or genetic deletion of the 5-HT3 receptors reduces taste nerve responses to acids and other taste stimuli compared with controls, but only when urethane was used as the anesthetic. We find that anesthetic levels of pentobarbital reduce taste nerve responses apparently by blocking the 5-HT3 receptors. Our results suggest that 5-HT released from type III cells activates gustatory nerve fibers via 5-HT3 receptors, accounting for a significant proportion of the neural taste response. PMID:26631478

  18. Temperature and voltage coupling to channel opening in transient receptor potential melastatin 8 (TRPM8).

    PubMed

    Raddatz, Natalia; Castillo, Juan P; Gonzalez, Carlos; Alvarez, Osvaldo; Latorre, Ramon

    2014-12-19

    Expressed in somatosensory neurons of the dorsal root and trigeminal ganglion, the transient receptor potential melastatin 8 (TRPM8) channel is a Ca(2+)-permeable cation channel activated by cold, voltage, phosphatidylinositol 4,5-bisphosphate, and menthol. Although TRPM8 channel gating has been characterized at the single channel and macroscopic current levels, there is currently no consensus regarding the extent to which temperature and voltage sensors couple to the conduction gate. In this study, we extended the range of voltages where TRPM8-induced ionic currents were measured and made careful measurements of the maximum open probability the channel can attain at different temperatures by means of fluctuation analysis. The first direct measurements of TRPM8 channel temperature-driven conformational rearrangements provided here suggest that temperature alone is able to open the channel and that the opening reaction is voltage-independent. Voltage is a partial activator of TRPM8 channels, because absolute open probability values measured with fully activated voltage sensors are less than 1, and they decrease as temperature rises. By unveiling the fast temperature-dependent deactivation process, we show that TRPM8 channel deactivation is well described by a double exponential time course. The fast and slow deactivation processes are temperature-dependent with enthalpy changes of 27.2 and 30.8 kcal mol(-1). The overall Q10 for the closing reaction is about 33. A three-tiered allosteric model containing four voltage sensors and four temperature sensors can account for the complex deactivation kinetics and coupling between voltage and temperature sensor activation and channel opening. PMID:25352597

  19. Evidence for Novel Pharmacological Sensitivities of Transient Receptor Potential (TRP) Channels in Schistosoma mansoni

    PubMed Central

    Bais, Swarna; Churgin, Matthew A.; Fang-Yen, Christopher; Greenberg, Robert M.

    2015-01-01

    Schistosomiasis, caused by parasitic flatworms of the genus Schistosoma, is a neglected tropical disease affecting hundreds of millions globally. Praziquantel (PZQ), the only drug currently available for treatment and control, is largely ineffective against juvenile worms, and reports of PZQ resistance lend added urgency to the need for development of new therapeutics. Ion channels, which underlie electrical excitability in cells, are validated targets for many current anthelmintics. Transient receptor potential (TRP) channels are a large family of non-selective cation channels. TRP channels play key roles in sensory transduction and other critical functions, yet the properties of these channels have remained essentially unexplored in parasitic helminths. TRP channels fall into several (7–8) subfamilies, including TRPA and TRPV. Though schistosomes contain genes predicted to encode representatives of most of the TRP channel subfamilies, they do not appear to have genes for any TRPV channels. Nonetheless, we find that the TRPV1-selective activators capsaicin and resiniferatoxin (RTX) induce dramatic hyperactivity in adult worms; capsaicin also increases motility in schistosomula. SB 366719, a highly-selective TRPV1 antagonist, blocks the capsaicin-induced hyperactivity in adults. Mammalian TRPA1 is not activated by capsaicin, yet knockdown of the single predicted TRPA1-like gene (SmTRPA) in S. mansoni effectively abolishes capsaicin-induced responses in adult worms, suggesting that SmTRPA is required for capsaicin sensitivity in these parasites. Based on these results, we hypothesize that some schistosome TRP channels have novel pharmacological sensitivities that can be targeted to disrupt normal parasite neuromuscular function. These results also have implications for understanding the phylogeny of metazoan TRP channels and may help identify novel targets for new or repurposed therapeutics. PMID:26655809

  20. Temperature and voltage coupling to channel opening in transient receptor potential melastatin 8 (TRPM8).

    PubMed

    Raddatz, Natalia; Castillo, Juan P; Gonzalez, Carlos; Alvarez, Osvaldo; Latorre, Ramon

    2014-12-19

    Expressed in somatosensory neurons of the dorsal root and trigeminal ganglion, the transient receptor potential melastatin 8 (TRPM8) channel is a Ca(2+)-permeable cation channel activated by cold, voltage, phosphatidylinositol 4,5-bisphosphate, and menthol. Although TRPM8 channel gating has been characterized at the single channel and macroscopic current levels, there is currently no consensus regarding the extent to which temperature and voltage sensors couple to the conduction gate. In this study, we extended the range of voltages where TRPM8-induced ionic currents were measured and made careful measurements of the maximum open probability the channel can attain at different temperatures by means of fluctuation analysis. The first direct measurements of TRPM8 channel temperature-driven conformational rearrangements provided here suggest that temperature alone is able to open the channel and that the opening reaction is voltage-independent. Voltage is a partial activator of TRPM8 channels, because absolute open probability values measured with fully activated voltage sensors are less than 1, and they decrease as temperature rises. By unveiling the fast temperature-dependent deactivation process, we show that TRPM8 channel deactivation is well described by a double exponential time course. The fast and slow deactivation processes are temperature-dependent with enthalpy changes of 27.2 and 30.8 kcal mol(-1). The overall Q10 for the closing reaction is about 33. A three-tiered allosteric model containing four voltage sensors and four temperature sensors can account for the complex deactivation kinetics and coupling between voltage and temperature sensor activation and channel opening.

  1. Characterization of ryanodine receptor type 1 single channel activity using "on-nucleus" patch clamp.

    PubMed

    Wagner, Larry E; Groom, Linda A; Dirksen, Robert T; Yule, David I

    2014-08-01

    In this study, we provide the first description of the biophysical and pharmacological properties of ryanodine receptor type 1 (RyR1) expressed in a native membrane using the on-nucleus configuration of the patch clamp technique. A stable cell line expressing rabbit RyR1 was established (HEK-RyR1) using the FLP-in 293 cell system. In contrast to untransfected cells, RyR1 expression was readily demonstrated by immunoblotting and immunocytochemistry in HEK-RyR1 cells. In addition, the RyR1 agonists 4-CMC and caffeine activated Ca(2+) release that was inhibited by high concentrations of ryanodine. On nucleus patch clamp was performed in nuclei prepared from HEK-RyR1 cells. Raising the [Ca(2+)] in the patch pipette resulted in the appearance of a large conductance cation channel with well resolved kinetics and the absence of prominent subconductance states. Current versus voltage relationships were ohmic and revealed a chord conductance of ∼750pS or 450pS in symmetrical 250mM KCl or CsCl, respectively. The channel activity was markedly enhanced by caffeine and exposure to ryanodine resulted in the appearance of a subconductance state with a conductance ∼40% of the full channel opening with a Po near unity. In total, these properties are entirely consistent with RyR1 channel activity. Exposure of RyR1 channels to cyclic ADP ribose (cADPr), nicotinic acid adenine dinucleotide phosphate (NAADP) or dantrolene did not alter the single channel activity stimulated by Ca(2+), and thus, it is unlikely these molecules directly modulate RyR1 channel activity. In summary, we describe an experimental platform to monitor the single channel properties of RyR channels. We envision that this system will be influential in characterizing disease-associated RyR mutations and the molecular determinants of RyR channel modulation.

  2. Temperature and Voltage Coupling to Channel Opening in Transient Receptor Potential Melastatin 8 (TRPM8)*♦

    PubMed Central

    Raddatz, Natalia; Castillo, Juan P.; Gonzalez, Carlos; Alvarez, Osvaldo; Latorre, Ramon

    2014-01-01

    Expressed in somatosensory neurons of the dorsal root and trigeminal ganglion, the transient receptor potential melastatin 8 (TRPM8) channel is a Ca2+-permeable cation channel activated by cold, voltage, phosphatidylinositol 4,5-bisphosphate, and menthol. Although TRPM8 channel gating has been characterized at the single channel and macroscopic current levels, there is currently no consensus regarding the extent to which temperature and voltage sensors couple to the conduction gate. In this study, we extended the range of voltages where TRPM8-induced ionic currents were measured and made careful measurements of the maximum open probability the channel can attain at different temperatures by means of fluctuation analysis. The first direct measurements of TRPM8 channel temperature-driven conformational rearrangements provided here suggest that temperature alone is able to open the channel and that the opening reaction is voltage-independent. Voltage is a partial activator of TRPM8 channels, because absolute open probability values measured with fully activated voltage sensors are less than 1, and they decrease as temperature rises. By unveiling the fast temperature-dependent deactivation process, we show that TRPM8 channel deactivation is well described by a double exponential time course. The fast and slow deactivation processes are temperature-dependent with enthalpy changes of 27.2 and 30.8 kcal mol−1. The overall Q10 for the closing reaction is about 33. A three-tiered allosteric model containing four voltage sensors and four temperature sensors can account for the complex deactivation kinetics and coupling between voltage and temperature sensor activation and channel opening. PMID:25352597

  3. Ca(2+)-BK channel clusters in olfactory receptor neurons and their role in odour coding.

    PubMed

    Bao, Guobin; de Jong, Daniëlle; Alevra, Mihai; Schild, Detlev

    2015-12-01

    Olfactory receptor neurons (ORNs) have high-voltage-gated Ca(2+) channels whose physiological impact has remained enigmatic since the voltage-gated conductances in this cell type were first described in the 1980s. Here we show that in ORN somata of Xenopus laevis tadpoles these channels are clustered and co-expressed with large-conductance potassium (BK) channels. We found approximately five clusters per ORN and twelve Ca(2+) channels per cluster. The action potential-triggered activation of BK channels accelerates the repolarization of action potentials and shortens interspike intervals during odour responses. This increases the sensitivity of individual ORNs to odorants. At the level of mitral cells of the olfactory bulb, odour qualities have been shown to be coded by first-spike-latency patterns. The system of Ca(2+) and BK channels in ORNs appears to be important for correct odour coding because the blockage of BK channels not only affects ORN spiking patterns but also changes the latency pattern representation of odours in the olfactory bulb.

  4. Transient receptor potential (TRP) channels as drug targets for diseases of the digestive system

    PubMed Central

    Holzer, Peter

    2011-01-01

    Approximately 20 of the 30 mammalian transient receptor potential (TRP) channel subunits are expressed by specific neurons and cells within the alimentary canal. They subserve important roles in taste, chemesthesis, mechanosensation, pain and hyperalgesia and contribute to the regulation of gastrointestinal motility, absorptive and secretory processes, blood flow, and mucosal homeostasis. In a cellular perspective, TRP channels operate either as primary detectors of chemical and physical stimuli, as secondary transducers of ionotropic or metabotropic receptors, or as ion transport channels. The polymodal sensory function of TRPA1, TRPM5, TRPM8, TRPP2, TRPV1, TRPV3 and TRPV4 enables the digestive system to survey its physical and chemical environment, which is relevant to all processes of digestion. TRPV5 and TRPV6 as well as TRPM6 and TRPM7 contribute to the absorption of Ca2+ and Mg2+, respectively. TRPM7 participates in intestinal pacemaker activity, and TRPC4 transduces muscarinic acetylcholine receptor activation to smooth muscle contraction. Changes in TRP channel expression or function are associated with a variety of diseases/disorders of the digestive system, notably gastro-esophageal reflux disease, inflammatory bowel disease, pain and hyperalgesia in heartburn, functional dyspepsia and irritable bowel syndrome, cholera, hypomagnesemia with secondary hypocalcemia, infantile hypertrophic pyloric stenosis, esophageal, gastrointestinal and pancreatic cancer, and polycystic liver disease. These implications identify TRP channels as promising drug targets for the management of a number of gastrointestinal pathologies. As a result, major efforts are put into the development of selective TRP channel agonists and antagonists and the assessment of their therapeutic potential. PMID:21420431

  5. Physiological functions of transient receptor potential channels in pulmonary arterial smooth muscle cells.

    PubMed

    Yang, Xiao-Ru; Lin, Mo-Jun; Sham, James S K

    2010-01-01

    The transient receptor potential (TRP) gene superfamily, which consists of 7 subfamilies with at least 28 mammalian homologues, is known to encode a wide variety of cation channels with diverse biophysical properties, activation mechanisms, and physiological functions. Recent studies have identified multiple TRP channel subtypes, belonging to the canonical (TRPC), melastatin-related (TRPM), and vanilloid-related (TRPV) subfamilies, in pulmonary arterial smooth muscle cells (PASMCs). They operate as specific Ca(2+) pathways responsive to stimuli, including Ca(2+) store depletion, receptor activation, reactive oxygen species, growth factors, and mechanical stress. Increasing evidence suggests that these channels play crucial roles in agonist-induced pulmonary vasoconstriction, hypoxic pulmonary vasoconstriction, smooth muscle cell proliferation, vascular remodeling, and pulmonary arterial hypertension. This chapter highlighted and discussed these putative physiological functions of TRP channels in pulmonary vasculatures. Since Ca(2+) ions regulate many cellular processes via specific Ca(2+) signals, future investigations of these novel channels will likely uncover more important regulatory mechanisms of pulmonary vascular functions in health and in disease states. PMID:20204726

  6. Transient Receptor Potential Channels Contribute to Pathological Structural and Functional Remodeling After Myocardial Infarction

    PubMed Central

    Davis, Jennifer; Correll, Robert N.; Trappanese, Danielle M.; Hoffman, Nicholas E.; Troupes, Constantine D.; Berretta, Remus M.; Kubo, Hajime; Madesh, Muniswamy; Chen, Xiongwen; Gao, Erhe; Molkentin, Jeffery D.; Houser, Steven R.

    2014-01-01

    Rationale The cellular and molecular basis for post myocardial infarction (MI) structural and functional remodeling is not well understood. Objective To determine if Ca2+ influx through transient receptor potential (canonical) (TRPC) channels contributes to post-MI structural and functional remodeling. Methods and Results TRPC1/3/4/6 channel mRNA increased after MI in mice and was associated with TRPC-mediated Ca2+ entry. Cardiac myocyte specific expression of a dominant negative (dn: loss of function) TRPC4 channel increased basal myocyte contractility and reduced hypertrophy and cardiac structural and functional remodeling after MI while increasing survival. We used adenovirus-mediated expression of TRPC3/4/6 channels in cultured adult feline myocytes (AFMs) to define mechanistic aspects of these TRPC-related effects. TRPC3/4/6 over expression in AFMs induced calcineurin (Cn)-Nuclear Factor of Activated T cells (NFAT) mediated hypertrophic signaling, which was reliant on caveolae targeting of TRPCs. TRPC3/4/6 expression in AFMs increased rested state contractions and increased spontaneous sarcoplasmic reticulum (SR) Ca2+ sparks mediated by enhanced phosphorylation of the ryanodine receptor. TRPC3/4/6 expression was associated with reduced contractility and response to catecholamines during steady state pacing, likely due to enhanced SR Ca2+ leak. Conclusions Ca2+ influx through TRPC channels expressed after MI activates pathological cardiac hypertrophy and reduces contractility reserve. Blocking post-MI TRPC activity improved post-MI cardiac structure and function. PMID:25047165

  7. An overview of trafficking and assembly of neurotransmitter receptors and ion channels (Review).

    PubMed

    Schwappach, Blanche

    2008-05-01

    Ionotropic neurotransmitter receptors and voltage-gated ion channels assemble from several homologous and non-homologous subunits. Assembly of these multimeric membrane proteins is a tightly controlled process subject to primary and secondary quality control mechanisms. An assembly pathway involving a dimerization of dimers has been demonstrated for a voltage-gated potassium channel and for different types of glutamate receptors. While many novel C-terminal assembly domains have been identified in various members of the voltage-gated cation channel superfamily, the assembly pathways followed by these proteins remain largely elusive. Recent progress on the recognition of polar residues in the transmembrane segments of membrane proteins by the retrieval factor Rer1 is likely to be relevant for the further investigation of trafficking defects in channelopathies. This mechanism might also contribute to controlling the assembly of ion channels by retrieving unassembled subunits to the endoplasmic reticulum. The endoplasmic reticulum is a metabolic compartment studded with small molecule transporters. This environment provides ligands that have recently been shown to act as pharmacological chaperones in the biogenesis of ligand-gated ion channels. Future progress depends on the improvement of tools, in particular the antibodies used by the field, and the continued exploitation of genetically tractable model organisms in screens and physiological experiments. PMID:18446613

  8. Interaction between positive allosteric modulators and trapping blockers of the NMDA receptor channel

    PubMed Central

    Emnett, Christine M; Eisenman, Lawrence N; Mohan, Jayaram; Taylor, Amanda A; Doherty, James J; Paul, Steven M; Zorumski, Charles F; Mennerick, Steven

    2015-01-01

    Background and Purpose Memantine and ketamine are clinically used, open-channel blockers of NMDA receptors exhibiting remarkable pharmacodynamic similarities despite strikingly different clinical profiles. Although NMDA channel gating constitutes an important difference between memantine and ketamine, it is unclear how positive allosteric modulators (PAMs) might affect the pharmacodynamics of these NMDA blockers. Experimental Approach We used two different PAMs: SGE-201, an analogue of an endogenous oxysterol, 24S-hydroxycholesterol, along with pregnenolone sulphate (PS), to test on memantine and ketamine responses in single cells (oocytes and cultured neurons) and networks (hippocampal slices), using standard electrophysiological techniques. Key Results SGE-201 and PS had no effect on steady-state block or voltage dependence of a channel blocker. However, both PAMs increased the actions of memantine and ketamine on phasic excitatory post-synaptic currents, but neither revealed underlying pharmacodynamic differences. SGE-201 accelerated the re-equilibration of blockers during voltage jumps. SGE-201 also unmasked differences among the blockers in neuronal networks – measured either by suppression of activity in multi-electrode arrays or by neuroprotection against a mild excitotoxic insult. Either potentiating NMDA receptors while maintaining the basal activity level or increasing activity/depolarization without potentiating NMDA receptor function is sufficient to expose pharmacodynamic blocker differences in suppressing network function and in neuroprotection. Conclusions and Implications Positive modulation revealed no pharmacodynamic differences between NMDA receptor blockers at a constant voltage, but did expose differences during spontaneous network activity. Endogenous modulator tone of NMDA receptors in different brain regions may underlie differences in the effects of NMDA receptor blockers on behaviour. PMID:25377730

  9. Transient Receptor Potential Ankyrin 1 (TRPA1) Channel and Neurogenic Inflammation in Pathogenesis of Asthma

    PubMed Central

    Yang, Hang; Li, ShuZhuang

    2016-01-01

    Asthma is characterized by airway inflammation, airway obstruction, and airway hyperresponsiveness (AHR), and it affects 300 million people worldwide. However, our current understanding of the molecular mechanisms that underlie asthma remains limited. Recent studies have suggested that transient receptor potential ankyrin 1 (TRPA1), one of the transient receptor potential cation channels, may be involved in airway inflammation in asthma. The present review discusses the relationship between TRPA1 and neurogenic inflammation in asthma, hoping to enhance our understanding of the mechanisms of airway inflammation in asthma. PMID:27539812

  10. Transient Receptor Potential Ankyrin 1 (TRPA1) Channel and Neurogenic Inflammation in Pathogenesis of Asthma.

    PubMed

    Yang, Hang; Li, ShuZhuang

    2016-01-01

    Asthma is characterized by airway inflammation, airway obstruction, and airway hyperresponsiveness (AHR), and it affects 300 million people worldwide. However, our current understanding of the molecular mechanisms that underlie asthma remains limited. Recent studies have suggested that transient receptor potential ankyrin 1 (TRPA1), one of the transient receptor potential cation channels, may be involved in airway inflammation in asthma. The present review discusses the relationship between TRPA1 and neurogenic inflammation in asthma, hoping to enhance our understanding of the mechanisms of airway inflammation in asthma. PMID:27539812

  11. A functional tandem between transient receptor potential canonical channels 6 and calcium-dependent chloride channels in human epithelial cells.

    PubMed

    Bertrand, Johanna; Dannhoffer, Luc; Antigny, Fabrice; Vachel, Laura; Jayle, Christophe; Vandebrouck, Clarisse; Becq, Frédéric; Norez, Caroline

    2015-10-15

    TRPC6 plays important human physiological functions, notably in artery and arterioles constriction, in regulation of vascular volume and in bronchial muscle constriction. It is implicated in pulmonary hypertension, cardiovascular disease, and focal segmental glomerulosclerosis and seems to play a role in cancer development. Previously, we identified Guanabenz, an α2-adrenergic agonist used for hypertension treatment (Wytensin®), as an activator of calcium-dependent chloride channels (CaCC) in human Cystic Fibrosis (CF) nasal epithelial cells by transiently increasing [Ca2+]i via an influx of extracellular Ca2+. In this study, using assays to measure chloride channel activity, we show that guanabenz is an activator of CaCC in freshly dissociated human bronchial epithelial cells from three CF patients with various genotypes (F508del/F508del, F508del/R1066C, F508del/H1085R). We further characterised the effect of guanabenz and show that it is independent of α-adrenergic receptors, is inhibited by the TRPC family inhibitor SKF-96365 but not by the TRPV family inhibitor ruthenium red. Using western-blotting, Ca2+ measurements and iodide efflux assay, we found that TRPC1 siRNA has no effect on guanabenz induced responses whereas TRPC6 siRNA prevented the guanabenz-dependent Ca2+ influx and the CaCC-dependent activity stimulated by guanabenz. In conclusion, we show that TRPC6 channel is pivotal for the activation of CaCC by guanabenz through a α2-adrenergic-independent pathway in human airway epithelial cells. We suggest propose a functional coupling between TRPC6 and CaCC and guanabenz as a potential TRPC6 activator for exploring TRPC6 and CaCC channel functions and corresponding channelopathies.

  12. Activation of human 5-hydroxytryptamine type 3 receptors via an allosteric transmembrane site.

    PubMed

    Lansdell, Stuart J; Sathyaprakash, Chaitra; Doward, Anne; Millar, Neil S

    2015-01-01

    In common with other members of the Cys-loop family of pentameric ligand-gated ion channels, 5-hydroxytryptamine type 3 receptors (5-HT3Rs) are activated by the binding of a neurotransmitter to an extracellular orthosteric site, located at the interface of two adjacent receptor subunits. In addition, a variety of compounds have been identified that modulate agonist-evoked responses of 5-HT3Rs, and other Cys-loop receptors, by binding to distinct allosteric sites. In this study, we examined the pharmacological effects of a group of monoterpene compounds on recombinant 5-HT3Rs expressed in Xenopus oocytes. Two phenolic monoterpenes (carvacrol and thymol) display allosteric agonist activity on human homomeric 5-HT3ARs (64 ± 7% and 80 ± 4% of the maximum response evoked by the endogenous orthosteric agonist 5-HT, respectively). In addition, at lower concentrations, where agonist effects are less apparent, carvacrol and thymol act as potentiators of responses evoked by submaximal concentrations of 5-HT. By contrast, carvacrol and thymol have no agonist or potentiating activity on the closely related mouse 5-HT3ARs. Using subunit chimeras containing regions of the human and mouse 5-HT3A subunits, and by use of site-directed mutagenesis, we have identified transmembrane amino acids that either abolish the agonist activity of carvacrol and thymol on human 5-HT3ARs or are able to confer this property on mouse 5-HT3ARs. By contrast, these mutations have no significant effect on orthosteric activation of 5-HT3ARs by 5-HT. We conclude that 5-HT3ARs can be activated by the binding of ligands to an allosteric transmembrane site, a conclusion that is supported by computer docking studies. PMID:25338672

  13. Ion permeation properties of the glutamate receptor channel in cultured embryonic Drosophila myotubes.

    PubMed Central

    Chang, H; Ciani, S; Kidokoro, Y

    1994-01-01

    Ion permeation properties of the glutamate receptor channel in cultured myotubes of Drosophila embryos were studied using the inside-out configuration of the patch-clamp technique. Lowering the NaCl concentration in the bath (intracellular solution), while maintaining that of the external solution constant, caused a shift of the reversal potential in the positive direction, thus indicating a higher permeability of the channel to Na+ than to Cl- (PCl/PNa < 0.04), and suggesting that the channel is cation selective. With 145 mM Na+ on both sides of the membrane, the single-channel current-voltage relation was almost linear in the voltage range between -80 and +80 mV, the conductance showing some variability in the range between 140 and 170 pS. All monovalent alkali cations tested, as well as NH4+, permeated the channel effectively. Using the Goldman-Hodgkin-Katz equation for the reversal potential, the permeability ratios with respect to Na+ were estimated to be: 1.32 for K+, 1.18 for NH4+, 1.15 for Rb+, 1.09 for Cs+, and 0.57 for Li+. Divalent cations, i.e. Mg2+ and Ca2+, in the external solution depressed not only the inward but also the outward Na+ currents, although reversal potential measurements indicated that both ions have considerably higher permeabilities than Na+ (PMg/PNa = 2.31; PCa/PNa = 9.55). The conductance-activity relation for Na+ was described by a hyperbolic curve. The maximal conductance was about 195 pS and the half-saturating activity 45 mM. This result suggests that Na+ ions bind to sites in the channel. All data were fitted by a model based on the Eyring's reaction rate theory, in which the receptor channel is a one-ion pore with three energy barriers and two internal sites. PMID:7519261

  14. Role of Src in C3 transient receptor potential channel function and evidence for a heterogeneous makeup of receptor- and store-operated Ca2+ entry channels.

    PubMed

    Kawasaki, Brian T; Liao, Yanhong; Birnbaumer, Lutz

    2006-01-10

    Receptor-operated Ca2+ entry (ROCE) and store-operated Ca2+ entry (SOCE) are known to be inhibited by tyrosine kinase inhibitors and activation of C-type transient receptor potential channel (TRPC) isoform 3 (TRPC3), a cation channel thought to be involved in SOCE and/or ROCE, was recently shown to depend on src tyrosine kinase activity. What is not known is the step at which src acts on TRPC3 and whether the role for tyrosine kinases in ROCE or SOCE is a general phenomenon. Using in vitro and in cell protein-protein interaction assays we now report that src phosphorylates TRPC3 at Y226 and that formation of phospho-Y226 is essential for TRPC3 activation. This requirement is unique for TRPC3 because (i) mutation of the cognate tyrosines of the closely related TRPC6 and TRPC7 had no effect; (ii) TRPC6 and TRPC7 were activated in src-, yes-, and fyn-deficient cells; and (iii) src, but not yes or fyn, rescued TRPC3 activation in src-, yes-, and fyn-deficient cells. The Src homology 2 domain of src was found to interact with either the N or the C termini of all TRPCs, suggesting that other tyrosine kinases may play a role in ion fluxes mediated by TRPCs other than TRPC3. A side-by-side comparison of the effects of genistein (a general tyrosine kinase inhibitor) on endogenous ROCE and SOCE in mouse fibroblasts, HEK and COS-7 cells, and ROCE in HEK cells mediated by TRPC3, TRPC6, TRPC7, and TRPC5 showed differences that argue for ROCE and SOCE channels to be heterogeneous.

  15. Modulation of defensive behavior by Transient Receptor Potential Vanilloid Type-1 (TRPV1) channels.

    PubMed

    Aguiar, D C; Moreira, F A; Terzian, A L; Fogaça, M V; Lisboa, S F; Wotjak, C T; Guimaraes, F S

    2014-10-01

    The Transient Receptor Potential Vanilloid Type-1 (TRPV1) was first characterized in primary afferent fibers as a receptor for capsaicin (the pungent ingredient of chili peppers). Later on, this cation-permeable ion channel was also described in the central nervous system, where its main putative endogenous ligand is N-arachidonoyl ethanolamide (an endocannabinoid, also known as anandamide). Recent results employing genetic, pharmacological and histochemical techniques indicate that TRPV1 tonically modulate anxiety, fear and panic responses in brain regions related to defensive responses, such as the dorsal periaqueductal gray, the hippocampus and the medial prefrontal cortex. Genetic deletion or antagonism of this ion channel induces anxiolytic-like effects in several animal models. The main mechanism responsible for TRPV1-mediated effects on anxiety seems to involve facilitation of glutamatergic neurotransmission. In addition, there is evidence for interactions with other neurotransmitter systems, such as nitric oxide and endocannabinoids. PMID:24726577

  16. Modulation of defensive behavior by Transient Receptor Potential Vanilloid Type-1 (TRPV1) channels.

    PubMed

    Aguiar, D C; Moreira, F A; Terzian, A L; Fogaça, M V; Lisboa, S F; Wotjak, C T; Guimaraes, F S

    2014-10-01

    The Transient Receptor Potential Vanilloid Type-1 (TRPV1) was first characterized in primary afferent fibers as a receptor for capsaicin (the pungent ingredient of chili peppers). Later on, this cation-permeable ion channel was also described in the central nervous system, where its main putative endogenous ligand is N-arachidonoyl ethanolamide (an endocannabinoid, also known as anandamide). Recent results employing genetic, pharmacological and histochemical techniques indicate that TRPV1 tonically modulate anxiety, fear and panic responses in brain regions related to defensive responses, such as the dorsal periaqueductal gray, the hippocampus and the medial prefrontal cortex. Genetic deletion or antagonism of this ion channel induces anxiolytic-like effects in several animal models. The main mechanism responsible for TRPV1-mediated effects on anxiety seems to involve facilitation of glutamatergic neurotransmission. In addition, there is evidence for interactions with other neurotransmitter systems, such as nitric oxide and endocannabinoids.

  17. Inhibition of transient receptor potential canonical channels impairs cytokinesis in human malignant gliomas

    PubMed Central

    Bomben, V. C.; Sontheimer, H. W.

    2009-01-01

    Objectives Glial-derived primary brain tumours, gliomas, are among the fastest growing malignancies and present a huge clinical challenge. Research suggests an important, yet poorly understood, role of ion channels in growth control of normal and malignant cells. In this study, we sought to functionally characterize Transient Receptor Potential Canoncial (TRPC) channels in glioma cell proliferation. TRPC channels form non-selective cation channels that have been suggested to represent a Ca2+ influx pathway impacting cellular growth. Materials and Methods Employing a combination of molecular, biochemical and biophysical techniques, we characterized TRPC channels in glioma cells. Results We showed consistent expression of four channel family members (TRPC-1, -3, -5, -6) in glioma cell lines and acute patient-derived tissues. These channels gave rise to small, non-voltage-dependent cation currents that were blocked by the TRPC inhibitors GdCl3, 2-APB, or SKF96365. Importantly, TRPC channels contributed to the resting conductance of glioma cells and their acute pharmacological inhibition caused an ~10 mV hyperpolarization of the cells’ resting potential. Additionally, chronic application of the TRPC inhibitor SKF96365 caused near complete growth arrest. A detailed analysis, by fluorescence-activated cell sorting and time-lapse microscopy, showed that growth inhibition occurred at the G2 + M phase of the cell cycle with cytokinesis defects. Cells underwent incomplete cell divisions and became multinucleate, enlarged cells. Conclusions Nuclear atypia and enlarged cells are histopathological hallmarks for glioblastoma multiforme, the highest grade glioma, suggesting that a defect in TRPC channel function may contribute to cellular abnormalities in these tumours. PMID:18211288

  18. Transient receptor potential ion channels in primary sensory neurons as targets for novel analgesics

    PubMed Central

    Sousa-Valente, J; Andreou, A P; Urban, L; Nagy, I

    2014-01-01

    The last decade has witnessed an explosion in novel findings relating to the molecules involved in mediating the sensation of pain in humans. Transient receptor potential (TRP) ion channels emerged as the greatest group of molecules involved in the transduction of various physical stimuli into neuronal signals in primary sensory neurons, as well as, in the development of pain. Here, we review the role of TRP ion channels in primary sensory neurons in the development of pain associated with peripheral pathologies and possible strategies to translate preclinical data into the development of effective new analgesics. Based on available evidence, we argue that nociception-related TRP channels on primary sensory neurons provide highly valuable targets for the development of novel analgesics and that, in order to reduce possible undesirable side effects, novel analgesics should prevent the translocation from the cytoplasm to the cell membrane and the sensitization of the channels rather than blocking the channel pore or binding sites for exogenous or endogenous activators. LINKED ARTICLES This article is part of a themed section on the pharmacology of TRP channels. To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2014.171.issue-10 PMID:24283624

  19. Micromolar-Affinity Benzodiazepine Receptors Regulate Voltage-Sensitive Calcium Channels in Nerve Terminal Preparations

    NASA Astrophysics Data System (ADS)

    Taft, William C.; Delorenzo, Robert J.

    1984-05-01

    Benzodiazepines in micromolar concentrations significantly inhibit depolarization-sensitive Ca2+ uptake in intact nerve-terminal preparations. Benzodiazepine inhibition of Ca2+ uptake is concentration dependent and stereospecific. Micromolar-affinity benzodiazepine receptors have been identified and characterized in brain membrane and shown to be distinct from nanomolar-affinity benzodiazepine receptors. Evidence is presented that micromolar, and not nanomolar, benzodiazepine binding sites mediate benzodiazepine inhibition of Ca2+ uptake. Irreversible binding to micromolar benzodiazepine binding sites also irreversibly blocked depolarization-dependent Ca2+ uptake in synaptosomes, indicating that these compounds may represent a useful marker for identifying the molecular components of Ca2+ channels in brain. Characterization of benzodiazepine inhibition of Ca2+ uptake demonstrates that these drugs function as Ca2+ channel antagonists, because benzodiazepines effectively blocked voltage-sensitive Ca2+ uptake inhibited by Mn2+, Co2+, verapamil, nitrendipine, and nimodipine. These results indicate that micromolar benzodiazepine binding sites regulate voltage-sensitive Ca2+ channels in brain membrane and suggest that some of the neuronal stabilizing effects of micromolar benzodiazepine receptors may be mediated by the regulation of Ca2+ conductance.

  20. Blockade of P2X7 receptors or pannexin-1 channels similarly attenuates postischemic damage

    PubMed Central

    Cisneros-Mejorado, Abraham; Gottlieb, Miroslav; Cavaliere, Fabio; Magnus, Tim; Koch-Nolte, Friederich; Scemes, Eliana; Pérez-Samartín, Alberto; Matute, Carlos

    2015-01-01

    The role of P2X7 receptors and pannexin-1 channels in ischemic damage remains controversial. Here, we analyzed their contribution to postanoxic depolarization after ischemia in cultured neurons and in brain slices. We observed that pharmacological blockade of P2X7 receptors or pannexin-1 channels delayed the onset of postanoxic currents and reduced their slope, and that simultaneous inhibition did not further enhance the effects of blocking either one. These results were confirmed in acute cortical slices from P2X7 and pannexin-1 knockout mice. Oxygen-glucose deprivation in cortical organotypic cultures caused neuronal death that was reduced with P2X7 and pannexin-1 blockers as well as in organotypic cultures derived from mice lacking P2X7 and pannexin 1. Subsequently, we used transient middle cerebral artery occlusion to monitor the neuroprotective effect of those drugs in vivo. We found that P2X7 and pannexin-1 antagonists, and their ablation in knockout mice, substantially attenuated the motor symptoms and reduced the infarct volume to ~50% of that in vehicle-treated or wild-type animals. These results show that P2X7 receptors and pannexin-1 channels are major mediators of postanoxic depolarization in neurons and of brain damage after ischemia, and that they operate in the same deleterious signaling cascade leading to neuronal and tissue demise. PMID:25605289

  1. Transient receptor potential (TRP) channels in the airway: role in airway disease

    PubMed Central

    Grace, M S; Baxter, M; Dubuis, E; Birrell, M A; Belvisi, M G

    2014-01-01

    Over the last few decades, there has been an explosion of scientific publications reporting the many and varied roles of transient receptor potential (TRP) ion channels in physiological and pathological systems throughout the body. The aim of this review is to summarize the existing literature on the role of TRP channels in the lungs and discuss what is known about their function under normal and diseased conditions. The review will focus mainly on the pathogenesis and symptoms of asthma and chronic obstructive pulmonary disease and the role of four members of the TRP family: TRPA1, TRPV1, TRPV4 and TRPM8. We hope that the article will help the reader understand the role of TRP channels in the normal airway and how their function may be changed in the context of respiratory disease. PMID:24286227

  2. [The action of ionotropic glutamate receptor channel blockers on effects of sleep deprivation in rats].

    PubMed

    Vataev, S I; Oganesian, G A; Lukomskaia, N Ia; Magazanik, L G

    2013-05-01

    The action of non-competitive glutamate receptor antagonists on the effects of sleep deprivation has been studied on Krushinskii-Molodkina rats having an inherited predisposition to audiogenic seizures and Wistar rats deprived to this respond. Two types of glutamate receptor open channels blockers were used: the selective blockers of NMDA-receptors (memantine and IEM-1921) and blockers of mixed type, impacting both on the NMDA- and presumably Ca(2+)-permeable AMPA/kainate receptors (IEM-1754 and IEM 1925). Rats were subjected to 12 hours long sleep deprivation. Immediatly after that memantine and IEM-1921 were injected, and during the first 3 hours the total or partial reduction of fast-wave (paradoxical) sleep and a significant increase of the representation of wakefulness at the cost of reducing the total time of slow-wave sleep were observed. These effects are most likely to be a consequence of the blockade of NMDA-receptors functioning in the systems of the rat brain responsible for the launch and maintenance of fast-wave sleep. Injection of IEM-1754 and IEM-1925 on background of sleep deprivation did not affect the organization of sleep during the first 3 hours of their action. During the second three-hour period the rebound effect was observed. The obtained results indicate the involvement of NMDA glutamate receptors in the functioning of various parts of the sleep system of both rat lines.

  3. Modulation of nicotinic receptor channels by adrenergic stimulation in rat pinealocytes

    PubMed Central

    Yoon, Jin-Young; Jung, Seung-Ryoung; Hille, Bertil

    2014-01-01

    Melatonin secretion from the pineal gland is triggered by norepinephrine released from sympathetic terminals at night. In contrast, cholinergic and parasympathetic inputs, by activating nicotinic cholinergic receptors (nAChR), have been suggested to counterbalance the noradrenergic input. Here we investigated whether adrenergic signaling regulates nAChR channels in rat pinealocytes. Acetylcholine or the selective nicotinic receptor agonist 1,1-dimethyl-4-phenylpiperazinium iodide (DMPP) activated large nAChR currents in whole cell patch-clamp experiments. Norepinephrine (NE) reduced the nAChR currents, an effect partially mimicked by a β-adrenergic receptor agonist, isoproterenol, and blocked by a β-adrenergic receptor antagonist, propranolol. Increasing intracellular cAMP levels using membrane-permeable 8-bromoadenosine (8-Br)-cAMP or 5,6-dichlorobenzimidazole riboside-3′,5′-cyclic monophosphorothioate (cBIMPS) also reduced nAChR activity, mimicking the effects of NE and isoproterenol. Further, removal of ATP from the intracellular pipette solution blocked the reduction of nAChR currents, suggesting involvement of protein kinases. Indeed protein kinase A inhibitors, H-89 and Rp-cAMPS, blocked the modulation of nAChR by adrenergic stimulation. After the downmodulation by NE, nAChR channels mediated a smaller Ca2+ influx and less membrane depolarization from the resting potential. Together these results suggest that NE released from sympathetic terminals at night attenuates nicotinic cholinergic signaling. PMID:24553185

  4. What we don't know about the structure of ryanodine receptor calcium release channels.

    PubMed

    Dulhunty, Angela F; Pouliquin, Pierre

    2003-10-01

    1. The ryanodine receptor (RyR) is the Ca2+ release channel in the sarcoplamic reticulum of skeletal and cardiac muscle and is essential for respiration and heart beat. The RyR channel releases Ca2+ from intracellular stores in a variety of other cell types, where it normally coexists with the inositiol 1,4,5-trisphosphate receptor (IP3R). The RyR and IP3R, forming a superfamily of homotetrameric ligand-gated intracellular Ca2+ channels, serve discrete functions: they can be located in independent Ca2+ stores with different activation mechanisms and can be coupled to different signalling pathways. 2. Although functional characteristics of the RyR have been investigated intensely, there remain major gaps in our knowledge about the structure of the protein, its ion-conducting pore, its ligand-binding sites and sites supporting the many protein/protein interactions that underlie the in vivo function of the channel. 3. Of particular importance are the transmembrane segments that form the membrane-spanning domain of the protein and the pore, define the conductance and selectivity of the channel and dictate the cytoplasmic and luminal domains and the overall protein structure. Hydropathy profiles predict between four and 12 transmembrane segments. One popular model shows four transmembrane segments in the C-terminal one-tenth of the protein. However, there is substantial evidence for a larger number of membrane-spanning segments located in both the C-terminal and central parts of the protein. 4. A model of the RyR pore based on the Streptomyces lividans KcsA channel structure is presented. Protein/protein interactions between the RyR and other regulatory proteins, as well as within the RyR subunit, are discussed. PMID:14516409

  5. Channel Gating Dependence on Pore Lining Helix Glycine Residues in Skeletal Muscle Ryanodine Receptor.

    PubMed

    Mei, Yingwu; Xu, Le; Mowrey, David D; Mendez Giraldez, Raul; Wang, Ying; Pasek, Daniel A; Dokholyan, Nikolay V; Meissner, Gerhard

    2015-07-10

    Type 1 ryanodine receptors (RyR1s) release Ca(2+) from the sarcoplasmic reticulum to initiate skeletal muscle contraction. The role of RyR1-G4934 and -G4941 in the pore-lining helix in channel gating and ion permeation was probed by replacing them with amino acid residues of increasing side chain volume. RyR1-G4934A, -G4941A, and -G4941V mutant channels exhibited a caffeine-induced Ca(2+) release response in HEK293 cells and bound the RyR-specific ligand [(3)H]ryanodine. In single channel recordings, significant differences in the number of channel events and mean open and close times were observed between WT and RyR1-G4934A and -G4941A. RyR1-G4934A had reduced K(+) conductance and ion selectivity compared with WT. Mutations further increasing the side chain volume at these positions (G4934V and G4941I) resulted in reduced caffeine-induced Ca(2+) release in HEK293 cells, low [(3)H]ryanodine binding levels, and channels that were not regulated by Ca(2+) and did not conduct Ca(2+) in single channel measurements. Computational predictions of the thermodynamic impact of mutations on protein stability indicated that although the G4934A mutation was tolerated, the G4934V mutation decreased protein stability by introducing clashes with neighboring amino acid residues. In similar fashion, the G4941A mutation did not introduce clashes, whereas the G4941I mutation resulted in intersubunit clashes among the mutated isoleucines. Co-expression of RyR1-WT with RyR1-G4934V or -G4941I partially restored the WT phenotype, which suggested lessening of amino acid clashes in heterotetrameric channel complexes. The results indicate that both glycines are important for RyR1 channel function by providing flexibility and minimizing amino acid clashes. PMID:25998124

  6. Structural requirements of steroidal agonists of transient receptor potential melastatin 3 (TRPM3) cation channels

    PubMed Central

    Drews, A; Mohr, F; Rizun, O; Wagner, T F J; Dembla, S; Rudolph, S; Lambert, S; Konrad, M; Philipp, S E; Behrendt, M; Marchais-Oberwinkler, S; Covey, D F; Oberwinkler, J

    2014-01-01

    Background and Purpose Transient receptor potential melastatin 3 (TRPM3) proteins form non-selective but calcium-permeable membrane channels, rapidly activated by extracellular application of the steroid pregnenolone sulphate and the dihydropyridine nifedipine. Our aim was to characterize the steroid binding site by analysing the structural chemical requirements for TRPM3 activation. Experimental Approach Whole-cell patch-clamp recordings and measurements of intracellular calcium concentrations were performed on HEK293 cells transfected with TRPM3 (or untransfected controls) during superfusion with pharmacological substances. Key Results Pregnenolone sulphate and nifedipine activated TRPM3 channels supra-additively over a wide concentration range. Other dihydropyridines inhibited TRPM3 channels. The natural enantiomer of pregnenolone sulphate was more efficient in activating TRPM3 channels than its synthetic mirror image. However, both enantiomers exerted very similar inhibitory effects on proton-activated outwardly rectifying anion channels. Epiallopregnanolone sulphate activated TRPM3 almost equally as well as pregnenolone sulphate. Exchanging the sulphate for other chemical moieties showed that a negative charge at this position is required for activating TRPM3 channels. Conclusions and Implications Our data demonstrate that nifedipine and pregnenolone sulphate act at different binding sites when activating TRPM3. The latter activates TRPM3 by binding to a chiral and thus proteinaceous binding site, as inferred from the differential effects of the enantiomers. The double bond between position C5 and C6 of pregnenolone sulphate is not strictly necessary for the activation of TRPM3 channels, but a negative charge at position C3 of the steroid is highly important. These results provide a solid basis for understanding mechanistically the rapid chemical activation of TRPM3 channels. PMID:24251620

  7. The unliganded long isoform of estrogen receptor beta stimulates brain ryanodine receptor single channel activity alongside with cytosolic Ca2+

    PubMed Central

    Rybalchenko, Volodymyr; Grillo, Michael A.; Gastinger, Matthew J.; Rybalchenko, Nataliya; Payne, Andrew J.; Koulen, Peter

    2010-01-01

    Ca2+ release from intracellular stores mediated by endoplasmic reticulum membrane ryanodine receptors (RyR) plays a key role in activating and synchronizing downstream Ca2+-dependent mechanisms, in different cells varying from apoptosis to nuclear transcription and development of defensive responses. Recently discovered, atypical “non-genomic” effects mediated by estrogen receptors (ER) include rapid Ca2+ release upon estrogen exposure in conditions implicitly suggesting involvement of RyRs. In the present study, we report various levels of co-localization between RyR type 2 (RyR2) and ER type β (ERβ) in the neuronal cell line HT-22, indicating a possible functional interaction. Electrophysiological analyses revealed a significant increase in single channel ionic currents generated by mouse brain RyRs after application of the soluble monomer of the long form ERβ (ERβ1). The effect was due to a strong increase in open probability of RyR higher open channel sublevels at cytosolic [Ca2+] concentrations of 100 nM, suggesting a synergistic action of ERβ1 and Ca2+ in RyR activation, and a potential contribution to Ca2+-induced Ca2+ release rather than to basal intracellular Ca2+ concentration level at rest. This RyR/ERβ interaction has potential effects on cellular physiology, including roles of shorter ERβ isoforms and modulation of the RyR/ERβ complexes by exogenous estrogens. PMID:19899956

  8. [Effects of ionotropic glutamate receptor channel blockers ON sleep-waking organization in rats].

    PubMed

    Vataev, S I; Oganesian, G A; Gmiro, V E; Lukomskaia, N Ia; Magazanik, L G

    2012-07-01

    The effects of non-competitive glutamate receptor antagonists on sleep-waking organization have been studied on Krushinskii-Molodkina rats having an inherited predisposition to audiogenic seizures and Wistar ones which are resistant to this action of sound. Two types of blockers of glutamate receptor open channels were used: selective blockers of NMDA receptors (memantine and IEM-1921) and blockers of mixed type, impacting both on the NMDA and Ca-permeable AMPA/ kainate receptors (IEM-1754 and IEM 1925). During the first 3 hours after administration of these glutamate antagonists the total or partial deprivation of fast-wave sleep was provoked. Additionally the selective NMDA receptor blocking drugs (memantine, IEM-1921) induced in the same period a significant increase of the representation of wakefulness at the cost of reducing of the total time of slow-wave sleep. These effects are most likely to be a consequence of the blockade of NMDA receptors responsible for the launch and maintenance of wakefulness, slow- and fast-wave sleep. In the same first 3 hours period after the administration of IEM-1754 and IEM-1925 the organization of sleep was not significantly affected. The evident reduction of wakefulness, total duration and increase of slow-wave sleep impact was observed, during the second three-hour period. It, apparently, can be caused by the blockade of AMPA/kainate receptors. The obtained results indicate the involvement of NMDA and AMPA/kainate receptors in the functioning of various parts of the sleep system of rats belonging to both lines.

  9. Transient receptor potential channel A1 and noxious cold responses in rat cutaneous nociceptors.

    PubMed

    Dunham, J P; Leith, J L; Lumb, B M; Donaldson, L F

    2010-02-17

    The role of transient receptor potential channel A1 (TRPA1) in noxious cold sensation remains unclear. Some data support the hypothesis that TRPA1 is a transducer of noxious cold whilst other data contest it. In this study we investigated the role of TRPA1 in cold detection in cutaneous nociceptors in vivo using complementary experimental approaches. We used noxious withdrawal reflex electromyography, and single fibre recordings in vivo, to test the hypothesis that TRPA1-expressing primary afferents mediate noxious cold responses in anaesthetised rats. TRPV1 and TRPM8 agonists sensitise their cognate receptors to heat and cold stimuli respectively. Herein we show that the TRPA1 agonist cinnamaldehyde applied to the skin in anaesthetised rats did not sensitise noxious cold evoked hind limb withdrawal. In contrast, cinnamaldehyde did sensitise the C fibre-mediated noxious heat withdrawal, indicated by a significant drop in the withdrawal temperature. TRPA1 agonist thus sensitised the noxious reflex withdrawal to heat, but not cold. Thermal stimuli also sensitise transient receptor potential (TRP) channels to agonist. Activity evoked by capsaicin in teased primary afferent fibres showed a significant positive correlation with receptive field temperature, in both normal and Freund's complete adjuvant-induced cutaneous inflammation. Altering the temperature of the receptive field did not modulate TRPA1 agonist evoked-activity in cutaneous primary afferents, in either normal or inflamed skin. In addition, block of the TRPA1 channel with Ruthenium Red did not inhibit cold evoked activity in either cinnamaldehyde sensitive or insensitive cold responsive nociceptors. In cinnamaldehyde-sensitive-cold-sensitive afferents, although TRPA1 agonist-evoked activity was totally abolished by Ruthenium Red, cold evoked activity was unaffected by channel blockade. We conclude that these results do not support the hypothesis that TRPA1-expressing cutaneous afferents play an important

  10. Functional characterization of transient receptor potential channels in mouse urothelial cells.

    PubMed

    Everaerts, Wouter; Vriens, Joris; Owsianik, Grzegorz; Appendino, Giovanni; Voets, Thomas; De Ridder, Dirk; Nilius, Bernd

    2010-03-01

    The bladder urothelium is currently believed to be a sensory structure, contributing to mechano- and chemosensation in the bladder. Transient receptor potential (TRP) cation channels act as polymodal sensors and may underlie some of the receptive properties of urothelial cells. However, the exact TRP channel expression profile of urothelial cells is unclear. In this study, we have performed a systematic analysis of the molecular and functional expression of various TRP channels in mouse urothelium. Urothelial cells from control and trpv4-/- mice were isolated, cultured (12-48 h), and used for quantitative real-time PCR, immunocytochemistry, calcium imaging, and whole cell patch-clamp experiments. At the mRNA level, TRPV4, TRPV2, and TRPM7 were the most abundantly expressed TRP genes. Immunohistochemistry showed a clear expression of TRPV4 in the plasma membrane, whereas TRPV2 was more prominent in the cytoplasm. TRPM7 was detected in the plasma membrane as well as cytoplasmic vesicles. Calcium imaging and patch-clamp experiments using TRP channel agonists and antagonists provided evidence for the functional expression of TRPV4, TRPV2, and TRPM7 but not of TRPA1, TRPV1, and TRPM8. In conclusion, we have demonstrated functional expression of TRPV4, TRPV2, and TRPM7 in mouse urothelial cells. These channels may contribute to the (mechano)sensory function of the urothelial layer and represent potential targets for the treatment of bladder dysfunction. PMID:20015940

  11. Ca sup 2+ channel blockers interact with. alpha. sub 2 -adrenergic receptors in rabbit ileum

    SciTech Connect

    Homaidan, F.R.; Donowitz, M.; Wicks, J.; Cusolito, S.; El Sabban, M.E.; Weiland, G.A.; Sharp, G.W.G. Tufts Univ. School of Medicine and New England Medical Center Hospital, Boston, MA )

    1988-04-01

    An interaction between Ca{sup 2+} channel blockers and {alpha}{sub 2}-adrenergic receptors has been demonstrated in rabbit ileum by studying the effect of clonidine on active electrolyte transport, under short-circuited conditions, in the presence and absence of several Ca{sup 2+} channel blocking agents. Clonidine, verapamil, diltiazem, cadmium, and nitrendipine all decrease short-circuit current and stimulate NaCl absorption to different extents with clonidine having the largest effect. Exposure to verapamil, diltiazem, and cadmium inhibited the effects of clonidine on transport, whereas nitrendipine had no such effect. Verapamil, diltiazem, and cadmium, but not nitrendipine, also decreased the specific binding of ({sup 3}H){alpha}{sub 2}-adrenergic agents to a preparation of ileal basolateral membranes explaining the observed decrease in the transport effects of clonidine. The effective concentrations of the Ca{sup 2+} channel blockers that inhibited the effects of clonidine on transport were fairly similar to the concentrations needed to inhibit its specific binding. The displacement of clonidine by calcium channel blockers is ascribed to a nonspecific effect of these agents, although the possibility that their effects are exerted via their binding to the calcium channels is not excluded.

  12. Energetics of divalent selectivity in a calcium channel: the ryanodine receptor case study.

    PubMed

    Gillespie, Dirk

    2008-02-15

    A model of the ryanodine receptor (RyR) calcium channel is used to study the energetics of binding selectivity of Ca(2+) versus monovalent cations. RyR is a calcium-selective channel with a DDDD locus in the selectivity filter, similar to the EEEE locus of the L-type calcium channel. While the affinity of RyR for Ca(2+) is in the millimolar range (as opposed to the micromolar range of the L-type channel), the ease of single-channel measurements compared to L-type and its similar selectivity filter make RyR an excellent candidate for studying calcium selectivity. A Poisson-Nernst-Planck/density functional theory model of RyR is used to calculate the energetics of selectivity. Ca(2+) versus monovalent selectivity is driven by the charge/space competition mechanism in which selectivity arises from a balance of electrostatics and the excluded volume of ions in the crowded selectivity filter. While electrostatic terms dominate the selectivity, the much smaller excluded-volume term also plays a substantial role. In the D4899N and D4938N mutations of RyR that are analyzed, substantial changes in specific components of the chemical potential profiles are found far from the mutation site. These changes result in the significant reduction of Ca(2+) selectivity found in both theory and experiments.

  13. Modulation of BK channel activities by calcium-sensing receptor in rat bronchopulmonary sensory neurons.

    PubMed

    Vysotskaya, Zhanna V; Moss, Charles R; Gilbert, Carolyn A; Gabriel, Sabry A; Gu, Qihai

    2014-11-01

    This study was carried out to investigate the expression of large-conductance Ca(2+)-activated potassium (BK) channels and to explore the possible modulation of BK channel activities by calcium-sensing receptors (CaSR) in rat bronchopulmonary sensory neurons. The expression of BK channels was demonstrated by immunohistochemistry and RT-PCR. Results from whole-cell patch-clamp recordings demonstrated that activation of CaSR with its agonist spermine or NPS R-568 showed a dual regulating effect on BK channel activities: it potentiated BK currents in cells exhibiting low baseline BK activity while slightly inhibited BK currents in cells with high baseline BK activity. Blocking CaSR with its antagonist NPS 2143 significantly inhibited BK currents. Our results further showed that the modulation of BK currents by CaSR activation or blockade was completely abolished when the intracellular Ca(2+) was chelated by BAPTA-AM. In summary, our data suggest that CaSR plays an integrative role in bronchopulmonary afferent signaling, at least partially through the regulation of BK channel activities.

  14. Cross-talk and co-trafficking between rho1/GABA receptors and ATP-gated channels.

    PubMed

    Boué-Grabot, Eric; Emerit, Michel B; Toulmé, Estelle; Séguéla, Philippe; Garret, Maurice

    2004-02-20

    Gamma-aminobutyric-acid (GABA) and ATP ionotropic receptors represent two structurally and functionally different classes of neurotransmitter-gated channels involved in fast synaptic transmission. We demonstrate here that, when the inhibitory rho1/GABA and the excitatory P2X2 receptor channels are co-expressed in Xenopus oocytes, activation of one channel reduces the currents mediated by the other one. This reciprocal inhibitory cross-talk is a receptor-mediated phenomenon independent of agonist cross-modulation, membrane potential, direction of ionic flux, or channel densities. Functional interaction is disrupted when the cytoplasmic C-terminal domain of P2X2 is deleted or in competition experiments with minigenes coding for the C-terminal domain of P2X2 or the main intracellular loop of rho1 subunits. We also show a physical interaction between P2X2 and rho1 receptors expressed in oocytes and the co-clustering of these receptors in transfected hippocampal neurons. Co-expression with P2X2 induces retargeting and recruitment of mainly intracellular rho1/GABA receptors to surface clusters. Therefore, molecular and functional cross-talk between inhibitory and excitatory ligand-gated channels may regulate synaptic strength both by activity-dependent current occlusion and synaptic receptors co-trafficking.

  15. Antibody probe study of Ca2+ channel regulation by interdomain interaction within the ryanodine receptor.

    PubMed Central

    Kobayashi, Shigeki; Yamamoto, Takeshi; Parness, Jerome; Ikemoto, Noriaki

    2004-01-01

    N-terminal and central domains of ryanodine receptor 1 (RyR1), where many reported malignant hyperthermia (MH) mutations are localized, represent putative channel regulatory domains. Recent domain peptide (DP) probe studies led us to the hypothesis that these domains interact to stabilize the closed state of channel (zipping), while weakening of domain-domain interactions (unzipping) by mutation de-stabilizes the channel, making it leaky to Ca2+ or sensitive to the agonists of RyR1. As shown previously, DP1 (N-terminal domain peptide) and DP4 (central domain peptide) produced MH-like channel activation/sensitization effects, presumably by peptide binding to sites critical to stabilizing domain-domain interactions and resultant loss of conformational constraints. Here we report that polyclonal anti-DP1 and anti-DP4 antibodies also produce MH-like channel activation and sensitization effects as evidenced by about 4-fold enhancement of high affinity [3H]ryanodine binding to RyR1 and by a significant left-shift of the concentration-dependence of activation of sarcoplasmic reticulum Ca2+ release by polylysine. Fluorescence quenching experiments demonstrate that the accessibility of a DP4-directed, conformationally sensitive fluorescence probe linked to the RyR1 N-terminal domain is increased in the presence of domain-specific antibodies, consistent with the view that these antibodies produce unzipping of interacting domains that are of hindered accessibility to the surrounding aqueous environment. Our results suggest that domain-specific antibody binding induces a conformational change resulting in channel activation, and are consistent with the hypothesis that interacting N-terminal and central domains are intimately involved in the regulation of RyR1 channel function. PMID:15027895

  16. Prolactin stimulates cell proliferation through a long form of prolactin receptor and K+ channel activation.

    PubMed Central

    Van Coppenolle, Fabien; Skryma, Roman; Ouadid-Ahidouch, Halima; Slomianny, Christian; Roudbaraki, Morad; Delcourt, Philippe; Dewailly, Etienne; Humez, Sandrine; Crépin, Alexandre; Gourdou, Isabelle; Djiane, Jean; Bonnal, Jean-Louis; Mauroy, Brigitte; Prevarskaya, Natalia

    2004-01-01

    PRL (prolactin) has been implicated in the proliferation and differentiation of numerous tissues, including the prostate gland. However, the PRL-R (PRL receptor) signal transduction pathway, leading to the stimulation of cell proliferation, remains unclear and has yet to be mapped. The present study was undertaken to develop a clear understanding of the mechanisms involved in this pathway and, in particular, to determine the role of K(+) channels. We used androgen-sensitive prostate cancer (LNCaP) cells whose proliferation is known to be stimulated by PRL. Reverse transcriptase PCR analysis showed that LNCaP cells express a long form of PRL-R, but do not produce its intermediate isoform. Patch-clamp techniques showed that the application of 5 nM PRL increased both the macroscopic K(+) current amplitude and the single K(+)-channel open probability. This single-channel activity increase was reduced by the tyrosine kinase inhibitors genistein, herbimycin A and lavandustine A, thereby indicating that tyrosine kinase phosphorylation is required in PRL-induced K(+) channel stimulation. PRL enhances p59( fyn ) phosphorylation by a factor of 2 after a 10 min application in culture. In addition, where an antip59( fyn ) antibody is present in the patch pipette, PRL no longer increases K(+) current amplitude. Furthermore, the PRL-stimulated proliferation is inhibited by the K(+) channel inhibitors alpha-dendrotoxin and tetraethylammonium. Thus, as K(+) channels are known to be involved in LNCaP cell proliferation, we suggest that K(+) channel modulation by PRL, via p59( fyn ) pathway, is the primary ionic event in PRL signal transduction, triggering cell proliferation. PMID:14565846

  17. Evolution of Pentameric Ligand-Gated Ion Channels: Pro-Loop Receptors.

    PubMed

    Jaiteh, Mariama; Taly, Antoine; Hénin, Jérôme

    2016-01-01

    Pentameric ligand-gated ion channels (pLGICs) are ubiquitous neurotransmitter receptors in Bilateria, with a small number of known prokaryotic homologues. Here we describe a new inventory and phylogenetic analysis of pLGIC genes across all kingdoms of life. Our main finding is a set of pLGIC genes in unicellular eukaryotes, some of which are metazoan-like Cys-loop receptors, and others devoid of Cys-loop cysteines, like their prokaryotic relatives. A number of such "Cys-less" receptors also appears in invertebrate metazoans. Together, those findings draw a new distribution of pLGICs in eukaryotes. A broader distribution of prokaryotic channels also emerges, including a major new archaeal taxon, Thaumarchaeota. More generally, pLGICs now appear nearly ubiquitous in major taxonomic groups except multicellular plants and fungi. However, pLGICs are sparsely present in unicellular taxa, suggesting a high rate of gene loss and a non-essential character, contrasting with their essential role as synaptic receptors of the bilaterian nervous system. Multiple alignments of these highly divergent sequences reveal a small number of conserved residues clustered at the interface between the extracellular and transmembrane domains. Only the "Cys-loop" proline is absolutely conserved, suggesting the more fitting name "Pro loop" for that motif, and "Pro-loop receptors" for the superfamily. The infered molecular phylogeny shows a Cys-loop and a Cys-less clade in eukaryotes, both containing metazoans and unicellular members. This suggests new hypotheses on the evolutionary history of the superfamily, such as a possible origin of the Cys-loop cysteines in an ancient unicellular eukaryote. Deeper phylogenetic relationships remain uncertain, particularly around the split between bacteria, archaea, and eukaryotes. PMID:26986966

  18. Metabotropic glutamate receptor modulation of voltage-gated Ca2+ channels involves multiple receptor subtypes in cortical neurons.

    PubMed

    Choi, S; Lovinger, D M

    1996-01-01

    Metabotropic glutamate receptor (mGluR) modulation of voltage-gated Ca2+ channels was examined in isolated deep layer frontoparietal cortical neurons under conditions designed to isolate calcium-independent modulatory pathways. Trans-1-aminocyclopentane-1,3-dicarboxylate (t-ACPD), a nonspecific mGluR agonist, produced rapid and reversible inhibition of Ca2+ channels. This effect was mimicked by agonists for group I and group II, but not group III, mGluRs. Effects of group I and II agonists often were observed in the same neurons, but separate subgroups of neurons were unresponsive to the group I agonist quisqualate or the group II agonist 2-(2,3-dicarboxycyclopropyl) glycine (DCG-IV). Inhibition by quisqualate and DCG-IV was nonocclusive in neurons responding to both agonists. These agonists thus appear to act on different mGluRs. The mGluR antagonist alpha-methyl-4-carboxylphenylglycine attenuated inhibition by t-ACPD, quisqualate, and DCG-IV. Inhibition by quisqualate and DCG-IV was voltage-dependent. Although the effects of both agonists were greatly reduced by N-ethylmaleimide (NEM), inhibition by DCG-IV was more sensitive to NEM than inhibition by quisqualate. t-ACPD-induced inhibition was reduced by omega-conotoxin GVIA (omega-CgTx) and omega-agatoxin IVA (omega-AgTx) but was affected little by nifedipine. Inhibition by DCG-IV and quisqualate also was reduced by omega-CgTx. We conclude that multiple mGluR subtypes inhibit Ca2+ channels in cortical neurons and that N- and possibly P-type channels are inhibited. Modulation is via a rapid-onset, voltage-dependent mechanism that likely involves a pertussis toxin (PTX)-sensitive G-protein. Type I mGluRs may work via additional PTX-insensitive pathways.

  19. Polymodal Transient Receptor Potential Vanilloid (TRPV) Ion Channels in Chondrogenic Cells

    PubMed Central

    Szűcs Somogyi, Csilla; Matta, Csaba; Foldvari, Zsofia; Juhász, Tamás; Katona, Éva; Takács, Ádám Roland; Hajdú, Tibor; Dobrosi, Nóra; Gergely, Pál; Zákány, Róza

    2015-01-01

    Mature and developing chondrocytes exist in a microenvironment where mechanical load, changes of temperature, osmolarity and acidic pH may influence cellular metabolism. Polymodal Transient Receptor Potential Vanilloid (TRPV) receptors are environmental sensors mediating responses through activation of linked intracellular signalling pathways. In chondrogenic high density cultures established from limb buds of chicken and mouse embryos, we identified TRPV1, TRPV2, TRPV3, TRPV4 and TRPV6 mRNA expression with RT-PCR. In both cultures, a switch in the expression pattern of TRPVs was observed during cartilage formation. The inhibition of TRPVs with the non-selective calcium channel blocker ruthenium red diminished chondrogenesis and caused significant inhibition of proliferation. Incubating cell cultures at 41 °C elevated the expression of TRPV1, and increased cartilage matrix production. When chondrogenic cells were exposed to mechanical load at the time of their differentiation into matrix producing chondrocytes, we detected increased mRNA levels of TRPV3. Our results demonstrate that developing chondrocytes express a full palette of TRPV channels and the switch in the expression pattern suggests differentiation stage-dependent roles of TRPVs during cartilage formation. As TRPV1 and TRPV3 expression was altered by thermal and mechanical stimuli, respectively, these are candidate channels that contribute to the transduction of environmental stimuli in chondrogenic cells. PMID:26262612

  20. Modulation of nociceptive ion channels and receptors via protein-protein interactions: implications for pain relief

    PubMed Central

    Rouwette, Tom; Avenali, Luca; Sondermann, Julia; Narayanan, Pratibha; Gomez-Varela, David; Schmidt, Manuela

    2015-01-01

    In the last 2 decades biomedical research has provided great insights into the molecular signatures underlying painful conditions. However, chronic pain still imposes substantial challenges to researchers, clinicians and patients alike. Under pathological conditions, pain therapeutics often lack efficacy and exhibit only minimal safety profiles, which can be largely attributed to the targeting of molecules with key physiological functions throughout the body. In light of these difficulties, the identification of molecules and associated protein complexes specifically involved in chronic pain states is of paramount importance for designing selective interventions. Ion channels and receptors represent primary targets, as they critically shape nociceptive signaling from the periphery to the brain. Moreover, their function requires tight control, which is usually implemented by protein-protein interactions (PPIs). Indeed, manipulation of such PPIs entails the modulation of ion channel activity with widespread implications for influencing nociceptive signaling in a more specific way. In this review, we highlight recent advances in modulating ion channels and receptors via their PPI networks in the pursuit of relieving chronic pain. Moreover, we critically discuss the potential of targeting PPIs for developing novel pain therapies exhibiting higher efficacy and improved safety profiles. PMID:26039491

  1. Acetylcholine receptors and sodium channels in denervated and botulinum-toxin-treated adult rat muscle.

    PubMed Central

    Bambrick, L; Gordon, T

    1987-01-01

    1. The number of acetylcholine (ACh) receptors and Na channels was measured in adult rat hind-limb muscles after denervation or injection of botulinum toxin type A (BoTX), using specific binding of radiolabelled neurotoxins. 2. Denervation by sciatic nerve section increased the number of [125I]iodo-alpha-bungarotoxin ([125I]BTX) binding sites from low, unmeasurable levels to 39 +/- 3 fmol of toxin bound per milligram muscle protein at 21 days. 3. Subcutaneous injection of BoTX produced complete neuromuscular blockade for 11-14 days over which time the number of [125I]BTX binding sites increased with the same time course and to the same extent as following denervation. 4. Neither denervation nor BoTX treatment significantly altered the number of tritiated saxitoxin ([3H]STX) binding sites from normal values of 7.8 fmol/mg muscle weight or 57 +/- 3 fmol/mg homogenate protein. This may, however, correspond to a lower density of [3H]STX sites in the muscle membrane. 5. It was concluded that neuromuscular blockade with BoTX is equivalent to denervation in its effects on synthesis of ACh receptors. Numbers of Na channels are more stable than ACh receptors but may also be modulated by neuromuscular activity. PMID:2442368

  2. Thromboxane A2 receptor and MaxiK-channel intimate interaction supports channel trans-inhibition independent of G-protein activation

    PubMed Central

    Tanaka, Yoshio; Alioua, Abderrahmane; Wu, Yong; Lu, Rong; Kundu, Pallob; Sanchez-Pastor, Enrique; Marijic, Jure; Stefani, Enrico; Toro, Ligia

    2010-01-01

    Large conductance voltage- and calcium-activated potassium channels (MaxiK, BKCa) are well known for sustaining cerebral and coronary arterial tone and for their linkage to vasodilator β-adrenergic receptors. However, how MaxiK channels are linked to counterbalancing vasoconstrictor receptors is unknown. Here, we show that vasopressive thromboxane A2 receptors (TP) can intimately couple with and inhibit MaxiK channels. Activation of the receptor with its agonist trans-inhibits MaxiK independently of G-protein activation. This unconventional mechanism is supported by independent lines of evidence: (i) inhibition of MaxiK current by thromboxane A2 mimetic, U46619, occurs even when G-protein activity is suppressed; (ii) MaxiK and TP physically associate and display a high degree of proximity; and (iii) Förster resonance energy transfer occurs between fluorescently labeled MaxiK and TP, supporting a direct interaction. The molecular mechanism of MaxiK–TP intimate interaction involves the receptor's first intracellular loop and C terminus, and it entails the voltage-sensing conduction cassette of MaxiK channel. Further, physiological evidence of MaxiK–TP physical interaction is given in human coronaries and rat aorta, and by confirming TP role (with antagonist SQ29,548) in the U46619-induced MaxiK inhibition in human coronaries. We propose that vasoconstrictor TP receptor and MaxiK-channel direct interaction facilitates G-protein–independent TP to MaxiK trans-inhibition, which would promote vasoconstriction. PMID:20959415

  3. Vascular smooth muscle cell dysfunction in diabetes: nuclear receptors channel to relaxation.

    PubMed

    Doyon, Geneviève; Bruemmer, Dennis

    2016-10-01

    Endothelial dysfunction and impaired vascular relaxation represent a common cause of microvascular disease in patients with diabetes. Although multiple mechanisms underlying altered endothelial cell function in diabetes have been described, there is currently no specific and approved pharmacological treatment. In this edition of Clinical Science, Morales-Cano et al. characterize voltage-dependent K(+) (Kv) channels as genes regulated by pharmacological activation of peroxisome proliferator-activated receptor-b/d (PPARb/d). Diabetes altered Kv channel function leading to impaired coronary artery relaxation, which was prevented by pharmacological activation of PPARb/d. These studies highlight an important mechanism of vascular dysfunction in diabetes and point to a potential approach for therapy, particularly considering that PPARb/d ligands have been developed and tested in small clinical trials.

  4. Vascular smooth muscle cell dysfunction in diabetes: nuclear receptors channel to relaxation.

    PubMed

    Doyon, Geneviève; Bruemmer, Dennis

    2016-10-01

    Endothelial dysfunction and impaired vascular relaxation represent a common cause of microvascular disease in patients with diabetes. Although multiple mechanisms underlying altered endothelial cell function in diabetes have been described, there is currently no specific and approved pharmacological treatment. In this edition of Clinical Science, Morales-Cano et al. characterize voltage-dependent K(+) (Kv) channels as genes regulated by pharmacological activation of peroxisome proliferator-activated receptor-b/d (PPARb/d). Diabetes altered Kv channel function leading to impaired coronary artery relaxation, which was prevented by pharmacological activation of PPARb/d. These studies highlight an important mechanism of vascular dysfunction in diabetes and point to a potential approach for therapy, particularly considering that PPARb/d ligands have been developed and tested in small clinical trials. PMID:27634843

  5. 5-HT3 receptor-channels coupled with Na+ influx in human T cells: role in T cell activation.

    PubMed

    Khan, N A; Poisson, J P

    1999-09-01

    The study was conducted on a human (Jurkat) T cell line, loaded with a Na+ fluorescent probe, SBFI/AM. Serotonin and an agonist of 5-HT3 receptor-channels, 2-methyl-5HT, evoked Na+ influx, whereas the agonists of other serotonergic receptor subtypes, i.e., 5-HT1A and 5-HT1B receptors, failed to induce Na+ influx in these cells. By using 3H-BRL43694, an agonist of 5-HT3 receptor-channels, we characterized 5-HT3 lymphocyte receptors which exhibited a density (Bmax) of 300 +/- 20 fmol/10(6) cells and a Kd of 30 nM in Jurkat T cells. The T-cell 5-HT3 receptor-channel is not regulated either by the protein kinase C or by the free intracellular calcium concentrations as the agents known to activate the PKC and to induce increases in intracellular free calcium concentrations failed to influence the free intracellular Na+ concentrations, [Na+]i, in these cells. Furthermore, an increase in [Na+]i, induced by 2-methyl-5HT, via 5-HT3 receptor-channels seems to stimulate T-cell activation by facilitating the progression of T cells from S to G2/M phase of the cell cycle.

  6. Evolution of Pentameric Ligand-Gated Ion Channels: Pro-Loop Receptors

    PubMed Central

    Jaiteh, Mariama; Taly, Antoine; Hénin, Jérôme

    2016-01-01

    Pentameric ligand-gated ion channels (pLGICs) are ubiquitous neurotransmitter receptors in Bilateria, with a small number of known prokaryotic homologues. Here we describe a new inventory and phylogenetic analysis of pLGIC genes across all kingdoms of life. Our main finding is a set of pLGIC genes in unicellular eukaryotes, some of which are metazoan-like Cys-loop receptors, and others devoid of Cys-loop cysteines, like their prokaryotic relatives. A number of such “Cys-less” receptors also appears in invertebrate metazoans. Together, those findings draw a new distribution of pLGICs in eukaryotes. A broader distribution of prokaryotic channels also emerges, including a major new archaeal taxon, Thaumarchaeota. More generally, pLGICs now appear nearly ubiquitous in major taxonomic groups except multicellular plants and fungi. However, pLGICs are sparsely present in unicellular taxa, suggesting a high rate of gene loss and a non-essential character, contrasting with their essential role as synaptic receptors of the bilaterian nervous system. Multiple alignments of these highly divergent sequences reveal a small number of conserved residues clustered at the interface between the extracellular and transmembrane domains. Only the “Cys-loop” proline is absolutely conserved, suggesting the more fitting name “Pro loop” for that motif, and “Pro-loop receptors” for the superfamily. The infered molecular phylogeny shows a Cys-loop and a Cys-less clade in eukaryotes, both containing metazoans and unicellular members. This suggests new hypotheses on the evolutionary history of the superfamily, such as a possible origin of the Cys-loop cysteines in an ancient unicellular eukaryote. Deeper phylogenetic relationships remain uncertain, particularly around the split between bacteria, archaea, and eukaryotes. PMID:26986966

  7. Evolution of vertebrate transient receptor potential vanilloid 3 channels: opposite temperature sensitivity between mammals and western clawed frogs.

    PubMed

    Saito, Shigeru; Fukuta, Naomi; Shingai, Ryuzo; Tominaga, Makoto

    2011-04-01

    Transient Receptor Potential (TRP) channels serve as temperature receptors in a wide variety of animals and must have played crucial roles in thermal adaptation. The TRP vanilloid (TRPV) subfamily contains several temperature receptors with different temperature sensitivities. The TRPV3 channel is known to be highly expressed in skin, where it is activated by warm temperatures and serves as a sensor to detect ambient temperatures near the body temperature of homeothermic animals such as mammals. Here we performed comprehensive comparative analyses of the TRPV subfamily in order to understand the evolutionary process; we identified novel TRPV genes and also characterized the evolutionary flexibility of TRPV3 during vertebrate evolution. We cloned the TRPV3 channel from the western clawed frog Xenopus tropicalis to understand the functional evolution of the TRPV3 channel. The amino acid sequences of the N- and C-terminal regions of the TRPV3 channel were highly diversified from those of other terrestrial vertebrate TRPV3 channels, although central portions were well conserved. In a heterologous expression system, several mammalian TRPV3 agonists did not activate the TRPV3 channel of the western clawed frog. Moreover, the frog TRPV3 channel did not respond to heat stimuli, instead it was activated by cold temperatures. Temperature thresholds for activation were about 16 °C, slightly below the lower temperature limit for the western clawed frog. Given that the TRPV3 channel is expressed in skin, its likely role is to detect noxious cold temperatures. Thus, the western clawed frog and mammals acquired opposite temperature sensitivity of the TRPV3 channel in order to detect environmental temperatures suitable for their respective species, indicating that temperature receptors can dynamically change properties to adapt to different thermal environments during evolution.

  8. Proteolytic fragmentation of inositol 1,4,5-trisphosphate receptors: a novel mechanism regulating channel activity?

    PubMed

    Wang, Liwei; Alzayady, Kamil J; Yule, David I

    2016-06-01

    Inositol 1,4,5-trisphosphate receptors (IP3 Rs) are a family of ubiquitously expressed intracellular Ca(2+) release channels. Regulation of channel activity by Ca(2+) , nucleotides, phosphorylation, protein binding partners and other cellular factors is thought to play a major role in defining the specific spatiotemporal characteristics of intracellular Ca(2+) signals. These properties are, in turn, believed pivotal for the selective and specific physiological activation of Ca(2+) -dependent effectors. IP3 Rs are also substrates for the intracellular cysteine proteases, calpain and caspase. Cleavage of the IP3 R has been proposed to play a role in apoptotic cell death by uncoupling regions important for IP3 binding from the channel domain, leaving an unregulated leaky Ca(2+) pore. Contrary to this hypothesis, we demonstrate following proteolysis that N- and C-termini of IP3 R1 remain associated, presumably through non-covalent interactions. Further, we show that complementary fragments of IP3 R1 assemble into tetrameric structures and retain their ability to be regulated robustly by IP3 . While peptide continuity is clearly not necessary for IP3 -gating of the channel, we propose that cleavage of the IP3 R peptide chain may alter other important regulatory events to modulate channel activity. In this scenario, stimulation of the cleaved IP3 R may support distinct spatiotemporal Ca(2+) signals and activation of specific effectors. Notably, in many adaptive physiological events, the non-apoptotic activities of caspase and calpain are demonstrated to be important, but the substrates of the proteases are poorly defined. We speculate that proteolytic fragmentation may represent a novel form of IP3 R regulation, which plays a role in varied adaptive physiological processes.

  9. (De)constructing the ryanodine receptor: modeling ion permeation and selectivity of the calcium release channel.

    PubMed

    Gillespie, Dirk; Xu, Le; Wang, Ying; Meissner, Gerhard

    2005-08-18

    Biological ion channels are proteins that passively conduct ions across membranes that are otherwise impermeable to ions. Here, we present a model of ion permeation and selectivity through a single, open ryanodine receptor (RyR) ion channel. Combining recent mutation data with electrodiffusion of finite-sized ions, the model reproduces the current/voltage curves of cardiac RyR (RyR2) in KCl, LiCl, NaCl, RbCl, CsCl, CaCl(2), MgCl(2), and their mixtures over large concentrations and applied voltage ranges. It also reproduces the reduced K(+) conductances and Ca(2+) selectivity of two skeletal muscle RyR (RyR1) mutants (D4899N and E4900Q). The model suggests that the selectivity filter of RyR contains the negatively charged residue D4899 that dominates the permeation and selectivity properties and gives RyR a DDDD locus similar to the EEEE locus of the L-type calcium channel. In contrast to previously applied barrier models, the current model describes RyR as a multi-ion channel with approximately three monovalent cations in the selectivity filter at all times. Reasons for the contradicting occupancy predictions are discussed. In addition, the model predicted an anomalous mole fraction effect for Na(+)/Cs(+) mixtures, which was later verified by experiment. Combining these results, the binding selectivity of RyR appears to be driven by the same charge/space competition mechanism of other highly charged channels.

  10. Pre-clinical studies in cough research: Role of Transient Receptor Potential (TRP) channels

    PubMed Central

    Grace, Megan S.; Dubuis, Eric; Birrell, Mark A.; Belvisi, Maria G.

    2013-01-01

    Cough is a protective reflex and defence mechanism in healthy individuals, which helps clear excessive secretions and foreign material from the lungs. Cough often presents as the first and most persistent symptom of many respiratory diseases and some non-respiratory disorders, but can also be idiopathic, and is a common respiratory complaint for which medical attention is sought. Chronic cough of various aetiologies is a regular presentation to specialist respiratory clinics, and is reported as a troublesome symptom by a significant proportion of the population. Despite this, the treatment options for cough are limited. The lack of effective anti-tussives likely stems from our incomplete understanding of how the tussive reflex is mediated. However, research over the last decade has begun to shed some light on the mechanisms which provoke cough, and may ultimately provide us with better anti-tussive therapies. This review will focus on the in vitro and in vivo models that are currently used to further our understanding of the sensory innervation of the respiratory tract, and how these nerves are involved in controlling the cough response. Central to this are the Transient Receptor Potential (TRP) ion channels, a family of polymodal receptors that can be activated by such diverse stimuli as chemicals, temperature, osmotic stress, and mechanical perturbation. These ion channels are thought to be molecular pain integrators and targets for novel analgesic agents for the treatment of various pain disorders but some are also being developed as anti-tussives. PMID:23474212

  11. Pre-clinical studies in cough research: role of Transient Receptor Potential (TRP) channels.

    PubMed

    Grace, Megan S; Dubuis, Eric; Birrell, Mark A; Belvisi, Maria G

    2013-10-01

    Cough is a protective reflex and defence mechanism in healthy individuals, which helps clear excessive secretions and foreign material from the lungs. Cough often presents as the first and most persistent symptom of many respiratory diseases and some non-respiratory disorders, but can also be idiopathic, and is a common respiratory complaint for which medical attention is sought. Chronic cough of various aetiologies is a regular presentation to specialist respiratory clinics, and is reported as a troublesome symptom by a significant proportion of the population. Despite this, the treatment options for cough are limited. The lack of effective anti-tussives likely stems from our incomplete understanding of how the tussive reflex is mediated. However, research over the last decade has begun to shed some light on the mechanisms which provoke cough, and may ultimately provide us with better anti-tussive therapies. This review will focus on the in vitro and in vivo models that are currently used to further our understanding of the sensory innervation of the respiratory tract, and how these nerves are involved in controlling the cough response. Central to this are the Transient Receptor Potential (TRP) ion channels, a family of polymodal receptors that can be activated by such diverse stimuli as chemicals, temperature, osmotic stress, and mechanical perturbation. These ion channels are thought to be molecular pain integrators and targets for novel analgesic agents for the treatment of various pain disorders but some are also being developed as anti-tussives. PMID:23474212

  12. Interaction between Ca/sup + +/-channel antagonists and. cap alpha. /sub 2/-adrenergic receptors in rabbit ileal cell membrane

    SciTech Connect

    Homeidan, F.R.; Wicks, J.; Cusolito, S.; El-Sabban, M.E.; Sharp, G.W.G.; Donowitz, M.

    1986-03-05

    An interaction between Ca/sup + +/-channel antagonists and the ..cap alpha../sub 2/-adrenergic receptor on active electrolyte transport was demonstrated in rabbit ileum. Clonidine, an ..cap alpha../sub 2/-agonist, stimulated NaCl absorption apparently by Ca/sup + +/-channel antagonism since it inhibited /sup 45/Ca/sup + +/ uptake across the basolateral membrane and decreased total ileal calcium content. This stimulation was inhibited by the Ca/sup + +/-channel antagonists dl- and l-verapamil and cadmium but not by nifedipine. The binding of /sup 3/H-yohimbine, a specific ..cap alpha../sub 2/-adrenergic antagonist, was studied on purified ileal cell membranes using a rapid filtration technique. dl-Verapamil and Cd/sup + +/ inhibited the specific binding of /sup 3/H-yohimbine over the same concentration range in which they affected transport. In contrast, nifedipine had no effect on binding, just as it had no effect on clonidine-stimulated NaCl absorption. These data demonstrate that there is an interaction between Ca/sup + +/-channels and ..cap alpha../sub 2/-adrenergic receptors in ileal basolateral membranes. Some Ca/sup + +/-channel antagonists alter ..cap alpha../sub 2/-adrenergic binding to the receptor and ..cap alpha../sub 2/-agonist binding leads to changes in Ca/sup + +/ entry. A close spatial relationship between the Ca/sup + +/-channel and the ..cap alpha../sub 2/-receptor could explain the data.

  13. Inhibitory effect of oleanolic acid from the rhizomes of Cyperus rotundus on transient receptor potential vanilloid 1 channel.

    PubMed

    Nam, Joo Hyun; Lee, Dong-Ung

    2015-01-01

    Cyperus rotundus is used as an analgesic and sedative in oriental medicine and has been reported to exhibit antinociceptive and anti-inflammatory effects. On the other hand, the transient receptor potential vanilloid 1 channel is a nonselective cation channel that senses various noxious chemical and thermal stimuli. However, it has recently been reported that the epidermally expressed transient receptor potential vanilloid 1 channel is involved in heat- and UV-induced skin aging. The aim of this study was to evaluate whether C. rotundus extract and its constituents can inhibit this channel. Ethylacetate and hexane fractions of the methanol extract were found to partially inhibit transient receptor potential vanilloid 1 channel activity, and at a concentration of 90 µM, oleanolic acid, which was one of three constituents isolated from the ethylacetate fraction, inhibited this activity by 61.4 ± 8.0 %. This is first electrophysiological study to be conducted on the effects of C. rotundus extract and its constituents on the transient receptor potential vanilloid 1 channel. The results obtained provide insight of the potential therapeutic effects of C. rotundus in the contexts of analgesia and UV-induced photoaging. PMID:25402944

  14. Membrane-delimited coupling between sigma receptors and K+ channels in rat neurohypophysial terminals requires neither G-protein nor ATP

    PubMed Central

    Lupardus, Patrick J; Wilke, Russell A; Aydar, Ebru; Palmer, Chris P; Chen, Yuenmu; Ruoho, Arnold E; Jackson, Meyer B

    2000-01-01

    Receptor-mediated modulation of ion channels generally involves G-proteins, phosphorylation, or both in combination. The sigma receptor, which modulates voltage-gated K+ channels, is a novel protein with no homology to other receptors known to modulate ion channels. In the present study patch clamp and photolabelling techniques were used to investigate the mechanism by which sigma receptors modulate K+ channels in peptidergic nerve terminals. The sigma receptor photoprobe iodoazidococaine labelled a protein with the same molecular mass (26 kDa) as the sigma receptor protein identified by cloning. The sigma receptor ligands pentazocine and SKF10047 modulated K+ channels, despite intra-terminal perfusion with GTP-free solutions, a G-protein inhibitor (GDPβS), a G-protein activator (GTPγS) or a non-hydrolysable ATP analogue (AMPPcP). Channels in excised outside-out patches were modulated by ligand, indicating that soluble cytoplasmic factors are not required. In contrast, channels within cell-attached patches were not modulated by ligand outside a patch, indicating that receptors and channels must be in close proximity for functional interactions. Channels expressed in oocytes without receptors were unresponsive to sigma receptor agonists, ruling out inhibition through a direct drug interaction with channels. These experiments indicate that sigma receptor-mediated signal transduction is membrane delimited, and requires neither G-protein activation nor protein phosphorylation. This novel transduction mechanism is mediated by membrane proteins in close proximity, possibly through direct interactions between the receptor and channel. This would allow for more rapid signal transduction than other ion channel modulation mechanisms, which in the present case of neurohypophysial nerve terminals would lead to the enhancement of neuropeptide release. PMID:10922005

  15. Ca(2+)-permeable AMPA and NMDA receptor channels in basket cells of rat hippocampal dentate gyrus.

    PubMed Central

    Koh, D S; Geiger, J R; Jonas, P; Sakmann, B

    1995-01-01

    1. Glutamate receptor (GluR) channels were studied in basket cells in the dentate gyrus of rat hippocampal slices. Basket cells were identified by their location, dendritic morphology and high frequency of action potentials generated during sustained current injection. 2. Dual-component currents were activated by fast application of glutamate to outside-out membrane patches isolated from basket cell somata (10 microM glycine, no external Mg2+). The fast component was selectively blocked by 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), the slow component by D-2-amino-5-phosphonopentanoic acid (D-AP5). This suggests that the two components were mediated by alpha-amino-3- hydroxy-5-methyl-4-isoxazolepropionate receptor (AMPAR)/kainate receptor and N-methyl-D-aspartate receptor (NMDAR) channels, respectively. The mean ratio of the peak current of the NMDAR component to that of the AMPAR/kainate receptor component was 0.22 (1 ms pulses of 10 mM glutamate). 3. The AMPAR/kainate receptor component, which was studied in isolation in the presence of D-AP5, was identified as AMPAR mediated on the basis of the preferential activation by AMPA as compared with kainate, the weak desensitization of kainate-activated currents, the cross-desensitization between AMPA and kainate, and the reduction of desensitization by cyclothiazide. 4. Deactivation of basket cell AMPARs following 1 ms pulses of glutamate occurred with a time constant (tau) of 1.2 +/- 0.1 ms (mean +/- S.E.M.). During 100 ms glutamate pulses AMPARs desensitized with a tau of 3.7 +/- 0.2ms. 5. The peak current-voltage (I-V) relation of AMPAR-mediated currents in Na(+)-rich extracellular solution showed a reversal potential of -4.0 +/- 2.6 mV and was characterized by a a doubly rectifying shape. The conductance of single AMPAR channels was estimated as 22.6 +/- 1.6 pS using non-stationary fluctuation analysis. AMPARs expressed in hippocampal basket cells were highly Ca2+ permeable (PCa/PK = 1.79). 6. NMDARs in

  16. GABA-A receptor inhibition of local calcium signaling in spines and dendrites.

    PubMed

    Marlin, Joseph J; Carter, Adam G

    2014-11-26

    Cortical interneurons activate GABA-A receptors to rapidly control electrical and biochemical signaling at pyramidal neurons. Different populations of interneurons are known to uniquely target the soma and dendrites of pyramidal neurons. However, the ability of these interneurons to inhibit Ca(2+) signaling at spines and dendrites is largely unexplored. Here we use whole-cell recordings, two-photon microscopy, GABA uncaging and optogenetics to study dendritic inhibition at layer 5 (L5) pyramidal neurons in slices of mouse PFC. We first show that GABA-A receptors strongly inhibit action potential (AP)-evoked Ca(2+) signals at both spines and dendrites. We find robust inhibition over tens of milliseconds that spreads along the dendritic branch. However, we observe no difference in the amount of inhibition at neighboring spines and dendrites. We then examine the influence of interneurons expressing parvalbumin (PV), somatostatin (SOM), or 5HT3a receptors. We determine that these populations of interneurons make unique contacts onto the apical and basal dendrites of L5 pyramidal neurons. We also show that SOM and 5HT3a but not PV interneurons potently inhibit AP Ca(2+) signals via GABA-A receptors at both spines and dendrites. These findings reveal how multiple interneurons regulate local Ca(2+) signaling in pyramidal neurons, with implications for cortical function and disease.

  17. Regulation of the transient receptor potential channel TRPM3 by phosphoinositides.

    PubMed

    Tóth, Balázs I; Konrad, Maik; Ghosh, Debapriya; Mohr, Florian; Halaszovich, Christian R; Leitner, Michael G; Vriens, Joris; Oberwinkler, Johannes; Voets, Thomas

    2015-07-01

    The transient receptor potential (TRP) channel TRPM3 is a calcium-permeable cation channel activated by heat and by the neurosteroid pregnenolone sulfate (PregS). TRPM3 is highly expressed in sensory neurons, where it plays a key role in heat sensing and inflammatory hyperalgesia, and in pancreatic β cells, where its activation enhances glucose-induced insulin release. However, despite its functional importance, little is known about the cellular mechanisms that regulate TRPM3 activity. Here, we provide evidence for a dynamic regulation of TRPM3 by membrane phosphatidylinositol phosphates (PIPs). Phosphatidylinositol 4,5-bisphosphate (PI[4,5]P2) and ATP applied to the intracellular side of excised membrane patches promote recovery of TRPM3 from desensitization. The stimulatory effect of cytosolic ATP on TRPM3 reflects activation of phosphatidylinositol kinases (PI-Ks), leading to resynthesis of PIPs in the plasma membrane. Various PIPs directly enhance TRPM3 activity in cell-free inside-out patches, with a potency order PI(3,4,5)P3 > PI(3,5)P2 > PI(4,5)P2 ≈ PI(3,4)P2 > PI(4)P. Conversely, TRPM3 activity is rapidly and reversibly inhibited by activation of phosphatases that remove the 5-phosphate from PIPs. Finally, we show that recombinant TRPM3, as well as the endogenous TRPM3 in insuloma cells, is rapidly and reversibly inhibited by activation of phospholipase C-coupled muscarinic acetylcholine receptors. Our results reveal basic cellular mechanisms whereby membrane receptors can regulate TRPM3 activity. PMID:26123194

  18. Analysis of factors influencing moxibustion efficacy by affecting heat-activated transient receptor potential vanilloid channels.

    PubMed

    Jiang, Jinfeng; Wang, Xinjun; Wu, Xiaojing; Yu, Zhi

    2016-04-01

    Moxibustion is an important component part of Traditional Chinese Medicine (TCM). Among differ- ent kinds of moxibustion methods, thermal stimulation seems to be a pivotal impact factor to the theraputic efficacy. Based on its thermal characteristic and treated area-skin, we hypothesize that the thermosensitive TRPV channels may involve in the mechanism of moxibustion. This study, by referring to various experimental and clinical data, analyzes the properties and features of transient receptor potential vanilloid (TRPV) subfamily 1-4 and the impact of moxibustion on these channels. The factors impacting the efficacy of moxibustion treatment were analyzed on three levels: the independent basic factors of moxibustion (temperature, space and time); moxibustion intensity (a compound factor achieved through comprehensive control of the three individual basic factors mentioned above); and moxibustion quantity (the amount of temperature stimulation applied within a certain unit of time, including the total amount of moxibustion treatment). The results from present study show that the effect of moxibustion therapy appears to be determined by the activation of TRPV1-4, mainly TRPV1 and TRPV2. Temperature (the degree of heat stimulation), time and area (how long the treatment lasts and how many TRPV1-4 channels are activated) affect the intensity of moxibustion treatment to form effective moxibustion quantity; this should be considered in clinical moxibustion application.

  19. Analysis of factors influencing moxibustion efficacy by affecting heat-activated transient receptor potential vanilloid channels.

    PubMed

    Jiang, Jinfeng; Wang, Xinjun; Wu, Xiaojing; Yu, Zhi

    2016-04-01

    Moxibustion is an important component part of Traditional Chinese Medicine (TCM). Among differ- ent kinds of moxibustion methods, thermal stimulation seems to be a pivotal impact factor to the theraputic efficacy. Based on its thermal characteristic and treated area-skin, we hypothesize that the thermosensitive TRPV channels may involve in the mechanism of moxibustion. This study, by referring to various experimental and clinical data, analyzes the properties and features of transient receptor potential vanilloid (TRPV) subfamily 1-4 and the impact of moxibustion on these channels. The factors impacting the efficacy of moxibustion treatment were analyzed on three levels: the independent basic factors of moxibustion (temperature, space and time); moxibustion intensity (a compound factor achieved through comprehensive control of the three individual basic factors mentioned above); and moxibustion quantity (the amount of temperature stimulation applied within a certain unit of time, including the total amount of moxibustion treatment). The results from present study show that the effect of moxibustion therapy appears to be determined by the activation of TRPV1-4, mainly TRPV1 and TRPV2. Temperature (the degree of heat stimulation), time and area (how long the treatment lasts and how many TRPV1-4 channels are activated) affect the intensity of moxibustion treatment to form effective moxibustion quantity; this should be considered in clinical moxibustion application. PMID:27400483

  20. A mutational analysis of the acetylcholine receptor channel transmitter binding site.

    PubMed Central

    Akk, G; Zhou, M; Auerbach, A

    1999-01-01

    Mutagenesis and single-channel kinetic analysis were used to investigate the roles of four acetylcholine receptor channel (AChR) residues that are candidates for interacting directly with the agonist. The EC50 of the ACh dose-response curve was increased following alpha-subunit mutations Y93F and Y198F and epsilon-subunit mutations D175N and E184Q. Single-channel kinetic modeling indicates that the increase was caused mainly by a reduced gating equilibrium constant (Theta) in alphaY198F and epsilonD175N, by an increase in the equilibrium dissociation constant for ACh (KD) and a reduction in Theta in alphaY93F, and only by a reduction in KD in epsilonE184Q. This mutation altered the affinity of only one of the two binding sites and was the only mutation that reduced competition by extracellular K+. Additional mutations of epsilonE184 showed that K+ competition was unaltered in epsilonE184D and was virtually eliminated in epsilonE184K, but that neither of these mutations altered the intrinsic affinity for ACh. Thus there is an apparent electrostatic interaction between the epsilonE184 side chain and K+ ( approximately 1.7kBT), but not ACh+. The results are discussed in terms of multisite and induced-fit models of ligand binding to the AChR. PMID:9876135

  1. Canonical transient receptor potential 1 channel is involved in contractile function of glomerular mesangial cells.

    PubMed

    Du, Juan; Sours-Brothers, Sherry; Coleman, Rashadd; Ding, Min; Graham, Sarabeth; Kong, De-Hu; Ma, Rong

    2007-05-01

    Contractility of mesangial cells (MC) is tightly controlled by [Ca(2+)](i). Ca(2+) influx across the plasma membrane constitutes a major component of mesangial responses to vasoconstrictors. Canonical transient receptor potential 1 (TRPC1) is a Ca(2+)-permeable cation channel in a variety of cell types. This study was performed to investigate whether TRPC1 takes part in vasoconstrictor-induced mesangial contraction by mediating Ca(2+) entry. It was found that angiotensin II (AngII) evoked remarkable contraction of the cultured MC. Downregulation of TRPC1 using RNA interference significantly attenuated the contractile response. Infusion of AngII or endothelin-1 in rats caused a decrease in GFR. The GFR decline was significantly reduced by infusion of TRPC1 antibody that targets an extracellular domain in the pore region of TRPC1 channel. However, the treatment of TRPC1 antibody did not affect the AngII-induced vasopressing effect. Electrophysiologic experiments revealed that functional or biologic inhibition of TRPC1 significantly depressed AngII-induced channel activation. Fura-2 fluorescence-indicated that Ca(2+) entry in response to AngII stimulation was also dramatically inhibited by TRPC1 antibody and TRPC1-specific RNA interference. These results suggest that TRPC1 plays an important role in controlling contractile function of MC. Mediation of Ca(2+) entry might be the underlying mechanism for the TRPC1-associated MC contraction. PMID:17389736

  2. Spontaneous opening of the acetylcholine receptor channel in developing muscle cells from normal and dystrophic mice

    SciTech Connect

    Franco-Obregon, A.; Lansman, J.B.

    1995-12-31

    Single-channel activity was recorded from cell-attached patches on skeletal muscle cells isolated from wild-type mice and from mice carrying the dy or mdx mutations. Spontaneous openings of the nicotinic acetylcholine receptor channel (nAChR) were detected in virtually all recordings from either 4v/dy or dyl + myotubes. but only infrequently from wild-type or mdx myotubes. Spontaneous openings were also present in most recordings from undifferentiated myoblasts from all of the mouse strains studied. The biophysical properties of the spontaneous activity were similar to those of the embryonic form of the nAChR in the presence of acetylcholine (ACh). Examination of the single-channel currents evoked by low concentrations of ACh showed a reduced sensitivity to the agonist in the dystrophic dy and mdx myotubes. but not in wild- type myotubes. The results suggest that alterations in nAChR function are associated with the pathogenesis of muscular dystrophy in the dy mouse.

  3. Upregulation of Transient Receptor Potential Canonical Channels Contributes to Endotoxin-Induced Pulmonary Arterial Stenosis

    PubMed Central

    Chen, Gui-Lan; Jiang, Hongni; Zou, Fangdong

    2016-01-01

    Background Septic shock is a pathologic condition caused by endotoxin-producing bacteria, and often associated with severe pulmonary hypertension. Inflammation is a major systemic response to endotoxin; however, it is unknown whether endotoxin has a direct impact on pulmonary arteries that contributes to pathogenesis of pulmonary hypertension. Material/Methods Rat pulmonary arteries and primary pulmonary arterial smooth muscle cells (PASMCs) were cultured in vitro and treated with lipopolysaccharide (LPS) and blockers of transient receptor potential canonical (TRPC) channels. Neointimal growth and arterial stenosis were observed on cryosections of cultured pulmonary arteries. Proliferation of PASMCs was examined by a WST-1 (water-soluble tetrazolium salt) assay. Expression of TRPC genes in pulmonary arteries and PASMCs were detected and quantified by real-time polymerase chain reaction and Western blotting. Results LPS significantly induced neointimal growth and stenosis of pulmonary arteries and promoted proliferation of PASMCs. TRPC channel blockers 2-aminoethoxydiphenyl borate and SKF-96365 inhibited LPS-induced remodeling of pulmonary arteries and PASMC proliferation. Expression of TRPC1/3/4/6 was detected in pulmonary arteries and PASMCs. LPS treatment dramatically increased the expression of TRPC3 and TRPC4 at both messenger RNA and protein levels. Conclusions LPS stimulates stenosis of pulmonary arteries through enhancement of TRPC-mediated Ca2+ entry into PASMCs, which is caused by upregulation of TRPC3 and TRPC4 channels. PMID:27471122

  4. Verrucotoxin inhibits KATP channels in cardiac myocytes through a muscarinic M3 receptor-PKC pathway.

    PubMed

    Wang, Jian-Wu; Yazawa, Kazuto; Hao, Li-Ying; Onoue, Yoshio; Kameyama, Masaki

    2007-06-01

    Verrucotoxin is the major component of venom from the stonefish (Synanceia verrucosa). Stings from the dorsal spines of the stonefish produce intensive pain, convulsions, hypotension, paralysis, respiratory weakness and collapse of the cardiovascular system, occasionally leading to death. It has been reported that verrucotoxin might modulate ATP-sensitive K+ (KATP) current in frog atrial fibers. However, the mechanism by which verrucotoxin acts on KATP current remains unclear. In this study, we examined whether verrucotoxin inhibited KATP current in guinea pig ventricular myocytes, using the patch clamp method. Verrucotoxin suppressed KATP current induced by pinacidil (KATP channel opener) in a concentration-dependent manner, with a half maximum concentration of 16.3 microg/ml. The effect of verrucotoxin on KATP current was suppressed by atropine (1 microM), a muscarinic receptor antagonist, or by 4-diphenylacetoxy-N-methylpiperidine (100 nM), a muscarinic M3 receptor antagonist. Furthermore, the effect of verrucotoxin on KATP current was attenuated by the protein kinase C (PKC) inhibitor chelerythrine (10 microM) and calphostin C (10 microM), yet not by the cAMP-dependent protein kinase (PKA) inhibitor H-89 (0.5 microM). These results suggest that verrucotoxin inhibits KATP current through the muscarinic M3 receptor-PKC pathway. These findings enhance our understanding of the toxic effects of verrucotoxin from the stonefish. PMID:17362922

  5. Dimensions and ion selectivity of recombinant AMPA and kainate receptor channels and their dependence on Q/R site residues.

    PubMed Central

    Burnashev, N; Villarroel, A; Sakmann, B

    1996-01-01

    1. Recombinant alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate receptor (AMPAR) subunits (GluR-A or GluR-B) and kainate receptor (KAR) subunit (GluR-6) in their unedited (Q)- and edited (R)-forms were expressed in HEK 293 cells. To estimate the dimensions of the narrow portion of these channels, biionic reversal potentials for organic cations of different mean diameters were determined with Cs+ as the internal reference ion. 2. Homomeric channels assembled from Q-form subunits were cation selective. The relation between the relative permeability and the mean size of different organic cations suggests that the diameter of the narrow portion of Q-form channels is approximately 0.78 nm for AMPAR and 0.75 nm for KAR channels. 3. Homomeric channels assembled from R-form subunits were permeant for anions and cations. When probed with CsC1 gradients the relative chloride permeability (PC1/PCs) was estimated as 0.14 for GluR-B(R) and 0.74 for GluR-6(R)-subunit channels. The permeability versus mean size relation for large cations measured with the weakly permeant F- as anion, indicates that for the R-form KAR channels the apparent pore diameter is close to 0.76 nm. 4. Heteromeric AMPAR and KAR channels co-assembled from Q- and R-form subunits were cation selective. The diameter of the narrow portion of these channels is estimated to be in the range between 0.70 and 0.74 nm. 5. The results indicated that the diameters of the narrow portion of AMPAR and KAR channels of different subunit composition and of widely different ion selectivity are comparable. Therefore, the differences in the anion versus cation selectivity, in Ca2+ permeability and in channel conductance are likely to be determined by the difference in charge density of the channel. PMID:8910205

  6. Evidence for glucagon-like peptide-1 receptor signaling to activate ATP-sensitive potassium channels in pancreatic beta cells.

    PubMed

    Kwon, Hye-Jung; Park, Hyun-Sun; Park, Sung-Hee; Park, Jae-Hyung; Shin, Su-Kyung; Song, Seung Eun; Hwang, Meeyul; Cho, Ho-Chan; Song, Dae-Kyu

    2016-01-01

    Glucagon-like peptide-1 (GLP-1) is a gut peptide that promotes insulin release from pancreatic beta cells. GLP-1 has been shown to confer glucose-insensitive beta cells with glucose sensitivity by modulation of the activity of the ATP-sensitive potassium (KATP) channel. The channel closing effect of GLP-1, interacting with corresponding G-protein-coupled receptors, has been well established; however, to our knowledge, no study has shown whether GLP-1 directly induces activation of beta-cell KATP channels. Here, we aimed to evaluate whether the activation of beta-cell KATP channels by GLP-1 exists and affects intracellular Ca(2+) levels ([Ca(2+)]i). KATP channel activity was measured in isolated rat pancreatic beta cells by whole-cell perforated patch-clamp recordings with a diazoxide-containing pipette solution. Changes in [Ca(2+)]i and the subcellular localization of KATP channels were observed using the calcium-sensitive dye fura-4/AM and anti-Kir6.2 antibodies in INS-1 beta cells, respectively. To eliminate the well-known inhibitory effects of GLP-1 on KATP channel activity, channels were fully inhibited by pretreatment with methyl pyruvate and epigallocatechin-3-gallate. In the pretreated beta cells, GLP-1 and exendin-4 promptly activated the channels, reducing [Ca(2+)]i. The phosphoinositide 3-kinase (PI3K) inhibitor LY294002 blocked the effects of GLP-1 on channel activity. Moreover, phosphatidylinositol-3,4,5-trisphosphate mimicked the effects of GLP-1. These results suggested that beta-cell GLP-1 receptor signaling involved activation of KATP channels via a PI3K-dependent pathway. This alternative mechanism of GLP-1 function may act as a negative feedback pathway, modulating the glucose-dependent GLP-1 inhibition on KATP channel activity. PMID:26655814

  7. Restricted usefulness of tetraethylammonium and 4-aminopyridine for the characterization of receptor-operated K+-channels.

    PubMed Central

    Drukarch, B.; Kits, K. S.; Leysen, J. E.; Schepens, E.; Stoof, J. C.

    1989-01-01

    1. Recently, we suggested that the D2-dopamine receptor involved in the inhibition of evoked [3H]-acetylcholine release from rat striatum is coupled to K+-channels. 2. In the present study, an attempt was made to elucidate further the role of these K+-channels, using the K+-channel blocking agents tetraethylammonium and 4-aminopyridine. With a superfusion method, the effects of both drugs on the D2-dopamine receptor-mediated inhibition of the electrically evoked release of [3H]-acetylcholine from rat striatal tissue slices was investigated. 3. Both tetraethylammonium (30 mM) and 4-aminopyridine (0.1 mM) significantly stimulated the electrically evoked release of [3H]-acetylcholine and completely abolished the effect of the selective D2-receptor agonist LY 171555 (1 microM) on evoked acetylcholine release. In addition, tetraethylammonium (0.03-30 mM) and 4-aminopyridine (0.003-1 mM) strongly increased the basal (non-evoked) release of radioactivity in a concentration-dependent manner. The results suggest that the effect of the drugs on the basal release of radioactivity and on the electrically evoked release of acetylcholine cannot exclusively be explained by their action on K+-channels. 4. Furthermore, with the use of a receptor binding assay, data were obtained on the affinity of tetraethylammonium and 4-aminopyridine for D2-receptors and various other neurotransmitter recognition sites. At concentrations in which both drugs are known to block K+-channels, they were found to inhibit the specific binding of selective radioligands to their respective recognition sites. 5. It is concluded that due to their 'side-effects', both tetraethylammonium and 4-aminopyridine are of only limited value in the investigation of the alleged interaction between neurotransmitter receptors and K+-channels. PMID:2553183

  8. Coarse architecture of the transient receptor potential vanilloid 1 (TRPV1) ion channel determined by fluorescence resonance energy transfer.

    PubMed

    De-la-Rosa, Víctor; Rangel-Yescas, Gisela E; Ladrón-de-Guevara, Ernesto; Rosenbaum, Tamara; Islas, León D

    2013-10-11

    The transient receptor potential vanilloid 1 ion channel is responsible for the perception of high temperatures and low extracellular pH, and it is also involved in the response to some pungent compounds. Importantly, it is also associated with the perception of pain and noxious stimuli. Here, we attempt to discern the molecular organization and location of the N and C termini of the transient receptor potential vanilloid 1 ion channel by measuring FRET between genetically attached enhanced yellow and cyan fluorescent protein to the N or C terminus of the channel protein, expressed in transfected HEK 293 cells or Xenopus laevis oocytes. The static measurements of the domain organization were mapped into an available cryo-electron microscopy density of the channel with good agreement. These measurements also provide novel insights into the organization of terminal domains and their proximity to the plasma membrane. PMID:23965996

  9. Evaluation of agonist selectivity for the NMDA receptor ion channel in bilayer lipid membranes based on integrated single-channel currents.

    PubMed

    Hirano, A; Sugawara, M; Umezawa, Y; Uchino, S; Nakajima-Iijima, S

    2000-06-01

    A new method for evaluating chemical selectivity of agonists to activate the N-methyl-D-aspartate (NMDA) receptor was presented by using typical agonists NMDA, L-glutamate and (2S, 3R, 4S)-2-(carboxycyclopropyl)glycine (L-CCG-IV) and the mouse epsilon1/zeta1 NMDA receptor incorporated in bilayer lipid membranes (BLMs) as an illustrative example. The method was based on the magnitude of an agonist-induced integrated single-channel current corresponding to the number of total ions passed through the open channel. The very magnitudes of the integrated single-channel currents were compared with the different BLMs as a new measure of agonist selectivity. The epsilon1/zeta1 NMDA receptor was partially purified from Chinese hamster ovary (CHO) cells expressing the epsilon1/zeta1 NMDA receptor and incorporated in BLMs formed by the tip-dip method. The agonist-induced integrated single-channel currents were obtained at 50 microM agonist concentration, where the integrated current for NMDA was shown to reach its saturated value. The obtained integrated currents were found to be (4.5 +/- 0.55) x 10(-13) C/s for NMDA, (5.8 +/- 0.72) x 10(-13) C/s for L-glutamate and (6.6 +/- 0.61) x 10(-13) C/s for L-CCG-IV, respectively. These results suggest that the agonist selectivity in terms of the total ion flux through the single epsilon1/zeta1 NMDA receptor is in the order of L-CCG-IV approximately = L-glutamate > NMDA.

  10. Transient Receptor Potential Canonical Type 3 Channels Control the Vascular Contractility of Mouse Mesenteric Arteries

    PubMed Central

    Yeon, Soo-In; Kim, Joo Young; Yeon, Dong-Soo; Abramowitz, Joel; Birnbaumer, Lutz; Muallem, Shmuel; Lee, Young-Ho

    2014-01-01

    Transient receptor potential canonical type 3 (TRPC3) channels are non-selective cation channels and regulate intracellular Ca2+ concentration. We examined the role of TRPC3 channels in agonist-, membrane depolarization (high K+)-, and mechanical (pressure)-induced vasoconstriction and vasorelaxation in mouse mesenteric arteries. Vasoconstriction and vasorelaxation of endothelial cells intact mesenteric arteries were measured in TRPC3 wild-type (WT) and knockout (KO) mice. Calcium concentration ([Ca2+]) was measured in isolated arteries from TRPC3 WT and KO mice as well as in the mouse endothelial cell line bEnd.3. Nitric oxide (NO) production and nitrate/nitrite concentrations were also measured in TRPC3 WT and KO mice. Phenylephrine-induced vasoconstriction was reduced in TRPC3 KO mice when compared to that of WT mice, but neither high K+- nor pressure-induced vasoconstriction was altered in TRPC3 KO mice. Acetylcholine-induced vasorelaxation was inhibited in TRPC3 KO mice and by the selective TRPC3 blocker pyrazole-3. Acetylcholine blocked the phenylephrine-induced increase in Ca2+ ratio and then relaxation in TRPC3 WT mice but had little effect on those outcomes in KO mice. Acetylcholine evoked a Ca2+ increase in endothelial cells, which was inhibited by pyrazole-3. Acetylcholine induced increased NO release in TRPC3 WT mice, but not in KO mice. Acetylcholine also increased the nitrate/nitrite concentration in TRPC3 WT mice, but not in KO mice. The present study directly demonstrated that the TRPC3 channel is involved in agonist-induced vasoconstriction and plays important role in NO-mediated vasorelaxation of intact mesenteric arteries. PMID:25310225

  11. Transient receptor potential channel ankyrin-1 is not a cold sensor for autonomic thermoregulation in rodents.

    PubMed

    de Oliveira, Cristiane; Garami, Andras; Lehto, Sonya G; Pakai, Eszter; Tekus, Valeria; Pohoczky, Krisztina; Youngblood, Beth D; Wang, Weiya; Kort, Michael E; Kym, Philip R; Pinter, Erika; Gavva, Narender R; Romanovsky, Andrej A

    2014-03-26

    The rodent transient receptor potential ankyrin-1 (TRPA1) channel has been hypothesized to serve as a temperature sensor for thermoregulation in the cold. We tested this hypothesis by using deletion of the Trpa1 gene in mice and pharmacological blockade of the TRPA1 channel in rats. In both Trpa1(-/-) and Trpa1(+/+) mice, severe cold exposure (8°C) resulted in decreases of skin and deep body temperatures to ∼8°C and 13°C, respectively, both temperatures being below the reported 17°C threshold temperature for TRPA1 activation. Under these conditions, Trpa1(-/-) mice had the same dynamics of body temperature as Trpa1(+/+) mice and showed no weakness in the tail skin vasoconstriction response or thermogenic response to cold. In rats, the effects of pharmacological blockade were studied by using two chemically unrelated TRPA1 antagonists: the highly potent and selective compound A967079, which had been characterized earlier, and the relatively new compound 43 ((4R)-1,2,3,4-tetrahydro-4-[3-(3-methoxypropoxy)phenyl]-2-thioxo-5H-indeno[1,2-d]pyrimidin-5-one), which we further characterized in the present study and found to be highly potent (IC50 against cold of ∼8 nm) and selective. Intragastric administration of either antagonist at 30 mg/kg before severe (3°C) cold exposure did not affect the thermoregulatory responses (deep body and tail skin temperatures) of rats, even though plasma concentrations of both antagonists well exceeded their IC50 value at the end of the experiment. In the same experimental setup, blocking the melastatin-8 (TRPM8) channel with AMG2850 (30 mg/kg) attenuated cold-defense mechanisms and led to hypothermia. We conclude that TRPA1 channels do not drive autonomic thermoregulatory responses to cold in rodents.

  12. Bisphenol A binds to the local anesthetic receptor site to block the human cardiac sodium channel.

    PubMed

    O'Reilly, Andrias O; Eberhardt, Esther; Weidner, Christian; Alzheimer, Christian; Wallace, B A; Lampert, Angelika

    2012-01-01

    Bisphenol A (BPA) has attracted considerable public attention as it leaches from plastic used in food containers, is detectable in human fluids and recent epidemiologic studies link BPA exposure with diseases including cardiovascular disorders. As heart-toxicity may derive from modified cardiac electrophysiology, we investigated the interaction between BPA and hNav1.5, the predominant voltage-gated sodium channel subtype expressed in the human heart. Electrophysiology studies of heterologously-expressed hNav1.5 determined that BPA blocks the channel with a K(d) of 25.4±1.3 µM. By comparing the effects of BPA and the local anesthetic mexiletine on wild type hNav1.5 and the F1760A mutant, we demonstrate that both compounds share an overlapping binding site. With a key binding determinant thus identified, an homology model of hNav1.5 was generated based on the recently-reported crystal structure of the bacterial voltage-gated sodium channel NavAb. Docking predictions position both ligands in a cavity delimited by F1760 and contiguous with the DIII-IV pore fenestration. Steered molecular dynamics simulations used to assess routes of ligand ingress indicate that the DIII-IV pore fenestration is a viable access pathway. Therefore BPA block of the human heart sodium channel involves the local anesthetic receptor and both BPA and mexiletine may enter the closed-state pore via membrane-located side fenestrations. PMID:22848561

  13. Serotonin stimulates lateral habenula via activation of the post-synaptic serotonin 2/3 receptors and transient receptor potential channels.

    PubMed

    Zuo, Wanhong; Zhang, Yong; Xie, Guiqin; Gregor, Danielle; Bekker, Alex; Ye, Jiang-Hong

    2016-02-01

    There is growing interest on the role of the lateral habenula (LHb) in depression, because it closely and bilaterally connects with the serotoninergic raphe nuclei. The LHb sends glutamate efferents to the raphe nuclei, while it receives serotoninergic afferents, and expresses a high density of serotonin (5-HT) receptors. Recent studies suggest that 5-HT receptors exist both in the presynaptic and postsynaptic sites of LHb neurons, and activation of these receptors may have different effects on the activity of LHb neurons. The current study focused on the effect of 5-HT on the postsynaptic membrane. We found that 5-HT initiated a depolarizing inward current (I((5-HTi))) and accelerated spontaneous firing in ∼80% of LHb neurons in rat brain slices. I((5-HTi)) was also induced by the 5-HT uptake blocker citalopram, indicating activity of endogenous 5-HT. I((5-HTi)) was diminished by 5-HT(2/3) receptor antagonists (ritanserin, SB-200646 or ondansetron), and activated by the selective 5-HT(2/3) agonists 1-(3-Chlorophenyl) piperazine hydrochloride or 1-(3-Chlorophenyl) biguanide hydrochloride. Furthermore, I((5-HTi)) was attenuated by 2-Aminoethyl diphenylborinate, a blocker of transient receptor potential channels, and an IP3 receptor inhibitor, indicating the involvement of transient receptor potential channels. These results demonstrate that the reciprocal connection between the LHb and the 5-HT system highlights a key role for 5-HT stimulation of LHb neurons that may be important in the pathogenesis of depression.

  14. Phylogenetic sequence analysis, recombinant expression, and tissue distribution of a channel catfish estrogen receptor beta

    USGS Publications Warehouse

    Xia, Zhenfang; Gale, William L.; Chang, Xiaotian; Langenau, David; Patino, Reynaldo; Maule, Alec G.; Densmore, Llewellyn D.

    2000-01-01

    An estrogen receptor β (ERβ) cDNA fragment was amplified by RT-PCR of total RNAextracted from liver and ovary of immature channel catfish. This cDNA fragment was used to screen an ovarian cDNA library made from an immature female fish. A clone was obtained that contained an open reading frame encoding a 575-amino-acid protein with a deduced molecular weight of 63.9 kDa. Maximum parsimony and Neighbor Joining analyses were used to generate a phylogenetic classification of channel catfish ERβ on the basis of 25 full-length teleost and tetrapod ER sequences. The consensus tree obtained indicated the existence of two major vertebrate ER subtypes, α and β. Within each subtype, and in accordance with established phylogenetic relationships, teleost and tetrapod ER were monophyletic confirming the results of a previous analysis (Z. Xiaet al., 1999, Gen. Comp. Endocrinol. 113, 360–368). Extracts of COS-7 cells transfectedwith channel catfish ERβ cDNA bound estrogen with high affinity (Kd = 0.21 nM) and specificity. The affinity of channel catfish ERβ for estrogen was higher than previously reported for channel catfish ERα. As determined by qualitative RT-PCR, the tissue distributions of ERα and ERβ were similar but not identical. Both ER subtypes were present in ovary and testis. ERα was found in all other tissues examined from juvenile and mature fish of both sexes. ERβ was also found in most tissues except, in most cases, whole blood and head kidney. Interestingly, the pattern of expression of ER subtypes in head kidney always corresponded to the pattern in whole blood. In conclusion, we isolated a channel catfish ERβ with ligand-binding affinity and tissue expression patterns different from ERα. Also, we confirmed the validity of our previously proposed general classification scheme for vertebrate ER into α and β subtypes and within each subtype, into teleost and tetrapod clades.

  15. Analysis of G-protein-activated inward rectifying K(+) (GIRK) channel currents upon GABAB receptor activation in rat supraoptic neurons.

    PubMed

    Harayama, Nobuya; Kayano, Tomohiko; Moriya, Taiki; Kitamura, Naoki; Shibuya, Izumi; Tanaka-Yamamoto, Keiko; Uezono, Yasuhito; Ueta, Yoichi; Sata, Takeyoshi

    2014-12-01

    While magnocellular neurons in the supraoptic nucleus (SON) possess rich Gi/o-mediated mechanisms, molecular and cellular properties of G-protein-activated inwardly rectifying K(+) (GIRK) channels have been controversial. Here, properties of GIRK channels are examined by RT-PCR and whole-cell patch-clamp techniques in rat SON neurons. Patch clamp experiments showed that the selective GABAB agonist, baclofen, enhanced currents in a high K(+) condition. The baclofen-enhanced currents exhibited evident inward rectification and were blocked by the selective GABAB antagonist, CGP55845A, the IRK channel blocker, Ba(2+), and the selective GIRK channel blocker, tertiapin, indicating that baclofen activates GIRK channels via GABAB receptors. The GIRK currents were abolished by N-ethylmaleimide pretreatment, and prolonged by GTPγS inclusion in the patch pipette, suggesting that Gi/o proteins are involved. RT-PCR analysis revealed mRNAs for all four GIRK 1-4 channels and for both GABABR1 and GABABR2 receptors in rat SON. However, the concentration-dependency of the baclofen-induced activation of GIRK currents had an EC50 of 110 µM, which is about 100 times higher than that of baclofen-induced inhibition of voltage-dependent Ca(2+) channels. Moreover, baclofen caused no significant changes in the membrane potential and the firing rate. These results suggest that although GIRK channels can be activated by GABAB receptors via the Gi/o pathway, this occurs at high agonist concentrations, and thus may not be a physiological mechanism regulating the function of SON neurons. This property that the membrane potential receives little influence from GIRK currents seems to be uncommon for CNS neurons possessing rich Gi/o-coupled receptors, and could be a special feature of rat SON neurons.

  16. Neurotransmitter receptors and voltage-dependent Ca2+ channels encoded by mRNA from the adult corpus callosum.

    PubMed

    Matute, C; Miledi, R

    1993-04-15

    The presence of mRNAs encoding neurotransmitter receptors and voltage-gated channels in the adult human and bovine corpus callosum was investigated using Xenopus oocytes. Oocytes injected with mRNA extracted from the corpus callosum expressed functional receptors to glutamate, acetylcholine, and serotonin, and also voltage-operated Ca2+ channels, all with similar properties in the two species studied. Acetylcholine and serotonin elicited oscillatory Cl- currents due to activation of the inositol phosphate-Ca2+ receptor-channel coupling system. Glutamate and its analogs N-methyl-D-aspartate (NMDA), kainate, quisqualate, and alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) induced smooth currents. The non-NMDA responses showed a strong inward rectification at positive potentials and were potently blocked by 6,7-dinitroquinoxaline-2,3-dione, as observed for the AMPA/kainate glutamate receptors GLUR1 and GLUR3. Furthermore, in situ hybridization experiments showed that GLUR1 and GLUR3 mRNAs are present in corpus callosum cells that were labeled with antiserum to glial fibrillary acid protein and that, in primary cell cultures, had the morphology of type 2 astrocytes. These results indicate that glial cells in the adult corpus callosum possess mRNA encoding functional neurotransmitter receptors and Ca2+ channels. These molecules may provide a mechanism for glial-neuronal interactions. PMID:7682696

  17. GLUCOSE-INDUCED INTESTINAL VASODILATION VIA ADENOSINE A1 RECEPTORS REQUIRES NITRIC OXIDE BUT NOT K+ATP CHANNELS

    PubMed Central

    Matheson, Paul J.; Li, Na; Harris, Patrick D.; Zakaria, El Rasheid; Garrison, R. Neal

    2010-01-01

    INTRODUCTION Both nitric oxide (NO) and adenosine A1 receptor activation mediate microvascular vasodilation during intestinal glucose absorption. Our overall hypothesis is that ATP utilization during glucose absorption would increase adenosine metabolite release, which acts on adenosine A1 receptors to alter endothelial production of NO and/or activate ATP-dependent potassium channels (K+ATP) to dilate intestinal microvessels. METHODS Intravital videomicroscopy of the rat jejunum was used to record the vascular responses of inflow (termed 1A) arterioles and proximal (p3A) and distal (d3A) premucosal arterioles during exposure to isotonic glucose or mannitol solutions alone or in the presence of the selective nitric oxide synthase (NOS) inhibitor (L-NMMA), an adenosine A1 receptor antagonist (8-cyclopentyl-1,3-dipropylxanthine (DPCPX)), or a K+ATP channel inhibitor (glibenclamide). RESULTS As expected, glucose exposure caused rapid dilation of both p3A and d3A arterioles, while mannitol exposure had no effect on microvascular diameters. Adenosine A1 receptor blockade completely prevented glucose-induced dilation of the premucosal arterioles. NOS inhibition significantly blunted the glucose-induced vasodilation of the premucosal arterioles, but had little effect in the mannitol group. Simultaneous application of both the NOS inhibitor and the adenosine A1 receptor antagonist gave the same reduction in glucose-induced dilation of the premucosal arterioles as the adenosine A1 receptor antagonist alone. Blockade of K+ATP channels with glibenclamide did not attenuate glucose-induced vasodilation of the premucosal arterioles. CONCLUSIONS These data suggest that glucose-induced vasodilation of premucosal jejunal arterioles is mediated through adenosine A1 receptors, and NO at least partially mediates the adenosine A1 receptor-induced vasodilation. In addition, K+ATP channels are not involved in premucosal arteriolar vasodilation during intestinal glucose exposure. PMID

  18. Transmembrane Communication: General Principles and Lessons from the Structure and Function of the M2 Proton Channel, K+ Channels, and Integrin Receptors

    PubMed Central

    Grigoryan, Gevorg; Moore, David T.; DeGrado, William F.

    2013-01-01

    Signal transduction across biological membranes is central to life. This process generally happens through communication between different domains and hierarchical coupling of information. Here, we review structural and thermodynamic principles behind transmembrane (TM) signal transduction and discuss common themes. Communication between signaling domains can be understood in terms of thermodynamic and kinetic principles, and complex signaling patterns can arise from simple wiring of thermodynamically coupled domains. We relate this to functions of several signal transduction systems: the M2 proton channel from influenza A virus, potassium channels, integrin receptors, and bacterial kinases. We also discuss key features in the structural rearrangements responsible for signal transduction in these systems. PMID:21548783

  19. Transmembrane communication: general principles and lessons from the structure and function of the M2 proton channel, K⁺ channels, and integrin receptors.

    PubMed

    Grigoryan, Gevorg; Moore, David T; DeGrado, William F

    2011-01-01

    Signal transduction across biological membranes is central to life. This process generally happens through communication between different domains and hierarchical coupling of information. Here, we review structural and thermodynamic principles behind transmembrane (TM) signal transduction and discuss common themes. Communication between signaling domains can be understood in terms of thermodynamic and kinetic principles, and complex signaling patterns can arise from simple wiring of thermodynamically coupled domains. We relate this to functions of several signal transduction systems: the M2 proton channel from influenza A virus, potassium channels, integrin receptors, and bacterial kinases. We also discuss key features in the structural rearrangements responsible for signal transduction in these systems.

  20. Activity-dependent bidirectional regulation of GABAA receptor channels by the 5-HT4 receptor-mediated signalling in rat prefrontal cortical pyramidal neurons

    PubMed Central

    Cai, Xiang; Flores-Hernandez, Jorge; Feng, Jian; Yan, Zhen

    2002-01-01

    Emerging evidence has implicated a potential role for 5-HT4 receptors in cognition and anxiolysis. One of the main target structures of 5-HT4 receptors on ‘cognitive and emotional’ pathways is the prefrontal cortex (PFC). As GABAergic signalling plays a key role in regulating PFC functions, we examined the effect of 5-HT4 receptors on GABAA receptor channels in PFC pyramidal neurons. Application of 5-HT4 receptor agonists produced either an enhancement or a reduction of GABA-evoked currents in PFC neurons, which are both mediated by anchored protein kinase A (PKA). Although PKA phosphorylation of GABAA receptor β3 or β1 subunits leads to current enhancement or reduction respectively in heterologous expression systems, we found that β3 and β1 subunits are co-expressed in PFC pyramidal neurons. Interestingly, altering PKA activation levels can change the direction of the dual effect, switching enhancement to reduction and vice versa. In addition, increased neuronal activity in PFC slices elevated the PKA activation level, changing the enhancing effect of 5-HT4 receptors on the amplitude of GABAergic inhibitory postsynaptic currents (IPSCs) to a reduction. These results suggest that 5-HT4 receptors can modulate GABAergic signalling bidirectionally, depending on the basal PKA activation levels that are determined by neuronal activity. This modulation provides a unique and flexible mechanism for 5-HT4 receptors to dynamically regulate synaptic transmission and neuronal excitability in the PFC network. PMID:11986365

  1. Age-related functional changes of the glutamate receptor channels in rat Meynert neurones.

    PubMed Central

    Akaike, N; Rhee, J S

    1997-01-01

    1. The developmental changes of glutamate receptors (GluRs) in acutely dissociated rat Meynert neurones were investigated using the conventional whole cell and nystatin perforated patch recording modes under voltage-clamp conditions. 2. The neurones became less responsive to N-methyl-D-aspartic acid (NMDA) with age, most dramatically between 1 day and 2 weeks, while the responses to kainic acid (KA) and L-alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) gradually increased. The metabotropic GluR response appeared a few days after birth, but thereafter no further change was observed. 3. The decrease in the NMDA response during postnatal development was due to an abrupt reduction in the number of receptors without affecting the affinity, voltage-dependent Mg2+ blockade or high Ca2+ permeability (PCa/PCs approximately 7.0). 4. PCa/PCs in the presence of KA decreased from 2.8 in the 1-day-old (1D) rat neurones to 1.1 and 0.44 in the 2-week-old (2W) and 6-month-old (6M) rat neurones, respectively. The concentration-response relationship for KA shifted to the left with age. The KA response was not affected by NS-102, a KA-selective antagonist, thus indicating that the increased affinity of the receptor for the ligand resulted from the change in the AMPA receptor channel subunits. 5. The AMPA response in the presence of 10(-4) M cyclothiazide showed a change in the inward rectifying current-voltage relationship with age. The KA response was strongly cross-desensitized by the addition of AMPA and was also blocked by 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), whereas a rapid desensitization of the AMPA response was removed in a concentration-dependent manner by cyclothiazide. These results indicate that the non-NMDA receptor channels are assembled from the subunits of the AMPA receptor family without the GluR-2 subunit, thus resulting in a high Ca2+ permeability. 6. The L-glutamate (Glu)-induced responses were more sensitive to DL-2-amino-5

  2. Discovery of functional monoclonal antibodies targeting G-protein-coupled receptors and ion channels.

    PubMed

    Wilkinson, Trevor C I

    2016-06-15

    The development of recombinant antibody therapeutics is a significant area of growth in the pharmaceutical industry with almost 50 approved monoclonal antibodies on the market in the US and Europe. Despite this growth, however, certain classes of important molecular targets have remained intractable to therapeutic antibodies due to complexity of the target molecules. These complex target molecules include G-protein-coupled receptors and ion channels which represent a large potential target class for therapeutic intervention with monoclonal antibodies. Although these targets have typically been addressed by small molecule approaches, the exquisite specificity of antibodies provides a significant opportunity to provide selective modulation of these target proteins. Given this opportunity, substantial effort has been applied to address the technical challenges of targeting these complex membrane proteins with monoclonal antibodies. In this review recent progress made in the strategies for discovery of functional monoclonal antibodies for these challenging membrane protein targets is addressed. PMID:27284048

  3. Changes in Membrane Receptors and Ion Channels as Potential Biomarkers for Osteoarthritis

    PubMed Central

    Lewis, Rebecca; Barrett-Jolley, Richard

    2015-01-01

    Osteoarthritis (OA), a degenerative joint condition, is currently difficult to detect early enough for any of the current treatment options to be completely successful. Early diagnosis of this disease could increase the numbers of patients who are able to slow its progression. There are now several diseases where membrane protein biomarkers are used for early diagnosis. The numbers of proteins in the membrane is vast and so it is a rich source of potential biomarkers for OA but we need more knowledge of these before they can be considered practical biomarkers. How are they best measured and are they selective to OA or even certain types of OA? The first step in this process is to identify membrane proteins that change in OA. Here, we summarize several ion channels and receptors that change in OA models and/or OA patients, and may thus be considered candidates as novel membrane biomarkers of OA. PMID:26648874

  4. Marine snail venoms: use and trends in receptor and channel neuropharmacology.

    PubMed

    Favreau, Philippe; Stöcklin, Reto

    2009-10-01

    Venoms are rich mixtures of mainly peptides and proteins evolved by nature to catch and digest preys or for protection against predators. They represent extensive sources of potent and selective bioactive compounds that can lead to original active ingredients, for use as drugs, as pharmacological tools in research and for the biotechnology industry. Among the most fascinating venomous animals, marine snails offer a unique set of pharmacologically active components, targeting a wide diversity of receptors and ion channels. Recent advances still continue to demonstrate their huge neuropharmacological potential. In the quest for interesting pharmacological profiles, researchers face a vast number of venom components to investigate within time and technological constraints. A brief perspective on marine snail venom's complexity and features is given followed by the different discovery strategies and pharmacological approaches, exemplified with some recent developments. These advances will hopefully help further uncovering new pharmacologically important venom molecules.

  5. Structural determinants of the transient receptor potential 1 (TRPV1) channel activation by phospholipid analogs.

    PubMed

    Morales-Lázaro, Sara L; Serrano-Flores, Barbara; Llorente, Itzel; Hernández-García, Enrique; González-Ramírez, Ricardo; Banerjee, Souvik; Miller, Duane; Gududuru, Veeresh; Fells, James; Norman, Derek; Tigyi, Gabor; Escalante-Alcalde, Diana; Rosenbaum, Tamara

    2014-08-29

    The transient receptor potential vanilloid 1 (TRPV1) ion channel is a polymodal protein that responds to various stimuli, including capsaicin (the pungent compound found in chili peppers), extracellular acid, and basic intracellular pH, temperatures close to 42 °C, and several lipids. Lysophosphatidic acid (LPA), an endogenous lipid widely associated with neuropathic pain, is an agonist of the TRPV1 channel found in primary afferent nociceptors and is activated by other noxious stimuli. Agonists or antagonists of lipid and other chemical natures are known to possess specific structural requirements for producing functional effects on their targets. To better understand how LPA and other lipid analogs might interact and affect the function of TRPV1, we set out to determine the structural features of these lipids that result in the activation of TRPV1. By changing the acyl chain length, saturation, and headgroup of these LPA analogs, we established strict requirements for activation of TRPV1. Among the natural LPA analogs, we found that only LPA 18:1, alkylglycerophosphate 18:1, and cyclic phosphatidic acid 18:1, all with a monounsaturated C18 hydrocarbon chain activate TRPV1, whereas polyunsaturated and saturated analogs do not. Thus, TRPV1 shows a more restricted ligand specificity compared with LPA G-protein-coupled receptors. We synthesized fatty alcohol phosphates and thiophosphates and found that many of them with a single double bond in position Δ9, 10, or 11 and Δ9 cyclopropyl group can activate TRPV1 with efficacy similar to capsaicin. Finally, we developed a pharmacophore and proposed a mechanistic model for how these lipids could induce a conformational change that activates TRPV1. PMID:25035428

  6. Structural Determinants of the Transient Receptor Potential 1 (TRPV1) Channel Activation by Phospholipid Analogs*

    PubMed Central

    Morales-Lázaro, Sara L.; Serrano-Flores, Barbara; Llorente, Itzel; Hernández-García, Enrique; González-Ramírez, Ricardo; Banerjee, Souvik; Miller, Duane; Gududuru, Veeresh; Fells, James; Norman, Derek; Tigyi, Gabor; Escalante-Alcalde, Diana; Rosenbaum, Tamara

    2014-01-01

    The transient receptor potential vanilloid 1 (TRPV1) ion channel is a polymodal protein that responds to various stimuli, including capsaicin (the pungent compound found in chili peppers), extracellular acid, and basic intracellular pH, temperatures close to 42 °C, and several lipids. Lysophosphatidic acid (LPA), an endogenous lipid widely associated with neuropathic pain, is an agonist of the TRPV1 channel found in primary afferent nociceptors and is activated by other noxious stimuli. Agonists or antagonists of lipid and other chemical natures are known to possess specific structural requirements for producing functional effects on their targets. To better understand how LPA and other lipid analogs might interact and affect the function of TRPV1, we set out to determine the structural features of these lipids that result in the activation of TRPV1. By changing the acyl chain length, saturation, and headgroup of these LPA analogs, we established strict requirements for activation of TRPV1. Among the natural LPA analogs, we found that only LPA 18:1, alkylglycerophosphate 18:1, and cyclic phosphatidic acid 18:1, all with a monounsaturated C18 hydrocarbon chain activate TRPV1, whereas polyunsaturated and saturated analogs do not. Thus, TRPV1 shows a more restricted ligand specificity compared with LPA G-protein-coupled receptors. We synthesized fatty alcohol phosphates and thiophosphates and found that many of them with a single double bond in position Δ9, 10, or 11 and Δ9 cyclopropyl group can activate TRPV1 with efficacy similar to capsaicin. Finally, we developed a pharmacophore and proposed a mechanistic model for how these lipids could induce a conformational change that activates TRPV1. PMID:25035428

  7. Structural determinants of the transient receptor potential 1 (TRPV1) channel activation by phospholipid analogs.

    PubMed

    Morales-Lázaro, Sara L; Serrano-Flores, Barbara; Llorente, Itzel; Hernández-García, Enrique; González-Ramírez, Ricardo; Banerjee, Souvik; Miller, Duane; Gududuru, Veeresh; Fells, James; Norman, Derek; Tigyi, Gabor; Escalante-Alcalde, Diana; Rosenbaum, Tamara

    2014-08-29

    The transient receptor potential vanilloid 1 (TRPV1) ion channel is a polymodal protein that responds to various stimuli, including capsaicin (the pungent compound found in chili peppers), extracellular acid, and basic intracellular pH, temperatures close to 42 °C, and several lipids. Lysophosphatidic acid (LPA), an endogenous lipid widely associated with neuropathic pain, is an agonist of the TRPV1 channel found in primary afferent nociceptors and is activated by other noxious stimuli. Agonists or antagonists of lipid and other chemical natures are known to possess specific structural requirements for producing functional effects on their targets. To better understand how LPA and other lipid analogs might interact and affect the function of TRPV1, we set out to determine the structural features of these lipids that result in the activation of TRPV1. By changing the acyl chain length, saturation, and headgroup of these LPA analogs, we established strict requirements for activation of TRPV1. Among the natural LPA analogs, we found that only LPA 18:1, alkylglycerophosphate 18:1, and cyclic phosphatidic acid 18:1, all with a monounsaturated C18 hydrocarbon chain activate TRPV1, whereas polyunsaturated and saturated analogs do not. Thus, TRPV1 shows a more restricted ligand specificity compared with LPA G-protein-coupled receptors. We synthesized fatty alcohol phosphates and thiophosphates and found that many of them with a single double bond in position Δ9, 10, or 11 and Δ9 cyclopropyl group can activate TRPV1 with efficacy similar to capsaicin. Finally, we developed a pharmacophore and proposed a mechanistic model for how these lipids could induce a conformational change that activates TRPV1.

  8. Calcium regulation by temperature-sensitive transient receptor potential channels in human uveal melanoma cells.

    PubMed

    Mergler, Stefan; Derckx, Raissa; Reinach, Peter S; Garreis, Fabian; Böhm, Arina; Schmelzer, Lisa; Skosyrski, Sergej; Ramesh, Niraja; Abdelmessih, Suzette; Polat, Onur Kerem; Khajavi, Noushafarin; Riechardt, Aline Isabel

    2014-01-01

    Uveal melanoma (UM) is both the most common and fatal intraocular cancer among adults worldwide. As with all types of neoplasia, changes in Ca(2+) channel regulation can contribute to the onset and progression of this pathological condition. Transient receptor potential channels (TRPs) and cannabinoid receptor type 1 (CB1) are two different types of Ca(2+) permeation pathways that can be dysregulated during neoplasia. We determined in malignant human UM and healthy uvea and four different UM cell lines whether there is gene and functional expression of TRP subtypes and CB1 since they could serve as drug targets to either prevent or inhibit initiation and progression of UM. RT-PCR, Ca(2+) transients, immunohistochemistry and planar patch-clamp analysis probed for their gene expression and functional activity, respectively. In UM cells, TRPV1 and TRPM8 gene expression was identified. Capsaicin (CAP), menthol or icilin induced Ca(2+) transients as well as changes in ion current behavior characteristic of TRPV1 and TRPM8 expression. Such effects were blocked with either La(3+), capsazepine (CPZ) or BCTC. TRPA1 and CB1 are highly expressed in human uvea, but TRPA1 is not expressed in all UM cell lines. In UM cells, the CB1 agonist, WIN 55,212-2, induced Ca(2+) transients, which were suppressed by La(3+) and CPZ whereas CAP-induced Ca(2+) transients could also be suppressed by CB1 activation. Identification of functional TRPV1, TRPM8, TRPA1 and CB1 expression in these tissues may provide novel drug targets for treatment of this aggressive neoplastic disease.

  9. Control of sensory neuron excitability by serotonin involves 5HT2C receptors and Ca(2+)-activated chloride channels.

    PubMed

    Salzer, Isabella; Gantumur, Enkhbileg; Yousuf, Arsalan; Boehm, Stefan

    2016-11-01

    Serotonin (5HT) is a constituent of the so-called "inflammatory soup" that sensitizes nociceptors during inflammation. Nevertheless, receptors and signaling mechanisms that mediate an excitation of dorsal root ganglion (DRG) neurons by 5HT remained controversial. Therefore, capsaicin-sensitive nociceptive neurons dissociated from rat DRGs were used to investigate effects of 5HT on membrane excitability and currents through ligand- as well as voltage-gated ion channels. In 58% of the neurons tested, 5HT increased action potential firing, an effect that was abolished by the 5HT2 receptor antagonist ritanserin, but not by the 5HT3 antagonist tropisetron. Unlike other algogenic mediators, such as PGE2 and bradykinin, 5HT did not affect currents through TTX-resistant Na(+) channels or Kv7 K(+) channels. In all neurons investigated, 5HT potentiated capsaicin-evoked currents through TRPV1 channels, an effect that was attenuated by antagonists at 5HT2A (4 F 4 PP), 5HT2B (SB 204741), as well as 5HT2C (RS 102221) receptors. 5HT triggered slowly arising inward Cl(-) currents in 53% of the neurons. This effect was antagonized by the 5HT2C receptor blocker only, and the current was prevented by an inhibitor of Ca(2+)-activated chloride channels (CaCC). The 5HT-induced increase in action potential firing was also abolished by this CaCC blocker and by the TRPV1 inhibitor capsazepine. Amongst the subtype selective 5HT2 antagonists, only RS 102221 (5HT2C-selectively) counteracted the rise in action potential firing elicited by 5HT. These results show that 5HT excites DRG neurons mainly via 5HT2C receptors which concomitantly mediate a sensitization of TRPV1 channels and an opening of CaCCs.

  10. Intracellular calcium level is an important factor influencing ion channel modulations by PLC-coupled metabotropic receptors in hippocampal neurons.

    PubMed

    Sugawara, Yuto; Echigo, Ryousuke; Kashima, Kousuke; Minami, Hanae; Watanabe, Megumi; Nishikawa, Yuiko; Muranishi, Miho; Yoneda, Mitsugu; Ohno-Shosaku, Takako

    2013-05-28

    Signaling pathways involving phospholipase C (PLC) are involved in various neural functions. Understanding how these pathways are regulated will lead to a better understanding of their roles in neural functions. Previous studies demonstrated that receptor-driven PLCβ activation depends on intracellular Ca(2+) concentration ([Ca(2+)]i), suggesting the possibility that PLCβ-dependent cellular responses are basically Ca(2+) dependent. To test this possibility, we examined whether modulations of ion channels driven by PLC-coupled metabotropic receptors are sensitive to [Ca(2+)]i using cultured hippocampal neurons. Muscarinic activation triggered an inward current at -100 mV (the equilibrium potential for K(+)) in a subpopulation of neurons. This current response was suppressed by pirenzepine (an M1-preferring antagonist), PLC inhibitor, non-selective cation channel blocker, and lowering [Ca(2+)]i. Using the neurons showing no response at -100 mV, effects of muscarinic activation on K(+) channels were examined at -40 mV. Muscarinic activation induced a transient decrease of the holding outward current. This current response was mimicked and occluded by XE991, an M-current K(+) channel blocker, suppressed by pirenzepine, PLC inhibitor and lowering [Ca(2+)]i, and enhanced by elevating [Ca(2+)]i. Similar results were obtained when group I metabotropic glutamate receptors were activated instead of muscarinic receptors. These results clearly show that ion channel modulations driven by PLC-coupled metabotropic receptors are dependent on [Ca(2+)]i, supporting the hypothesis that cellular responses induced by receptor-driven PLCβ activation are basically Ca(2+) dependent.

  11. Purification and subunit structure of the [3H]phenamil receptor associated with the renal apical Na+ channel.

    PubMed Central

    Barbry, P; Chassande, O; Vigne, P; Frelin, C; Ellory, C; Cragoe, E J; Lazdunski, M

    1987-01-01

    Sodium crosses the apical membrane of tight epithelia through a sodium channel, which is inhibited by the diuretic amiloride and by analogs such as phenamil. Target size analysis indicated that the functional size of the [3H]phenamil binding sites associated with the epithelial Na+ channel from pig kidney is 92 +/- 10 kDa. The [3H]phenamil receptor was solubilized by using 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate. The solubilized material displayed the same properties of interaction with amiloride and its derivatives as the membrane-bound receptor. A two-step purification of the epithelial Na+ channel was achieved by using QAE Sephadex chromatography and affinity chromatography on a Bandeiraea simplicifolia lectin column. It results in an 1100-fold purification of the Na+ channel as compared to pig kidney microsomes with a yield of 15% +/- 5%. The maximal specific activity was 3.7 nmol/mg of protein. NaDodSO4/poly-acrylamide gel electrophoresis of the purified Na+ channel under nonreducing conditions showed the presence of a single major polypeptide chain of apparent molecular mass 185 kDa. Under disulfide-reducing conditions, the purified epithelial Na+ channel migrated as a single band of apparent molecular mass 105 kDa. It is suggested that the epithelial Na+ channel from pig kidney has a total molecular mass of 185 kDa and consists of two nearly identical 90- to 105-kDa polypeptide chains crosslinked by disulfide bridges. Images PMID:2440032

  12. Transient receptor potential vanilloid type-1 (TRPV-1) channels contribute to cutaneous thermal hyperaemia in humans.

    PubMed

    Wong, Brett J; Fieger, Sarah M

    2010-11-01

    The initial, rapid increase in skin blood flow in response to direct application of heat is thought to be mediated by an axon reflex, which is dependent on intact cutaneous sensory nerves. We tested the hypothesis that inhibition of transient receptor potential vanilloid type 1 (TRPV-1) channels, which are putative channels located on sensory nerves, would attenuate the skin blood flow response to local heating in humans. Ten subjects were equipped with four microdialysis fibres which were randomly assigned one of four treatments: (1) vehicle control (90% propylene glycol + 10% lactated Ringer solution); (2) 20 mm capsazepine to inhibit TRPV-1 channels; (3) 10 mm l-NAME to inhibit NO synthase; and (4) combined 20 mm capsazepine + 10 mm l-NAME. Following baseline measurements, the temperature of skin heaters was increased from 33°C to 42°C at a rate of 1.0°C every 10 s and local temperature was held at 42°C for 20-30 min until a stable plateau in skin blood flow was achieved. An index of skin blood flow was measured directly over each microdialysis site via laser-Doppler flowmetry (LDF). Beat-by-beat blood pressure was measured via photoplethysmography and verified via automated brachial auscultation. At the end of the local heating protocol, temperature of the heaters was increased to 43°C and 28 mm nitroprusside was infused to achieve maximal vasodilatation. Cutaneous vascular conductance (CVC) was calculated as LDF/mean arterial pressure and normalized to maximal values (%CVCmax). Initial peak in capsazepine (44 ± 4%CVCmax), l-NAME (56 ± 4%CVCmax) and capsazepine + l-NAME (32 ± 6%CVCmax) sites was significantly attenuated compared to control (87 ± 5%CVCmax; P < 0.001 for all conditions). The plateau phase of thermal hyperaemia was significantly attenuated in capsazepine (73 ± 6%CVCmax), l-NAME (47 ± 5%CVCmax) and capsazepine + l-NAME (31 ± 7%CVCmax) sites compared to control (92 ± 5%CVCmax; P < 0.001 for all conditions). These data suggest TRPV-1

  13. Diabetes Stimulates Osteoclastogenesis by Acidosis-Induced Activation of Transient Receptor Potential Cation Channels

    PubMed Central

    Reni, Carlotta; Mangialardi, Giuseppe; Meloni, Marco; Madeddu, Paolo

    2016-01-01

    Patients with type 1 diabetes have lower bone mineral density and higher risk of fractures. The role of osteoblasts in diabetes-related osteoporosis is well acknowledged whereas the role of osteoclasts (OCLs) is still unclear. We hypothesize that OCLs participate in pathological bone remodeling. We conducted studies in animals (streptozotocin-induced type 1 diabetic mice) and cellular models to investigate canonical and non-canonical mechanisms underlying excessive OCL activation. Diabetic mice show an increased number of active OCLs. In vitro studies demonstrate the involvement of acidosis in OCL activation and the implication of transient receptor potential cation channel subfamily V member 1 (TRPV1). In vivo studies confirm the establishment of local acidosis in the diabetic bone marrow (BM) as well as the ineffectiveness of insulin in correcting the pH variation and osteoclast activation. Conversely, treatment with TRPV1 receptor antagonists re-establishes a physiological OCL availability. These data suggest that diabetes causes local acidosis in the BM that in turn increases osteoclast activation through the modulation of TRPV1. The use of clinically available TRPV1 antagonists may provide a new means to combat bone problems associated with diabetes. PMID:27468810

  14. The intrinsic energy of the gating isomerization of a neuromuscular acetylcholine receptor channel.

    PubMed

    Nayak, Tapan K; Purohit, Prasad G; Auerbach, Anthony

    2012-05-01

    Nicotinic acetylcholine receptor (AChR) channels at neuromuscular synapses rarely open in the absence of agonists, but many different mutations increase the unliganded gating equilibrium constant (E0) to generate AChRs that are active constitutively. We measured E0 for two different sets of mutant combinations and by extrapolation estimated E0 for wild-type AChRs. The estimates were 7.6 and 7.8×10(-7) in adult-type mouse AChRs (-100 mV at 23°C). The values are in excellent agreement with one obtained previously by using a completely different method (6.5×10(-7), from monoliganded gating). E0 decreases with depolarization to the same extent as does the diliganded gating equilibrium constant, e-fold with ∼60 mV. We estimate that at -100 mV the intrinsic energy of the unliganded gating isomerization is +8.4 kcal/mol (35 kJ/mol), and that in the absence of a membrane potential, the intrinsic chemical energy of this global conformational change is +9.4 kcal/mol (39 kJ/mol). Na+ and K+ in the extracellular solution have no measureable effect on E0, which suggests that unliganded gating occurs with only water occupying the transmitter binding sites. The results are discussed with regard to the energy changes in receptor activation and the competitive antagonism of ions in agonist binding.

  15. Neurosteroids shift partial agonist activation of GABA(A) receptor channels from low- to high-efficacy gating patterns.

    PubMed

    Bianchi, Matt T; Macdonald, Robert L

    2003-11-26

    Although GABA activates synaptic (alphabetagamma) GABA(A) receptors with high efficacy, partial agonist activation of alphabetagamma isoforms and GABA activation of the primary extrasynaptic (alphabetadelta) GABA(A) receptors are limited to low-efficacy activity, characterized by minimal desensitization and brief openings. The unusual sensitivity of alphabetadelta receptor channels to neurosteroid modulation prompted investigation of whether this high sensitivity was dependent on the delta subunit or the low-efficacy channel function that it confers. We show that the isoform specificity (alphabetadelta > alphabetagamma) of neurosteroid modulation could be reversed by conditions that reversed isoform-specific activity modes, including the use of beta-alanine to achieve increased efficacy with alphabetadelta receptors and taurine to render alphabetagamma receptors low efficacy. We suggest that neurosteroids preferentially enhance low-efficacy GABA(A) receptor activity independent of subunit composition. Allosteric conversion of partial to full agonism may be a general mechanism for reversibly scaling the efficacy of GABA(A) receptors to endogenous partial agonists.

  16. Transient Receptor Potential Ankyrin 1 Channel Involved in Atherosclerosis and Macrophage-Foam Cell Formation

    PubMed Central

    Zhao, Jin-Feng; Shyue, Song-Kun; Kou, Yu Ru; Lu, Tse-Min; Lee, Tzong-Shyuan

    2016-01-01

    Transient receptor potential ankyrin 1 channel (TRPA1) plays an important role in the pathogenesis of inflammatory diseases, yet its role and the underlying mechanism in atherosclerosis remain unclear. We aimed to investigate the role of TRPA1 in atherosclerosis and foam-cell formation in vivo in mice and in vitro in mouse macrophages. Histopathology was examined by hematoxylin and eosin staining, levels of cytokines and lipid profile were evaluated by assay kits, and protein expression was determined by western blot analysis. TRPA1 expression was increased in macrophage foam cells in atherosclerotic aortas of apolipoprotein E-deficient (apoE-/-) mice. Atherosclerotic lesions, hyperlipidemia and systemic inflammation were worsened with chronic administration of the TRPA1 channel antagonist HC030031 or genetic ablation of TRPA1 (TRPA1-/-) in apoE-/- mice. Treatment with allyl isothiocyanate (AITC, a TRPA1 agonist) retarded the progression of atherosclerosis in apoE-/- mice but not apoE-/-TRPA1-/- mice. Mouse macrophages showed oxidized low-density lipoprotein (oxLDL) activated TRPA1 channels. OxLDL-induced lipid accumulation of macrophages was exacerbated by HC030031 or loss of function of TRPA1. Inhibition of TRPA1 activity did not alter oxLDL internalization but impaired cholesterol efflux by downregulating the ATP-binding cassette transporters. Furthermore, tumor necrosis factor-α-induced inflammatory response was attenuated in AITC-activated macrophages. TRPA1 may be a pivotal regulator in the pathogenesis of atherosclerosis and cholesterol metabolism of macrophage foam cells. PMID:27313495

  17. A new cytoplasmic interaction between junctin and ryanodine receptor Ca2+ release channels.

    PubMed

    Li, Linwei; Mirza, Shamaruh; Richardson, Spencer J; Gallant, Esther M; Thekkedam, Chris; Pace, Suzy M; Zorzato, Francesco; Liu, Dan; Beard, Nicole A; Dulhunty, Angela F

    2015-03-01

    Junctin, a non-catalytic splice variant encoded by the aspartate-β-hydroxylase (Asph) gene, is inserted into the membrane of the sarcoplasmic reticulum (SR) Ca(2+) store where it modifies Ca(2+) signalling in the heart and skeletal muscle through its regulation of ryanodine receptor (RyR) Ca(2+) release channels. Junctin is required for normal muscle function as its knockout leads to abnormal Ca(2+) signalling, muscle dysfunction and cardiac arrhythmia. However, the nature of the molecular interaction between junctin and RyRs is largely unknown and was assumed to occur only in the SR lumen. We find that there is substantial binding of RyRs to full junctin, and the junctin luminal and, unexpectedly, cytoplasmic domains. Binding of these different junctin domains had distinct effects on RyR1 and RyR2 activity: full junctin in the luminal solution increased RyR channel activity by ∼threefold, the C-terminal luminal interaction inhibited RyR channel activity by ∼50%, and the N-terminal cytoplasmic binding produced an ∼fivefold increase in RyR activity. The cytoplasmic interaction between junctin and RyR is required for luminal binding to replicate the influence of full junctin on RyR1 and RyR2 activity. The C-terminal domain of junctin binds to residues including the S1-S2 linker of RyR1 and N-terminal domain of junctin binds between RyR1 residues 1078 and 2156. PMID:25609705

  18. Structural Studies of Inositol 1,4,5-Trisphosphate Receptor COUPLING LIGAND BINDING TO CHANNEL GATING

    SciTech Connect

    Chan, Jenny; Yamazaki, Haruka; Ishiyama, Noboru; Seo, Min-Duk; Mal, Tapas K.; Michikawa, Takayuki; Mikoshiba, Katsuhiko; Ikura, Mitsuhiko

    2010-11-22

    The three isoforms of the inositol 1,4,5-trisphosphate receptor (IP{sub 3}R) exhibit distinct IP{sub 3} sensitivities and cooperativities in calcium (Ca{sup 2+}) channel function. The determinants underlying this isoform-specific channel gating mechanism have been localized to the N-terminal suppressor region of IP3R. We determined the 1.9 {angstrom} crystal structure of the suppressor domain from type 3 IP{sub 3}R (IP{sub 3}R3{sub SUP}, amino acids 1-224) and revealed structural features contributing to isoform-specific functionality of IP{sub 3}R by comparing it with our previously determined structure of the type 1 suppressor domain (IP{sub 3}R1{sub SUP}). The molecular surface known to associate with the ligand binding domain (amino acids 224-604) showed marked differences between IP{sub 3}R3{sub SUP} and IP{sub 3}R1{sub SUP}. Our NMR and biochemical studies showed that three spatially clustered residues (Glu-20, Tyr-167, and Ser-217 in IP{sub 3}R1 and Glu-19, Trp-168, and Ser-218 in IP{sub 3}R3) within the N-terminal suppressor domains of IP{sub 3}R1{sub SUP} and IP{sub 3}R3{sub SUP} interact directly with their respective C-terminal fragments. Together with the accompanying paper (Yamazaki, H., Chan, J., Ikura, M., Michikawa, T., and Mikoshiba, K. (2010) J. Biol. Chem. 285, 36081-36091), we demonstrate that the single aromatic residue in this region (Tyr-167 in IP{sub 3}R1 and Trp-168 in IP{sub 3}R3) plays a critical role in the coupling between ligand binding and channel gating.

  19. Thermosensitive transient receptor potential channels (thermo-TRPs) in human corneal epithelial cells

    PubMed Central

    Mergler, Stefan; Garreis, Fabian; Sahlmüller, Monika; Reinach, Peter S.; Paulsen, Friedrich; Pleyer, Uwe

    2010-01-01

    Thermosensitive transient receptor potential proteins (TRPs) such as TRPV1-TRPV4 are all heat-activated non-selective cation channels that are modestly permeable to Ca2+. TRPV1, TRPV3 and TRPV4 functional expression were previously identified in human corneal epithelial cells (HCEC). However, the membrane currents were not described underlying their activation by either selective agonists or thermal variation. This study characterized the membrane currents and [Ca 2+]i transients induced by thermal and agonist TRPV1 and 4 stimulation. TRPV1 and 4 expressions were confirmed by RT-PCR and TRPV2 transcripts were also detected. In fura2-loaded HCEC, a TRPV1-3 selective agonist, 100 µM 2-aminoethoxydiphenyl borate (2-APB), induced intracellular Ca2+ transients and an increase in non-selective cation outward currents that were suppressed by ruthenium-red (RuR) (10–20 µM), a nonselective TRPV channel blocker. These changes were also elicited by rises in ambient temperature from 25 °C to over 40 °C. RuR (5 µM) and a selective TRPV1 channel blocker capsazepine (CPZ) (10 µM) or another related blocker, lanthanum chloride (La3+) (100 µM) suppressed these temperature-induced Ca2+ increases. Planar patch-clamp technique was used to characterize the currents underlying Ca2+ transients. Increasing the temperature to over 40 °C induced reversible rises in non-selective cation currents. Moreover, a hypotonic challenge (25 %) increased non-selective cation currents confirming TRPV4 activity. We conclude that HCEC possess in addition to thermo-sensitive TRPV3 activity TRPV1, TRPV2 and TRPV4 activity. Their activation confers temperature sensitivity at the ocular surface, which may protect the cornea against such stress. PMID:21506114

  20. A new cytoplasmic interaction between junctin and ryanodine receptor Ca2+ release channels

    PubMed Central

    Li, Linwei; Mirza, Shamaruh; Richardson, Spencer J.; Gallant, Esther M.; Thekkedam, Chris; Pace, Suzy M.; Zorzato, Francesco; Liu, Dan; Beard, Nicole A.; Dulhunty, Angela F.

    2015-01-01

    ABSTRACT Junctin, a non-catalytic splice variant encoded by the aspartate-β-hydroxylase (Asph) gene, is inserted into the membrane of the sarcoplasmic reticulum (SR) Ca2+ store where it modifies Ca2+ signalling in the heart and skeletal muscle through its regulation of ryanodine receptor (RyR) Ca2+ release channels. Junctin is required for normal muscle function as its knockout leads to abnormal Ca2+ signalling, muscle dysfunction and cardiac arrhythmia. However, the nature of the molecular interaction between junctin and RyRs is largely unknown and was assumed to occur only in the SR lumen. We find that there is substantial binding of RyRs to full junctin, and the junctin luminal and, unexpectedly, cytoplasmic domains. Binding of these different junctin domains had distinct effects on RyR1 and RyR2 activity: full junctin in the luminal solution increased RyR channel activity by ∼threefold, the C-terminal luminal interaction inhibited RyR channel activity by ∼50%, and the N-terminal cytoplasmic binding produced an ∼fivefold increase in RyR activity. The cytoplasmic interaction between junctin and RyR is required for luminal binding to replicate the influence of full junctin on RyR1 and RyR2 activity. The C-terminal domain of junctin binds to residues including the S1–S2 linker of RyR1 and N-terminal domain of junctin binds between RyR1 residues 1078 and 2156. PMID:25609705

  1. Propacetamol-Induced Injection Pain Is Associated with Activation of Transient Receptor Potential Vanilloid 1 Channels.

    PubMed

    Schillers, Florian; Eberhardt, Esther; Leffler, Andreas; Eberhardt, Mirjam

    2016-10-01

    Propacetamol (PPCM) is a prodrug of paracetamol (PCM), which was generated to increase water solubility of PCM for intravenous delivery. PPCM is rapidly hydrolyzed by plasma esterases to PCM and diethylglycine and shares some structural and metabolic properties with lidocaine. Although PPCM is considered to be comparable to PCM regarding its analgesic properties, injection pain is a common side effect described for PPCM but not PCM. Injection pain is a frequent and unpleasant side effect of numerous drugs in clinical use, and previous reports have indicated that the ligand gated ion channels transient receptor potential ankyrin 1 (TRPA1) and transient receptor potential vanilloid 1 (TRPV1) can mediate this effect on sensory neurons. This study aimed to investigate molecular mechanisms by which PPCM, in contrast to PCM, causes injection pain. Therefore, human TRPV1 and TRPA1 receptors were expressed in human embryonic kidney 293 cells and investigated by means of whole-cell patch clamp and ratiometric calcium imaging. PPCM (but not PCM) activated TRPV1, sensitized heat-induced currents, and caused an increase in intracellular calcium. In TRPA1-expressing cells however, both PPCM and PCM evoked calcium responses but failed to induce inward currents. Intracutaneous injection of PPCM, but not of PCM, in human volunteers induced an intense and short-lasting pain and an increase in superficial blood flow, indicating activation of nociceptive C fibers and subsequent neuropeptide release. In conclusion, activation of human TRPV1 by PPCM seems to be a relevant mechanism for induction of pain upon intracutaneous injection and thus also for pain reported as an adverse side effect upon intravenous administration. PMID:27457427

  2. Propacetamol-Induced Injection Pain Is Associated with Activation of Transient Receptor Potential Vanilloid 1 Channels.

    PubMed

    Schillers, Florian; Eberhardt, Esther; Leffler, Andreas; Eberhardt, Mirjam

    2016-10-01

    Propacetamol (PPCM) is a prodrug of paracetamol (PCM), which was generated to increase water solubility of PCM for intravenous delivery. PPCM is rapidly hydrolyzed by plasma esterases to PCM and diethylglycine and shares some structural and metabolic properties with lidocaine. Although PPCM is considered to be comparable to PCM regarding its analgesic properties, injection pain is a common side effect described for PPCM but not PCM. Injection pain is a frequent and unpleasant side effect of numerous drugs in clinical use, and previous reports have indicated that the ligand gated ion channels transient receptor potential ankyrin 1 (TRPA1) and transient receptor potential vanilloid 1 (TRPV1) can mediate this effect on sensory neurons. This study aimed to investigate molecular mechanisms by which PPCM, in contrast to PCM, causes injection pain. Therefore, human TRPV1 and TRPA1 receptors were expressed in human embryonic kidney 293 cells and investigated by means of whole-cell patch clamp and ratiometric calcium imaging. PPCM (but not PCM) activated TRPV1, sensitized heat-induced currents, and caused an increase in intracellular calcium. In TRPA1-expressing cells however, both PPCM and PCM evoked calcium responses but failed to induce inward currents. Intracutaneous injection of PPCM, but not of PCM, in human volunteers induced an intense and short-lasting pain and an increase in superficial blood flow, indicating activation of nociceptive C fibers and subsequent neuropeptide release. In conclusion, activation of human TRPV1 by PPCM seems to be a relevant mechanism for induction of pain upon intracutaneous injection and thus also for pain reported as an adverse side effect upon intravenous administration.

  3. Stable expression of a functional GluR6 homomeric glutamate receptor channel in mammalian cells.

    PubMed Central

    Tygesen, C K; Rasmussen, J S; Jones, S V; Hansen, A; Hansen, K; Andersen, P H

    1994-01-01

    This study demonstrates the stable expression of a functional ionotropic glutamate receptor in a mammalian cell line of non-neuronal origin. The kainate-selective glutamate receptor GluR6 was constitutively expressed under the control of a metallothionein promoter. Clones were isolated expressing approximately 3 pmol of receptor per mg of protein. Functionality of the recombinant GluR6 was demonstrated both by electrophysiology and by Ca2+ imaging. Application of kainate to the GluR6-transfected cells activated an inward current response at a holding potential of -60 mV. The kainate concentration needed to evoke 50% of the maximal response (EC50) was calculated to be 0.82 +/- 0.39 microM. The current-voltage relationship was found to be almost linear, with a reversal potential of -2.5 +/- 4.8 mV. Application of kainate also resulted in an increase in the intracellular Ca2+ concentration measured by Ca2+ imaging. The pharmacological profile of [3H]kainate binding to the recombinant GluR6 resembled the high-affinity [3H]kainate binding sites in rat brain, showing high affinity for domoate (Ki = 5.1 +/- 3.0 nM) and kainate (Kd = 12.9 +/- 2.4 nM). No decrease in GluR6 expression level was observed over > 75 passages of the transfected cells. When domoate, a slowly desensitizing GluR6 agonist, was included in the growth medium for 3 weeks, the number of GluR6 binding sites decreased by 30%, indicating the importance of complete channel closure for stable expression. Images Fig. 1 Fig. 4 PMID:7528929

  4. Drosophila Photoreceptor Cells Exploited for the Production of Eukaryotic Membrane Proteins: Receptors, Transporters and Channels

    PubMed Central

    Panneels, Valérie; Kock, Ines; Krijnse-Locker, Jacomine; Rezgaoui, Meriem; Sinning, Irmgard

    2011-01-01

    Background Membrane proteins (MPs) play key roles in signal transduction. However, understanding their function at a molecular level is mostly hampered by the lack of protein in suitable amount and quality. Despite impressive developments in the expression of prokaryotic MPs, eukaryotic MP production has lagged behind and there is a need for new expression strategies. In a pilot study, we produced a Drosophila glutamate receptor specifically in the eyes of transgenic flies, exploiting the naturally abundant membrane stacks in the photoreceptor cells (PRCs). Now we address the question whether the PRCs also process different classes of medically relevant target MPs which were so far notoriously difficult to handle with conventional expression strategies. Principal Findings We describe the homologous and heterologous expression of 10 different targets from the three major MP classes - G protein-coupled receptors (GPCRs), transporters and channels in Drosophila eyes. PRCs offered an extraordinary capacity to produce, fold and accommodate massive amounts of MPs. The expression of some MPs reached similar levels as the endogenous rhodopsin, indicating that the PRC membranes were almost unsaturable. Expression of endogenous rhodopsin was not affected by the target MPs and both could coexist in the membrane stacks. Heterologous expression levels reached about 270 to 500 pmol/mg total MP, resulting in 0.2–0.4 mg purified target MP from 1 g of fly heads. The metabotropic glutamate receptor and human serotonin transporter - both involved in synaptic transmission - showed native pharmacological characteristics and could be purified to homogeneity as a prerequisite for further studies. Significance We demonstrate expression in Drosophila PRCs as an efficient and inexpensive tool for the large scale production of functional eukaryotic MPs. The fly eye system offers a number of advantages over conventional expression systems and paves the way for in-depth analyses of

  5. A leukocyte immune-type receptor (LITR) subset is a marker of antiviral cytotoxic cells in channel catfish, Ictalurus punctatus

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Channel catfish, Ictalurus punctatus, leukocyte immune type-receptors (LITRs) represent a multigene family that encodes immunoglobulin superfamily proteins that mediate activating or inhibitory signaling. Here we demonstrate the utility of mAb CC41 to monitor viral cytotoxic responses in catfish an...

  6. Single-channel recording of inositol trisphosphate receptor in the isolated nucleus of a muscle cell line.

    PubMed

    Kusnier, Carlos; Cárdenas, César; Hidalgo, Jorge; Jaimovich, Enrique

    2006-01-01

    Nuclear calcium appears to have an important role in the regulation of gene expression in many cells, but the mechanisms involved in controlling nuclear Ca2+ signaling are controversial and still poorly understood. We have described the presence of inositol 1,4,5 trisphosphate (IP3) receptors in the nuclei of skeletal muscle cells. Now, we have characterized the properties of the IP3 receptors channels present in the nuclei of the 1B5 cell line, which do not express any isoforms of the ryanodine receptor. Immunocytochemistry of isolated nuclei confirmed the presence of IP3R in the nuclear envelope and fluorescence measurements in nuclei suspensions allowed us to document ATP-dependent calcium loading by the nucleus and its release upon IP3 addition. Patch clamp of nuclear membranes was performed, and single-channel activity recorded was dependent on the presence of IP3 in the pipette; single-channel conductance was in the range reported in the literature for these channels, and the open probability was shown to be dependent on IP3 concentration. The presence of functional IP3 receptors in the nuclear envelope membrane is likely to have an important function in the regulation of nucleoplasmic calcium concentration and consequently in the regulation of transcription in muscle cells. PMID:17106585

  7. Channel catfish (Ictalurus punctatus) leukocytes express estrogen receptor isoforms ERα and ERβ2 and are functionally modulated by estrogens.

    PubMed

    Iwanowicz, Luke R; Stafford, James L; Patiño, Reynaldo; Bengten, Eva; Miller, Norman W; Blazer, Vicki S

    2014-09-01

    Estrogens are recognized as modulators of immune responses in mammals and teleosts. While it is known that the effects of estrogens are mediated via leukocyte-specific estrogen receptors (ERs) in humans and mice, leucocyte-specific estrogen receptor expression and the effects of estrogens on this cell population is less explored and poorly understood in teleosts. Here in, we verify that channel catfish (Ictalurus punctaus) leukocytes express ERα and ERβ2. Transcripts of these isoforms were detected in tissue-associated leukocyte populations by PCR, but ERβ2 was rarely detected in PBLs. Expression of these receptors was temporally regulated in PBLs following polyclonal activation by concanavalin A, lipopolysaccharide or alloantigen based on evaluation by quantitative and end-point PCR. Examination of long-term leukocyte cell lines demonstrated that these receptors are differentially expressed depending on leukocyte lineage and phenotype. Expression of ERs was also temporally dynamic in some leukocyte lineages and may reflect stage of cell maturity. Estrogens affect the responsiveness of channel catfish peripheral blood leukocytes (PBLs) to mitogens in vitro. Similarly, bactericidal activity and phorbol 12-myristate 13-acetate induced respiratory burst was modulated by 17β-estradiol. These actions were blocked by the pure ER antagonist ICI 182780 indicating that response is, in part, mediated via ERα. In summary, estrogen receptors are expressed in channel catfish leukocytes and participate in the regulation of the immune response. This is the first time leukocyte lineage expression has been reported in teleost cell lines.

  8. Channel catfish (Ictalurus punctatus) leukocytes express estrogen receptor isoforms ERα and ERβ2 and are functionally modulated by estrogens

    USGS Publications Warehouse

    Iwanowicz, Luke R.; Stafford, James L.; Patiño, Reynaldo; Bengten, Eva; Miller, Norman W.; Blazer, Vicki

    2014-01-01

    Estrogens are recognized as modulators of immune responses in mammals and teleosts. While it is known that the effects of estrogens are mediated via leukocyte-specific estrogen receptors (ERs) in humans and mice, leucocyte-specific estrogen receptor expression and the effects of estrogens on this cell population is less explored and poorly understood in teleosts. Here in, we verify that channel catfish (Ictalurus punctaus) leukocytes express ERα and ERβ2. Transcripts of these isoforms were detected in tissue-associated leukocyte populations by PCR, but ERβ2 was rarely detected in PBLs. Expression of these receptors was temporally regulated in PBLs following polyclonal activation by concanavalin A, lipopolysaccharide or alloantigen based on evaluation by quantitative and end-point PCR. Examination of long-term leukocyte cell lines demonstrated that these receptors are differentially expressed depending on leukocyte lineage and phenotype. Expression of ERs was also temporally dynamic in some leukocyte lineages and may reflect stage of cell maturity. Estrogens affect the responsiveness of channel catfish peripheral blood leukocytes (PBLs) to mitogens in vitro. Similarly, bactericidal activity and phorbol 12-myristate 13-acetate induced respiratory burst was modulated by 17β-estradiol. These actions were blocked by the pure ER antagonist ICI 182780 indicating that response is, in part, mediated via ERα. In summary, estrogen receptors are expressed in channel catfish leukocytes and participate in the regulation of the immune response. This is the first time leukocyte lineage expression has been reported in teleost cell lines.

  9. Ion-channel-coupled receptor-based platform for a real-time measurement of G-protein-coupled receptor activities.

    PubMed

    Lim, Jong Hyun; Oh, Eun Hae; Park, Juhun; Hong, Seunghun; Park, Tai Hyun

    2015-02-24

    A simple but efficient measurement platform based on ion-channel-coupled receptors and nanovesicles was developed for monitoring the real-time activity of G-protein-coupled receptors (GPCRs). In this work, an olfactory receptor (OR), the most common class A GPCR, was covalently fused with a Kir6.2 channel so that the GPCR action directly induced the opening of the ion channels and changes in the electrical membrane potential without complex cellular signaling processes. This strategy reduced the measurement errors caused by instability of various cellular components. In addition, rather than using whole cells, a cell-surface-derived nanovesicle was used to preserve the membrane-integrated structure of GPCRs and to exclude case-dependent cellular conditions. Another merit of using the nanovesicle is that nanovesicles can be easily combined with nanomaterial-based field-effect transistors (FETs) to build a sensitive and stable measurement platform to monitor GPCR activities with high sensitivity in real-time. Using a platform based on carbon nanotube FETs and nanovesicles carrying Kir6.2-channel-coupled ORs, we monitored the real-time response of ORs to their ligand molecules. Significantly, since this platform does not rely on rather unstable cell signaling pathways, our platform could be utilized for a rather long time period without losing its functionality. This system can be utilized extensively for simple and sensitive analysis of the activities of various GPCRs and should enable various academic and practical applications.

  10. G protein-coupled receptor signaling via Src kinase induces endogenous human transient receptor potential vanilloid type 6 (TRPV6) channel activation.

    PubMed

    Spehr, Jennifer; Gelis, Lian; Osterloh, Markus; Oberland, Sonja; Hatt, Hanns; Spehr, Marc; Neuhaus, Eva M

    2011-04-15

    Ca(2+) homeostasis plays a critical role in a variety of cellular processes. We showed previously that stimulation of the prostate-specific G protein-coupled receptor (PSGR) enhances cytosolic Ca(2+) and inhibits proliferation of prostate cells. Here, we analyzed the signaling mechanisms underlying the PSGR-mediated Ca(2+) increase. Using complementary molecular, biochemical, electrophysiological, and live-cell imaging techniques, we found that endogenous Ca(2+)-selective transient receptor potential vanilloid type 6 (TRPV6) channels are critically involved in the PSGR-induced Ca(2+) signal. Biophysical characterization of the current activated by PSGR stimulation revealed characteristic properties of TRPV6. The molecular identity of the involved channel was confirmed using RNA interference targeting TrpV6. TRPV6-mediated Ca(2+) influx depended on Src kinase activity. Src kinase activation occurred independently of G protein activation, presumably by direct interaction with PSGR. Taken together, we report that endogenous TRPV6 channels are activated downstream of a G protein-coupled receptor and present the first physiological characterization of these channels in situ. PMID:21349844

  11. The Roles of Rasd1 small G proteins and leptin in the activation of TRPC4 transient receptor potential channels

    PubMed Central

    Wie, Jinhong; Kim, Byung Joo; Myeong, Jongyun; Ha, Kotdaji; Jeong, Seung Joo; Yang, Dongki; Kim, Euiyong; Jeon, Ju-Hong; So, Insuk

    2015-01-01

    TRPC4 is important regulators of electrical excitability in gastrointestinal myocytes, pancreatic β-cells and neurons. Much is known regarding the assembly and function of these channels including TRPC1 as a homotetramer or a heteromultimer and the roles that their interacting proteins play in controlling these events. Further, they are one of the best-studied targets of G protein-coupled receptors and growth factors in general and Gαi/o and Gαq protein coupled receptor or epidermal growth factor and leptin in particular. However, our understanding of the roles of small G proteins and leptin on TRPC4 channels is still rudimentary. We discuss potential roles for Rasd1 small G protein and leptin in channel activation in addition to their known role in cellular signaling. PMID:26083271

  12. Phytochemicals from Ruta graveolens Activate TAS2R Bitter Taste Receptors and TRP Channels Involved in Gustation and Nociception.

    PubMed

    Mancuso, Giuseppe; Borgonovo, Gigliola; Scaglioni, Leonardo; Bassoli, Angela

    2015-10-16

    Ruta graveolens (rue) is a spontaneous plant in the Mediterranean area with a strong aroma and a very intense bitter taste, used in gastronomy and in folk medicine. From the leaves, stems and fruits of rue, we isolated rutin, rutamarin, three furanocoumarins, two quinolinic alkaloids, a dicoumarin and two long chain ketones. Bitter taste and chemesthetic properties have been evaluated by in vitro assays with twenty receptors of the TAS2R family and four TRP ion channels involved in gustation and nociception. Among the alkaloids, skimmianine was active as a specific agonist of T2R14, whereas kokusaginin did not activate any of the tested receptors. The furanocoumarins activates TAS2R10, 14, and 49 with different degrees of selectivity, as well as the TRPA1 somatosensory ion channel. Rutamarin is an agonist of TRPM5 and TRPV1 and a strong antagonist of TRPM8 ion channels.

  13. Phytochemicals from Ruta graveolens Activate TAS2R Bitter Taste Receptors and TRP Channels Involved in Gustation and Nociception.

    PubMed

    Mancuso, Giuseppe; Borgonovo, Gigliola; Scaglioni, Leonardo; Bassoli, Angela

    2015-01-01

    Ruta graveolens (rue) is a spontaneous plant in the Mediterranean area with a strong aroma and a very intense bitter taste, used in gastronomy and in folk medicine. From the leaves, stems and fruits of rue, we isolated rutin, rutamarin, three furanocoumarins, two quinolinic alkaloids, a dicoumarin and two long chain ketones. Bitter taste and chemesthetic properties have been evaluated by in vitro assays with twenty receptors of the TAS2R family and four TRP ion channels involved in gustation and nociception. Among the alkaloids, skimmianine was active as a specific agonist of T2R14, whereas kokusaginin did not activate any of the tested receptors. The furanocoumarins activates TAS2R10, 14, and 49 with different degrees of selectivity, as well as the TRPA1 somatosensory ion channel. Rutamarin is an agonist of TRPM5 and TRPV1 and a strong antagonist of TRPM8 ion channels. PMID:26501253

  14. Regulation of transient receptor potential channels of melastatin type 8 (TRPM8): effect of cAMP, cannabinoid CB(1) receptors and endovanilloids.

    PubMed

    De Petrocellis, Luciano; Starowicz, Katarzyna; Moriello, Aniello Schiano; Vivese, Marta; Orlando, Pierangelo; Di Marzo, Vincenzo

    2007-05-15

    The transient receptor potential channel of melastatin type 8 (TRPM8), which is gated by low (<25 degrees C) temperature and chemical compounds, is regulated by protein kinase C-mediated phosphorylation in a way opposite to that observed with the transient receptor potential channel of vanilloid type 1 (TRPV1), i.e. by being desensitized and not sensitized. As TRPV1 is sensitized also by protein kinase A (PKA)-mediated phosphorylation, we investigated the effect of two activators of the PKA pathway, 8-Br-cAMP and forskolin, on the activity of menthol and icilin at TRPM8 in HEK-293 cells stably overexpressing the channel (TRPM8-HEK-293 cells). We also studied the effect on TRPM8 of: (1) a series of compounds previously shown to activate or antagonize TRPV1, and (2) co-stimulation of transiently co-expressed cannabinoid CB(1) receptors. Both 8-Br-cAMP (100 microM) and forskolin (10 microM) right-shifted the dose-response curves for the TRPM8-mediated effect of icilin and menthol on intracellular Ca(2+). The inhibitory effects of 8-Br-cAMP and forskolin were attenuated by the selective PKA inhibitor Rp-cAMP-S. Stimulation of human CB(1) receptors transiently co-expressed in TRPM8-HEK-293 cells also inhibited TRPM8 response to icilin. Finally, some TRPV1 agonists and antagonists, but not iodinated antagonists, antagonized icilin- and much less so menthol-, induced TRPM8 activation. Importantly, the endovanilloids/endocannabinoids, anandamide and NADA, also antagonized TRPM8 at submicromolar concentrations. Although these findings need to be confirmed by experiments directly measuring TRPM8 activity in natively TRPM8-expressing cells, they support the notion that the same regulatory events have opposing actions on TRPM8 and TRPV1 receptors and identify anandamide and NADA as the first potential endogenous functional antagonists of TRPM8 channels.

  15. Menthol derivative WS-12 selectively activates transient receptor potential melastatin-8 (TRPM8) ion channels.

    PubMed

    Ma, Sherkheli; G, Gisselmann; Ak, Vogt-Eisele; Jf, Doerner; H, Hatt

    2008-10-01

    Transient receptor potential melastatin-8 (TRPM8), a cationic ion channel is involved in detection of normal cooling-sensation in mammals. TRPM8 activation by cooling or chemical agonists have been shown to produce profound, mechanistically novel analgesia in chronic pain states such as neuropathic pain in rodents. Known TRPM8 agonists such as menthol and icilin have a relatively low potency and cross-activate nociceptors like TRPA1; thus bearing a limited therapeutic usefulness. For that reason, characterising ligands, which selectively activate TRPM8, presents a clinical need. Using Xenopus laevis oocytes as expression system, we evaluated WS-12, a menthol derivative, for its potential interaction with all six thermo-sensitive TRP ion channels. Oocytes were injected with cRNA of gene of interest and incubated for 3-5 days (at 16 degrees C) before testing for functional characterisation of the recombinant ion channels. Oocytes were superfused with the test and standard substances respectively. Responses were measured by two-electrode voltage clamp technique and the amplitudes of evoked currents were compared with baseline values. WS-12 robustly activated TRPM8 in low micromolar concentrations (EC50 12+/-5 microM) thereby displaying a higher potency and efficacy compared to menthol (EC50 196+/-22 microM). Any of the other described thermo-sensitive TRP ion channel including TRPV1, TRPV2, TRPV3, TRPV4 and TRPA1 were not activated at a concentration (1 mM) optimally effective for TRPM8 responses; a characteristic which is in sharp contrast to menthol as it activates TRPA1 and TRPV3 in addition to TRPM8. Unlike icilin (75% reduction; p<0.001, n=6), WS-12 does not induce tachyphylaxis (4+/-2.3% increase in responses; p<0.08, n=6) of TRPM8 mediated currents to repeated exposure of 1 mM doses. In addition, acidosis or variations in extracellular calcium have no influence on potency/efficacy of WS-12 for TRPM8. The selectivity profile of WS-12, its several-fold higher

  16. Heterologously-expressed and Liposome-reconstituted Human Transient Receptor Potential Melastatin 4 Channel (TRPM4) is a Functional Tetramer

    PubMed Central

    Constantine, Maryrose; Liew, Chu Kong; Lo, Victor; Macmillan, Alex; Cranfield, Charles G.; Sunde, Margaret; Whan, Renee; Graham, Robert M.; Martinac, Boris

    2016-01-01

    Mutation, irregular expression and sustained activation of the Transient Receptor Potential Channel, type Melastatin 4 (TRPM4), have been linked to various cardiovascular diseases. However, much remains unknown about the structure of this important ion channel. Here, we have purified a heterologously expressed TRPM4-eGFP fusion protein and investigated the oligomeric state of TRPM4-eGFP in detergent micelles using crosslinking, native gel electrophoresis, multi-angle laser light scattering and electron microscopy. Our data indicate that TRPM4 is tetrameric, like other TRP channels studied to date. Furthermore, the functionality of liposome reconstituted TRPM4-eGFP was examined using electrophysiology. Single-channel recordings from TRPM4-eGFP proteoliposomes showed inhibition of the channel using Flufenamic acid, a well-established inhibitor of TRPM4, suggesting that the channels are functional upon reconstitution. Our characterisation of the oligomeric structure of TRPM4 and the ability to reconstitute functional channels in liposomes should facilitate future studies into the structure, function and pharmacology of this therapeutically relevant channel. PMID:26785754

  17. Heterologously-expressed and Liposome-reconstituted Human Transient Receptor Potential Melastatin 4 Channel (TRPM4) is a Functional Tetramer.

    PubMed

    Constantine, Maryrose; Liew, Chu Kong; Lo, Victor; Macmillan, Alex; Cranfield, Charles G; Sunde, Margaret; Whan, Renee; Graham, Robert M; Martinac, Boris

    2016-01-01

    Mutation, irregular expression and sustained activation of the Transient Receptor Potential Channel, type Melastatin 4 (TRPM4), have been linked to various cardiovascular diseases. However, much remains unknown about the structure of this important ion channel. Here, we have purified a heterologously expressed TRPM4-eGFP fusion protein and investigated the oligomeric state of TRPM4-eGFP in detergent micelles using crosslinking, native gel electrophoresis, multi-angle laser light scattering and electron microscopy. Our data indicate that TRPM4 is tetrameric, like other TRP channels studied to date. Furthermore, the functionality of liposome reconstituted TRPM4-eGFP was examined using electrophysiology. Single-channel recordings from TRPM4-eGFP proteoliposomes showed inhibition of the channel using Flufenamic acid, a well-established inhibitor of TRPM4, suggesting that the channels are functional upon reconstitution. Our characterisation of the oligomeric structure of TRPM4 and the ability to reconstitute functional channels in liposomes should facilitate future studies into the structure, function and pharmacology of this therapeutically relevant channel. PMID:26785754

  18. Antagonism of ligand-gated ion channel receptors: two domains of the glycine receptor alpha subunit form the strychnine-binding site.

    PubMed Central

    Vandenberg, R J; French, C R; Barry, P H; Shine, J; Schofield, P R

    1992-01-01

    The inhibitory glycine receptor (GlyR) is a member of the ligand-gated ion channel receptor superfamily. Glycine activation of the receptor is antagonized by the convulsant alkaloid strychnine. Using in vitro mutagenesis and functional analysis of the cDNA encoding the alpha 1 subunit of the human GlyR, we have identified several amino acid residues that form the strychnine-binding site. These residues were identified by transient expression of mutated cDNAs in mammalian (293) cells and examination of resultant [3H]strychnine binding, glycine displacement of [3H]strychnine, and electrophysiological responses to the application of glycine and strychnine. This mutational analysis revealed that residues from two separate domains within the alpha 1 subunit form the binding site for the antagonist strychnine. The first domain includes the amino acid residues Gly-160 and Tyr-161, and the second domain includes the residues Lys-200 and Tyr-202. These results, combined with analyses of other ligand-gated ion channel receptors, suggest a conserved tertiary structure and a common mechanism for antagonism in this receptor superfamily. PMID:1311851

  19. Inter- and intrasubunit interactions between transmembrane helices in the open state of P2X receptor channels

    PubMed Central

    Heymann, Gabriel; Dai, Jian; Silberberg, Shai D.; Zhou, Huan-Xiang; Swartz, Kenton J.

    2013-01-01

    P2X receptor channels open in response to the binding of extracellular ATP, a property that is essential for purinergic sensory signaling. Apo and ATP-bound X-ray structures of the detergent-solubilized zebrafish P2X4 receptor provide a blueprint for receptor mechanisms but unexpectedly showed large crevices between subunits within the transmembrane (TM) domain of the ATP-bound structure. Here we investigate both intersubunit and intrasubunit interactions between TM helices of P2X receptors in membranes using both computational and functional approaches. Our results suggest that intersubunit crevices found in the TM domain of the ATP-bound crystal structure are not present in membrane-embedded receptors but substantiate helix interactions within individual subunits and identify a hot spot at the internal end of the pore where both the gating and permeation properties of P2X receptors can be tuned. We propose a model for the structure of the open state that has stabilizing intersubunit interactions and that is compatible with available structural constraints from functional channels in membrane environments. PMID:24082111

  20. Spinal transient receptor potential ankyrin 1 channel contributes to central pain hypersensitivity in various pathophysiological conditions in the rat.

    PubMed

    Wei, Hong; Koivisto, Ari; Saarnilehto, Marja; Chapman, Hugh; Kuokkanen, Katja; Hao, Bin; Huang, Jin-Lu; Wang, Yong-Xiang; Pertovaara, Antti

    2011-03-01

    The transient receptor potential ankyrin 1 (TRPA1) ion channel is expressed on nociceptive primary afferent neurons. On the proximal nerve ending within the spinal dorsal horn, TRPA1 regulates transmission to spinal interneurons, and thereby pain hypersensitivity. Here we assessed whether the contribution of the spinal TRPA1 channel to pain hypersensitivity varies with the experimental pain model, properties of test stimulation or the behavioral pain response. The antihypersensitivity effect of intrathecally (i.t.) administered Chembridge-5861528 (CHEM; a selective TRPA1 channel antagonist; 5-10μg) was determined in various experimental models of pain hypersensitivity in the rat. In spinal nerve ligation and rapid eye movement (REM) sleep deprivation models, i.t. CHEM attenuated mechanical hypersensitivity. Capsaicin-induced secondary (central) but not primary (peripheral) mechanical hypersensitivity was also reduced by i.t. administration of CHEM or A-967079, another TRPA1 channel antagonist. Formalin-induced secondary mechanical hypersensitivity, but not spontaneous pain, was suppressed by i.t. CHEM. Moreover, mechanical hypersensitivity induced by cholekystokinin in the rostroventromedial medulla was attenuated by i.t. pretreatment with CHEM. Independent of the model, the antihypersensitivity effect induced by i.t. CHEM was predominant on responses evoked by low-intensity stimuli (⩽6g). CHEM (10μg i.t.) failed to attenuate pain behavior in healthy controls or mechanical hypersensitivities induced by i.t. administrations of a GABA(A) receptor antagonist, or NMDA or 5-HT(3) receptor agonists. Conversely, i.t. administration of a TRPA1 channel agonist, cinnamon aldehyde, induced mechanical hypersensitivity. The results indicate that the spinal TRPA1 channel exerts an important role in secondary (central) pain hypersensitivity to low-intensity mechanical stimulation in various pain hypersensitivity conditions. The spinal TRPA1 channel provides a promising target

  1. Use of Label-free Optical Biosensors to Detect Modulation of Potassium Channels by G-protein Coupled Receptors

    PubMed Central

    Fleming, Matthew R.; Shamah, Steven M.; Kaczmarek, Leonard K.

    2014-01-01

    Ion channels control the electrical properties of neurons and other excitable cell types by selectively allowing ions to flow through the plasma membrane1. To regulate neuronal excitability, the biophysical properties of ion channels are modified by signaling proteins and molecules, which often bind to the channels themselves to form a heteromeric channel complex2,3. Traditional assays examining the interaction between channels and regulatory proteins require exogenous labels that can potentially alter the protein's behavior and decrease the physiological relevance of the target, while providing little information on the time course of interactions in living cells. Optical biosensors, such as the X-BODY Biosciences BIND Scanner system, use a novel label-free technology, resonance wavelength grating (RWG) optical biosensors, to detect changes in resonant reflected light near the biosensor. This assay allows the detection of the relative change in mass within the bottom portion of living cells adherent to the biosensor surface resulting from ligand induced changes in cell adhesion and spreading, toxicity, proliferation, and changes in protein-protein interactions near the plasma membrane. RWG optical biosensors have been used to detect changes in mass near the plasma membrane of cells following activation of G protein-coupled receptors (GPCRs), receptor tyrosine kinases, and other cell surface receptors. Ligand-induced changes in ion channel-protein interactions can also be studied using this assay. In this paper, we will describe the experimental procedure used to detect the modulation of Slack-B sodium-activated potassium (KNa) channels by GPCRs. PMID:24562095

  2. C-terminal Domains of N-Methyl-d-aspartic Acid Receptor Modulate Unitary Channel Conductance and Gating*

    PubMed Central

    Maki, Bruce A.; Aman, Teresa K.; Amico-Ruvio, Stacy A.; Kussius, Cassandra L.; Popescu, Gabriela K.

    2012-01-01

    NMDA receptors (NRs) are glutamate-gated calcium-permeable channels that are essential for normal synaptic transmssion and contribute to neurodegeneration. Tetrameric proteins consist of two obligatory GluN1 (N1) and two GluN2 (N2) subunits, of which GluN2A (2A) and GluN2B (2B) are prevalent in adult brain. The intracellularly located C-terminal domains (CTDs) make a significant portion of mass of the receptors and are essential for plasticity and excitotoxicity, but their functions are incompletely defined. Recent evidence shows that truncation of the N2 CTD alters channel kinetics; however, the mechanism by which this occurs is unclear. Here we recorded activity from individual NRs lacking the CTDs of N1, 2A, or 2B and determined the gating mechanisms of these receptors. Receptors lacking the N1 CTDs had larger unitary conductance and faster deactivation kinetics, receptors lacking the 2A or 2B CTDs had longer openings and longer desensitized intervals, and the first 100 amino acids of the N2 CTD were essential for these changes. In addition, receptors lacking the CTDs of either 2A or 2B maintained isoform-specific kinetic differences and swapping CTDs between 2A and 2B had no effect on single-channel properties. Based on these results, we suggest that perturbations in the CTD can modify the NR-mediated signal in a subunit-dependent manner, in 2A these effects are most likely mediated by membrane-proximal residues, and the isoform-specific biophysical properties conferred by 2A and 2B are CTD-independent. The kinetic mechanisms we developed afford a quantitative approach to understanding how the intracellular domains of NR subunits can modulate the responses of the receptor. PMID:22948148

  3. Mode switching is the major mechanism of ligand regulation of InsP3 receptor calcium release channels.

    PubMed

    Ionescu, Lucian; White, Carl; Cheung, King-Ho; Shuai, Jianwei; Parker, Ian; Pearson, John E; Foskett, J Kevin; Mak, Don-On Daniel

    2007-12-01

    The inositol 1,4,5-trisphosphate (InsP(3)) receptor (InsP(3)R) plays a critical role in generation of complex Ca(2+) signals in many cell types. In patch clamp recordings of isolated nuclei from insect Sf9 cells, InsP(3)R channels were consistently detected with regulation by cytoplasmic InsP(3) and free Ca(2+) concentrations ([Ca(2+)](i)) very similar to that observed for vertebrate InsP(3)R. Long channel activity durations of the Sf9-InsP(3)R have now enabled identification of a novel aspect of InsP(3)R gating: modal gating. Using a novel algorithm to analyze channel modal gating kinetics, InsP(3)R gating can be separated into three distinct modes: a low activity mode, a fast kinetic mode, and a burst mode with channel open probability (P(o)) within each mode of 0.007 +/- 0.002, 0.24 +/- 0.03, and 0.85 +/- 0.02, respectively. Channels reside in each mode for long periods (tens of opening and closing events), and transitions between modes can be discerned with high resolution (within two channel opening and closing events). Remarkably, regulation of channel gating by [Ca(2+)](i) and [InsP(3)] does not substantially alter channel P(o) within a mode. Instead, [Ca(2+)](i) and [InsP(3)] affect overall channel P(o) primarily by changing the relative probability of the channel being in each mode, especially the high and low P(o) modes. This novel observation therefore reveals modal switching as the major mechanism of physiological regulation of InsP(3)R channel activity, with implications for the kinetics of Ca(2+) release events in cells.

  4. Yeast gain-of-function mutations reveal structure-function relationships conserved among different subfamilies of transient receptor potential channels.

    PubMed

    Su, Zhenwei; Zhou, Xinliang; Haynes, W John; Loukin, Stephen H; Anishkin, Andriy; Saimi, Yoshiro; Kung, Ching

    2007-12-01

    Transient receptor potential (TRP) channels found in animals, protists, and fungi are primary chemo-, thermo-, or mechanosensors. Current research emphasizes the characteristics of individual channels in each animal TRP subfamily but not the mechanisms common across subfamilies. A forward genetic screen of the TrpY1, the yeast TRP channel, recovered gain-of-function (GOF) mutations with phenotype in vivo and in vitro. Single-channel patch-clamp analyses of these GOF-mutant channels show prominent aberrations in open probability and channel kinetics. These mutations revealed functionally important aromatic amino acid residues in four locations: at the intracellular end of the fifth transmembrane helix (TM5), at both ends of TM6, and at the immediate extension of TM6. These aromatics have counterparts in most TRP subfamilies. The one in TM5 (F380L) aligns precisely with an exceptional Drosophila mutant allele (F550I) that causes constitutive activity in the canonical TRP channel, resulting in rapid and severe retinal degeneration beyond mere loss of phototaxis. Thus, this phenylalanine maintains the balance of various functional states (conformations) of a channel for insect phototransduction as well as one for fungal mechanotransduction. This residue is among a small cluster of phenylalanines found in all known subfamilies of TRP channels. This unique case illustrates that GOF mutations can reveal structure-function principles that can be generalized across different TRP subfamilies. It appears that the conserved aromatics in the four locations have conserved functions in most TRP channels. The possible mechanistic roles of these aromatics and the further use of yeast genetics to dissect TRP channels are discussed.

  5. Raloxifene inhibits cloned Kv4.3 channels in an estrogen receptor-independent manner.

    PubMed

    Chae, Yun Ju; Kim, Dae Hun; Lee, Hong Joon; Sung, Ki-Wug; Kwon, Oh-Joo; Hahn, Sang June

    2015-08-01

    Raloxifene is widely used for the treatment and prevention of postmenopausal osteoporosis. We examined the effects of raloxifene on the Kv4.3 currents expressed in Chinese hamster ovary (CHO) cells using the whole-cell patch-clamp technique and on the long-term modulation of Kv4.3 messenger RNA (mRNA) by real-time PCR analysis. Raloxifene decreased the Kv4.3 currents with an IC50 of 2.0 μM and accelerated the inactivation and activation kinetics in a concentration-dependent manner. The inhibitory effects of raloxifene on Kv4.3 were time-dependent: the association and dissociation rate constants for raloxifene were 9.5 μM(-1) s(-1) and 23.0 s(-1), respectively. The inhibition by raloxifene was voltage-dependent (δ = 0.13). Raloxifene shifted the steady-state inactivation curves in a hyperpolarizing direction and accelerated the closed-state inactivation of Kv4.3. Raloxifene slowed the time course of recovery from inactivation, thus producing a use-dependent inhibition of Kv4.3. β-Estradiol and tamoxifen had little effect on Kv4.3. A preincubation of ICI 182,780, an estrogen receptor antagonist, for 1 h had no effect on the inhibitory effect of raloxifene on Kv4.3. The metabolites of raloxifene, raloxifene-4'-glucuronide and raloxifene-6'-glucuronide, had little or no effect on Kv4.3. Coexpression of KChIP2 subunits did not alter the drug potency and steady-state inactivation of Kv4.3 channels. Long-term exposure to raloxifene (24 h) significantly decreased the expression level of Kv4.3 mRNA. This effect was not abolished by the coincubation with ICI 182,780. Raloxifene inhibited Kv4.3 channels by interacting with their open state during depolarization and with the closed state at subthreshold potentials. This effect was not mediated via an estrogen receptor. PMID:25231973

  6. Dopamine D1 receptor modulation of calcium channel currents in horizontal cells of mouse retina.

    PubMed

    Liu, Xue; Grove, James C R; Hirano, Arlene A; Brecha, Nicholas C; Barnes, Steven

    2016-08-01

    Horizontal cells form the first laterally interacting network of inhibitory interneurons in the retina. Dopamine released onto horizontal cells under photic and circadian control modulates horizontal cell function. Using isolated, identified horizontal cells from a connexin-57-iCre × ROSA26-tdTomato transgenic mouse line, we investigated dopaminergic modulation of calcium channel currents (ICa) with whole cell patch-clamp techniques. Dopamine (10 μM) blocked 27% of steady-state ICa, an action blunted to 9% in the presence of the L-type Ca channel blocker verapamil (50 μM). The dopamine type 1 receptor (D1R) agonist SKF38393 (20 μM) inhibited ICa by 24%. The D1R antagonist SCH23390 (20 μM) reduced dopamine and SKF38393 inhibition. Dopamine slowed ICa activation, blocking ICa by 38% early in a voltage step. Enhanced early inhibition of ICa was eliminated by applying voltage prepulses to +120 mV for 100 ms, increasing ICa by 31% and 11% for early and steady-state currents, respectively. Voltage-dependent facilitation of ICa and block of dopamine inhibition after preincubation with a Gβγ-blocking peptide suggested involvement of Gβγ proteins in the D1R-mediated modulation. When the G protein activator guanosine 5'-O-(3-thiotriphosphate) (GTPγS) was added intracellularly, ICa was smaller and showed the same slowed kinetics seen during D1R activation. With GTPγS in the pipette, additional block of ICa by dopamine was only 6%. Strong depolarizing voltage prepulses restored the GTPγS-reduced early ICa amplitude by 36% and steady-state ICa amplitude by 3%. These results suggest that dopaminergic inhibition of ICa via D1Rs is primarily mediated through the action of Gβγ proteins in horizontal cells. PMID:27193322

  7. Cloning, in Vitro expression, and novel phylogenetic classification of a channel catfish estrogen receptor

    USGS Publications Warehouse

    Xia, Z.; Patino, R.; Gale, W.L.; Maule, A.G.; Densmore, L.D.

    1999-01-01

    We obtained two channel catfish estrogen receptor (ccER) cDNA from liver of female fish using RT–PCR. The two fragments were identical in sequence except that the smaller one had an out-of-frame deletion in the E domain, suggesting the existence of ccER splice variants. The larger fragment was used to screen a cDNA library from liver of a prepubescent female. A cDNA was obtained that encoded a 581-amino-acid ER with a deduced molecular weight of 63.8 kDa. Extracts of COS-7 cells transfected with ccER cDNA bound estrogen with high affinity (Kd = 4.7 nM) and specificity. Maximum parsimony and Neighbor Joining analyses were used to generate a phylogenetic classification of ccER on the basis of 18 full-length ER sequences. The tree suggested the existence of two major ER branches. One branch contained two clearly divergent clades which included all piscine ER (except Japanese eel ER) and all tetrapod ERα, respectively. The second major branch contained the eel ER and the mammalian ERβ. The high degree of divergence between the eel ER and mammalian ERβ suggested that they also represent distinct piscine and tetrapod ER. These data suggest that ERα and ERβ are present throughout vertebrates and that these two major ER types evolved by duplication of an ancestral ER gene. Sequence alignments with other members of the nuclear hormone receptor superfamily indicated the presence of 8 amino acids in the E domain that align exclusively among ER. Four of these amino acids have not received prior research attention and their function is unknown. The novel finding of putative ER splice variants in a nonmammalian vertebrate and the novel phylogenetic classification of ER offer new perspectives in understanding the diversification and function of ER.

  8. Trapping of glutamate and glycine during open channel block of rat hippocampal neuron NMDA receptors by 9-aminoacridine.

    PubMed Central

    Benveniste, M; Mayer, M L

    1995-01-01

    1. N-methyl-D-aspartate (NMDA) receptor responses were recorded from rat hippocampal neurons grown in dissociated culture, using whole-cell, outside-out and nucleated patch recording techniques. Rapid perfusion was used to study voltage-dependent block of NMDA receptors by 9-aminoacridine (9-AA) and by Mg2+. 2. Large amplitude tail currents were evoked on depolarization to +60 mV after application at -100 mV of NMDA and 9-AA but not NMDA and Mg2+. These tail currents were resistant to block by competitive antagonists to the glutamate and glycine binding sites on NMDA receptors and were not evoked when either NMDA or 9-AA were applied alone. 3. The decay kinetics of the tail current were dependent on agonist affinity; the time required for 80% charge transfer was 10-fold briefer for NMDA than for glutamate and 7-fold briefer for L-alanine than for glycine. These results are in accord with a sequential model for block of NMDA receptors by 9-AA, in which neither glutamate nor glycine can dissociate from the open-blocked state of the receptor. 4. Tail current responses had amplitudes 2- to 4-fold larger than responses to maximally effective concentrations of glutamate and glycine, indicating that NMDA receptor channels accumulate in the open-blocked state during co-application of agonist and 9-AA. The rise time and decay kinetics of tail current responses were faster than the response to brief applications of a maximally effective concentration of glutamate. Together, these results suggest that at +60 mV recovery from block by 9-AA occurs faster than the rate of opening of NMDA receptors in response to glutamate. 5. Our experiments suggest that open channel block of NMDA receptors can provide a novel approach for measurement of both open probability and the first latency distribution for ion channel opening in response to the binding of agonists, and provide additional evidence suggesting that the delayed opening of NMDA receptor channels underlies slow activation and

  9. Heat Avoidance Is Regulated by Transient Receptor Potential (TRP) Channels and a Neuropeptide Signaling Pathway in Caenorhabditis elegans

    PubMed Central

    Glauser, Dominique A.; Chen, Will C.; Agin, Rebecca; MacInnis, Bronwyn L.; Hellman, Andrew B.; Garrity, Paul A.; Tan, Man-Wah; Goodman, Miriam B.

    2011-01-01

    The ability to avoid noxious extremes of hot and cold is critical for survival and depends on thermal nociception. The TRPV subset of transient receptor potential (TRP) channels is heat activated and proposed to be responsible for heat detection in vertebrates and fruit flies. To gain insight into the genetic and neural basis of thermal nociception, we developed assays that quantify noxious heat avoidance in the nematode Caenorhabditis elegans and used them to investigate the genetic basis of this behavior. First, we screened mutants for 18 TRP channel genes (including all TRPV orthologs) and found only minor defects in heat avoidance in single and selected double and triple mutants, indicating that other genes are involved. Next, we compared two wild isolates of C. elegans that diverge in their threshold for heat avoidance and linked this phenotypic variation to a polymorphism in the neuropeptide receptor gene npr-1. Further analysis revealed that loss of either the NPR-1 receptor or its ligand, FLP-21, increases the threshold for heat avoidance. Cell-specific rescue of npr-1 implicates the interneuron RMG in the circuit regulating heat avoidance. This neuropeptide signaling pathway operates independently of the TRPV genes, osm-9 and ocr-2, since mutants lacking npr-1 and both TRPV channels had more severe defects in heat avoidance than mutants lacking only npr-1 or both osm-9 and ocr-2. Our results show that TRPV channels and the FLP-21/NPR-1 neuropeptide signaling pathway determine the threshold for heat avoidance in C. elegans. PMID:21368276

  10. Properties of transient K+ currents and underlying single K+ channels in rat olfactory receptor neurons

    PubMed Central

    1991-01-01

    The transient potassium current, IK(t), of enzymatically dissociated rat olfactory receptor neurons was studied using patch-clamp techniques. Upon depolarization from negative holding potentials, IK(t) activated rapidly and then inactivated with a time course described by the sum of two exponential components with time constants of 22.4 and 143 ms. Single-channel analysis revealed a further small component with a time constant of several seconds. Steady-state inactivation was complete at -20 mV and completely removed at -80 mV (midpoint -45 mV). Activation was significant at -40 mV and appeared to reach a maximum conductance at +40 mV (midpoint -13 mV). Deactivation was described by the sum of two voltage-dependent exponential components. Recovery from inactivation was extraordinarily slow (50 s at -100 mV) and the underlying processes appeared complex. IK(t) was reduced by 4- aminopyridine and tetraethylammonium applied externally. Increasing the external K+ concentration ([K+]o) from 5 to 25 mM partially removed IK(t) inactivation, usually without affecting activation kinetics. The elevated [K+]o also hyperpolarized the steady-state inactivation curve by 9 mV and significantly depolarized the voltage dependence of activation. Single transient K+ channels, with conductances of 17 and 26 pS, were observed in excised patches and often appeared to be localized into large clusters. These channels were similar to IK(t) in their kinetic, pharmacological, and voltage-dependent properties and their inactivation was also subject to modulation by [K+]o. The properties of IK(t) imply a role in action potential repolarization and suggest it may also be important in modulating spike parameters during neuronal burst firing. A simple method is also presented to correct for errors in the measurement of whole-cell resistance (Ro) that can result when patch-clamping very small cells. The analysis revealed a mean corrected Ro of 26 G omega for these cells. PMID:1865174

  11. Novel transcripts of the estrogen receptor α gene in channel catfish

    USGS Publications Warehouse

    Patino, Reynaldo; Xia, Zhenfang; Gale, William L.; Wu, Chunfa; Maule, Alec G.; Chang, Xiaotian

    2000-01-01

    Complementary DNA libraries from liver and ovary of an immature female channel catfish were screened with a homologous ERα cDNA probe. The hepatic library yielded two new channel catfish ER cDNAs that encode N-terminal ERα variants of different sizes. Relative to the catfish ERα (medium size; 581 residues) previously reported, these new cDNAs encode Long-ERα (36 residues longer) and Short-ERα (389 residues shorter). The 5′-end of Long-ERα cDNA is identical to that of Medium-ERα but has an additional 503-bp segment with an upstream, in-frame translation-start codon. Recombinant Long-ERα binds estrogen with high affinity (Kd = 3.4 nM), similar to that previously reported for Medium-ERα but lower than reported for catfish ERβ. Short-ERα cDNA encodes a protein that lacks most of the receptor protein and does not bind estrogen. Northern hybridization confirmed the existence of multiple hepatic ERα RNAs that include the size range of the ERα cDNAs obtained from the libraries as well as additional sizes. Using primers for RT-PCR that target locations internal to the protein-coding sequence, we also established the presence of several ERα cDNA variants with in-frame insertions in the ligand-binding and DNA-binding domains and in-frame or out-of-frame deletions in the ligand-binding domain. These internal variants showed patterns of expression that differed between the ovary and liver. Further, the ovarian library yielded a full-length, ERα antisense cDNA containing a poly(A) signal and tail. A limited survey of histological preparations from juvenile catfish by in situ hybridization using directionally synthesized cRNA probes also suggested the expression of ERα antisense RNA in a tissue-specific manner. In conclusion, channel catfish seemingly have three broad classes of ERα mRNA variants: those encoding N-terminal truncated variants, those encoding internal variants (including C-terminal truncated variants), and antisense mRNA. The sense variants may

  12. Batrachotoxin changes the properties of the muscarinic receptor in rat brain and heart: possible interaction(s) between muscarinic receptors and sodium channels.

    PubMed Central

    Cohen-Armon, M; Kloog, Y; Henis, Y I; Sokolovsky, M

    1985-01-01

    The effects of Na+-channel activator batrachotoxin (BTX) on the binding properties of muscarinic receptors in homogenates of rat brain and heart were studied. BTX enhanced the affinity for the binding of the agonists carbamoylcholine and acetylcholine to the muscarinic receptors in brainstem and ventricle, but not in the cerebral cortex. Analysis of the data according to a two-site model for agonist binding indicated that the effect of BTX was to increase the affinity of the agonists to the high-affinity site. Guanyl nucleotides, known to induce interconversion of high-affinity agonist binding sites to the low-affinity state, canceled the effect of BTX on carbamoylcholine and acetylcholine binding. BTX had no effect on the binding of the agonist oxotremorine or on the binding of the antagonist [3H]-N-methyl-4-piperidyl benzilate. The local anesthetics dibucaine and tetracaine antagonized the effect of BTX on the binding of muscarinic agonists at concentrations known to inhibit the activation of Na+ channels by BTX. On the basis of these findings, we propose that in specific tissues the muscarinic receptors may interact with the BTX binding site (Na+ channels). PMID:2582418

  13. ThermoTRP Channels in Nociceptors: Taking a Lead from Capsaicin Receptor TRPV1

    PubMed Central

    Mandadi, Sravan; Roufogalis, Basil D.

    2008-01-01

    Nociceptors with peripheral and central projections express temperature sensitive transient receptor potential (TRP) ion channels, also called thermoTRP’s. Chemosensitivity of thermoTRP’s to certain natural compounds eliciting pain or exhibiting thermal properties has proven to be a good tool in characterizing these receptors. Capsaicin, a pungent chemical in hot peppers, has assisted in the cloning of the first thermoTRP, TRPV1. This discovery initiated the search for other receptors encoding the response to a wide range of temperatures encountered by the body. Of these, TRPV1 and TRPV2 encode unique modalities of thermal pain when exposed to noxious heat. The ability of TRPA1 to encode noxious cold is presently being debated. The role of TRPV1 in peripheral inflammatory pain and central sensitization during chronic pain is well known. In addition to endogenous agonists, a wide variety of chemical agonists and antagonists have been discovered to activate and inhibit TRPV1. Efforts are underway to determine conditions under which agonist-mediated desensitization of TRPV1 or inhibition by antagonists can produce analgesia. Also, identification of specific second messenger molecules that regulate phosphorylation of TRPV1 has been the focus of intense research, to exploit a broader approach to pain treatment. The search for a role of TRPV2 in pain remains dormant due to the lack of suitable experimental models. However, progress into TRPA1’s role in pain has received much attention recently. Another thermoTRP, TRPM8, encoding for the cool sensation and also expressed in nociceptors, has recently been shown to reduce pain via a central mechanism, thus opening a novel strategy for achieving analgesia. The role of other thermoTRP’s (TRPV3 and TRPV4) encoding for detection of warm temperatures and expressed in nociceptors cannot be excluded. This review will discuss current knowledge on the role of nociceptor thermoTRPs in pain and therapy and describes the

  14. Receptor-mediated activation of a plant Ca2+-permeable ion channel involved in pathogen defense

    PubMed Central

    Zimmermann, Sabine; Nürnberger, Thorsten; Frachisse, Jean-Marie; Wirtz, Wolfgang; Guern, Jean; Hedrich, Rainer; Scheel, Dierk

    1997-01-01

    Pathogen recognition at the plant cell surface typically results in the initiation of a multicomponent defense response. Transient influx of Ca2+ across the plasma membrane is postulated to be part of the signaling chain leading to pathogen resistance. Patch-clamp analysis of parsley protoplasts revealed a novel Ca2+-permeable, La3+-sensitive plasma membrane ion channel of large conductance (309 pS in 240 mM CaCl2). At an extracellular Ca2+ concentration of 1 mM, which is representative of the plant cell apoplast, unitary channel conductance was determined to be 80 pS. This ion channel (LEAC, for large conductance elicitor-activated ion channel) is reversibly activated upon treatment of parsley protoplasts with an oligopeptide elicitor derived from a cell wall protein of Phytophthora sojae. Structural features of the elicitor found previously to be essential for receptor binding, induction of defense-related gene expression, and phytoalexin formation are identical to those required for activation of LEAC. Thus, receptor-mediated stimulation of this channel appears to be causally involved in the signaling cascade triggering pathogen defense in parsley. PMID:11038609

  15. Electrophysiology-based analysis of human histamine H(4) receptor pharmacology using GIRK channel coupling in Xenopus oocytes.

    PubMed

    Sahlholm, Kristoffer; Nilsson, Johanna; Marcellino, Daniel; Fuxe, Kjell; Arhem, Peter

    2008-09-01

    The recently cloned histamine H(4) receptor is expressed predominantly in haematopoietic cells and has been found to modulate the function of mast cells, eosinophils, dendritic cells and T lymphocytes. It represents an attractive target for pharmacological interventions against a number of inflammatory and autoimmune disorders. In the present work we used two-electrode voltage-clamp to demonstrate histamine H(4) receptor modulation of G protein-coupled inward rectifier potassium (GIRK) channels heterologously expressed in Xenopus oocytes. In accordance with earlier findings in other effector systems, full agonism by histamine and (R)-alpha-methylhistamine, partial agonism by clobenpropit and inverse agonism by thioperamide were observed. Furthermore, in oocytes injected with low amounts of receptor cRNA, clobenpropit apparently acted as a neutral antagonist. We also used the high temporal resolution afforded by this system to study the differential time courses of response deactivation upon ligand washout for clobenpropit and (R)-alpha-methylhistamine. GIRK channels represent a novel effector system for histamine H(4) receptor modulation, which may be of physiological relevance and prove useful in the development of compounds targeting this receptor.

  16. Phenylalanine in the pore of the Erwinia ligand-gated ion channel modulates picrotoxinin potency but not receptor function.

    PubMed

    Thompson, Andrew J; Alqazzaz, Mona; Price, Kerry L; Weston, David A; Lummis, Sarah C R

    2014-10-01

    The Erwinia ligand-gated ion channel (ELIC) is a bacterial homologue of eukaryotic Cys-loop ligand-gated ion channels. This protein has the potential to be a useful model for Cys-loop receptors but is unusual in that it has an aromatic residue (Phe) facing into the pore, leading to some predictions that this protein is incapable of ion flux. Subsequent studies have shown this is not the case, so here we probe the role of this residue by examining the function of the ELIC in cases in which the Phe has been substituted with a range of alternative amino acids, expressed in Xenopus oocytes and functionally examined. Most of the mutations have little effect on the GABA EC50, but the potency of the weak pore-blocking antagonist picrotoxinin at F16'A-, F16'D-, F16'S-, and F16'T-containing receptors was increased to levels comparable with those of Cys-loop receptors, suggesting that this antagonist can enter the pore only when residue 16' is small. T6'S has no effect on picrotoxinin potency when expressed alone but abolishes the increased potency when combined with F16'S, indicating that the inhibitor binds at position 6', as in Cys-loop receptors, if it can enter the pore. Overall, the data support the proposal that the ELIC pore is a good model for Cys-loop receptor pores if the role of F16' is taken into consideration.

  17. Transient receptor potential melastatin 4 channel contributes to migration of androgen-insensitive prostate cancer cells

    PubMed Central

    Kilch, Tatiana; Jochum, Marcus Martin; Urban, Sabine Katharina; Jung, Volker; Stöckle, Michael; Rother, Karen; Greiner, Markus; Peinelt, Christine

    2015-01-01

    Impaired Ca2+ signaling in prostate cancer contributes to several cancer hallmarks, such as enhanced proliferation and migration and a decreased ability to induce apoptosis. Na+ influx via transient receptor potential melastatin 4 channel (TRPM4) can reduce store-operated Ca2+ entry (SOCE) by decreasing the driving force for Ca2+. In patients with prostate cancer, gene expression of TRPM4 is elevated. Recently, TRPM4 was identified as a cancer driver gene in androgen-insensitive prostate cancer. We investigated TRPM4 protein expression in cancer tissue samples from 20 patients with prostate cancer. We found elevated TRPM4 protein levels in prostatic intraepithelial neoplasia (PIN) and prostate cancer tissue compared to healthy tissue. In primary human prostate epithelial cells (hPEC) from healthy tissue and in the androgen-insensitive prostate cancer cell lines DU145 and PC3, TRPM4 mediated large Na+ currents. We demonstrated significantly increased SOCE after siRNA targeting of TRPM4 in hPEC and DU145 cells. In addition, knockdown of TRPM4 reduced migration but not proliferation of DU145 and PC3 cells. Taken together, our data identify TRPM4 as a regulator of SOCE in hPEC and DU145 cells, demonstrate a role for TRPM4 in cancer cell migration and suggest that TRPM4 is a promising potential therapeutic target. PMID:26496025

  18. Receptor channel TRPC6 orchestrate the activation of human hepatic stellate cell under hypoxia condition

    SciTech Connect

    Iyer, Soumya C; Kannan, Anbarasu; Gopal, Ashidha; Devaraj, Niranjali; Halagowder, Devaraj

    2015-08-01

    Hepatic stellate cells (HSCs), a specialized stromal cytotype have a great impact on the biological behaviors of liver diseases. Despite this fact, the underlying mechanism that regulates HSC still remains poorly understood. The aim of the present study was to understand the role of TRPC6 signaling in regulating the molecular mechanism of HSCs in response to hypoxia. In the present study we showed that under hypoxia condition, the upregulated Hypoxia Inducible Factor 1α (HIF1α) increases NICD activation, which in turn induces the expression of transient receptor potential channel 6 (TRPC6) in HSC line lx-2. TRPC6 causes a sustained elevation of intracellular calcium which is coupled with the activation of the calcineurin-nuclear factor of activated T-cell (NFAT) pathway which activates the synthesis of extracellular matrix proteins. TRPC6 also activates SMAD2/3 dependent TGF-β signaling in facilitating upregulated expression of αSMA and collagen. As activated HSCs may be a suitable target for HCC therapy and targeting these cells rather than the HCC cells may result in a greater response. Collectively, our studies indicate for the first time the detailed mechanism of activation of HSC through TRPC6 signaling and thus being a promising therapeutic target. - Highlights: • HIF1α increases NICD, induces TRPC6 in lx2 cells. • TRPC6 a novel regulator in the activation of HSC. • HSCs as target for HCC therapy.

  19. Involvement of Transient Receptor Potential Cation Channel Vanilloid 1 (TRPV1) in Myoblast Fusion.

    PubMed

    Kurosaka, Mitsutoshi; Ogura, Yuji; Funabashi, Toshiya; Akema, Tatsuo

    2016-10-01

    The mechanisms that underlie the complex process of muscle regeneration after injury remain unknown. Transient receptor potential cation channel vanilloid 1 (TRPV1) is expressed in several cell types, including skeletal muscle, and is activated by high temperature and by certain molecules secreted during tissue inflammation. Severe inflammation and local temperature perturbations are induced during muscle regeneration, which suggests that TRPV1 might be activated and involved in the process. The aim of this study, was to clarify the role of TRPV1 in the myogenic potential of satellite cells responsible for muscle regeneration. We found that mRNA and protein levels of TRPV1 increased during regeneration after cardiotoxin (CTX)-induced muscle injury in mice. Using isolated mouse satellite cells (i.e., myoblasts), we observed that activation of TRPV1 by its agonist capsaicin (CAP) augmented myogenin protein levels. Whereas CAP did not alter myoblast proliferation, it facilitated myoblast fusion (evaluated using myonucleii number per myotube and fusion index). In contrast, suppression of TRPV1 by siRNA impaired myoblast fusion. Using mice, we also demonstrated that intramuscular injection of CAP facilitated muscle repair after CTX-induced muscle injury. Moreover, we showed that these roles of TRPV1 might be mediated by interleukin-4 and calcium signaling during myoblast fusion. Collectively, these results suggest that TRPV1 underlies normal myogenesis through promotion of myoblast fusion. J. Cell. Physiol. 231: 2275-2285, 2016. © 2016 Wiley Periodicals, Inc. PMID:26892397

  20. Involvement of Transient Receptor Potential Cation Channel Vanilloid 1 (TRPV1) in Myoblast Fusion.

    PubMed

    Kurosaka, Mitsutoshi; Ogura, Yuji; Funabashi, Toshiya; Akema, Tatsuo

    2016-10-01

    The mechanisms that underlie the complex process of muscle regeneration after injury remain unknown. Transient receptor potential cation channel vanilloid 1 (TRPV1) is expressed in several cell types, including skeletal muscle, and is activated by high temperature and by certain molecules secreted during tissue inflammation. Severe inflammation and local temperature perturbations are induced during muscle regeneration, which suggests that TRPV1 might be activated and involved in the process. The aim of this study, was to clarify the role of TRPV1 in the myogenic potential of satellite cells responsible for muscle regeneration. We found that mRNA and protein levels of TRPV1 increased during regeneration after cardiotoxin (CTX)-induced muscle injury in mice. Using isolated mouse satellite cells (i.e., myoblasts), we observed that activation of TRPV1 by its agonist capsaicin (CAP) augmented myogenin protein levels. Whereas CAP did not alter myoblast proliferation, it facilitated myoblast fusion (evaluated using myonucleii number per myotube and fusion index). In contrast, suppression of TRPV1 by siRNA impaired myoblast fusion. Using mice, we also demonstrated that intramuscular injection of CAP facilitated muscle repair after CTX-induced muscle injury. Moreover, we showed that these roles of TRPV1 might be mediated by interleukin-4 and calcium signaling during myoblast fusion. Collectively, these results suggest that TRPV1 underlies normal myogenesis through promotion of myoblast fusion. J. Cell. Physiol. 231: 2275-2285, 2016. © 2016 Wiley Periodicals, Inc.

  1. Novel role for the transient receptor potential channel TRPM2 in prostate cancer cell proliferation

    PubMed Central

    Zeng, X; Sikka, S C; Huang, L; Sun, C; Xu, C; Jia, D; Abdel-Mageed, A B; Pottle, J E; Taylor, J T; Li, M

    2009-01-01

    We have identified a novel function for a member of the transient receptor potential (TRP) protein super-family, TRPM2, in prostate cancer cell proliferation. TRPM2 encodes a non-selective cation-permeable ion channel. We found that selectively knocking down TRPM2 with the small interfering RNA technique inhibited the growth of prostate cancer cells but not of non-cancerous cells. The subcellular localization of this protein is also remarkably different between cancerous and non-cancerous cells. In BPH-1 (benign), TRPM2 protein is homogenously located near the plasma membrane and in the cytoplasm, whereas in the cancerous cells (PC-3 and DU-145), a significant amount of the TRPM2 protein is located in the nuclei in a clustered pattern. Furthermore, we have found that TRPM2 inhibited nuclear ADP-ribosylation in prostate cancer cells. However, TRPM2 knockdown-induced inhibition of proliferation is independent of the activity of poly(ADP-ribose) polymerases. We conclude that TRPM2 is essential for prostate cancer cell proliferation and may be a potential target for the selective treatment of prostate cancer. PMID:20029400

  2. Methylglyoxal Activates Nociceptors through Transient Receptor Potential Channel A1 (TRPA1)

    PubMed Central

    Eberhardt, Mirjam J.; Filipovic, Milos R.; Leffler, Andreas; de la Roche, Jeanne; Kistner, Katrin; Fischer, Michael J.; Fleming, Thomas; Zimmermann, Katharina; Ivanovic-Burmazovic, Ivana; Nawroth, Peter P.; Bierhaus, Angelika; Reeh, Peter W.; Sauer, Susanne K.

    2012-01-01

    Neuropathic pain can develop as an agonizing sequela of diabetes mellitus and chronic uremia. A chemical link between both conditions of altered metabolism is the highly reactive compound methylglyoxal (MG), which accumulates in all cells, in particular neurons, and leaks into plasma as an index of the severity of the disorder. The electrophilic structure of this cytotoxic ketoaldehyde suggests TRPA1, a receptor channel deeply involved in inflammatory and neuropathic pain, as a molecular target. We demonstrate that extracellularly applied MG accesses specific intracellular binding sites of TRPA1, activating inward currents and calcium influx in transfected cells and sensory neurons, slowing conduction velocity in unmyelinated peripheral nerve fibers, and stimulating release of proinflammatory neuropeptides from and action potential firing in cutaneous nociceptors. Using a model peptide of the N terminus of human TRPA1, we demonstrate the formation of disulfide bonds based on MG-induced modification of cysteines as a novel mechanism. In conclusion, MG is proposed to be a candidate metabolite that causes neuropathic pain in metabolic disorders and thus is a promising target for medicinal chemistry. PMID:22740698

  3. The Role of 5-HT3 Receptors in Signaling from Taste Buds to Nerves

    PubMed Central

    Vandenbeuch, Aurelie; Voigt, Anja; Meyerhof, Wolfgang; Kinnamon, Sue C.; Finger, Thomas E.

    2015-01-01

    Activation of taste buds triggers the release of several neurotransmitters, including ATP and serotonin (5-hydroxytryptamine; 5-HT). Type III taste cells release 5-HT directly in response to acidic (sour) stimuli and indirectly in response to bitter and sweet tasting stimuli. Although ATP is necessary for activation of nerve fibers for all taste stimuli, the role of 5-HT is unclear. We investigated whether gustatory afferents express functional 5-HT3 receptors and, if so, whether these receptors play a role in transmission of taste information from taste buds to nerves. In mice expressing GFP under the control of the 5-HT3A promoter, a subset of cells in the geniculate ganglion and nerve fibers in taste buds are GFP-positive. RT-PCR and in situ hybridization confirmed the presence of 5-HT3A mRNA in the geniculate ganglion. Functional studies show that only those geniculate ganglion cells expressing 5-HT3A-driven GFP respond to 10 μm 5-HT and this response is blocked by 1 μm ondansetron, a 5-HT3 antagonist, and mimicked by application of 10 μm m-chlorophenylbiguanide, a 5-HT3 agonist. Pharmacological blockade of 5-HT3 receptors in vivo or genetic deletion of the 5-HT3 receptors reduces taste nerve responses to acids and other taste stimuli compared with controls, but only when urethane was used as the anesthetic. We find that anesthetic levels of pentobarbital reduce taste nerve responses apparently by blocking the 5-HT3 receptors. Our results suggest that 5-HT released from type III cells activates gustatory nerve fibers via 5-HT3 receptors, accounting for a significant proportion of the neural taste response. SIGNIFICANCE STATEMENT Historically, serotonin (5-hydroxytryptamine; 5-HT) has been described as a candidate neurotransmitter in the gustatory system and recent studies show that type III taste receptor cells release 5-HT in response to various taste stimuli. In the present study, we demonstrate that a subset of gustatory sensory neurons express functional

  4. Conformational Dynamics of Kir3.1/Kir3.2 Channel Activation Via δ-Opioid Receptors

    PubMed Central

    Richard-Lalonde, Melissa; Nagi, Karim; Audet, Nicolas; Sleno, Rory; Amraei, Mohammad; Hogue, Mireille; Balboni, Gianfranco; Schiller, Peter W.; Bouvier, Michel; Hébert, Terence E.; Pineyro, Graciela

    2013-01-01

    This study assessed how conformational information encoded by ligand binding to δ-opioid receptors (DORs) is transmitted to Kir3.1/Kir3.2 channels. Human embryonic kidney 293 cells were transfected with bioluminescence resonance energy transfer (BRET) donor/acceptor pairs that allowed us to evaluate independently reciprocal interactions among signaling partners. These and coimmunoprecipitation studies indicated that DORs, Gβγ, and Kir3 subunits constitutively interacted with one another. GαoA associated with DORs and Gβγ, but despite being part of the complex, no evidence of its direct association with the channel was obtained. DOR activation by different ligands left DOR-Kir3 interactions unmodified but modulated BRET between DOR-GαoA, DOR-Gβγ, GαoA-Gβγ, and Gβγ-Kir3 interfaces. Ligand-induced BRET changes assessing Gβγ-Kir3.1 subunit interaction 1) followed similar kinetics to those monitoring the GαoA-Gβγ interface, 2) displayed the same order of efficacy as those observed at the DOR-Gβγ interface, 3) were sensitive to pertussis toxin, and 4) were predictive of whether a ligand could evoke channel currents. Conformational changes at the Gβγ/Kir3 interface were lost when Kir3.1 subunits were replaced by a mutant lacking essential sites for Gβγ-mediated activation. Thus, conformational information encoded by agonist binding to the receptor is relayed to the channel via structural rearrangements that involve repositioning of Gβγ with respect to DORs, GαoA, and channel subunits. Further, the fact that BRET changes at the Gβγ-Kir3 interface are predictive of a ligand’s ability to induce channel currents points to these conformational biosensors as screening tools for identifying GPCR ligands that induce Kir3 channel activation. PMID:23175530

  5. The Ca2+-release channel/ryanodine receptor is localized in junctional and corbular sarcoplasmic reticulum in cardiac muscle

    PubMed Central

    1993-01-01

    The subcellular distribution of the Ca(2+)-release channel/ryanodine receptor in adult rat papillary myofibers has been determined by immunofluorescence and immunoelectron microscopical studies using affinity purified antibodies against the ryanodine receptor. The receptor is confined to the sarcoplasmic reticulum (SR) where it is localized to interior and peripheral junctional SR and the corbular SR, but it is absent from the network SR where the SR-Ca(2+)-ATPase and phospholamban are densely distributed. Immunofluorescence labeling of sheep Purkinje fibers show that the ryanodine receptor is confined to discrete foci while the SR-Ca(2+)-ATPase is distributed in a continuous network-like structure present at the periphery as well as throughout interior regions of these myofibers. Because Purkinje fibers lack T- tubules, these results indicate that the ryanodine receptor is localized not only to the peripheral junctional SR but also to corbular SR densely distributed in interfibrillar spaces of the I-band regions. We have previously identified both corbular SR and junctional SR in cardiac muscle as potential Ca(2+)-storage/Ca(2+)-release sites by demonstrating that the Ca2+ binding protein calsequestrin and calcium are very densely distributed in these two specialized domains of cardiac SR in situ. The results presented here provide strong evidence in support of the hypothesis that corbular SR is indeed a site of Ca(2+)-induced Ca2+ release via the ryanodine receptor during excitation contraction coupling in cardiac muscle. Furthermore, these results indicate that the function of the cardiac Ca(2+)-release channel/ryanodine receptor is not confined to junctional complexes between SR and the sarcolemma. PMID:8381786

  6. Estradiol activates epithelial sodium channels in rat alveolar cells through the G protein-coupled estrogen receptor

    PubMed Central

    Mitzelfelt, Jeremiah D.; Yu, Ling; Yue, Qiang; Duke, Billie Jeanne; Harrell, Constance S.; Neigh, Gretchen N.; Eaton, Douglas C.

    2013-01-01

    Female sex predisposes individuals to poorer outcomes during respiratory disorders like cystic fibrosis and influenza-associated pneumonia. A common link between these disorders is dysregulation of alveolar fluid clearance via disruption of epithelial sodium channel (ENaC) activity. Recent evidence suggests that female sex hormones directly regulate expression and activity of alveolar ENaC. In our study, we identified the mechanism by which estradiol (E2) or progesterone (P4) independently regulates alveolar ENaC. Using cell-attached patch clamp, we measured ENaC single-channel activity in a rat alveolar cell line (L2) in response to overnight exposure to either E2 or P4. In contrast to P4, E2 increased ENaC channel activity (NPo) through an increase in channel open probability (Po) and an increased number of patches with observable channel activity. Apical plasma membrane abundance of the ENaC α-subunit (αENaC) more than doubled in response to E2 as determined by cell surface biotinylation. αENaC membrane abundance was approximately threefold greater in lungs from female rats in proestrus, when serum E2 is greatest, compared with diestrus, when it is lowest. Our results also revealed a significant role for the G protein-coupled estrogen receptor (Gper) to mediate E2's effects on ENaC. Overall, our results demonstrate that E2 signaling through Gper selectively activates alveolar ENaC through an effect on channel gating and channel density, the latter via greater trafficking of channels to the plasma membrane. The results presented herein implicate E2-mediated regulation of alveolar sodium channels in the sex differences observed in the pathogenesis of several pulmonary diseases. PMID:24097558

  7. Estradiol activates epithelial sodium channels in rat alveolar cells through the G protein-coupled estrogen receptor.

    PubMed

    Greenlee, Megan M; Mitzelfelt, Jeremiah D; Yu, Ling; Yue, Qiang; Duke, Billie Jeanne; Harrell, Constance S; Neigh, Gretchen N; Eaton, Douglas C

    2013-12-01

    Female sex predisposes individuals to poorer outcomes during respiratory disorders like cystic fibrosis and influenza-associated pneumonia. A common link between these disorders is dysregulation of alveolar fluid clearance via disruption of epithelial sodium channel (ENaC) activity. Recent evidence suggests that female sex hormones directly regulate expression and activity of alveolar ENaC. In our study, we identified the mechanism by which estradiol (E2) or progesterone (P4) independently regulates alveolar ENaC. Using cell-attached patch clamp, we measured ENaC single-channel activity in a rat alveolar cell line (L2) in response to overnight exposure to either E2 or P4. In contrast to P4, E2 increased ENaC channel activity (NPo) through an increase in channel open probability (Po) and an increased number of patches with observable channel activity. Apical plasma membrane abundance of the ENaC α-subunit (αENaC) more than doubled in response to E2 as determined by cell surface biotinylation. αENaC membrane abundance was approximately threefold greater in lungs from female rats in proestrus, when serum E2 is greatest, compared with diestrus, when it is lowest. Our results also revealed a significant role for the G protein-coupled estrogen receptor (Gper) to mediate E2's effects on ENaC. Overall, our results demonstrate that E2 signaling through Gper selectively activates alveolar ENaC through an effect on channel gating and channel density, the latter via greater trafficking of channels to the plasma membrane. The results presented herein implicate E2-mediated regulation of alveolar sodium channels in the sex differences observed in the pathogenesis of several pulmonary diseases. PMID:24097558

  8. Channel

    NASA Technical Reports Server (NTRS)

    2006-01-01

    [figure removed for brevity, see original site] Context image for PIA03693 Channel

    This channel is located south of Iani Chaos.

    Image information: VIS instrument. Latitude -10.9N, Longitude 345.5E. 17 meter/pixel resolution.

    Note: this THEMIS visual image has not been radiometrically nor geometrically calibrated for this preliminary release. An empirical correction has been performed to remove instrumental effects. A linear shift has been applied in the cross-track and down-track direction to approximate spacecraft and planetary motion. Fully calibrated and geometrically projected images will be released through the Planetary Data System in accordance with Project policies at a later time.

    NASA's Jet Propulsion Laboratory manages the 2001 Mars Odyssey mission for NASA's Office of Space Science, Washington, D.C. The Thermal Emission Imaging System (THEMIS) was developed by Arizona State University, Tempe, in collaboration with Raytheon Santa Barbara Remote Sensing. The THEMIS investigation is led by Dr. Philip Christensen at Arizona State University. Lockheed Martin Astronautics, Denver, is the prime contractor for the Odyssey project, and developed and built the orbiter. Mission operations are conducted jointly from Lockheed Martin and from JPL, a division of the California Institute of Technology in Pasadena.

  9. Calcium-dependent expression of transient receptor potential canonical type 3 channels in patients with chronic kidney disease.

    PubMed

    Liu, Ying; Krueger, Katharina; Hovsepian, Anahit; Tepel, Martin; Thilo, Florian

    2011-10-01

    It is unknown whether extracellular calcium may regulate the expression of transient receptor potential canonical type 3 (TRPC3) channels in patients with chronic kidney disease. Using quantitative in-cell Western assay we compared the expression of TRPC3 channel protein in monocytes from 20 patients with chronic kidney disease and 19 age- and sex-matched healthy control subjects. TRPC3 channels were identified by immunoblotting using specific antibodies and TRPC3 protein was further confirmed by mass spectrometry. We observed a significant increase of TRPC3 channel protein expression in patients with chronic kidney disease compared to healthy control subjects (normalized expression, 0.42±0.06 vs. 0.19±0.03; p<0.01). Expression of TRPC3 was significantly inversely correlated with estimated glomerular filtration rates (Spearman r=-0.41) or serum calcium concentration (Spearman r=-0.34). During a hemodialysis session serum calcium concentrations significantly increased, whereas the expression of TRPC3 channels and calcium influx significantly decreased. In vitro studies confirmed that higher calcium concentrations but not magnesium, barium nor sodium concentrations significantly decreased TRPC3 expression in human monocytes. This study indicates that reduced extracellular calcium concentrations up-regulate TRPC3 channel protein expression in patients with chronic kidney disease.

  10. The Pyrexia transient receptor potential channel mediates circadian clock synchronization to low temperature cycles in Drosophila melanogaster.

    PubMed

    Wolfgang, Werner; Simoni, Alekos; Gentile, Carla; Stanewsky, Ralf

    2013-10-01

    Circadian clocks are endogenous approximately 24 h oscillators that temporally regulate many physiological and behavioural processes. In order to be beneficial for the organism, these clocks must be synchronized with the environmental cycles on a daily basis. Both light : dark and the concomitant daily temperature cycles (TCs) function as Zeitgeber ('time giver') and efficiently entrain circadian clocks. The temperature receptors mediating this synchronization have not been identified. Transient receptor potential (TRP) channels function as thermo-receptors in animals, and here we show that the Pyrexia (Pyx) TRP channel mediates temperature synchronization in Drosophila melanogaster. Pyx is expressed in peripheral sensory organs (chordotonal organs), which previously have been implicated in temperature synchronization. Flies deficient for Pyx function fail to synchronize their behaviour to TCs in the lower range (16-20°C), and this deficit can be partially rescued by introducing a wild-type copy of the pyx gene. Synchronization to higher TCs is not affected, demonstrating a specific role for Pyx at lower temperatures. In addition, pyx mutants speed up their clock after being exposed to TCs. Our results identify the first TRP channel involved in temperature synchronization of circadian clocks.

  11. Inhibition of cation channel function at the nicotinic acethylcholine receptor from Torpedo: Agonist self-inhibition and anesthetic drugs

    SciTech Connect

    Forman, S.A.

    1989-01-01

    Modulation of the nicotinic acethylcholine receptor from Torpedo by cholinergic agonists, local anesthetics, and n-alkanols was studied using {sup 86}Rb{sup +} flux studies in sealed native Torpedo electroplaque membrane vesicles. Reliable concentration-response and kinetic data were obtained using manual ten sec filtration assays in vesicles partially blocked with alpha-bungarotoxin to remove spare receptors and quenched-flow assays to assess initial {sup 86}Rb{sup +} flux rates or the rate of drug-induced receptor inactivation. Concentration response relationships for the agonists acetylcholine, carbamylcholine, suberyldicholine, phenyltrimethylammonium, and (-)-nicotine are all bell-shape due to stimulation of cation channel opening at low concentrations and inhibition of channels at higher concentrations. The rate of agonist-induced fast desensitization (k{sub d}) increases with (acetylcholine) in parallel with channel activation, suggesting that desensitization proceeds from the open state and/or states in rapid equilibrium with it. At self-inhibitory acetylcholine concentrations, a new rapid inactivation (rate = k{sub f}) is observed before fast desensitization. The rate and extent of rapid inactivation is compatible with bimolecular association between acethylcholine and inhibitory site with K{sub B} = 40 mM.

  12. Role of transient receptor potential channels in intestinal inflammation and visceral pain: novel targets in inflammatory bowel diseases.

    PubMed

    Zielińska, Marta; Jarmuż, Agata; Wasilewski, Andrzej; Sałaga, Maciej; Fichna, Jakub

    2015-02-01

    Transient receptor potential (TRP) channels are a large group of ion channels that are prevalent in mammalian tissues. They are widely distributed in the central and peripheral nervous systems, and in nonneuronal cells, where they are implicated in sensing temperature, noxious substances, and pain. TRPs play an important role in immune response and nociception and, therefore, may be involved in the pathogenesis of inflammatory bowel diseases, whose major symptoms include chronic inflammatory state and abdominal pain. In this review, we summarize what is known on TRP channels in inflammatory bowel disease and visceral pain; we focus in particular on TRPV1, TRPV4, TRPA1, and TRPM. We also analyze scientific reports that evidence potential use of TRP regulators in future inflammatory bowel disease treatment.

  13. Subunit composition of G(o) proteins functionally coupling galanin receptors to voltage-gated calcium channels.

    PubMed Central

    Kalkbrenner, F; Degtiar, V E; Schenker, M; Brendel, S; Zobel, A; Heschler, J; Wittig, B; Schultz, G

    1995-01-01

    The neuropeptide galanin is widely expressed in the central nervous system and other tissues and induces different cellular reactions, e.g. hormone release from pituitary and inhibition of insulin release from pancreatic B cells. By microinjection of antisense oligonucleotides we studied the question as to which G proteins mediate the galanin-induced inhibition of voltage-gated Ca2+ channels in the rat pancreatic B-cell line RINm5F and in the rat pituitary cell line GH3. Injection of antisense oligonucleotides directed against alpha 01, beta 2, beta 3, gamma 2 and gamma 4 G protein subunits reduced the inhibition of Ca2+ channel current which was induced by galanin, whereas no change was seen after injection of cells with antisense oligonucleotides directed against alpha i, alpha q, alpha 11, alpha 14, alpha 15, beta 1, beta 4, gamma 1, gamma 3, gamma 5, or gamma 7 G protein subunits or with sense control oligonucleotides. In view of these data and of previous results, we conclude that the galanin receptors in GH3 and in RINm5F cells couple mainly to the G(0) protein consisting of alpha 01 beta 2 gamma 2 to inhibit Ca2+ channels and use alpha 01beta 3 gamma 4 less efficiently. The latter G protein composition was previously shown to be used by muscarinic M4 receptors to inhibit Ca2+ channels. Images PMID:7588602

  14. The foot structure from the type 1 ryanodine receptor is required for functional coupling to store-operated channels.

    PubMed

    Sampieri, Alicia; Diaz-Muñoz, Mauricio; Antaramian, Anaid; Vaca, Luis

    2005-07-01

    In the present study we have explored structural determinants of the functional interaction between skeletal muscle ryanodine receptor (RyR1) and transient receptor potential channel 1 (TRPC1) channels expressed in Chinese hamster ovary cells. We have illustrated a functional interaction between TRPC1 channels and RyR1 for the regulation of store-operated calcium entry (SOCE) initiated after releasing calcium from a caffeine-sensitive intracellular calcium pool. RNA interference experiments directed to reduce the amount of TRPC1 protein indicate that RyR1 associates to at least two different types of store-operated channels (SOCs), one dependent and one independent of TRPC1. In contrast, bradykinin-induced SOCE is completely dependent on the presence of TRPC1 protein, as we have previously illustrated. Removing the foot structure from RyR1 results in normal caffeine-induced release of calcium from internal stores but abolishes the activation of SOCE, indicating that this structure is require for functional coupling to SOCs. The footless RyR1 protein shows a different cellular localization when compared with wild type RyR1. The later protein shows a higher percentage of colocalization with FM-464, a marker of plasma membrane. The implications of the foot structure for the functional and physical coupling to TRPC and SOCs is discussed.

  15. Transient Receptor Potential Vanilloid 4-Induced Modulation of Voltage-Gated Sodium Channels in Hippocampal Neurons.

    PubMed

    Hong, Zhiwen; Jie, Pinghui; Tian, Yujing; Chen, Tingting; Chen, Lei; Chen, Ling

    2016-01-01

    Transient receptor potential vanilloid 4 (TRPV4) is reported to control the resting membrane potential and increase excitability in many types of cells. Voltage-gated sodium channels (VGSCs) play an important role in initiating action potentials in neurons. However, whether VGSCs can be modulated by the activation of TRPV4 in hippocampal pyramidal neurons remains unknown. In this study, we tested the effect of TRPV4 agonists (GSK1016790A and 4α-PDD) on voltage-gated sodium current (I Na) in hippocampal CA1 pyramidal neurons and the protein levels of α/β-subunit of VGSCs in the hippocampus of mice subjected to intracerebroventricular (icv.) injection of GSK1016790A (GSK-injected mice). Herein, we report that I Na was inhibited by acute application of GSK1016790A or 4α-PDD. In the presence of TRPV4 agonists, the voltage-dependent inactivation curve shifted to the hyperpolarization, whereas the voltage-dependent activation curve remained unchanged. The TRPV4 agonist-induced inhibition of I Na was blocked by the TRPV4 antagonist or tetrodotoxin. Moreover, blocking protein kinase A (PKA) markedly attenuated the GSK1016790A-induced inhibition of I Na, whereas antagonism of protein kinase C or p38 mitogen-activated protein kinase did not change GSK1016790A action. Finally, the protein levels of Nav1.1, Nav1.2, and Nav1.6 in the hippocampus increased in GSK-injected mice, whereas those of Nav1.3 and Navβ1 remained nearly unchanged. We conclude that I Na is inhibited by the acute activation of TRPV4 through PKA signaling pathway in hippocampal pyramidal neurons, but protein expression of α-subunit of VGSCs is increased by sustained TRPV4 activation, which may compensate for the acute inhibition of I Na and provide a possibility for hyper-excitability upon sustained TRPV4 activation.

  16. Crystal structures of the glutamate receptor ion channel GluK3 and GluK5 amino terminal domains

    PubMed Central

    Kumar, Janesh; Mayer, Mark L.

    2010-01-01

    Ionotropic glutamate receptors (iGluRs) mediate the majority of fast excitatory synaptic neurotransmission in the central nervous system. The selective assembly of iGluRs into the AMPA, kainate and NMDA receptor subtypes is regulated by their extracellular amino terminal domains (ATD). Kainate receptors are further classified into low-affinity (GluK1-3) and high-affinity (GluK4-5) receptor families based on their affinity for the neurotoxin kainic acid. These two families share 42% sequence identity for the intact receptor but only 28% sequence identity at the level of ATD. We have determined for the first time high-resolution crystal structures for the GluK3 and GluK5 ATDs, both of which crystallize as dimers, but with a strikingly different dimer assembly at the R1 interface. By contrast, for both GluK3 and GluK5 the R2 domain dimer assembly is similar to that reported previously for other non-NMDA iGluRs. This observation is consistent with the reports that GluK4-5 cannot form functional homomeric ion channels and require obligate coassembly with GluK1-3. Our analysis also reveals that the relative orientation of domains R1 and R2 in individual non-NMDA receptor ATDs varies by up to 10°, in contrast to the 50° difference reported for the NMDA receptor GluN2B subunit. This restricted domain movement in non-NMDA receptor ATDs seems to result from both extensive intramolecular contacts between domains R1 and R2, and from their assembly as dimers which interact at both the R1 and R2 domains. Our results provide the first insights into the structure and function for GluK4-5, the least understood family of iGluRs. PMID:20951142

  17. Antagonism of Nav channels and α1-adrenergic receptors contributes to vascular smooth muscle effects of ranolazine

    PubMed Central

    Virsolvy, Anne; Farah, Charlotte; Pertuit, Nolwenn; Kong, Lingyan; Lacampagne, Alain; Reboul, Cyril; Aimond, Franck; Richard, Sylvain

    2015-01-01

    Ranolazine is a recently developed drug used for the treatment of patients with chronic stable angina. It is a selective inhibitor of the persistent cardiac Na+ current (INa), and is known to reduce the Na+-dependent Ca2+ overload that occurs in cardiomyocytes during ischemia. Vascular effects of ranolazine, such as vasorelaxation,have been reported and may involve multiple pathways. As voltage-gated Na+ channels (Nav) present in arteries play a role in contraction, we hypothesized that ranolazine could target these channels. We studied the effects of ranolazine in vitro on cultured aortic smooth muscle cells (SMC) and ex vivo on rat aortas in conditions known to specifically activate or promote INa. We observed that in the presence of the Nav channel agonist veratridine, ranolazine inhibited INa and intracellular Ca2+ calcium increase in SMC, and arterial vasoconstriction. In arterial SMC, ranolazine inhibited the activity of tetrodotoxin-sensitive voltage-gated Nav channels and thus antagonized contraction promoted by low KCl depolarization. Furthermore, the vasorelaxant effects of ranolazine, also observed in human arteries and independent of the endothelium, involved antagonization of the α1-adrenergic receptor. Combined α1-adrenergic antagonization and inhibition of SMCs Nav channels could be involved in the vascular effects of ranolazine. PMID:26655634

  18. Antagonism of Nav channels and α1-adrenergic receptors contributes to vascular smooth muscle effects of ranolazine.

    PubMed

    Virsolvy, Anne; Farah, Charlotte; Pertuit, Nolwenn; Kong, Lingyan; Lacampagne, Alain; Reboul, Cyril; Aimond, Franck; Richard, Sylvain

    2015-01-01

    Ranolazine is a recently developed drug used for the treatment of patients with chronic stable angina. It is a selective inhibitor of the persistent cardiac Na(+) current (INa), and is known to reduce the Na(+)-dependent Ca(2+) overload that occurs in cardiomyocytes during ischemia. Vascular effects of ranolazine, such as vasorelaxation,have been reported and may involve multiple pathways. As voltage-gated Na(+) channels (Nav) present in arteries play a role in contraction, we hypothesized that ranolazine could target these channels. We studied the effects of ranolazine in vitro on cultured aortic smooth muscle cells (SMC) and ex vivo on rat aortas in conditions known to specifically activate or promote INa. We observed that in the presence of the Nav channel agonist veratridine, ranolazine inhibited INa and intracellular Ca(2+) calcium increase in SMC, and arterial vasoconstriction. In arterial SMC, ranolazine inhibited the activity of tetrodotoxin-sensitive voltage-gated Nav channels and thus antagonized contraction promoted by low KCl depolarization. Furthermore, the vasorelaxant effects of ranolazine, also observed in human arteries and independent of the endothelium, involved antagonization of the α1-adrenergic receptor. Combined α1-adrenergic antagonization and inhibition of SMCs Nav channels could be involved in the vascular effects of ranolazine. PMID:26655634

  19. Yeast screens show aromatic residues at the end of the sixth helix anchor transient receptor potential channel gate.

    PubMed

    Zhou, Xinliang; Su, Zhenwei; Anishkin, Andriy; Haynes, W John; Friske, Eric M; Loukin, Stephen H; Kung, Ching; Saimi, Yoshiro

    2007-09-25

    Transient receptor potential (TRP) channels are first elements in sensing chemicals, heat, and force and are widespread among protists and fungi as well as animals. Despite their importance, the arrangement and roles of the amino acids that constitute the TRP channel gate are unknown. The yeast TRPY1 is activated in vivo by osmotically induced vacuolar membrane deformation and by cytoplasmic Ca(2+). After a random mutagenesis, we isolated TRPY1 mutants that responded more strongly to mild osmotic upshocks. One such gain-of-function mutant has a Y458H substitution at the C terminus of the predicted sixth transmembrane helix. Direct patch-clamp examination of vacuolar membranes showed that Y458H channels were already active with little stimulus and showed marked flickers between the open and intraburst closed states. They remained responsive to membrane stretch force and to Ca(2+), indicating primary defects in the gate region but not in the sensing of gating principles. None of the other 18 amino acid replacements engineered here showed normal channel kinetics except the two aromatic substitutions, Y458F and Y458W. The Y458 of TRPY1 has its aromatic counterpart in mammalian TRPM. Furthermore, conserved aromatics one alpha-helical turn downstream from this point are also found in animal TRPC, TRPN, TRPP, and TRPML, suggesting that gate anchoring with aromatics may be common among many TRP channels. The possible roles of aromatics at the end of the sixth transmembrane helix are discussed.

  20. Structural and Single-Channel Results Indicate that the Rates of Ligand Binding Domain Closing and Opening Directly Impact AMPA Receptor Gating

    SciTech Connect

    Zhang,W.; Cho, Y.; Lolis, E.; Howe, J.

    2008-01-01

    At most excitatory central synapses, glutamate is released from presynaptic terminals and binds to postsynaptic AMPA receptors, initiating a series of conformational changes that result in ion channel opening. Efficient transmission at these synapses requires that glutamate binding to AMPA receptors results in rapid and near-synchronous opening of postsynaptic receptor channels. In addition, if the information encoded in the frequency of action potential discharge is to be transmitted faithfully, glutamate must dissociate from the receptor quickly, enabling the synapse to discriminate presynaptic action potentials that are spaced closely in time. The current view is that the efficacy of agonists is directly related to the extent to which ligand binding results in closure of the binding domain. For glutamate to dissociate from the receptor, however, the binding domain must open. Previously, we showed that mutations in glutamate receptor subunit 2 that should destabilize the closed conformation not only sped deactivation but also altered the relative efficacy of glutamate and quisqualate. Here we present x-ray crystallographic and single-channel data that support the conclusions that binding domain closing necessarily precedes channel opening and that the kinetics of conformational changes at the level of the binding domain importantly influence ion channel gating. Our findings suggest that the stability of the closed-cleft conformation has been tuned during evolution so that glutamate dissociates from the receptor as rapidly as possible but remains an efficacious agonist.

  1. Design and Characterization of Superpotent Bivalent Ligands Targeting Oxytocin Receptor Dimers via a Channel-Like Structure.

    PubMed

    Busnelli, Marta; Kleinau, Gunnar; Muttenthaler, Markus; Stoev, Stoytcho; Manning, Maurice; Bibic, Lucka; Howell, Lesley A; McCormick, Peter J; Di Lascio, Simona; Braida, Daniela; Sala, Mariaelvina; Rovati, G Enrico; Bellini, Tommaso; Chini, Bice

    2016-08-11

    Dimeric/oligomeric states of G-protein coupled receptors have been difficult to target. We report here bivalent ligands consisting of two identical oxytocin-mimetics that induce a three order magnitude boost in G-protein signaling of oxytocin receptors (OTRs) in vitro and a 100- and 40-fold gain in potency in vivo in the social behavior of mice and zebrafish. Through receptor mutagenesis and interference experiments with synthetic peptides mimicking transmembrane helices (TMH), we show that such superpotent behavior follows from the binding of the bivalent ligands to dimeric receptors based on a TMH1-TMH2 interface. Moreover, in this arrangement, only the analogues with a well-defined spacer length (∼25 Å) precisely fit inside a channel-like passage between the two protomers of the dimer. The newly discovered oxytocin bivalent ligands represent a powerful tool for targeting dimeric OTR in neurodevelopmental and psychiatric disorders and, in general, provide a framework to untangle specific arrangements of G-protein coupled receptor dimers. PMID:27420737

  2. 17β-Estradiol regulates the gene expression of voltage-gated sodium channels: role of estrogen receptor α and estrogen receptor β.

    PubMed

    Hu, Fang; Wang, Qiang; Wang, Peizhi; Wang, Wenjuan; Qian, Wenyi; Xiao, Hang; Wang, Lin

    2012-04-01

    Estradiol (E2) plays a key role in pain modulation, and the biological effects of E2 are transduced by binding estrogen receptors (ERs). Voltage-gated sodium (Nav) channels are responsible for the generation and propagation of action potentials in the membranes of most neurons and excitable cells. Adult dorsal root ganglion (DRG) neurons can express the ERs (ERα and ERβ), and Nav channels (TTX-S: Nav1.1, Nav1.6, and Nav1.7; and TTX-R: Nav1.8, and Nav1.9). Although E2 modulates Nav channel currents, little is known about the molecular mechanisms involved. In this study, we investigate the mRNA expressions of Nav channel subtypes mediated differentially by the ERs in the DRGs of wild-type (WT) and estrogen receptor knockout (αERKO and βERKO) mice. By means of quantitative real-time PCR, we found that the expressions of Nav1.1, Nav1.7, Nav1.8, and Nav1.9 subtypes were elevated in αERKO and βERKO mice, whereas Nav1.6 mRNA decreased in αERKO, but not in βERKO mice. The mRNA expressions of Nav subtypes were increased in E2-treated WT ovariectomized animals. We also found that E2-regulation of Nav1.1 and Nav1.9 mRNA expressions is dependent on ERα, ERβ, and another ER, whereas E2-regulation of Nav1.8 appears to be in an ERβ-dependent manner. PMID:22169964

  3. G(i)-dependent localization of beta(2)-adrenergic receptor signaling to L-type Ca(2+) channels.

    PubMed Central

    Chen-Izu, Y; Xiao, R P; Izu, L T; Cheng, H; Kuschel, M; Spurgeon, H; Lakatta, E G

    2000-01-01

    A plausible determinant of the specificity of receptor signaling is the cellular compartment over which the signal is broadcast. In rat heart, stimulation of beta(1)-adrenergic receptor (beta(1)-AR), coupled to G(s)-protein, or beta(2)-AR, coupled to G(s)- and G(i)-proteins, both increase L-type Ca(2+) current, causing enhanced contractile strength. But only beta(1)-AR stimulation increases the phosphorylation of phospholamban, troponin-I, and C-protein, causing accelerated muscle relaxation and reduced myofilament sensitivity to Ca(2+). beta(2)-AR stimulation does not affect any of these intracellular proteins. We hypothesized that beta(2)-AR signaling might be localized to the cell membrane. Thus we examined the spatial range and characteristics of beta(1)-AR and beta(2)-AR signaling on their common effector, L-type Ca(2+) channels. Using the cell-attached patch-clamp technique, we show that stimulation of beta(1)-AR or beta(2)-AR in the patch membrane, by adding agonist into patch pipette, both activated the channels in the patch. But when the agonist was applied to the membrane outside the patch pipette, only beta(1)-AR stimulation activated the channels. Thus, beta(1)-AR signaling to the channels is diffusive through cytosol, whereas beta(2)-AR signaling is localized to the cell membrane. Furthermore, activation of G(i) is essential to the localization of beta(2)-AR signaling because in pertussis toxin-treated cells, beta(2)-AR signaling becomes diffusive. Our results suggest that the dual coupling of beta(2)-AR to both G(s)- and G(i)-proteins leads to a highly localized beta(2)-AR signaling pathway to modulate sarcolemmal L-type Ca(2+) channels in rat ventricular myocytes. PMID:11053129

  4. Sensitivity of bronchopulmonary receptors to cold and heat mediated by transient receptor potential cation channel subtypes in an ex vivo rat lung preparation.

    PubMed

    Zhou, Yun; Sun, Biying; Li, Qian; Luo, Pin; Dong, Li; Rong, Weifang

    2011-08-15

    Changes in airway temperature can result in respiratory responses such as cough, bronchoconstriction and mucosal secretion after cold exposure and hyperventilation after heat exposure. In the present investigation, we examined the activity of bronchopulmonary receptors in response to activators of thermo-sensitive transient receptor potential (TS-TRP) cation channels using an ex vivo rat lung preparation. Receptive fields in small bronchioles were probed with von Frey hair monofilaments, warm (50°C) or cold (8°C) saline or saline containing TS-TRP agonists. Among 233 fibers tested, 159 (68.2%) responded to heat (50°C). A large proportion of heat-responsive receptors (107/145) were also activated by capsaicin. Heat and capsaicin-evoked responses were both blocked by TRPV1 antagonist, capsazepine. Only 15.3% of airway receptors responded to cold, which was associated with sensitivity to TRPM8 agonist menthol but not to TRPA1 agonist cinnamaldehyde (CA). Moreover, cold-evoked responses was unaffected by TRPA1 antagonist HC-03001. Our observations suggest that TRPV1 and TRPM8 are involved in transducing heat and cold in the lower respiratory tract, respectively.

  5. Calcium-sensing receptor activation contributed to apoptosis stimulates TRPC6 channel in rat neonatal ventricular myocytes

    SciTech Connect

    Sun, Yi-hua; Li, Yong-quan; Feng, Shan-li; Li, Bao-xin; Pan, Zhen-wei; Xu, Chang-qing; Li, Ting-ting; Yang, Bao-feng

    2010-04-16

    Capacitative calcium entry (CCE) refers to the influx of calcium through plasma membrane channels activated on depletion of endoplasmic sarcoplasmic/reticulum (ER/SR) Ca{sup 2+} stores, which is performed mainly by the transient receptor potential (TRP) channels. TRP channels are expressed in cardiomyocytes. Calcium-sensing receptor (CaR) is also expressed in rat cardiac tissue and plays an important role in mediating cardiomyocyte apoptosis. However, there are no data regarding the link between CaR and TRP channels in rat heart. In this study, in rat neonatal myocytes, by Ca{sup 2+} imaging, we found that the depletion of ER/SR Ca{sup 2+} stores by thapsigargin (TG) elicited a transient rise in cytoplasmic Ca{sup 2+} ([Ca{sup 2+}]{sub i}), followed by sustained increase depending on extracellular Ca{sup 2+}. But, TRP channels inhibitor (SKF96365), not L-type channels or the Na{sup +}/Ca{sup 2+} exchanger inhibitors, inhibited [Ca{sup 2+}]{sub i} relatively high. Then, we found that the stimulation of CaR with its activator gadolinium chloride (GdCl{sub 3}) or by an increased extracellular Ca{sup 2+}([Ca{sup 2+}]{sub o}) increased the concentration of intracelluar Ca{sup 2+}, whereas, the sustained elevation of [Ca{sup 2+}]{sub i} was reduced in the presence of SKF96365. Similarly, the duration of [Ca{sup 2+}]{sub i} increase was also shortened in the absence of extracellular Ca{sup 2+}. Western blot analysis showed that GdCl{sub 3} increased the expression of TRPC6, which was reversed by SKF96365. Additionally, SKF96365 reduced cardiomyocyte apoptosis induced by GdCl{sub 3}. Our results suggested that CCE exhibited in rat neonatal myocytes and CaR activation induced Ca{sup 2+}-permeable cationic channels TRPCs to gate the CCE, for which TRPC6 was one of the most likely candidates. TRPC6 channel was functionally coupled with CaR to enhance the cardiomyocyte apoptosis.

  6. Molecular Insights into the Local Anesthetic Receptor within Voltage-Gated Sodium Channels Using Hydroxylated Analogs of Mexiletine.

    PubMed

    Desaphy, Jean-François; Dipalma, Antonella; Costanza, Teresa; Carbonara, Roberta; Dinardo, Maria Maddalena; Catalano, Alessia; Carocci, Alessia; Lentini, Giovanni; Franchini, Carlo; Camerino, Diana Conte

    2012-01-01

    We previously showed that the β-adrenoceptor modulators, clenbuterol and propranolol, directly blocked voltage-gated sodium channels, whereas salbutamol and nadolol did not (Desaphy et al., 2003), suggesting the presence of two hydroxyl groups on the aromatic moiety of the drugs as a molecular requisite for impeding sodium channel block. To verify such an hypothesis, we synthesized five new mexiletine analogs by adding one or two hydroxyl groups to the aryloxy moiety of the sodium channel blocker and tested these compounds on hNav1.4 channels expressed in HEK293 cells. Concentration-response relationships were constructed using 25-ms-long depolarizing pulses at -30 mV applied from an holding potential of -120 mV at 0.1 Hz (tonic block) and 10 Hz (use-dependent block) stimulation frequencies. The half-maximum inhibitory concentrations (IC(50)) were linearly correlated to drug lipophilicity: the less lipophilic the drug, minor was the block. The same compounds were also tested on F1586C and Y1593C hNav1.4 channel mutants, to gain further information on the molecular interactions of mexiletine with its receptor within the sodium channel pore. In particular, replacement of Phe1586 and Tyr1593 by non-aromatic cysteine residues may help in the understanding of the role of π-π or π-cation interactions in mexiletine binding. Alteration of tonic block suggests that the aryloxy moiety of mexiletine may interact either directly or indirectly with Phe1586 in the closed sodium channel to produce low-affinity binding block, and that this interaction depends on the electrostatic potential of the drug aromatic tail. Alteration of use-dependent block suggests that addition of hydroxyl groups to the aryloxy moiety may modify high-affinity binding of the drug amine terminal to Phe1586 through cooperativity between the two pharmacophores, this effect being mainly related to drug lipophilicity. Mutation of Tyr1593 further impaired such cooperativity. In conclusion, these

  7. Modulation of Voltage-Gated Sodium Channels by Activation of Tumor Necrosis Factor Receptor-1 and Receptor-2 in Small DRG Neurons of Rats.

    PubMed

    Leo, M; Argalski, S; Schäfers, M; Hagenacker, T

    2015-01-01

    Tumor necrosis factor- (TNF-) α is a proinflammatory cytokine involved in the development and maintenance of inflammatory and neuropathic pain. Its effects are mediated by two receptors, TNF receptor-1 (TNFR-1) and TNF receptor-2 (TNFR-2). These receptors play a crucial role in the sensitization of voltage-gated sodium channels (VGSCs), a key mechanism in the pathogenesis of chronic pain. Using the whole-cell patch-clamp technique, we examined the influence of TNFR-1 and TNFR-2 on VGSCs and TTX-resistant NaV1.8 channels in isolated rat dorsal root ganglion neurons by using selective TNFR agonists. The TNFR-1 agonist R32W (10 pg/mL) caused an increase in the VGSC current (I(Na(V))) by 27.2 ± 5.1%, while the TNFR-2 agonist D145 (10 pg/mL) increased the current by 44.9 ± 2.6%. This effect was dose dependent. Treating isolated NaV1.8 with R32W (100 pg/mL) resulted in an increase in I(NaV(1.8)) by 18.9 ± 1.6%, while treatment with D145 (100 pg/mL) increased the current by 14.5 ± 3.7%. Based on the current-voltage relationship, 10 pg of R32W or D145 led to an increase in I(Na(V)) in a bell-shaped, voltage-dependent manner with a maximum effect at -30 mV. The effects of TNFR activation on VGSCs promote excitation in primary afferent neurons and this might explain the sensitization mechanisms associated with neuropathic and inflammatory pain. PMID:26504355

  8. Modulation of Voltage-Gated Sodium Channels by Activation of Tumor Necrosis Factor Receptor-1 and Receptor-2 in Small DRG Neurons of Rats

    PubMed Central

    Leo, M.; Argalski, S.; Schäfers, M.; Hagenacker, T.

    2015-01-01

    Tumor necrosis factor- (TNF-) α is a proinflammatory cytokine involved in the development and maintenance of inflammatory and neuropathic pain. Its effects are mediated by two receptors, TNF receptor-1 (TNFR-1) and TNF receptor-2 (TNFR-2). These receptors play a crucial role in the sensitization of voltage-gated sodium channels (VGSCs), a key mechanism in the pathogenesis of chronic pain. Using the whole-cell patch-clamp technique, we examined the influence of TNFR-1 and TNFR-2 on VGSCs and TTX-resistant NaV1.8 channels in isolated rat dorsal root ganglion neurons by using selective TNFR agonists. The TNFR-1 agonist R32W (10 pg/mL) caused an increase in the VGSC current (INa(V)) by 27.2 ± 5.1%, while the TNFR-2 agonist D145 (10 pg/mL) increased the current by 44.9 ± 2.6%. This effect was dose dependent. Treating isolated NaV1.8 with R32W (100 pg/mL) resulted in an increase in INaV(1.8) by 18.9 ± 1.6%, while treatment with D145 (100 pg/mL) increased the current by 14.5 ± 3.7%. Based on the current-voltage relationship, 10 pg of R32W or D145 led to an increase in INa(V) in a bell-shaped, voltage-dependent manner with a maximum effect at −30 mV. The effects of TNFR activation on VGSCs promote excitation in primary afferent neurons and this might explain the sensitization mechanisms associated with neuropathic and inflammatory pain. PMID:26504355

  9. Single channel properties of P2X ATP receptors in outside-out patches from rat hippocampal granule cells

    PubMed Central

    Wong, Adrian YC; Burnstock, Geoffrey; Gibb, Alasdair J

    2000-01-01

    The single channel properties of P2X ATP receptors were investigated in outside-out patches from hippocampal granule cells in brain slices from 12-day-old rats. The results demonstrate that functional P2X ATP receptors are expressed in hippocampal granule cells and, combined with previously published information on the P2X subunits expressed in the hippocampus, suggest that the receptors may be heteromers of the P2X4 and P2X6 subunits or P2X1, P2X2, P2X4 and P2X6 subunits. Two distinct types of P2X channel openings were observed. A flickery P2X receptor channel was observed in three patches with a mean chord conductance of 32 ± 6 pS, a mean open time of 1.0 ± 0.3 ms and a mean burst length of 11 ± 5 ms at a membrane potential of −60 mV. A large conductance P2X receptor was observed in 19 out of 98 patches with a mean conductance of 56 ± 1.8 pS, a linear current-voltage relationship between −80 and +60 mV with a reversal potential around 0 mV, a mean open time of 2.6 ± 0.2 ms and a mean burst length of 8.8 ± 1.8 ms at −60 mV. At an ATP concentration of 1 mm, these channels exhibited a low steady-state open probability (Popen, 0.07 ± 0.008; n = 15), little apparent desensitisation and were also activated by α,β-methylene ATP (α,β-meATP, 40 μm; Popen, 0.007 ± 0.0002; conductance, 57 ± 1.1 pS; n = 3). No decrease in the single channel conductance was observed on increasing the free extracellular calcium concentration from 0.3 to 0.85 mm. Channel closed time distributions were fitted with five exponential components with time constants (and relative areas) of 90 μs (20%), 0.77 ms (32%), 10 ms (15%), 90 ms (18%) and 403 ms (15%) at 1 mm ATP. Of these, the first two components are suggested to represent gaps within single activations of the receptor based on the lack of agonist concentration dependence of these two shut time components between 1 μm and 1 mm ATP. Suramin (40 μm) significantly increased the single channel conductance (19 ± 7%; n = 5

  10. [Effects of ionotropic glutamate receptor channel blockers on the development of audiogenic seizures in Krushinski-Molodkina rats].

    PubMed

    Lukomskaia, N Ia; Vataev, S I; Zhabko, E P; Magazanik, L G

    2012-04-01

    The action of noncompetitive blockers of glutamate receptors has been investigated on Krushinski-Molodkina rats genetically-prone to audiogenic seizures. The selective blockers of NMDA receptor channels, memantine and IEM-1921, and their dicationic homologues, IEM-1925 and IEM-1754, capable of blocking in varying degrees both NMDA and Ca-permeable AMPA receptor channels, were studied. The drugs were injected intramuscularly to rats with the different time intervals (30 min, 1, 2 or 3 hours) before sound signal. The effects of the drugs on latent period of initial locomotor activity provoked by audio stimulation (8 kHz sine-wave tone, 90 dB volume), the appearance of clonic convulsions of different intensities, and, finally, tonic convulsions with limb and tail extension were evaluated. Within 30 min after injection IEM-1921 at a dose of 5 mg/kg, 33% of rats manifested a complete absence of convulsive reactions to sound, and in 59% of rats audiogenic seizures occured only in the form of motor excitation without a generalized clonic-tonic convulsions. Memantine at a dose of 5 mg/kg did not cause a complete blockade of seizures, but after 1 h of injection in 50% of the rats and after 2 h in 70% of rats a weakening of the audiogenic seizures to the level of motor excitation only was observed. After 3 hrs after administration of blockers its anticonvulsive action weakened significantly (p < 0.01). Dicationic blockers that block both NMDA and AMPA/kainate receptors, IEM-1925 (in doses of 0.001-20.0 mg/kg) and IEM-1754 (0.025-50.0 mg/kg), did not affect audiogenic clonic-tonic convulsive reactions. The involvement of activation of NMDA and calcium permeable AMPA/kainate receptors in the pathogenesis of audiogenic seizures is discussed.

  11. Inositol triphosphate-mediated Ca2+ signals direct purinergic P2Y receptor regulation of neuronal ion channels.

    PubMed

    Zaika, Oleg; Tolstykh, Gleb P; Jaffe, David B; Shapiro, Mark S

    2007-08-15

    Purinergic P2Y receptors are one of four types of G(q/11)-coupled receptors in rat superior cervical ganglia (SCG) sympathetic neurons. In cultured SCG neurons, purinergic and bradykinin suppression of I(M) were similar in magnitude and somewhat less than that by muscarinic agonists. The effects of the P2Y receptor agonist UTP on neuronal excitability and discharge properties were studied. Under current clamp, UTP increased action potential (AP) firing in response to depolarizing current steps, depolarized the resting potential, decreased the threshold current required to fire an AP, and decreased spike-frequency adaptation. These effects were very similar to those resulting from bradykinin stimulation and not as profound as from muscarinic stimulation or full M-current blockade. We then examined the P2Y mechanism of action. Like bradykinin, but unlike muscarinic, purinergic stimulation induced rises in intracellular [Ca(2+)](i). Tests using expression of IP(3)"sponge" or IP(3) phosphatase constructs implicated IP(3) accumulation as necessary for purinergic suppression of I(M). Overexpression of wild-type or dominant-negative calmodulin (CaM) implicated Ca(2+)/CaM in the purinergic action. Both sets of results were similar to bradykinin, and opposite to muscarinic, suppression. We also examined modulation of Ca(2+) channels. As for bradykinin, purinergic stimulation did not suppress I(Ca), unless neuronal calcium sensor-1 (NCS-1) activity was blocked by a dominant-negative NCS-1 construct. Our results indicate that P2Y receptors modulate M-type channels in SCG cells via IP(3)-mediated [Ca(2+)](i) signals in concert with CaM and not by depletion of phosphatidylinositol-4, 5-biphosphate. We group purinergic P2Y and bradykinin B(2) receptors together as having a common mode of action.

  12. Sequence analysis, characterization and mRNA distribution of channel catfish (Ictalurus punctatus Rafinesque, 1818) chemokine (C-X-C Motif) receptor 4 (CXCR4) cDNA

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Chemokine receptor CXCR4, a member of the G protein-coupled receptor superfamily, binds selectively CXCL12. This protein plays many important roles in immunological as well as pathophysiological functions. In this study, we identified and characterized the channel catfish CXCR4 transcript. The fu...

  13. Coxsackievirus and Adenovirus Receptor (CAR) Mediates Trafficking of Acid-Sensing Ion Channel 3 (ASIC3) via PSD-95

    PubMed Central

    Excoffon, Katherine J.D.A.; Kolawole, Abimbola O.; Kusama, Nobuyoshi; Gansemer, Nicholas D.; Sharma, Priyanka; Hruska-Hageman, Alesia M.; Petroff, Elena; Benson, Christopher J.

    2012-01-01

    We have previously shown that the Coxsackievirus and adenovirus receptor (CAR) can interact with post-synaptic density 95 (PSD-95) and localize PSD-95 to cell-cell junctions. We have also shown that activity of the acid-sensing ion channel (ASIC3), a H+-gated cation channel that plays a role in mechanosensation and pain signaling, is negatively modulated by PSD-95 through a PDZ-based interaction. We asked whether CAR and ASIC3 simultaneously interact with PSD-95, and if so, whether co-expression of these proteins alters their cellular distribution and localization. Results indicate that CAR and ASIC3 co-immunoprecipitate only when co-expressed with PSD-95. CAR also brings both PSD-95 and ASIC3 to the junctions of heterologous cells. Moreover, CAR rescues PSD-95-mediated inhibition of ASIC3 currents. These data suggest that, in addition to activity as a viral receptor and adhesion molecule, CAR can play a role in trafficking proteins, including ion channels, in a PDZ-based scaffolding complex. PMID:22809504

  14. Transient Receptor Potential Melastatin 7 Cation Channel Kinase: New Player in Angiotensin II-Induced Hypertension.

    PubMed

    Antunes, Tayze T; Callera, Glaucia E; He, Ying; Yogi, Alvaro; Ryazanov, Alexey G; Ryazanova, Lillia V; Zhai, Alexander; Stewart, Duncan J; Shrier, Alvin; Touyz, Rhian M

    2016-04-01

    Transient receptor potential melastatin 7 (TRPM7) is a bifunctional protein comprising a magnesium (Mg(2+))/cation channel and a kinase domain. We previously demonstrated that vasoactive agents regulate vascular TRPM7. Whether TRPM7 plays a role in the pathophysiology of hypertension and associated cardiovascular dysfunction is unknown. We studied TRPM7 kinase-deficient mice (TRPM7Δkinase; heterozygous for TRPM7 kinase) and wild-type (WT) mice infused with angiotensin II (Ang II; 400 ng/kg per minute, 4 weeks). TRPM7 kinase expression was lower in heart and aorta from TRPM7Δkinase versus WT mice, effects that were further reduced by Ang II infusion. Plasma Mg(2+) was lower in TRPM7Δkinase versus WT mice in basal and stimulated conditions. Ang II increased blood pressure in both strains with exaggerated responses in TRPM7Δkinase versus WT groups (P<0.05). Acetylcholine-induced vasorelaxation was reduced in Ang II-infused TRPM7Δkinase mice, an effect associated with Akt and endothelial nitric oxide synthase downregulation. Vascular cell adhesion molecule-1 expression was increased in Ang II-infused TRPM7 kinase-deficient mice. TRPM7 kinase targets, calpain, and annexin-1, were activated by Ang II in WT but not in TRPM7Δkinase mice. Echocardiographic and histopathologic analysis demonstrated cardiac hypertrophy and left ventricular dysfunction in Ang II-treated groups. In TRPM7 kinase-deficient mice, Ang II-induced cardiac functional and structural effects were amplified compared with WT counterparts. Our data demonstrate that in TRPM7Δkinase mice, Ang II-induced hypertension is exaggerated, cardiac remodeling and left ventricular dysfunction are amplified, and endothelial function is impaired. These processes are associated with hypomagnesemia, blunted TRPM7 kinase expression/signaling, endothelial nitric oxide synthase downregulation, and proinflammatory vascular responses. Our findings identify TRPM7 kinase as a novel player in Ang II-induced hypertension

  15. Transient Receptor Potential Melastatin 7 Cation Channel Kinase: New Player in Angiotensin II-Induced Hypertension.

    PubMed

    Antunes, Tayze T; Callera, Glaucia E; He, Ying; Yogi, Alvaro; Ryazanov, Alexey G; Ryazanova, Lillia V; Zhai, Alexander; Stewart, Duncan J; Shrier, Alvin; Touyz, Rhian M

    2016-04-01

    Transient receptor potential melastatin 7 (TRPM7) is a bifunctional protein comprising a magnesium (Mg(2+))/cation channel and a kinase domain. We previously demonstrated that vasoactive agents regulate vascular TRPM7. Whether TRPM7 plays a role in the pathophysiology of hypertension and associated cardiovascular dysfunction is unknown. We studied TRPM7 kinase-deficient mice (TRPM7Δkinase; heterozygous for TRPM7 kinase) and wild-type (WT) mice infused with angiotensin II (Ang II; 400 ng/kg per minute, 4 weeks). TRPM7 kinase expression was lower in heart and aorta from TRPM7Δkinase versus WT mice, effects that were further reduced by Ang II infusion. Plasma Mg(2+) was lower in TRPM7Δkinase versus WT mice in basal and stimulated conditions. Ang II increased blood pressure in both strains with exaggerated responses in TRPM7Δkinase versus WT groups (P<0.05). Acetylcholine-induced vasorelaxation was reduced in Ang II-infused TRPM7Δkinase mice, an effect associated with Akt and endothelial nitric oxide synthase downregulation. Vascular cell adhesion molecule-1 expression was increased in Ang II-infused TRPM7 kinase-deficient mice. TRPM7 kinase targets, calpain, and annexin-1, were activated by Ang II in WT but not in TRPM7Δkinase mice. Echocardiographic and histopathologic analysis demonstrated cardiac hypertrophy and left ventricular dysfunction in Ang II-treated groups. In TRPM7 kinase-deficient mice, Ang II-induced cardiac functional and structural effects were amplified compared with WT counterparts. Our data demonstrate that in TRPM7Δkinase mice, Ang II-induced hypertension is exaggerated, cardiac remodeling and left ventricular dysfunction are amplified, and endothelial function is impaired. These processes are associated with hypomagnesemia, blunted TRPM7 kinase expression/signaling, endothelial nitric oxide synthase downregulation, and proinflammatory vascular responses. Our findings identify TRPM7 kinase as a novel player in Ang II-induced hypertension

  16. Angiotensin-II receptor antagonist combined with calcium channel blocker or diuretic for essential hypertension.

    PubMed

    Ishimitsu, Toshihiko; Numabe, Atsushi; Masuda, Toshihide; Akabane, Tomoyuki; Okamura, Atsushi; Minami, Junichi; Matsuoka, Hiroaki

    2009-11-01

    To achieve the target blood pressure recommended by the latest guidelines, multiple antihypertensive drugs are needed in most patients. In this study, the efficacy of treatment using an angiotensin II receptor antagonist (ARB) combined with a calcium channel blocker (CCB) or a diuretic was compared from multiple perspectives in patients with hypertension. Twenty-nine patients with essential hypertension, who had failed to achieve their target blood pressure (<130/85 mm Hg for patients <65 years old and <140/90 mm Hg for those >/=65 years) when treated with the ARB olmesartan at 20 mg day(-1), were additionally given 8-16 mg day(-1) of the CCB azelnidipine or 1-2 mg day(-1) of trichlormethiazide (a thiazide diuretic) in a randomized crossover manner for 4 months each. At the end of each combination therapy period, blood and urine samples were collected and arterial stiffness was evaluated by measuring the cardio-ankle pulse wave velocity. Compared with monotherapy, the blood pressure was reduced similarly by adding azelnidipine (-12/-10 mm Hg) or trichlormethiazide (-14/-9 mm Hg). The heart rate was decreased with the CCB by 4 b.p.m. (P<0.05), whereas it was unchanged with the thiazide. Serum K, lipids and blood glucose were not significantly changed with either combination, whereas serum uric acid was increased with the thiazide (P<0.01) but was unchanged with azelnidipine. Plasma levels of renin, angiotensin II and aldosterone were also increased with the thiazide period, whereas high-sensitivity C-reactive protein and oxidized low-density lipoprotein were decreased with azelnidipine. In addition, the cardio-ankle vascular index, a parameter of arterial stiffness, was decreased with the azelnidipine period but was unchanged with the thiazide period (P<0.01). It is suggested that the combination of olmesartan and azelnidipine has advantages over the combination of olmesartan and a thiazide with respect to avoiding hyperuricemia, sympathetic activation, renin

  17. Contribution of the respiratory network to rhythm and motor output revealed by modulation of GIRK channels, somatostatin and neurokinin-1 receptors

    NASA Astrophysics Data System (ADS)

    Montandon, Gaspard; Liu, Hattie; Horner, Richard L.

    2016-09-01

    Breathing is generated by a respiratory network in the brainstem. At its core, a population of neurons expressing neurokinin-1 receptors (NK1R) and the peptide somatostatin (SST) form the preBötzinger Complex (preBötC), a site essential for the generation of breathing. PreBötC interneurons generate rhythm and follower neurons shape motor outputs by activating upper airway respiratory muscles. Since NK1R-expressing preBötC neurons are preferentially inhibited by μ-opioid receptors via activation of GIRK channels, NK1R stimulation may also involve GIRK channels. Hence, we identify the contribution of GIRK channels to rhythm, motor output and respiratory modulation by NK1Rs and SST. In adult rats, GIRK channels were identified in NK1R-expressing preBötC cells. Their activation decreased breathing rate and genioglossus muscle activity, an important upper airway muscle. NK1R activation increased rhythmic breathing and genioglossus muscle activity in wild-type mice, but not in mice lacking GIRK2 subunits (GIRK2‑/‑). Conversely, SST decreased rhythmic breathing via SST2 receptors, reduced genioglossus muscle activity likely through SST4 receptors, but did not involve GIRK channels. In summary, NK1R stimulation of rhythm and motor output involved GIRK channels, whereas SST inhibited rhythm and motor output via two SST receptor subtypes, therefore revealing separate circuits mediating rhythm and motor output.

  18. Contribution of the respiratory network to rhythm and motor output revealed by modulation of GIRK channels, somatostatin and neurokinin-1 receptors

    PubMed Central

    Montandon, Gaspard; Liu, Hattie; Horner, Richard L.

    2016-01-01

    Breathing is generated by a respiratory network in the brainstem. At its core, a population of neurons expressing neurokinin-1 receptors (NK1R) and the peptide somatostatin (SST) form the preBötzinger Complex (preBötC), a site essential for the generation of breathing. PreBötC interneurons generate rhythm and follower neurons shape motor outputs by activating upper airway respiratory muscles. Since NK1R-expressing preBötC neurons are preferentially inhibited by μ-opioid receptors via activation of GIRK channels, NK1R stimulation may also involve GIRK channels. Hence, we identify the contribution of GIRK channels to rhythm, motor output and respiratory modulation by NK1Rs and SST. In adult rats, GIRK channels were identified in NK1R-expressing preBötC cells. Their activation decreased breathing rate and genioglossus muscle activity, an important upper airway muscle. NK1R activation increased rhythmic breathing and genioglossus muscle activity in wild-type mice, but not in mice lacking GIRK2 subunits (GIRK2−/−). Conversely, SST decreased rhythmic breathing via SST2 receptors, reduced genioglossus muscle activity likely through SST4 receptors, but did not involve GIRK channels. In summary, NK1R stimulation of rhythm and motor output involved GIRK channels, whereas SST inhibited rhythm and motor output via two SST receptor subtypes, therefore revealing separate circuits mediating rhythm and motor output. PMID:27599866

  19. Contribution of the respiratory network to rhythm and motor output revealed by modulation of GIRK channels, somatostatin and neurokinin-1 receptors.

    PubMed

    Montandon, Gaspard; Liu, Hattie; Horner, Richard L

    2016-01-01

    Breathing is generated by a respiratory network in the brainstem. At its core, a population of neurons expressing neurokinin-1 receptors (NK1R) and the peptide somatostatin (SST) form the preBötzinger Complex (preBötC), a site essential for the generation of breathing. PreBötC interneurons generate rhythm and follower neurons shape motor outputs by activating upper airway respiratory muscles. Since NK1R-expressing preBötC neurons are preferentially inhibited by μ-opioid receptors via activation of GIRK channels, NK1R stimulation may also involve GIRK channels. Hence, we identify the contribution of GIRK channels to rhythm, motor output and respiratory modulation by NK1Rs and SST. In adult rats, GIRK channels were identified in NK1R-expressing preBötC cells. Their activation decreased breathing rate and genioglossus muscle activity, an important upper airway muscle. NK1R activation increased rhythmic breathing and genioglossus muscle activity in wild-type mice, but not in mice lacking GIRK2 subunits (GIRK2(-/-)). Conversely, SST decreased rhythmic breathing via SST2 receptors, reduced genioglossus muscle activity likely through SST4 receptors, but did not involve GIRK channels. In summary, NK1R stimulation of rhythm and motor output involved GIRK channels, whereas SST inhibited rhythm and motor output via two SST receptor subtypes, therefore revealing separate circuits mediating rhythm and motor output. PMID:27599866

  20. FKBP12 activates the cardiac ryanodine receptor Ca2+-release channel and is antagonised by FKBP12.6.

    PubMed

    Galfré, Elena; Pitt, Samantha J; Venturi, Elisa; Sitsapesan, Mano; Zaccai, Nathan R; Tsaneva-Atanasova, Krasimira; O'Neill, Stephen; Sitsapesan, Rebecca

    2012-01-01

    Changes in FKBP12.6 binding to cardiac ryanodine receptors (RyR2) are implicated in mediating disturbances in Ca(2+)-homeostasis in heart failure but there is controversy over the functional effects of FKBP12.6 on RyR2 channel gating. We have therefore investigated the effects of FKBP12.6 and another structurally similar molecule, FKBP12, which is far more abundant in heart, on the gating of single sheep RyR2 channels incorporated into planar phospholipid bilayers and on spontaneous waves of Ca(2+)-induced Ca(2+)-release in rat isolated permeabilised cardiac cells. We demonstrate that FKBP12 is a high affinity activator of RyR2, sensitising the channel to cytosolic Ca(2+), whereas FKBP12.6 has very low efficacy, but can antagonise the effects of FKBP12. Mathematical modelling of the data shows the importance of the relative concentrations of FKBP12 and FKBP12.6 in determining RyR2 activity. Consistent with the single-channel results, physiological concentrations of FKBP12 (3 µM) increased Ca(2+)-wave frequency and decreased the SR Ca(2+)-content in cardiac cells. FKBP12.6, itself, had no effect on wave frequency but antagonised the effects of FKBP12.We provide a biophysical analysis of the mechanisms by which FK-binding proteins can regulate RyR2 single-channel gating. Our data indicate that FKBP12, in addition to FKBP12.6, may be important in regulating RyR2 function in the heart. In heart failure, it is possible that an alteration in the dual regulation of RyR2 by FKBP12 and FKBP12.6 may occur. This could contribute towards a higher RyR2 open probability, 'leaky' RyR2 channels and Ca(2+)-dependent arrhythmias. PMID:22363773

  1. Novel role for the transient potential receptor melastatin 4 channel in guinea pig detrusor smooth muscle physiology.

    PubMed

    Smith, Amy C; Hristov, Kiril L; Cheng, Qiuping; Xin, Wenkuan; Parajuli, Shankar P; Earley, Scott; Malysz, John; Petkov, Georgi V

    2013-03-01

    Members of the transient receptor potential (TRP) channel superfamily, including the Ca(2+)-activated monovalent cation-selective TRP melastatin 4 (TRPM4) channel, have been recently identified in the urinary bladder. However, their expression and function at the level of detrusor smooth muscle (DSM) remain largely unexplored. In this study, for the first time we investigated the role of TRPM4 channels in guinea pig DSM excitation-contraction coupling using a multidisciplinary approach encompassing protein detection, electrophysiology, live-cell Ca(2+) imaging, DSM contractility, and 9-phenanthrol, a recently characterized selective inhibitor of the TRPM4 channel. Western blot and immunocytochemistry experiments demonstrated the expression of the TRPM4 channel in whole DSM tissue and freshly isolated DSM cells with specific localization on the plasma membrane. Perforated whole cell patch-clamp recordings and real-time Ca(2+) imaging experiments with fura 2-AM, both using freshly isolated DSM cells, revealed that 9-phenanthrol (30 μM) significantly reduced the cation current and decreased intracellular Ca(2+) levels. 9-Phenanthrol (0.1-30 μM) significantly inhibited spontaneous, 0.1 μM carbachol-induced, 20 mM KCl-induced, and nerve-evoked contractions in guinea pig DSM-isolated strips with IC50 values of 1-7 μM and 70-80% maximum inhibition. 9-Phenanthrol also reduced nerve-evoked contraction amplitude induced by continuous repetitive electrical field stimulation of 10-Hz frequency and shifted the frequency-response curve (0.5-50 Hz) relative to the control. Collectively, our data demonstrate the novel finding that TRPM4 channels are expressed in guinea pig DSM and reveal their critical role in the regulation of guinea pig DSM excitation-contraction coupling.

  2. [Application of Brownian dynamics to the description of transmembrane ion flow as exemplified by the chloride channel of glycine receptor].

    PubMed

    Boronovskiĭ, S E; Nartsissov, Ia R

    2009-01-01

    Using the Brownian dynamics of the movement of hydrated ion in a viscous water solution, a mathematical model has been built, which describes the transport of charged particles through a single protein pore in a lipid membrane. The dependences of transmembrane ion currents on ion concentrations in solution have been obtained. It was shown that, if the geometry of a membrane pore is identical to that of the inner part of the glycine receptor channel and there is no ion selectivity, then the values of both chloride and sodium currents are not greater than 0.5 pA at the physiological concentrations of these ions. If local charge heterogeneity caused by charged amino acid residues of transmembrane protein segments is included into the model calculations, the chloride current increases to about 3.7 pA, which exceeds more than seven times the value for sodium ions under the conditions of the complex channel geometry in the range of physiological concentrations of ions in the solution. The model takes changes in the density of charge distribution both inside the channel and near the protein surface into account. The alteration of pore geometry can be also considered as a parameter at the researcher's option. Thus, the model appears as an effective tool for the description of transmembrane currents for other types of membrane channels.

  3. Transient receptor potential (TRP) channels as a therapeutic target for intervention of respiratory effects and lethality from phosgene.

    PubMed

    Andres, Devon; Keyser, Brian; Benton, Betty; Melber, Ashley; Olivera, Dorian; Holmes, Wesley; Paradiso, Danielle; Anderson, Dana; Ray, Radharaman

    2016-02-26

    Phosgene (CG), a toxic inhalation and industrial hazard, causes bronchoconstriction, vasoconstriction and associated pathological effects that could be life threatening. Ion channels of the transient receptor potential (TRP) family have been identified to act as specific chemosensory molecules in the respiratory tract in the detection, control of adaptive responses and initiation of detrimental signaling cascades upon exposure to various toxic inhalation hazards (TIH); their activation due to TIH exposure may result in broncho- and vasoconstriction. We studied changes in the regulation of intracellular free Ca(2+) concentration ([Ca(2+)]i) in cultures of human bronchial smooth muscle cells (BSMC) and human pulmonary microvascular endothelial cells (HPMEC) exposed to CG (16ppm, 8min), using an air/liquid interface exposure system. CG increased [Ca(2+)]i (p<0.05) in both cell types, The CG-induced [Ca(2+)]i was blocked (p<0.05) by two types of TRP channel blockers, SKF-96365, a general TRP channel blocker, and RR, a general TRPV (vanilloid type) blocker, in both BSMC and HPMEC. These effects correlate with the in vivo efficacies of these compounds to protect against lung injury and 24h lethality from whole body CG inhalation exposure in mice (8-10ppm×20min). Thus the TRP channel mechanism appears to be a potential target for intervention in CG toxicity. PMID:26562769

  4. Homomers of alpha 8 and alpha 7 subunits of nicotinic receptors exhibit similar channel but contrasting binding site properties.

    PubMed

    Gerzanich, V; Anand, R; Lindstrom, J

    1994-02-01

    alpha 8 subunits of alpha-bungarotoxin-sensitive chick neuronal nicotinic acetylcholine receptors expressed in Xenopus oocytes from cRNA are shown to form homomeric, acetylcholine-gated, rapidly desensitizing, inwardly rectifying, Ca(2+)-permeable cation channels similar to those of alpha 7 homomers. alpha 8 forms oligomers of several sizes, of which < 14% are expressed on the oocyte surface, which is less efficient than for alpha 7 homomers. alpha 8 homomers are more sensitive to agonists but less sensitive to antagonists than are alpha 7 homomers, and some agonists for alpha 8 homomers are partial agonists or antagonists for alpha 7 homomers. The pharmacological properties of homomers of alpha 8 and alpha 7 subunits generally reflect those of native alpha 8 and alpha 7 receptors.

  5. Transcranial random noise stimulation-induced plasticity is NMDA-receptor independent but sodium-channel blocker and benzodiazepines sensitive

    PubMed Central

    Chaieb, Leila; Antal, Andrea; Paulus, Walter

    2015-01-01

    Background: Application of transcranial random noise stimulation (tRNS) between 0.1 and 640 Hz of the primary motor cortex (M1) for 10 min induces a persistent excitability increase lasting for at least 60 min. However, the mechanism of tRNS-induced cortical excitability alterations is not yet fully understood. Objective: The main aim of this study was to get first efficacy data with regard to the possible neuronal effect of tRNS. Methods: Single-pulse transcranial magnetic stimulation (TMS) was used to measure levels of cortical excitability before and after combined application of tRNS at an intensity of 1 mA for 10 min stimulation duration and a pharmacological agent (or sham) on eight healthy male participants. Results: The sodium channel blocker carbamazepine showed a tendency toward inhibiting MEPs 5–60 min poststimulation. The GABAA agonist lorazepam suppressed tRNS-induced cortical excitability increases at 0–20 and 60 min time points. The partial NMDA receptor agonist D-cycloserine, the NMDA receptor antagonist dextromethorphan and the D2/D3 receptor agonist ropinirole had no significant effects on the excitability increases seen with tRNS. Conclusions: In contrast to transcranial direct current stimulation (tDCS), aftereffects of tRNS are seem to be not NMDA receptor dependent and can be suppressed by benzodiazepines suggesting that tDCS and tRNS depend upon different mechanisms. PMID:25914617

  6. Prostacyclin attenuates oxidative damage of myocytes by opening mitochondrial ATP-sensitive K+ channels via the EP3 receptor.

    PubMed

    Shinmura, Ken; Tamaki, Kayoko; Sato, Toshiaki; Ishida, Hideyuki; Bolli, Roberto

    2005-05-01

    Prostacyclin (PGI2) and the PGE family alleviate myocardial ischemia-reperfusion injury and limit oxidative damage. The cardioprotective effects of PGI2 have been traditionally ascribed to activation of IP receptors. Recent advances in prostanoid research have revealed that PGI2 can bind not only to IP, but also to EP, receptors, suggesting cross talk between PGI2 and PGEs. The mechanism(s) whereby PGI2 protects myocytes from oxidative damage and the specific receptors involved remain unknown. Thus fresh isolated adult rat myocytes were exposed to 200 microM H2O2 with or without carbaprostacyclin (cPGI2), IP-selective agonists, and ONO-AE-248 (an EP3-selective agonist). Cell viability was assessed by trypan blue exclusion after 30 min of H2O2 superfusion. cPGI2 and ONO-AE-248 significantly improved cell survival during H2O2 superfusion; IP-selective agonists did not. The protective effect of cPGI2 and ONO-AE-248 was completely abrogated by pretreatment with 5-hydroxydecanoate or glibenclamide. In the second series of experiments, the mitochondrial ATP-sensitive K+ (K(ATP)) channel opener diazoxide (Dx) reversibly oxidized flavoproteins in control myocytes. Exposure to prostanoid analogs alone had no effect on flavoprotein fluorescence. A second application of Dx in the presence of cPGI2 or ONO-AE-248 significantly increased flavoprotein fluorescence compared with Dx alone, but IP-selective agonists did not. This study demonstrates that PGI2 analogs protect cardiac myocytes from oxidative stress mainly via activation of EP3. The data also indicate that activation of EP3 receptors primes the opening of mitochondrial K(ATP) channels and that this mechanism is essential for EP3-dependent protection.

  7. Single-channel currents from diethylpyrocarbonate-modified NMDA receptors in cultured rat brain cortical neurons

    PubMed Central

    1995-01-01

    The role of histidine residues in the function of N-methyl-D-aspartate (NMDA)-activated channels was tested with the histidine-modifying reagent diethylpyrocarbonate (DEP) applied to cells and membrane patches from rat brain cortical neurons in culture. Channels in excised outside-out patches that were treated with 3 mM DEP for 15-30 s (pH 6.5) showed an average 3.4-fold potentiation in steady state open probability when exposed to NMDA and glycine. Analysis of the underlying alterations in channel gating revealed no changes in the numbers of kinetic states: distributions of open intervals were fitted with three exponential components, and four components described the shut intervals, in both control and DEP-modified channels. However, the distribution of shut intervals was obviously different after DEP treatment, consistent with the single-channel current record. After modification, the proportion of long shut states was decreased while the time constants were largely unaffected. Burst kinetics reflected these effects with an increase in the average number of openings/burst from 1.5 (control) to 2.2 (DEP), and a decrease in the average interburst interval from 54.1 to 38.2 ms. These effects were most likely due to histidine modification because other reagents (n- acetylimidazole and 2,4,6-trinitrobenzene 1-sulfonic acid) that are specific for residues other than histidine failed to reproduce the effects of DEP, whereas hydroxylamine could restore channel open probability to control levels. In contrast to these effects on channel gating, DEP had no effect on average single-channel conductance or reversal potential under bi-ionic (Na+:Cs+) conditions. Inhibition by zinc was also unaffected by DEP. We propose a channel gating model in which transitions between single- and multi-opening burst modes give rise to the channel activity observed under steady state conditions. When adjusted to account for the effects of DEP, this model suggests that one or more

  8. T Cell Receptor Mediated Calcium Entry Requires Alternatively Spliced Cav1.1 Channels.

    PubMed

    Matza, Didi; Badou, Abdallah; Klemic, Kathryn G; Stein, Judith; Govindarajulu, Usha; Nadler, Monica J; Kinet, Jean-Pierre; Peled, Amnon; Shapira, Oz M; Kaczmarek, Leonard K; Flavell, Richard A

    2016-01-01

    The process of calcium entry in T cells is a multichannel and multi-step process. We have studied the requirement for L-type calcium channels (Cav1.1) α1S subunits during calcium entry after TCR stimulation. High expression levels of Cav1.1 channels were detected in activated T cells. Sequencing and cloning of Cav1.1 channel cDNA from T cells revealed that a single splice variant is expressed. This variant lacks exon 29, which encodes the linker region adjacent to the voltage sensor, but contains five new N-terminal exons that substitute for exons 1 and 2, which are found in the Cav1.1 muscle counterpart. Overexpression studies using cloned T cell Cav1.1 in 293HEK cells (that lack TCR) suggest that the gating of these channels was altered. Knockdown of Cav1.1 channels in T cells abrogated calcium entry after TCR stimulation, suggesting that Cav1.1 channels are controlled by TCR signaling. PMID:26815481

  9. Enhanced activation of the transient receptor potential channel TRPA1 by ajoene, an allicin derivative.

    PubMed

    Yassaka, Ricardo Tsuneo; Inagaki, Hidetoshi; Fujino, Tsuchiyoshi; Nakatani, Kei; Kubo, Tai

    2010-01-01

    TRPA1 is a calcium-permeable, nonselective cation channel expressed in the dorsal root ganglion and trigeminal ganglia nociceptive neurons. It is activated by the pungent compounds in mustard oil (AITC, allyl isothiocyanate), cinnamon (cinnamaldehyde), garlic (allicin), and is believed to mediate the inflammatory actions of environmental irritants and proalgesic agents. Thiosulfinate (allicin) and isothiocyanate (AITC) compounds contain reactive electrophilic chemical groups that react with cysteine residues within the TRPA1 channel N terminus, leading to channel activation. Ajoene also contains reactive electrophilic chemical groups likely to target TRPA1 channel. Here, we have used voltage-clamp recordings to show that TRPA1-responses are enhanced by ajoene application in a Xenopus oocyte expression system. Though ajoene alone did not activate TRPA1, subsequent application of ajoene enhanced the AITC-, allicin- and depolarization-induced responses of TRPA1. Moreover, when increasing concentrations of ajoene were applied along with constant concentrations of allicin or AITC, stronger responses were elicited. These findings suggest that ajoene is a novel TRPA1 channel enhancer, operating in a channel-opening-dependent manner.

  10. Molecular Bases of Multimodal Regulation of a Fungal Transient Receptor Potential (TRP) Channel*

    PubMed Central

    Ihara, Makoto; Hamamoto, Shin; Miyanoiri, Yohei; Takeda, Mitsuhiro; Kainosho, Masatsune; Yabe, Isamu; Uozumi, Nobuyuki; Yamashita, Atsuko

    2013-01-01

    Multimodal activation by various stimuli is a fundamental characteristic of TRP channels. We identified a fungal TRP channel, TRPGz, exhibiting activation by hyperosmolarity, temperature increase, cytosolic Ca2+ elevation, membrane potential, and H2O2 application, and thus it is expected to represent a prototypic multimodal TRP channel. TRPGz possesses a cytosolic C-terminal domain (CTD), primarily composed of intrinsically disordered regions with some regulatory modules, a putative coiled-coil region and a basic residue cluster. The CTD oligomerization mediated by the coiled-coil region is required for the hyperosmotic and temperature increase activations but not for the tetrameric channel formation or other activation modalities. In contrast, the basic cluster is responsible for general channel inhibition, by binding to phosphatidylinositol phosphates. The crystal structure of the presumed coiled-coil region revealed a tetrameric assembly in an offset spiral rather than a canonical coiled-coil. This structure underlies the observed moderate oligomerization affinity enabling the dynamic assembly and disassembly of the CTD during channel functions, which are compatible with the multimodal regulation mediated by each functional module. PMID:23553631

  11. T Cell Receptor Mediated Calcium Entry Requires Alternatively Spliced Cav1.1 Channels

    PubMed Central

    Matza, Didi; Badou, Abdallah; Klemic, Kathryn G.; Stein, Judith; Govindarajulu, Usha; Nadler, Monica J.; Kinet, Jean-Pierre; Peled, Amnon; Shapira, Oz M.; Kaczmarek, Leonard K.; Flavell, Richard A.

    2016-01-01

    The process of calcium entry in T cells is a multichannel and multi-step process. We have studied the requirement for L-type calcium channels (Cav1.1) α1S subunits during calcium entry after TCR stimulation. High expression levels of Cav1.1 channels were detected in activated T cells. Sequencing and cloning of Cav1.1 channel cDNA from T cells revealed that a single splice variant is expressed. This variant lacks exon 29, which encodes the linker region adjacent to the voltage sensor, but contains five new N-terminal exons that substitute for exons 1 and 2, which are found in the Cav1.1 muscle counterpart. Overexpression studies using cloned T cell Cav1.1 in 293HEK cells (that lack TCR) suggest that the gating of these channels was altered. Knockdown of Cav1.1 channels in T cells abrogated calcium entry after TCR stimulation, suggesting that Cav1.1 channels are controlled by TCR signaling. PMID:26815481

  12. Inhibitory effects of Tyrphostin AG-related compounds on oxidative stress-sensitive transient receptor potential channel activation.

    PubMed

    Toda, Takahiro; Yamamoto, Shinichiro; Yonezawa, Ryo; Mori, Yasuo; Shimizu, Shunichi

    2016-09-01

    Some transient receptor potential (TRP) proteins including TRPA1, TPRM2 and TRPV1 are oxidative stress-sensitive Ca(2+)-permeable channels. Ca(2+) signaling via these TRP channels activated by oxidative stress has been implicated in the aggravation of various inflammatory diseases and pain sensation. We recently reported that Tyrphostin AG490 exerted inhibitory effects on H2O2-induced TRPM2 activation by scavenging the hydroxyl radical. In order to identify stronger inhibitors of oxidative stress-sensitive TRP channels than AG490, we examined the inhibitory effects of Tyrphostin AG-related compounds on H2O2-induced TRP channel activation in human embryonic kidney 293 cells expressing TRP channels. AG555 and AG556 blocked the activation of TRPM2 by H2O2 more strongly than AG490. Regarding TRPV1 and TRPA1, none of the three compounds tested affected H2O2-induced TRPV1 activation; however, AG555 and AG556 reduced H2O2-induced TRPA1 activation more than AG490. Thus, we herein identified AG555 and AG556 as new compounds that exert stronger inhibitory effects on H2O2-induced TRPM2 and TRPA1 activation than AG490. Edaravone, a hydroxyl radical scavenger used in the treatment of cerebral hemorrhage and cerebral infarction, did not affect H2O2-induced TRPM2 or TRPA1 activation. AG555 and AG556 may be useful seed compounds as therapeutic agents for several TRP-related diseases associated with oxidative stress. PMID:27238971

  13. Adenosine A1 receptor signaling inhibits BK channels through a PKCα-dependent mechanism in mouse aortic smooth muscle.

    PubMed

    Kunduri, Ss; Dick, Gm; Nayeem, Ma; Mustafa, Sj

    2013-09-01

    Adenosine receptors (AR; A1, A2A, A2B, and A3) contract and relax smooth muscle through different signaling mechanisms. Deciphering these complex responses remains difficult because relationships between AR subtypes and various end-effectors (e.g., enzymes and ion channels) remain to be identified. A1AR stimulation is associated with the production of 20-hydroxyeicosatetraenoic acid (20-HETE) and activation of protein kinase C (PKC). 20-HETE and PKC can inhibit large conductance Ca(2+)/voltage-sensitive K(+) (BK) channels that regulate smooth muscle contraction. We tested the hypothesis that activation of A1AR inhibits BK channels via a PKC-dependent mechanism. Patch clamp recordings and Western blots were performed using aortae of wild type (WT) and A1AR knockout (A1KO) mice. There were no differences in whole-cell K(+) current or α and β1 subunits expression between WT and A1KO. 20-HETE (100 nM) inhibited BK current similarly in WT and A1KO mice. NECA (5'-N-ethylcarboxamidoadenosine; 10 μM), a non-selective AR agonist, increased BK current in myocytes from both WT and A1KO mice, but the increase was greater in A1KO (52±15 vs. 17±3%; p<0.05). This suggests that A1AR signaling negatively regulates BK channel activity. Accordingly, CCPA (2-chloro-N(6)-cyclopentyladenosine; 100 nM), an A1AR-selective agonist, inhibited BK current in myocytes from WT but not A1KO mice (81±4 vs. 100±7% of control; p<0.05). Gö6976 (100 nM), a PKCα inhibitor, abolished the effect of CCPA to inhibit BK current (99±3% of control). These data lead us to conclude that, in aortic smooth muscle, A1AR inhibits BK channel activity and that this occurs via a mechanism involving PKCα.

  14. Ischemia enhances activation by Ca2+ and redox modification of ryanodine receptor channels from rat brain cortex.

    PubMed

    Bull, Ricardo; Finkelstein, José Pablo; Gálvez, Jorge; Sánchez, Gina; Donoso, Paulina; Behrens, María Isabel; Hidalgo, Cecilia

    2008-09-17

    Cerebral ischemia stimulates Ca2+ influx and thus increases neuronal intracellular free [Ca2+]. Using a rat model of cerebral ischemia without recirculation, we tested whether ischemia enhances the activation by Ca2+ of ryanodine receptor (RyR) channels, a requisite feature of RyR-mediated Ca2+-induced Ca2+ release (CICR). To this aim, we evaluated how single RyR channels from endoplasmic reticulum vesicles, fused into planar lipid bilayers, responded to cytoplasmic [Ca2+] changes. Endoplasmic reticulum vesicles were isolated from the cortex of rat brains incubated without blood flow for 5 min at 37 degrees C (ischemic) or at 4 degrees C (control). Ischemic brains displayed increased oxidative intracellular conditions, as evidenced by a lower ratio (approximately 130:1) of reduced/oxidized glutathione than controls (approximately 200:1). Single RyR channels from ischemic or control brains displayed the same three responses to Ca2+ reported previously, characterized by low, moderate, or high maximal activity. Relative to controls, RyR channels from ischemic brains displayed with increased frequency the high activity response and with lower frequency the low activity response. Both control and ischemic cortical vesicles contained the RyR2 and RyR3 isoforms in a 3:1 proportion, with undetectable amounts of RyR1. Ischemia reduced [3H]ryanodine binding and total RyR protein content by 35%, and increased at least twofold endogenous RyR2 S-nitrosylation and S-glutathionylation without affecting the corresponding RyR3 endogenous levels. In vitro RyR S-glutathionylation but not S-nitrosylation favored the emergence of high activity channels. We propose that ischemia, by enhancing RyR2 S-glutathionylation, allows RyR2 to sustain CICR; the resulting amplification of Ca2+ entry signals may contribute to cortical neuronal death. PMID:18799678

  15. Glutamate receptor-like channels in plants: a role as amino acid sensors in plant defence?

    PubMed Central

    Roberts, Michael R.

    2014-01-01

    Plant glutamate receptor-like genes (GLRs) are homologous to the genes for mammalian ionotropic glutamate receptors (iGluRs), after which they were named, but in the 16 years since their existence was first revealed, progress in elucidating their biological role has been disappointingly slow. Recently, however, studies from a number of laboratories focusing on the model plant species Arabidopsis thaliana (L.) have thrown new light on the functional properties of some members of the GLR gene family. One important finding has been that plant GLR receptors have a much broader ligand specificity than their mammalian iGluR counterparts, with evidence that some individual GLR receptors can be gated by as many as seven amino acids. These results, together with the ubiquity of their expression throughout the plant, open up the possibility that GLR receptors could have a pervasive role in plants as non-specific amino acid sensors in diverse biological processes. Addressing what one of these roles could be, recent studies examining the wound response and disease susceptibility in GLR knockout mutants have provided evidence that some members of clade 3 of the GLR gene family encode important components of the plant's defence response. Ways in which this family of amino acid receptors might contribute to the plant's ability to respond to an attack from pests and pathogens are discussed. PMID:24991414

  16. Mechanisms of activation of nucleus accumbens neurons by cocaine via sigma-1 receptor-inositol 1,4,5-trisphosphate-transient receptor potential canonical channel pathways.

    PubMed

    Barr, Jeffrey L; Deliu, Elena; Brailoiu, G Cristina; Zhao, Pingwei; Yan, Guang; Abood, Mary E; Unterwald, Ellen M; Brailoiu, Eugen

    2015-08-01

    Cocaine promotes addictive behavior primarily by blocking the dopamine transporter, thus increasing dopamine transmission in the nucleus accumbens (nAcc); however, additional mechanisms are continually emerging. Sigma-1 receptors (σ1Rs) are known targets for cocaine, yet the mechanisms underlying σ1R-mediated effects of cocaine are incompletely understood. The present study examined direct effects of cocaine on dissociated nAcc neurons expressing phosphatidylinositol-linked D1 receptors. Endoplasmic reticulum-located σ1Rs and inositol 1,4,5-trisphosphate (IP3) receptors (IP3Rs) were targeted using intracellular microinjection. IP3 microinjection robustly elevated intracellular Ca(2+) concentration, [Ca(2+)]i. While cocaine alone was devoid of an effect, the IP3-induced response was σ1R-dependently enhanced by cocaine co-injection. Likewise, cocaine augmented the [Ca(2+)]i increase elicited by extracellularly applying an IP3-generating molecule (ATP), via σ1Rs. The cocaine-induced enhancement of the IP3/ATP-mediated Ca(2+) elevation occurred at pharmacologically relevant concentrations and was mediated by transient receptor potential canonical channels (TRPC). IP3 microinjection elicited a slight, transient depolarization, further converted to a greatly enhanced, prolonged response, by cocaine co-injection. The cocaine-triggered augmentation was σ1R-dependent, TRPC-mediated and contingent on [Ca(2+)]i elevation. ATP-induced depolarization was similarly enhanced by cocaine. Thus, we identify a novel mechanism by which cocaine promotes activation of D1-expressing nAcc neurons: enhancement of IP3R-mediated responses via σ1R activation at the endoplasmic reticulum, resulting in augmented Ca(2+) release and amplified depolarization due to subsequent stimulation of TRPC. In vivo, intra-accumbal blockade of σ1R or TRPC significantly diminished cocaine-induced hyperlocomotion and locomotor sensitization, endorsing a physio-pathological significance of the pathway

  17. Sildenafil prevents the up-regulation of transient receptor potential canonical channels in the development of cardiomyocyte hypertrophy

    SciTech Connect

    Kiso, Hironori; Ohba, Takayoshi; Iino, Kenji; Sato, Kazuhiro; Terata, Yutaka; Murakami, Manabu; Ono, Kyoichi; Watanabe, Hiroyuki; Ito, Hiroshi

    2013-07-05

    Highlights: •Transient receptor potential canonical (TRPC1, 3 and 6) are up-regulated by ET-1. •Sildenafil inhibited hypertrophic responses (BNP, Ca entry, NFAT activation). •Sildenafil suppressed TRPC1, 3 and 6 expression. -- Abstract: Background: Transient receptor potential canonical (TRPCs) channels are up-regulated in the development of cardiac hypertrophy. Sildenafil inhibits TRPC6 activation and expression, leading to the prevention of cardiac hypertrophy. However, the effects of sildenafil on the expression of other TRPCs remain unknown. We hypothesized that in addition to its effects of TRPC6, sildenafil blocks the up-regulation of other TRPC channels to suppress cardiomyocyte hypertrophy. Methods and results: In cultured neonatal rat cardiomyocytes, a 48 h treatment with 10 nM endothelin (ET)-1 induced hypertrophic responses characterized by nuclear factor of activated T cells activation and enhancement of brain natriuretic peptide expression and cell surface area. Co-treatment with sildenafil (1 μM, 48 h) inhibited these ET-1-induced hypertrophic responses. Although ET-1 enhanced the gene expression of TRPCs, sildenafil inhibited the enhanced gene expression of TRPC1, C3 and C6. Moreover, co-treatment with sildenafil abolished the augmentation of SOCE in the hypertrophied cardiomyocytes. Conclusions: These results suggest that sildenafil inhibits cardiomyocyte hypertrophy by suppressing the up-regulation of TRPC expression.

  18. Schedule of NMDA receptor subunit expression and functional channel formation in the course of in vitro-induced neurogenesis.

    PubMed

    Varju, P; Schlett, K; Eisel, U; Madarász, E

    2001-06-01

    NE-7C2 neuroectodermal cells derived from forebrain vesicles of p53-deficient mouse embryos (E9) produce neurons and astrocytes in vitro if induced by all-trans retinoic acid. The reproducible morphological stages of neurogenesis were correlated with the expression of various NMDA receptor subunits. RT-PCR studies revealed that GluRepsilon1 and GluRepsilon4 subunit mRNAs were transcribed by both non-induced and neuronally differentiated cells. GluRepsilon3 subunit mRNAs were not synthesized by NE-7C2 cells and increased numbers of messages from the GluRepsilon2 gene were detected only after neural network formation. The presence of the GluRzeta1 protein was detected throughout neural induction, whereas retinoic acid-induced neuron formation elevated the amount of exon 21 (C1)- and exon 22 (C2)-containing GluRzeta1 mRNAs and resulted in the appearance of exon 5 (N1)-containing transcripts. NMDA-elicited Ca(2+)-signals were detected only in cells displaying neuronal morphology, but preceding the appearance of synapsin-I immunoreactivity. Our findings demonstrated that, in spite of the presence of subunits necessary for channel formation, functional channels were formed by NE-7C2 cells no sooner than the time of neurite maturation. The data show that the cell line provides a suitable model to analyse the mechanisms involved in NMDA receptor gene expression before the appearance of synaptic communication.

  19. D2 dopamine receptors modulate neuronal resonance in subthalamic nucleus and cortical high-voltage spindles through HCN channels.

    PubMed

    Yang, Chen; Yan, Zhiqiang; Zhao, Bo; Wang, Julei; Gao, Guodong; Zhu, Junling; Wang, Wenting

    2016-06-01

    The high-voltage spindles (HVSs), one of the characteristic oscillations that include theta frequencies in the basal ganglia (BG)-cortical system, are involved in immobile behavior and show increasing power in Parkinson's disease (PD). Our previous results suggested that the D2 dopamine receptor might be involved in HVSs modulations in a rat model of PD. Membrane resonance is one of the cellular mechanisms of network oscillation; therefore, we investigated how dopamine modulates the theta frequency membrane resonance of neurons in the subthalamic nucleus (STN), a central pacemaker of BG, and whether such changes in STN neurons subsequently alter HVSs in the BG-cortical system. In particular, we tested whether dopamine modulates HVSs through hyperpolarization-activated cyclic nucleotide-gated (HCN) channels-dependent membrane resonance in STN neurons. We found that an antagonist of D2 receptors, but not of D1 receptors, inhibited membrane resonance and HCN currents of STN neurons through a G-protein activity in acute brain slices. Our further in vivo experiments using local injection of a D2 receptor antagonist or an HCN blocker in STNs of free-moving rats showed an increase in HVSs power and correlation in the BG-cortical system. Local injection of lamotrigine, an HCN agonist, counteracted the effect induced by the D2 antagonist. Taken together, our results revealed a potential cellular mechanism underlying HVSs activity modulation in the BG-cortical system, i.e. tuning HCN activities in STN neurons through dopamine D2 receptors. Our findings might lead to a new direction in PD treatment by providing promising new drug targets for HVSs activity modulation.

  20. 5-Hydroxytryptamine1A receptor-activation hyperpolarizes pyramidal cells and suppresses hippocampal gamma oscillations via Kir3 channel activation

    PubMed Central

    Johnston, April; McBain, Chris J; Fisahn, André

    2014-01-01

    Rhythmic cortical neuronal oscillations in the gamma frequency band (30–80 Hz, gamma oscillations) have been associated with cognitive processes such as sensory perception and integration, attention, learning, and memory. Gamma oscillations are disrupted in disorders for which cognitive deficits are hallmark symptoms such as schizophrenia and Alzheimer's disease. In vitro, various neurotransmitters have been found to modulate gamma oscillations. Serotonin (5-HT) has long been known to be important for both behavioural and cognitive functions such as learning and memory. Multiple 5-HT receptor subtypes are expressed in the CA3 region of the hippocampus and high doses of 5-HT reduce the power of induced gamma oscillations. Hypothesizing that 5-HT may have cell- and receptor subtype-specific modulatory effects, we investigated the receptor subtypes, cell types and cellular mechanisms engaged by 5-HT in the modulation of gamma oscillations in mice and rats. We found that 5-HT decreases the power of kainate-induced hippocampal gamma oscillations in both species via the 5-HT1A receptor subtype. Whole-cell patch clamp recordings demonstrated that this decrease was caused by a hyperpolarization of CA3 pyramidal cells and a reduction of their firing frequency, but not by alteration of inhibitory neurotransmission. Finally, our results show that the effect on pyramidal cells is mediated via the G protein-coupled receptor inwardly rectifying potassium channel Kir3. Our findings suggest this novel cellular mechanism as a potential target for therapies that are aimed at alleviating cognitive decline by helping the brain to maintain or re-establish normal gamma oscillation levels in neuropsychiatric and neurodegenerative disorders. PMID:25107925

  1. Multiplexing Ligand–Receptor Binding Measurements by Chemically Patterning Microfluidic Channels

    PubMed Central

    Shi, Jinjun; Yang, Tinglu; Cremer, Paul S.

    2012-01-01

    A method has been designed for patterning supported phospholipid bilayers (SLBs) on planar substrates and inside microfluidic channels. To do this, bovine serum albumin (BSA) monolayers were formed via adsorption at the liquid/solid interface. Next, this interfacial protein film was selectively patterned by using deep UV lithography. Subsequently, SLBs could be deposited in the patterned locations by vesicle fusion. By cycling through this process several times, spatially addressed bilayer arrays could be formed with intervening protein molecules serving as two-dimensional corrals. By employing this method, phospholipid bilayers containing various concentrations of ganglioside GM1 were addressed along the length of individual microfluidic channels. Therefore, the binding of GM1 with pentameric cholera toxin B (CTB) subunits could be probed. A seven-channel microfluidic device was fabricated for this purpose. Each channel was simultaneously patterned with four chemically distinct SLBs containing 0, 0.2, 0.5, and 2.0 mol % GM1, respectively. Varying concentrations of CTB were then introduced into each of the channels. With the use of total internal reflection fluorescence microscopy, it was possible to simultaneously abstract multiple equilibrium dissociation constants as a function of ligand density for the CTB-GM1 system in a single shot. PMID:18570383

  2. The Phospholipid-binding Protein SESTD1 Is a Novel Regulator of the Transient Receptor Potential Channels TRPC4 and TRPC5

    PubMed Central

    Miehe, Susanne; Bieberstein, Andrea; Arnould, Isabelle; Ihdene, Orhia; Rütten, Hartmut; Strübing, Carsten

    2010-01-01

    TRPC4 and TRPC5 are two closely related members of the mammalian transient receptor potential cation channel family that have been implicated in important physiological functions, such as growth cone guidance and smooth muscle contraction. To further unravel the role of TRPC4 and TRPC5 in these processes in vivo, detailed information about the molecular composition of native channel complexes and their association with cellular signaling networks is needed. We therefore searched a human aortic cDNA library for novel TRPC4-interacting proteins using a modified yeast two-hybrid assay. This screen identified SESTD1, a previously uncharacterized protein containing a lipid-binding SEC14-like domain as well as spectrin-type cytoskeleton interaction domains. SESTD1 was found to associate with TRPC4 and TRPC5 via the channel's calmodulin- and inositol 1,4,5-trisphosphate receptor-binding domain. In functional studies, we demonstrate that SESTD1 binds several phospholipid species in vitro and is essential for efficient receptor-mediated activation of TRPC5. Notably, phospholipid binding to SESTD1 was Ca2+-dependent. Because TRPC4 and -5 conduct Ca2+, SESTD1-channel signaling may be bidirectional and also couple TRPC activity to lipid signaling through SESTD1. The modulation of TRPC channel function by specific lipid-binding proteins, such as SESTD1, adds another facet to the complex regulation of these channels complementary to the previously described effects of direct channel-phospholipid interaction. PMID:20164195

  3. Point mutations at the local anesthetic receptor site modulate the state-dependent block of rat Na v1.4 sodium channels by pyrazoline-type insecticides.

    PubMed

    Silver, Kristopher S; Soderlund, David M

    2007-05-01

    Pyrazoline-type insecticides (PTIs) selectively block sodium channels at membrane potentials that promote slow sodium channel inactivation and are proposed to interact with a site that overlaps the local anesthetic (LA) receptor site. Mutagenesis studies identified two amino acid residues in the S6 segment of homology domain IV (Phe-1579 and Tyr-1586 in the rat Na(v)1.4 sodium channel) as principal elements of the LA receptor. To test the hypothesis that PTIs bind to the LA receptor, we constructed mutated Na(v)1.4/F1579A and Na(v)1.4/Y1586A cDNAs, expressed native and mutated channels in Xenopus oocytes, and examined the effects of these mutations on channel block by three PTIs (indoxacarb, its bioactivation product DCJW, and RH3421) by two-electrode voltage clamp. DCJW and RH3421 had no effect on Na(v)1.4 channels held at -120mV but caused a slowly developing block upon depolarization to -30mV. Estimated IC(50) values following 15min of exposure were 1 and 4muM for DCJW and RH3421, respectively. Indoxacarb failed to block Na(v)1.4 channels under all experimental conditions. Sensitivity to block by DCJW and RH3421 at -30mV was significantly reduced in Na(v)1.4/F1579A channels, a finding that is consistent with the impact of this mutation on drug binding. In contrast to its effect on drug binding, the Y1586A mutation increased the sensitivity of Na(v)1.4 channels held at -30mV to all three compounds, conferring modest sensitivity to indoxacarb and increasing sensitivity to DCJW and RH3421 by 58- and 16-fold, respectively. These results provide direct evidence for the action of PTIs at the LA receptor.

  4. The apparent voltage dependence of GABAA receptor activation and modulation is inversely related to channel open probability.

    PubMed

    O'Toole, Kate K; Jenkins, Andrew

    2012-02-01

    The GABA type A receptor (GABA(A)R) is expressed ubiquitously throughout the brain and is a target for many therapeutic agents, including general anesthetics and benzodiazepines, which enhance receptor function by increasing the open probability (P(o)) of the ion channel. It is commonplace for in vitro studies of receptor pharmacological characteristics to use negative membrane holding potentials to mimic the resting potential of neurons and symmetrical chloride to eliminate Goldman rectification, which results in chloride flow in the opposite direction, compared with in vivo conditions. This critical difference is usually overlooked because the GABA(A)R has been reported to behave as an ohmic pore, but our results show that the current-voltage relationship is nonlinear with respect to P(o). Specifically, we found that currents were outwardly rectifying at low P(o) and linear at high P(o). We confirmed the correlation between P(o) and rectification with a partial agonist, piperidine-4-sulfonic acid, and a gating-impaired mutation, α1(L277A); both exhibited enhanced outward rectification. Furthermore, this correlation was independent of Goldman rectification and persisted under altered chloride gradient conditions, which suggests that rectification is linked to the direction of chloride flux. Finally, our results showed that the degree of potentiation by general anesthetics (etomidate, propofol, and isoflurane) was greater at negative membrane potentials. Traditional in vitro experiments thus overestimate the action of positive allosteric modulators of the GABA(A)R. Our results show that the direction of the driving force on the permeant ion, as well as P(o), must be considered together for a complete understanding of drug actions on ligand-gated ion channels.

  5. Mutations in ionotropic AMPA receptor 3 alter channel properties and are associated with moderate cognitive impairment in humans.

    PubMed

    Wu, Ye; Arai, Amy C; Rumbaugh, Gavin; Srivastava, Anand K; Turner, Gillian; Hayashi, Takashi; Suzuki, Erika; Jiang, Yuwu; Zhang, Lilei; Rodriguez, Jayson; Boyle, Jackie; Tarpey, Patrick; Raymond, F Lucy; Nevelsteen, Joke; Froyen, Guy; Stratton, Mike; Futreal, Andy; Gecz, Jozef; Stevenson, Roger; Schwartz, Charles E; Valle, David; Huganir, Richard L; Wang, Tao

    2007-11-13

    Ionotropic alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors (iGluRs) mediate the majority of excitatory synaptic transmission in the CNS and are essential for the induction and maintenance of long-term potentiation and long-term depression, two cellular models of learning and memory. We identified a genomic deletion (0.4 Mb) involving the entire GRIA3 (encoding iGluR3) by using an X-array comparative genomic hybridization (CGH) and four missense variants (G833R, M706T, R631S, and R450Q) in functional domains of iGluR3 by sequencing 400 males with X-linked mental retardation (XLMR). Three variants were found in males with moderate MR and were absent in 500 control males. Expression studies in HEK293 cells showed that G833R resulted in a 78% reduction of iGluR3 due to protein misfolding. Whole-cell recording studies of iGluR3 homomers in HEK293 cells revealed that neither iGluR3-M706T (S2 domain) nor iGluR3-R631S (near channel core) had substantial channel function, whereas R450Q (S1 domain) was associated with accelerated receptor desensitization. When forming heteromeric receptors with iGluR2 in HEK293 cells, all four iGluR3 variants had altered desensitization kinetics. Our study provides the genetic and functional evidence that mutant iGluR3 with altered kinetic properties is associated with moderate cognitive impairment in humans.

  6. Mutations in ionotropic AMPA receptor 3 alter channel properties and are associated with moderate cognitive impairment in humans

    PubMed Central

    Wu, Ye; Arai, Amy C.; Rumbaugh, Gavin; Srivastava, Anand K.; Turner, Gillian; Hayashi, Takashi; Suzuki, Erika; Jiang, Yuwu; Zhang, Lilei; Rodriguez, Jayson; Boyle, Jackie; Tarpey, Patrick; Raymond, F. Lucy; Nevelsteen, Joke; Froyen, Guy; Stratton, Mike; Futreal, Andy; Gecz, Jozef; Stevenson, Roger; Schwartz, Charles E.; Valle, David; Huganir, Richard L.; Wang, Tao

    2007-01-01

    Ionotropic α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors (iGluRs) mediate the majority of excitatory synaptic transmission in the CNS and are essential for the induction and maintenance of long-term potentiation and long-term depression, two cellular models of learning and memory. We identified a genomic deletion (0.4 Mb) involving the entire GRIA3 (encoding iGluR3) by using an X-array comparative genomic hybridization (CGH) and four missense variants (G833R, M706T, R631S, and R450Q) in functional domains of iGluR3 by sequencing 400 males with X-linked mental retardation (XLMR). Three variants were found in males with moderate MR and were absent in 500 control males. Expression studies in HEK293 cells showed that G833R resulted in a 78% reduction of iGluR3 due to protein misfolding. Whole-cell recording studies of iGluR3 homomers in HEK293 cells revealed that neither iGluR3-M706T (S2 domain) nor iGluR3-R631S (near channel core) had substantial channel function, whereas R450Q (S1 domain) was associated with accelerated receptor desensitization. When forming heteromeric receptors with iGluR2 in HEK293 cells, all four iGluR3 variants had altered desensitization kinetics. Our study provides the genetic and functional evidence that mutant iGluR3 with altered kinetic properties is associated with moderate cognitive impairment in humans. PMID:17989220

  7. Angiotensin II induces membrane trafficking of natively expressed transient receptor potential vanilloid type 4 channels in hypothalamic 4B cells.

    PubMed

    Saxena, Ashwini; Bachelor, Martha; Park, Yong H; Carreno, Flavia R; Nedungadi, T Prashant; Cunningham, J Thomas

    2014-10-15

    Transient receptor potential vanilloid family type 4 (TRPV4) channels are expressed in central neuroendocrine neurons and have been shown to be polymodal in other systems. We previously reported that in the rodent, a model of dilutional hyponatremia associated with hepatic cirrhosis, TRPV4 expression is increased in lipid rafts from the hypothalamus and that this effect may be angiotensin dependent. In this study, we utilized the immortalized neuroendocrine rat hypothalamic 4B cell line to more directly test the effects of angiotensin II (ANG II) on TRPV4 expression and function. Our results demonstrate the expression of corticotropin-releasing factor (CRF) transcripts, for sex-determining region Y (SRY) (male genotype), arginine vasopressin (AVP), TRPV4, and ANG II type 1a and 1b receptor in 4B cells. After a 1-h incubation in ANG II (100 nM), 4B cells showed increased TRPV4 abundance in the plasma membrane fraction, and this effect was prevented by the ANG II type 1 receptor antagonist losartan (1 μM) and by a Src kinase inhibitor PP2 (10 μM). Ratiometric calcium imaging experiments demonstrated that ANG II incubation potentiated TRPV4 agonist (GSK 1016790A, 20 nM)-induced calcium influx (control 18.4 ± 2.8% n = 5 and ANG II 80.5 ± 2.4% n = 5). This ANG II-induced increase in calcium influx was also blocked by 1 μM losartan and 10 μM PP2 (losartan 26.4 ± 3.8% n = 5 and PP2 19.7 ± 3.9% n = 5). Our data suggests that ANG II can increase TRPV4 channel membrane expression in 4B cells through its action on AT1R involving a Src kinase pathway. PMID:25080500

  8. The stress protein heat shock cognate 70 (Hsc70) inhibits the Transient Receptor Potential Vanilloid type 1 (TRPV1) channel

    PubMed Central

    Iftinca, Mircea; Flynn, Robyn; Basso, Lilian; Melo, Helvira; Aboushousha, Reem; Taylor, Lauren

    2016-01-01

    Background Specialized cellular defense mechanisms prevent damage from chemical, biological, and physical hazards. The heat shock proteins have been recognized as key chaperones that maintain cell survival against a variety of exogenous and endogenous stress signals including noxious temperature. However, the role of heat shock proteins in nociception remains poorly understood. We carried out an expression analysis of the constitutively expressed 70 kDa heat-shock cognate protein, a member of the stress-induced HSP70 family in lumbar dorsal root ganglia from a mouse model of Complete Freund’s Adjuvant-induced chronic inflammatory pain. We used immunolabeling of dorsal root ganglion neurons, behavioral analysis and patch clamp electrophysiology in both dorsal root ganglion neurons and HEK cells transfected with Hsc70 and Transient Receptor Potential Channels to examine their functional interaction in heat shock stress condition. Results We report an increase in protein levels of Hsc70 in mouse dorsal root ganglia, 3 days post Complete Freund’s Adjuvant injection in the hind paw. Immunostaining of Hsc70 was observed in most of the dorsal root ganglion neurons, including the small size nociceptors immunoreactive to the TRPV1 channel. Standard whole-cell patch-clamp technique was used to record Transient Receptor Potential Vanilloid type 1 current after exposure to heat shock. We found that capsaicin-evoked currents are inhibited by heat shock in dorsal root ganglion neurons and transfected HEK cells expressing Hsc70 and TRPV1. Blocking Hsc70 with matrine or spergualin compounds prevented heat shock-induced inhibition of the channel. We also found that, in contrast to TRPV1, both the cold sensor channels TRPA1 and TRPM8 were unresponsive to heat shock stress. Finally, we show that inhibition of TRPV1 depends on the ATPase activity of Hsc70 and involves the rho-associated protein kinase. Conclusions Our work identified Hsc70 and its ATPase activity as a central

  9. Potential role of transient receptor potential channel M5 in sensing putative pheromones in mouse olfactory sensory neurons.

    PubMed

    Oshimoto, Arisa; Wakabayashi, Yoshihiro; Garske, Anna; Lopez, Roberto; Rolen, Shane; Flowers, Michael; Arevalo, Nicole; Restrepo, Diego

    2013-01-01

    Based on pharmacological studies of chemosensory transduction in transient receptor potential channel M5 (TRPM5) knockout mice it was hypothesized that this channel is involved in transduction for a subset of putative pheromones in mouse olfactory sensory neurons (OSNs). Yet, in the same study an electroolfactogram (EOG) in the mouse olfactory epithelium showed no significant difference in the responses to pheromones (and odors) between wild type and TRPM5 knockout mice. Here we show that the number of OSNs expressing TRPM5 is increased by unilateral naris occlusion. Importantly, EOG experiments show that mice lacking TRPM5 show a decreased response in the occluded epithelia to putative pheromones as opposed to wild type mice that show no change upon unilateral naris occlusion. This evidence indicates that under decreased olfactory sensory input TRPM5 plays a role in mediating putative pheromone transduction. Furthermore, we demonstrate that cyclic nucleotide gated channel A2 knockout (CNGA2-KO) mice that show substantially decreased or absent responses to odors and pheromones also have elevated levels of TRPM5 compared to wild type mice. Taken together, our evidence suggests that TRPM5 plays a role in mediating transduction for putative pheromones under conditions of reduced chemosensory input.

  10. The proposed channel-enzyme transient receptor potential melastatin 2 does not possess ADP ribose hydrolase activity

    PubMed Central

    Iordanov, Iordan; Mihályi, Csaba; Tóth, Balázs; Csanády, László

    2016-01-01

    Transient Receptor Potential Melastatin 2 (TRPM2) is a Ca2+-permeable cation channel essential for immunocyte activation, insulin secretion, and postischemic cell death. TRPM2 is activated by ADP ribose (ADPR) binding to its C-terminal cytosolic NUDT9-homology (NUDT9H) domain, homologous to the soluble mitochondrial ADPR pyrophosphatase (ADPRase) NUDT9. Reported ADPR hydrolysis classified TRPM2 as a channel-enzyme, but insolubility of isolated NUDT9H hampered further investigations. Here we developed a soluble NUDT9H model using chimeric proteins built from complementary polypeptide fragments of NUDT9H and NUDT9. When expressed in E.coli, chimeras containing up to ~90% NUDT9H sequence remained soluble and were affinity-purified. In ADPRase assays the conserved Nudix-box sequence of NUDT9 proved essential for activity (kcat~4-9s-1), that of NUDT9H did not support catalysis. Replacing NUDT9H in full-length TRPM2 with soluble chimeras retained ADPR-dependent channel gating (K1/2~1-5 μM), confirming functionality of chimeric domains. Thus, TRPM2 is not a 'chanzyme'. Chimeras provide convenient soluble NUDT9H models for structural/biochemical studies. DOI: http://dx.doi.org/10.7554/eLife.17600.001 PMID:27383051

  11. The proposed channel-enzyme transient receptor potential melastatin 2 does not possess ADP ribose hydrolase activity.

    PubMed

    Iordanov, Iordan; Mihályi, Csaba; Tóth, Balázs; Csanády, László

    2016-01-01

    Transient Receptor Potential Melastatin 2 (TRPM2) is a Ca(2+)-permeable cation channel essential for immunocyte activation, insulin secretion, and postischemic cell death. TRPM2 is activated by ADP ribose (ADPR) binding to its C-terminal cytosolic NUDT9-homology (NUDT9H) domain, homologous to the soluble mitochondrial ADPR pyrophosphatase (ADPRase) NUDT9. Reported ADPR hydrolysis classified TRPM2 as a channel-enzyme, but insolubility of isolated NUDT9H hampered further investigations. Here we developed a soluble NUDT9H model using chimeric proteins built from complementary polypeptide fragments of NUDT9H and NUDT9. When expressed in E.coli, chimeras containing up to ~90% NUDT9H sequence remained soluble and were affinity-purified. In ADPRase assays the conserved Nudix-box sequence of NUDT9 proved essential for activity (kcat~4-9s(-1)), that of NUDT9H did not support catalysis. Replacing NUDT9H in full-length TRPM2 with soluble chimeras retained ADPR-dependent channel gating (K1/2~1-5 μM), confirming functionality of chimeric domains. Thus, TRPM2 is not a 'chanzyme'. Chimeras provide convenient soluble NUDT9H models for structural/biochemical studies. PMID:27383051

  12. Fragmented inositol 1,4,5-trisphosphate receptors retain tetrameric architecture and form functional Ca2+ release channels.

    PubMed

    Alzayady, Kamil J; Chandrasekhar, Rahul; Yule, David I

    2013-04-19

    Inositol 1,4,5-trisphosphate receptor isoforms are a family of ubiquitously expressed ligand-gated channels encoded by three individual genes. The proteins are localized to membranes of intracellular Ca(2+) stores and play pivotal roles in Ca(2+) homeostasis. Previous studies have demonstrated that IP3R1 is cleaved by the intracellular proteases calpain and caspase both in vivo and in vitro. However, the resultant cleavage products are poorly defined, and the functional consequences of these proteolytic events are not fully understood. We demonstrate that IP3R1 is cleaved during staurosporine-induced apoptosis, yielding N-terminal fragments encompassing the ligand-binding domain and the majority of the central modulatory domain together with a C-terminal fragment containing the channel domain and cytosolic tail. Notably, these fragments remain associated with the membrane after initiation of apoptotic cleavage. Furthermore, when recombinant IP3R1 fragments, corresponding to those predicted to be generated by caspase or calpain cleavage, are stably coexpressed in cells, they physically associate and form functional channels. These data provide novel insights regarding the regulation of IP3R1 during proteolysis and provide direct evidence that polypeptide continuity is not required for IP3R activation and Ca(2+) release.

  13. TREK1 channel blockade induces an antidepressant-like response synergizing with 5-HT1A receptor signaling.

    PubMed

    Ye, Dongqing; Li, Yang; Zhang, Xiangrong; Guo, Fei; Geng, Leiyu; Zhang, Qi; Zhang, Zhijun

    2015-12-01

    Current antidepressants often remain the inadequate efficacy for many depressive patients, which warrant the necessary endeavor to develop the new molecules and targets for treating depression. Recently, the two-pore domain potassium channel TREK1 has been implicated in mood regulation and TREK-1 antagonists could be the promising antidepressant. This study has screened a TREK1 blocker (SID1900) with a satisfactory blood-brain barrier permeation and bioavailability. Electrophysiological research has shown that SID1900 and the previously reported TREK1 blocker (spadin) efficiently blocked TREK-1 current in HEK293 cells and specifically blocked two-pore domain potassium channels in primary-cultured rat hippocampal neurons. SID1900 and spadin induced a significant antidepressant-like response in the rat model of chronic unpredictable mild stress (CUMS). Both two TREK1 blockers substantially increased the firing rate of 5-HT-ergic neurons in the dorsal raphe nuclei (DRN) and PFC of CUMS rats. SID1900 and spadin significantly up-regulated the expression of PKA-pCREB-BDNF signaling in DRN, hippocampus and PFC of CUMS rats, which were enhanced and reversed by a 5-HTR1A agonist (8-OH-DPAT) and antagonist (WAY100635) respectively. The present findings suggested that TREK1 channel blockers posses the substantial antidepressant-like effect and have the potential synergistic effect with 5-HT1A receptor activation through the common CREB-BDNF signal transduction. PMID:26441141

  14. Spexin Enhances Bowel Movement through Activating L-type Voltage-dependent Calcium Channel via Galanin Receptor 2 in Mice

    PubMed Central

    Lin, Cheng-yuan; Zhang, Man; Huang, Tao; Yang, Li-ling; Fu, Hai-bo; Zhao, Ling; Zhong, Linda LD; Mu, Huai-xue; Shi, Xiao-ke; Leung, Christina FP; Fan, Bao-min; Jiang, Miao; Lu, Ai-ping; Zhu, Li-xin; Bian, Zhao-xiang

    2015-01-01

    A novel neuropeptide spexin was found to be broadly expressed in various endocrine and nervous tissues while little is known about its functions. This study investigated the role of spexin in bowel movement and the underlying mechanisms. In functional constipation (FC) patients, serum spexin levels were significantly decreased. Consistently, in starved mice, the mRNA of spexin was significantly decreased in intestine and colon. Spexin injection increased the velocity of carbon powder propulsion in small intestine and decreased the glass beads expulsion time in distal colon in mice. Further, spexin dose-dependently stimulated the intestinal/colonic smooth muscle contraction. Galanin receptor 2 (GALR2) antagonist M871, but not Galanin receptor 3 (GALR3) antagonist SNAP37899, effectively suppressed the stimulatory effects of spexin on intestinal/colonic smooth muscle contraction, which could be eliminated by extracellular [Ca2+] removal and L-type voltage-dependentCa2+ channel (VDCC) inhibitor nifedipine. Besides, spexin dramatically increased the [Ca2+]i in isolated colonic smooth muscle cells. These data indicate that spexin can act on GALR2 receptor to regulate bowel motility by activating L-type VDCC. Our findings provide evidence for important physiological roles of spexin in GI functions. Selective action on spexin pathway might have therapeutic effects on GI diseases with motility disorders. PMID:26160593

  15. TRPM5, a taste-signaling transient receptor potential ion-channel, is a ubiquitous signaling component in chemosensory cells

    PubMed Central

    Kaske, Silke; Krasteva, Gabriele; König, Peter; Kummer, Wolfgang; Hofmann, Thomas; Gudermann, Thomas; Chubanov, Vladimir

    2007-01-01

    Background A growing number of TRP channels have been identified as key players in the sensation of smell, temperature, mechanical forces and taste. TRPM5 is known to be abundantly expressed in taste receptor cells where it participates in sweet, amino acid and bitter perception. A role of TRPM5 in other sensory systems, however, has not been studied so far. Results Here, we systematically investigated the expression of TRPM5 in rat and mouse tissues. Apart from taste buds, where we found TRPM5 to be predominantly localized on the basolateral surface of taste receptor cells, TRPM5 immunoreactivity was seen in other chemosensory organs – the main olfactory epithelium and the vomeronasal organ. Most strikingly, we found solitary TRPM5-enriched epithelial cells in all parts of the respiratory and gastrointestinal tract. Based on their tissue distribution, the low cell density, morphological features and co-immunostaining with different epithelial markers, we identified these cells as brush cells (also known as tuft, fibrillovesicular, multivesicular or caveolated cells). In terms of morphological characteristics, brush cells resemble taste receptor cells, while their origin and biological role are still under intensive debate. Conclusion We consider TRPM5 to be an intrinsic signaling component of mammalian chemosensory organs, and provide evidence for brush cells being an important cellular correlate in the periphery. PMID:17610722

  16. Functional assay for T4 lysozyme-engineered G Protein-Coupled Receptors with an ion channel reporter

    PubMed Central

    Niescierowicz, Katarzyna; Caro, Lydia; Cherezov, Vadim; Vivaudou, Michel; Moreau, Christophe J.

    2013-01-01

    Summary: Structural studies of G protein-coupled receptors (GPCRs) extensively use the insertion of globular soluble protein domains in order to facilitate their crystallization. However, when inserted in the third intracellular loop (i3 loop), the soluble protein domain disrupts their coupling to G proteins and impedes the GPCRs functional characterization by standard G protein-based assays. Therefore, activity tests of crystallization-optimized GPCRs are essentially limited to their ligand binding properties using radioligand binding assays. Functional characterization of additional thermostabilizing mutations requires the insertion of similar mutations in the wild-type receptor to allow G protein-activation tests. We demonstrate that Ion Channel-Coupled Receptor technology is a complementary approach for a comprehensive functional characterization of crystallization-optimized GPCRs and potentially of any engineered GPCR. Ligand-induced conformational changes of the GPCRs are translated into electrical signal and detected by simple current recordings, even though binding of G proteins is sterically blocked by the added soluble protein domain. PMID:24268646

  17. Mechanosensitive ion channels in chara: influence of water channel inhibitors, HgCl2 and ZnCl2, on generation of receptor potential.

    PubMed

    Iwabuchi, Kosei; Kaneko, Toshiyuki; Kikuyama, Munehiro

    2008-01-01

    Characean internodal cells generate receptor potential (DeltaE (m)) in response to mechanical stimuli. Upon a long-lasting stimulus, the cells generated DeltaE (m) at the moment of both compression and decompression, and the amplitude of DeltaE (m) at the moment of decompression, (DeltaE (m))(E), was larger than that at compression. The long-lasting stimulus caused a membrane deformation (DeltaD (m)) having two components, a rapid one, (DeltaD (m))(rapid), at the moment of compression and a slower one, (DeltaD (m))(slow), during the long-lasting compression. We assumed that (DeltaD (m))(slow) might have some causal relation with the larger DeltaE (m) at (DeltaE (m))(E). We treated internodal cells with either HgCl(2) or ZnCl(2), water channel inhibitors, to decrease (DeltaD (m))(slow). Both inhibitors attenuated (DeltaD (m))(slow) during compression. Cells treated with HgCl(2) generated smaller (DeltaE (m))(E) compared to nontreated cells. On the other hand, cells treated with ZnCl(2) never attenuated (DeltaE (m))(E) but, rather, amplified it. Thus, the amplitude of (DeltaD (m))(slow) did not always show tight correlation with the amplitude of (DeltaE (m))(E). Furthermore, when a constant deformation was applied to an internodal cell in a medium with higher or lower osmotic value, a cell having higher turgor always showed a larger (DeltaE (m))(E). Thus, we concluded that changes in tension at the membrane may be the most important factor to induce activation of mechanosensitive Ca(2+) channel.

  18. Serotonin contracts the rat mesenteric artery by inhibiting 4-aminopyridine-sensitive Kv channels via the 5-HT2A receptor and Src tyrosine kinase.

    PubMed

    Sung, Dong Jun; Noh, Hyun Ju; Kim, Jae Gon; Park, Sang Woong; Kim, Bokyung; Cho, Hana; Bae, Young Min

    2013-01-01

    Serotonin (5-hydroxytryptamine (5-HT)) is a neurotransmitter that regulates a variety of functions in the nervous, gastrointestinal and cardiovascular systems. Despite such importance, 5-HT signaling pathways are not entirely clear. We demonstrated previously that 4-aminopyridine (4-AP)-sensitive voltage-gated K(+) (Kv) channels determine the resting membrane potential of arterial smooth muscle cells and that the Kv channels are inhibited by 5-HT, which depolarizes the membranes. Therefore, we hypothesized that 5-HT contracts arteries by inhibiting Kv channels. Here we studied 5-HT signaling and the detailed role of Kv currents in rat mesenteric arteries using patch-clamp and isometric tension measurements. Our data showed that inhibiting 4-AP-sensitive Kv channels contracted arterial rings, whereas inhibiting Ca(2+)-activated K(+), inward rectifier K(+) and ATP-sensitive K(+) channels had little effect on arterial contraction, indicating a central role of Kv channels in the regulation of resting arterial tone. 5-HT-induced arterial contraction decreased significantly in the presence of high KCl or the voltage-gated Ca(2+) channel (VGCC) inhibitor nifedipine, indicating that membrane depolarization and the consequent activation of VGCCs mediate the 5-HT-induced vasoconstriction. The effects of 5-HT on Kv currents and arterial contraction were markedly prevented by the 5-HT2A receptor antagonists ketanserin and spiperone. Consistently, α-methyl 5-HT, a 5-HT2 receptor agonist, mimicked the 5-HT action on Kv channels. Pretreatment with a Src tyrosine kinase inhibitor, 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine, prevented both the 5-HT-mediated vasoconstriction and Kv current inhibition. Our data suggest that 4-AP-sensitive Kv channels are the primary regulator of the resting tone in rat mesenteric arteries. 5-HT constricts the arteries by inhibiting Kv channels via the 5-HT2A receptor and Src tyrosine kinase pathway. PMID:24336234

  19. Functional link between muscarinic receptors and large-conductance Ca2+ -activated K+ channels in freshly isolated human detrusor smooth muscle cells.

    PubMed

    Parajuli, Shankar P; Hristov, Kiril L; Cheng, Qiuping; Malysz, John; Rovner, Eric S; Petkov, Georgi V

    2015-04-01

    Activation of muscarinic acetylcholine receptors (mAChRs) constitutes the primary mechanism for enhancing excitability and contractility of human detrusor smooth muscle (DSM). Since the large-conductance Ca(2+)-activated K(+) (KCa1.1) channels are key regulators of human DSM function, we investigated whether mAChR activation increases human DSM excitability by inhibiting KCa1.1 channels. We used the mAChR agonist, carbachol, to determine the changes in KCa1.1 channel activity upon mAChR activation in freshly isolated human DSM cells obtained from open bladder surgeries using the perforated whole cell and single KCa1.1 channel patch-clamp recordings. Human DSM cells were collected from 29 patients (23 males and 6 females, average age of 65.9 ± 1.5 years). Carbachol inhibited the amplitude and frequency of KCa1.1 channel-mediated spontaneous transient outward currents and spontaneous transient hyperpolarizations, which are triggered by the release of Ca(2+) from ryanodine receptors. Carbachol also caused membrane potential depolarization, which was not observed in the presence of iberiotoxin, a KCa1.1 channel inhibitor, indicating the critical role of the KCa1.1 channels. The potential direct carbachol effects on KCa1.1 channels were examined under conditions of removing the major cellular Ca(2+) sources for KCa1.1 channel activation with pharmacological inhibitors (thapsigargin, ryanodine, and nifedipine). In the presence of these inhibitors, carbachol did not affect the single KCa1.1 channel open probability and mean KCa1.1 channel conductance (cell-attached configuration) or depolarization-induced whole cell steady-state KCa1.1 currents. The data support the concept that mAChR activation triggers indirect functional KCa1.1 channel inhibition mediated by intracellular Ca(2+), thus increasing the excitability in human DSM cells.

  20. Acetylcholine receptor in planar lipid bilayers. Characterization of the channel properties of the purified nicotinic acetylcholine receptor from Torpedo californica reconstituted in planar lipid bilayers.

    PubMed

    Labarca, P; Lindstrom, J; Montal, M

    1984-04-01

    The properties of the channel of the purified acetylcholine receptor (AChR) were investigated after reconstitution in planar lipid bilayers. The time course of the agonist-induced conductance exhibits a transient peak that relaxes to a steady state value. The macroscopic steady state membrane conductance increases with agonist concentration, reaching saturation at 10(-5) M for carbamylcholine (CCh). The agonist-induced membrane conductance was inhibited by d-tubocurarine (50% inhibition, IC50, at approximately 10(-6) M) and hexamethonium (IC50 approximately 10(-5) M). The single channel conductance, gamma, is ohmic and independent of the agonist. At 0.3 M monovalent salt concentrations, gamma = 28 pS for Na+, 30 pS for Rb+, 38 pS for Cs+, and 50 pS for NH+4. The distribution of channel open times was fit by a sum of two exponentials, reflecting the existence of two distinct open states. tau o1 and tau o2, the fast and slow components of the distribution of open times, are independent of the agonist concentration: for CCh this was verified in the range of 10(-6) M less than C less than 10(-3)M. tau 01 and tau o2 are approximately three times longer for suberyldicholine ( SubCh ) than for CCh. tau o1 and tau o2 are moderately voltage dependent, increasing as the applied voltage in the compartment containing agonist is made more positive with respect to the other. At desensitizing concentrations of agonist, the AChR channel openings occurred in a characteristic pattern of sudden paroxysms of channel activity followed by quiescent periods. A local anesthetic derivative of lidocaine ( QX -222) reduced both tau o1 and tau o2. This effect was dependent on both the concentration of QX -222 and the applied voltage. Thus, the AChR purified from Torpedo electric organ and reconstituted in planar lipid bilayers exhibits ion conduction and kinetic and pharmacological properties similar to AChR in intact muscle postsynaptic membranes.

  1. Pharmacological characterization of the Haemonchus contortus GABA-gated chloride channel, Hco-UNC-49: modulation by macrocyclic lactone anthelmintics and a receptor for piperazine.

    PubMed

    Brown, David D R; Siddiqui, Salma Z; Kaji, Mark D; Forrester, Sean G

    2012-04-30

    Invertebrate ligand-gated chloride channels are well recognized as important targets for several insecticides and anthelmintics. Hco-UNC-49 is a GABA-gated chloride channel from the parasitic nematode Haemonchus contortus and is an orthologue to the neuromuscular receptor (Cel-UNC-49) from the free-living nematode Caenorhabditis elegans. While the receptors from the two nematodes are similar in sequence, they exhibit different sensitivities to GABA which may reflect differences in in vivo function. The aim of the current study was to further characterize the pharmacology of the Hco-UNC-49 receptor by examining its sensitivity to various insecticides and anthelmintics using two-electrode voltage clamp. Specifically, the insecticides fipronil and picrotoxin appear to inhibit the channel in a similar manner. The IC(50) of picrotoxin on the homomeric channel was 3.65 ± 0.64 μM and for the heteromeric channel was 134.56 ± 44.12 μM. On the other hand, dieldrin, a well-known insect GABA receptor blocker, had little effect on the UNC-49 channel. The anthelmintics ivermectin and moxidectin both moderately potentiated the activation of Hco-UNC-49 by GABA, while piperazine was able to directly activate both the Hco-UNC-49 homomeric and heteromeric channels with EC(50) values of 6.23 ± 0.45 mM and 5.09 ± 0.32 mM, respectively. This piperazine current was reversibly blocked by picrotoxin which demonstrates that the anthelmintic specifically targets Hco-UNC-49. These results demonstrate that Hco-UNC-49 exhibits binding sites for several molecules including piperazine and macrocyclic lactone anthelmintics. In addition, this is the first report of the heterologous expression and subsequent characterization of a receptor for piperazine.

  2. Blockade of brain mineralocorticoid receptors or Na+ channels prevents sympathetic hyperactivity and improves cardiac function in rats post-MI.

    PubMed

    Huang, Bing S; Leenen, Frans H H

    2005-05-01

    In rats post-myocardial infarction (MI), sympathetic hyperactivity can be prevented by blockade of brain mineralocorticoid receptors (MR). Stimulatory responses to central infusion of aldosterone can be blocked by benzamil and therefore appear to be mediated via Na+ channels, presumably epithelial Na+ channels (ENaC), in the brain. To evaluate this concept of endogenous mineralocorticoids in Wistar rats post-MI, we examined effects of blockade of MR and Na+ channels in the brain. At 3 days after coronary artery ligation, intracerebroventricular infusions were started with spironolactone (400 ng.kg(-1).h(-1)) or its vehicle, or with benzamil (4 microg.kg(-1).h(-1)) or its vehicle, using osmotic minipumps. Rats with sham ligation served as control. After 4 wk, in conscious rats, mean arterial pressure, heart rate, and renal sympathetic nerve activity were recorded at rest and in response to air-jet stress, intracerebroventricular injection of the alpha2-adrenoceptor agonist guanabenz, and intravenous infusion of phenylephrine and nitroprusside for baroreflex function. MI size was similar among the four groups of rats (approximately 31%). In rats treated post-MI with vehicles, cardiac function was decreased, sympathetic reactivity was enhanced, and baroreflex function was impaired. Blockade of brain Na+ channels or brain MR similarly prevented sympathetic hyperactivity and impairment of baroreflex function and improved cardiac function. These findings suggest that in rats post-MI, increased binding of endogenous agonists to MR increases ENaC activity in the brain and thereby leads to sympathetic hyperactivity and progressive left ventricular dysfunction.

  3. Stimulation of calcium-sensing receptors induces endothelium-dependent vasorelaxations via nitric oxide production and activation of IKCa channels

    PubMed Central

    Greenberg, Harry Z.E.; Shi, Jian; Jahan, Kazi S.; Martinucci, Matthew C.; Gilbert, Steven J.; Vanessa Ho, W.-S.; Albert, Anthony P.

    2016-01-01

    Stimulation of vascular calcium-sensing receptors (CaSRs) is reported to induce both constrictions and relaxations. However, cellular mechanisms involved in these responses remain unclear. The present study investigates the effect of stimulating CaSRs on vascular contractility and focuses on the role of the endothelium, nitric oxide (NO) and K+ channels in these responses. In wire myography studies, increasing [Ca2 +]o from 1 mM to 6 mM induced concentration-dependent relaxations of methoxamine pre-contracted rabbit mesenteric arteries. [Ca2 +]o-induced relaxations were dependent on a functional endothelium, and were inhibited by the negative allosteric CaSR modulator Calhex-231. [Ca2 +]o-induced relaxations were reduced by inhibitors of endothelial NO synthase, guanylate cyclase, and protein kinase G. CaSR activation also induced NO production in freshly isolated endothelial cells (ECs) in experiments using the fluorescent NO indicator DAF-FM. Pre-treatment with inhibitors of large (BKCa) and intermediate (IKCa) Ca2 +-activated K+ channels (iberiotoxin and charybdotoxin), and Kv7 channels (linopirdine) also reduced [Ca2 +]o-induced vasorelaxations. Increasing [Ca2 +]o also activated IKCa currents in perforated-patch recordings of isolated mesenteric artery ECs. These findings indicate that stimulation of CaSRs induces endothelium-dependent vasorelaxations which are mediated by two separate pathways involving production of NO and activation of IKCa channels. NO stimulates PKG leading to BKCa activation in vascular smooth muscle cells, whereas IKCa activity contributes to endothelium-derived hyperpolarisations. PMID:26772767

  4. Glycine activated ion channel subunits encoded by ctenophore glutamate receptor genes.

    PubMed

    Alberstein, Robert; Grey, Richard; Zimmet, Austin; Simmons, David K; Mayer, Mark L

    2015-11-01

    Recent genome projects for ctenophores have revealed the presence of numerous ionotropic glutamate receptors (iGluRs) in Mnemiopsis leidyi and Pleurobrachia bachei, among our earliest metazoan ancestors. Sequence alignments and phylogenetic analysis show that these form a distinct clade from the well-characterized AMPA, kainate, and NMDA iGluR subtypes found in vertebrates. Although annotated as glutamate and kainate receptors, crystal structures of the ML032222a and PbiGluR3 ligand-binding domains (LBDs) reveal endogenous glycine in the binding pocket, whereas ligand-binding assays show that glycine binds with nanomolar affinity; biochemical assays and structural analysis establish that glutamate is occluded from the binding cavity. Further analysis reveals ctenophore-specific features, such as an interdomain Arg-Glu salt bridge, present only in subunits that bind glycine, but also a conserved disulfide in loop 1 of the LBD that is found in all vertebrate NMDA but not AMPA or kainate receptors. We hypothesize that ctenophore iGluRs are related to an early ancestor of NMDA receptors, suggesting a common evolutionary path for ctenophores and bilaterian species, and suggest that future work should consider both glycine and glutamate as candidate neurotransmitters in ctenophore species. PMID:26460032

  5. Glycine activated ion channel subunits encoded by ctenophore glutamate receptor genes

    PubMed Central

    Alberstein, Robert; Grey, Richard; Zimmet, Austin; Simmons, David K.; Mayer, Mark L.

    2015-01-01

    Recent genome projects for ctenophores have revealed the presence of numerous ionotropic glutamate receptors (iGluRs) in Mnemiopsis leidyi and Pleurobrachia bachei, among our earliest metazoan ancestors. Sequence alignments and phylogenetic analysis show that these form a distinct clade from the well-characterized AMPA, kainate, and NMDA iGluR subtypes found in vertebrates. Although annotated as glutamate and kainate receptors, crystal structures of the ML032222a and PbiGluR3 ligand-binding domains (LBDs) reveal endogenous glycine in the binding pocket, whereas ligand-binding assays show that glycine binds with nanomolar affinity; biochemical assays and structural analysis establish that glutamate is occluded from the binding cavity. Further analysis reveals ctenophore-specific features, such as an interdomain Arg-Glu salt bridge, present only in subunits that bind glycine, but also a conserved disulfide in loop 1 of the LBD that is found in all vertebrate NMDA but not AMPA or kainate receptors. We hypothesize that ctenophore iGluRs are related to an early ancestor of NMDA receptors, suggesting a common evolutionary path for ctenophores and bilaterian species, and suggest that future work should consider both glycine and glutamate as candidate neurotransmitters in ctenophore species. PMID:26460032

  6. Glycine activated ion channel subunits encoded by ctenophore glutamate receptor genes.

    PubMed

    Alberstein, Robert; Grey, Richard; Zimmet, Austin; Simmons, David K; Mayer, Mark L

    2015-11-01

    Recent genome projects for ctenophores have revealed the presence of numerous ionotropic glutamate receptors (iGluRs) in Mnemiopsis leidyi and Pleurobrachia bachei, among our earliest metazoan ancestors. Sequence alignments and phylogenetic analysis show that these form a distinct clade from the well-characterized AMPA, kainate, and NMDA iGluR subtypes found in vertebrates. Although annotated as glutamate and kainate receptors, crystal structures of the ML032222a and PbiGluR3 ligand-binding domains (LBDs) reveal endogenous glycine in the binding pocket, whereas ligand-binding assays show that glycine binds with nanomolar affinity; biochemical assays and structural analysis establish that glutamate is occluded from the binding cavity. Further analysis reveals ctenophore-specific features, such as an interdomain Arg-Glu salt bridge, present only in subunits that bind glycine, but also a conserved disulfide in loop 1 of the LBD that is found in all vertebrate NMDA but not AMPA or kainate receptors. We hypothesize that ctenophore iGluRs are related to an early ancestor of NMDA receptors, suggesting a common evolutionary path for ctenophores and bilaterian species, and suggest that future work should consider both glycine and glutamate as candidate neurotransmitters in ctenophore species.

  7. Enhanced cytotoxicity in triple-negative and estrogen receptor-positive breast adenocarcinoma cells due to inhibition of the transient receptor potential melastatin-2 channel

    PubMed Central

    KOH, DAVID W.; POWELL, DANIEL P.; BLAKE, STEVEN D.; HOFFMAN, JOY L.; HOPKINS, MANDI M.; FENG, XIAOXING

    2015-01-01

    We previously demonstrated a unique protective role for the transient receptor potential, melastatin-2 (TRPM2) cation channel in breast cancer cells. In the present study, we investigated the chemotherapeutic effects elicited by inhibiting this protective role in metastatic breast adenocarcinoma cells. TRPM2 inhibition led to dose-dependent increases in MDA-MB-231 breast adenocarcinoma cell death after treatment with doxorubicin or the DNA-methylating agent, N-methyl-N'-nitro-N-nitrosoguanidine. Similar results were observed after RNAi silencing of TRPM2 in these cells after doxorubicin treatment. However, TRPM2 RNAi silencing also led to increased MCF-7 breast adenocarcinoma cell death after tamoxifen treatment, yet not in non-cancerous human mammary epithelial cells. These results thus revealed that TRPM2 inhibition selectively increased cytotoxicity in a triple-negative and an estrogen receptor-positive breast cancer cell line, with minimal deleterious effects in non-cancerous breast cells. Analysis of DNA damage revealed enhanced DNA damage levels in MCF-7 cells treated with doxorubicin due to TRPM2 inhibition. Analysis of cell death demonstrated that inhibition of apoptosis, caspase-independent cell death or autophagy failed to significantly reduce cell death induced by TRPM2 inhibition and chemotherapy. These results indicate that TRPM2 inhibition activates alternative pathways of cell death in breast cancer cells. Taken together, our results provide significant evidence that TRPM2 inhibition is a potential strategy to induce triple-negative and estrogen receptor-positive breast adenocarcinoma cell death via alternative cell death pathways. This is expected to provide a basis for inhibiting TRPM2 for the improved treatment of breast cancer, which potentially includes treating breast tumors that are resistant to chemotherapy due to their evasion of apoptosis. PMID:26178079

  8. 2-(1-Hexyn-1-yl)adenosine-induced intraocular hypertension is mediated via K+ channel opening through adenosine A2A receptor in rabbits.

    PubMed

    Konno, Takashi; Uchibori, Takehiro; Nagai, Akihiko; Kogi, Kentaro; Nakahata, Norimichi

    2005-08-22

    The present study was performed to clarify the mechanism of change in intraocular pressure by 2-(1-hexyn-1-yl)adenosine (2-H-Ado), a selective adenosine A2 receptor agonist, in rabbits. 2-H-Ado (0.1%, 50 microl)-induced ocular hypertension (E(max): 7.7 mm Hg) was inhibited by an adenosine A2A receptor antagonist 1,3,7-trimethyl-8-(3-chlorostyryl)xanthine, ATP-sensitive K+ channel blocker glibenclamide or 5-hydroxydecanoic acid, but not by an adenosine A1 receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine, an adenosine A2B receptor antagonist alloxazine or a cyclooxygenase inhibitor indomethacin. The outflow facility induced by 2-H-Ado seems to be independent of increase in intraocular pressure or ATP-sensitive K+ channel. In contrast, the recovery rate in intraocular pressure decreased by hypertonic saline was accelerated by 2-H-Ado, and this response was dependent on ATP-sensitive K+ channel. These results suggest that 2-H-Ado-induced ocular hypertension is mediated via K+ channel opening through adenosine A2A receptor, and this is probably due to aqueous formation, but independent of change in outflow facility or prostaglandin production.

  9. Intracellular P2X receptors as novel calcium release channels and modulators of osmoregulation in Dictyostelium: a comparison of two common laboratory strains.

    PubMed

    Sivaramakrishnan, Venketesh; Fountain, Samuel J

    2013-01-01

    P2X receptors are calcium permeable ligand-gated ion channels activated by ATP. Their role as cell surface receptors for extracellular ATP released physiologically by mammalian cells is well established. However, the cellular function of P2X receptor subtypes that populate the membranes of intracellular compartments is not defined. An initial report described how intracellular P2X receptors control the function of the contractile vacuole, an osmoregulatory organelle in Dictyostelium and other protists, and that genetic disruption of P2X receptors severely impaired cell volume control during hypotonic stress. However, later studies refuted a functional role of intracellular P2X receptors in Dictyostelium. Here we provide evidence that the discrepancies reported between the studies are due to the laboratory strain of Dictyostelium employed, which display different phenotypes in response to hypotonic stress and a varied dependency upon P2X receptors for osmoregulation. We use the recent discovery that intracellular P2X receptors are novel calcium release channels to provide some mechanistic insight in an effort to explain why the strain variance may exist.

  10. PYR/PYL/RCAR Abscisic Acid Receptors Regulate K+ and Cl− Channels through Reactive Oxygen Species-Mediated Activation of Ca2+ Channels at the Plasma Membrane of Intact Arabidopsis Guard Cells1[W][OPEN

    PubMed Central

    Wang, Yizhou; Chen, Zhong-Hua; Zhang, Ben; Hills, Adrian; Blatt, Michael R.

    2013-01-01

    The discovery of the START family of abscisic acid (ABA) receptors places these proteins at the front of a protein kinase/phosphatase signal cascade that promotes stomatal closure. The connection of these receptors to Ca2+ signals evoked by ABA has proven more difficult to resolve, although it has been implicated by studies of the pyrbactin-insensitive pyr1/pyl1/pyl2/pyl4 quadruple mutant. One difficulty is that flux through plasma membrane Ca2+ channels and Ca2+ release from endomembrane stores coordinately elevate cytosolic free Ca2+ concentration ([Ca2+]i) in guard cells, and both processes are facilitated by ABA. Here, we describe a method for recording Ca2+ channels at the plasma membrane of intact guard cells of Arabidopsis (Arabidopsis thaliana). We have used this method to resolve the loss of ABA-evoked Ca2+ channel activity at the plasma membrane in the pyr1/pyl1/pyl2/pyl4 mutant and show the consequent suppression of [Ca2+]i increases in vivo. The basal activity of Ca2+ channels was not affected in the mutant; raising the concentration of Ca2+ outside was sufficient to promote Ca2+ entry, to inactivate current carried by inward-rectifying K+ channels and to activate current carried by the anion channels, both of which are sensitive to [Ca2+]i elevations. However, the ABA-dependent increase in reactive oxygen species (ROS) was impaired. Adding the ROS hydrogen peroxide was sufficient to activate the Ca2+ channels and trigger stomatal closure in the mutant. These results offer direct evidence of PYR/PYL/RCAR receptor coupling to the activation by ABA of plasma membrane Ca2+ channels through ROS, thus affecting [Ca2+]i and its regulation of stomatal closure. PMID:23899646

  11. P2Y2 receptor activation decreases blood pressure via intermediate conductance potassium channels and connexin 37

    PubMed Central

    Dominguez Rieg, J. A.; Burt, J. M.; Ruth, P.; Rieg, T.

    2015-01-01

    Aims Nucleotides are important paracrine regulators of vascular tone. We previously demonstrated that activation of P2Y2 receptors causes an acute, NO-independent decrease in blood pressure, indicating this signalling pathway requires an endothelial-derived hyperpolarization (EDH) response. To define the mechanisms by which activation of P2Y2 receptors initiates EDH and vasodilation, we studied intermediate-conductance (KCa3.1, expressed in endothelial cells) and big-conductance potassium channels (KCa1.1, expressed in smooth muscle cells) as well as components of the myoendothelial gap junction, connexins 37 and 40 (Cx37, Cx40), all hypothesized to be part of the EDH response. Methods We compared the effects of a P2Y2/4 receptor agonist in wild-type (WT) mice and in mice lacking KCa3.1, KCa1.1, Cx37 or Cx40 under anaesthesia, while monitoring intra-arterial blood pressure and heart rate. Results Acute activation of P2Y2/4 receptors (0.01–3 mg kg−1 body weight i.v.) caused a biphasic blood pressure response characterized by a dose-dependent and rapid decrease in blood pressure in WT (maximal response % of baseline at 3 mg kg−1: −38 ± 1%) followed by a consecutive increase in blood pressure (+44 ± 11%). The maximal responses in KCa3.1−/− and Cx37−/− were impaired (−13 ± 5, +17 ± 7 and −27 ± 1, +13 ± 3% respectively), whereas the maximal blood pressure decrease in response to acetylcholine at 3 µg kg−1 was not significantly different (WT: −53 ± 3%; KCa3.1−/−: −52 ± 3; Cx37−/−: −53 ± 3%). KCa1.1−/− and Cx40−/− showed an identical biphasic response to P2Y2/4 receptor activation compared to WT. Conclusions The data suggest that the P2Y2/4 receptor activation elicits blood pressure responses via distinct mechanisms involving KCa3.1 and Cx37. PMID:25545736

  12. Polyamine spider toxins and mammalian N-methyl-D-aspartate receptors. Structural basis for channel blocking and binding of argiotoxin636.

    PubMed

    Raditsch, M; Geyer, M; Kalbitzer, H R; Jahn, W; Ruppersberg, J P; Witzemann, V

    1996-09-01

    Recombinant N-methyl-D-aspartate receptors composed of NR1/NR2A subunits were expressed in Xenopus oocytes to analyse the voltage-dependent and use-dependent channel blocking activity of argiotoxin636. Functional assays demonstrate that the toxin competes with other open channel blockers such as Mg2+ and MK-801. Direct binding or competition assays using radiolabeled ligands and isolated rat brain membranes, in contrast, reveal no specific binding or yield binding constants which differ by orders of magnitude from the IC50 values of the functional assays. One explanation is that argiotoxin636 does not bind with high affinity to the inhibitory site in the N-methyl-D-aspartate-receptor channel under in vitro conditions when membranes are depolarised. The structure of argiotoxin636 was investigated by NMR spectroscopy. In solution the positively charged argiotoxin636 acquires an extended conformation and its dimensions might allow permeation deep into the channel. In the absence of direct structural information on the channel protein, the detailed analysis of blockade in conjunction with structural information, as provided here, may be of aid in the deduction of structural features of glutamate-receptor channel ion pores.

  13. N-Methyl-d-Aspartate Receptor Channel Blocker–Like Discriminative Stimulus Effects of Nitrous Oxide Gas

    PubMed Central

    Richardson, Kellianne J.

    2015-01-01

    Nitrous oxide (N2O) gas is a widely used anesthetic adjunct in dentistry and medicine that is also commonly abused. Studies have shown that N2O alters the function of the N-methyl-d-aspartate (NMDA), GABAA, opioid, and serotonin receptors among others. However, the receptors systems underlying the abuse-related central nervous system effects of N2O are unclear. The present study explores the receptor systems responsible for producing the discriminative stimulus effects of N2O. B6SJLF1/J male mice trained to discriminate 10 minutes of exposure to 60% N2O + 40% oxygen versus 100% oxygen served as subjects. Both the high-affinity NMDA receptor channel blocker (+)-MK-801 maleate [(5S,10R)-(+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine maleate] and the low-affinity blocker memantine partially mimicked the stimulus effects of N2O. Neither the competitive NMDA antagonist, CGS-19755 (cis-4-[phosphomethyl]-piperidine-2-carboxylic acid), nor the NMDA glycine-site antagonist, L701-324 [7-chloro-4-hydroxy-3-(3-phenoxy)phenyl-2(1H)-quinolinone], produced N2O-like stimulus effects. A range of GABAA agonists and positive modulators, including midazolam, pentobarbital, muscimol, and gaboxadol (4,5,6,7-tetrahydroisoxazolo[4,5-c]pyridine-3-ol), all failed to produce N2O-like stimulus effects. The μ-, κ-, and δ-opioid agonists, as well as 5-hydroxytryptamine (serotonin) 1B/2C (5-HT1B/2C) and 5-HT1A agonists, also failed to produce N2O-like stimulus effects. Ethanol partially substituted for N2O. Both (+)-MK-801 and ethanol but not midazolam pretreatment also significantly enhanced the discriminative stimulus effects of N2O. Our results support the hypothesis that the discriminative stimulus effects of N2O are at least partially mediated by NMDA antagonist effects similar to those produced by channel blockers. However, as none of the drugs tested fully mimicked the stimulus effects of N2O, other mechanisms may also be involved. PMID:25368340

  14. Anticonvulsant effects of N-arachidonoyl-serotonin, a dual fatty acid amide hydrolase enzyme and transient receptor potential vanilloid type-1 (TRPV1) channel blocker, on experimental seizures: the roles of cannabinoid CB1 receptors and TRPV1 channels.

    PubMed

    Vilela, Luciano R; Medeiros, Daniel C; de Oliveira, Antonio Carlos P; Moraes, Marcio F; Moreira, Fabricio A

    2014-10-01

    Selective blockade of anandamide hydrolysis, through the inhibition of the FAAH enzyme, has anticonvulsant effects, which are mediated by CB1 receptors. Anandamide, however, also activates TRPV1 channels, generally with an opposite outcome on neuronal modulation. Thus, we suggested that the dual FAAH and TRPV1 blockade with N-arachidonoyl-serotonin (AA-5-HT) would be efficacious in inhibiting pentylenetetrazole (PTZ)-induced seizures in mice. We also investigated the contribution of CB1 activation and TRPV1 blockade to the overt effect of AA-5-HT. In the first experiment, injection of AA-5-HT (0.3-3.0 mg/kg) delayed the onset and reduced the duration of PTZ (60 mg)-induced seizures in mice. These effects were reversed by pre-treatment with the CB1 antagonist, AM251 (1.0-3.0 mg/kg). Finally, we observed that administration of the selective TRPV1 antagonist, SB366791 (0.1-1 mg/kg), did not entirely mimic AA-5-HT effects. In conclusion, AA-5-HT alleviates seizures in mice, an effect inhibited by CB1 antagonism, but not completely mimicked by TRPV1 blockage, indicating that the overall effect of AA-5-HT seems to depend mainly on CB1 receptors. This may represent a new strategy for the development of drugs against seizures, epilepsies and related syndromes.

  15. Inhibition of in vivo [(3)H]MK-801 binding by NMDA receptor open channel blockers and GluN2B antagonists in rats and mice.

    PubMed

    Fernandes, Alda; Wojcik, Trevor; Baireddy, Praveena; Pieschl, Rick; Newton, Amy; Tian, Yuan; Hong, Yang; Bristow, Linda; Li, Yu-Wen

    2015-11-01

    N-methyl-D-aspartate (NMDA) receptor antagonists, including open channel blockers and GluN2B receptor subtype selective antagonists, have been developed for the treatment of depression. The current study investigated effects of systemically administered NMDA channel blockers and GluN2B receptor antagonists on NMDA receptor activity in rodents using in vivo [(3)H]MK-801 binding. The receptor occupancy of GluN2B antagonists was measured using ex vivo [(3)H]Ro 25-6981 binding. Ketamine, a NMDA receptor channel blocker, produced a dose/exposure- and time-dependent inhibition of in vivo [(3)H]MK-801 binding that was maximal at ~100%. The complete inhibition of in vivo [(3)H]MK-801 binding was also observed with NMDA receptor channel blockers, AZD6765 (Lanicemine) and MK-801 (Dizocilpine). CP-101,606 (Traxoprodil), a GluN2B antagonist, produced a dose/exposure- and time-dependent inhibition of in vivo [(3)H]MK-801 binding that was maximal at ~60%. Partial inhibition was also observed with other GluN2B antagonists including MK-0657 (CERC-301), EVT-101, Ro 25-6981 and radiprodil. For all GluN2B antagonists tested, partial [(3)H]MK-801 binding inhibition was achieved at doses saturating GluN2B receptor occupancy. Combined treatment with ketamine (10mg/kg, i.p.) and Ro 25-6981(10mg/kg, i.p.) produced a level of inhibition of in vivo [(3)H]MK-801 binding that was similar to treatment with either agent alone. In conclusion, this in vivo [(3)H]MK-801 binding study shows that NMDA receptor activity in the rodent forebrain can be inhibited completely by channel blockers, but only partially (~60%) by GluN2B receptor antagonists. At doses effective in preclinical models of depression, ketamine may preferentially inhibit the same population of NMDA receptors as Ro 25-6981, namely those containing the GluN2B subunit. PMID:26325093

  16. Relationships of agonist properties to the single channel kinetics of nicotinic acetylcholine receptors.

    PubMed Central

    Papke, R L; Millhauser, G; Lieberman, Z; Oswald, R E

    1988-01-01

    The effects of the systematic variations of the acetylcholine molecule on the microscopic kinetics of channel activation were studied using the patch clamp technique. The modifications consisted of adding either halogens or a methyl group to the acetyl carbon of acetylcholine, which results in a change in both the steric and ionic character of that portion of the molecule. The ionic character of the bond affected both the opening and closing rates of the channel. An increase in the ionicity decreased the opening rate and increased the closing rate of the channel, suggesting that the open state was destabilized. Increasing the size of the substituent decreased both the association and dissociation rates for agonist binding but had little effect on the equilibrium constant. This indicates that the energy barrier for binding and unbinding was increased without a major change in the energy of the bound and unbound states. These results suggest that it is possible to assign changes in the structural characteristics of the ligand to changes in individual steps in a reaction scheme, which can lead to specific predictions for the properties of related compounds. PMID:2449251

  17. Peripheral benzodiazepine receptor regulates vascular endothelial activations via suppression of the voltage-dependent anion channel-1.

    PubMed

    Joo, Hee Kyoung; Lee, Yu Ran; Lim, Sun Young; Lee, Eun Ji; Choi, Sunga; Cho, Eun Jung; Park, Myoung Soo; Ryoo, Sungwoo; Park, Jin Bong; Jeon, Byeong Hwa

    2012-05-01

    Peripheral benzodiazepine receptor (PBR) is a multifunctional protein mainly found on the outer mitochondrial membrane. PBR expression is increased by tumor necrosis factor-α (TNF-α) in endothelial cells. Adenoviral overexpression of PBR inhibits monocyte adhesion, VCAM-1, and ICAM-1 expression in TNF-α-activated endothelial cells. Rotenone, cyclosporine A, and bongkrekic acid suppress TNF-α-induced VCAM-1 expression. Overexpression of PBR inhibits voltage-dependent anion channel-1 (VDAC-1) expression and the silencing of PBR increases VDAC-1 expression in endothelial cells. Moreover, TNF-α-induced VCAM-1 expression is suppressed by VDAC-1 gene silencing. PBR overexpression significantly decreases TNF-α-induced mitochondrial reactive oxygen species and MnSOD expression. These results suggest that PBR can inhibit endothelial activation and this action is related to the inhibition of mitochondrial ROS and/or VDAC-1 expression in endothelial cells.

  18. 20-Hydroxyeicosatetraenoic acid (20-HETE) is a novel activator of transient receptor potential vanilloid 1 (TRPV1) channel.

    PubMed

    Wen, Hairuo; Östman, Johan; Bubb, Kristen J; Panayiotou, Catherine; Priestley, John V; Baker, Mark D; Ahluwalia, Amrita

    2012-04-20

    TRPV1 is a member of the transient receptor potential ion channel family and is gated by capsaicin, the pungent component of chili pepper. It is expressed predominantly in small diameter peripheral nerve fibers and is activated by noxious temperatures >42 °C. 20-Hydroxyeicosatetraenoic acid (20-HETE) is a cytochrome P-450 4A/4F-derived metabolite of the membrane phospholipid arachidonic acid. It is a powerful vasoconstrictor and has structural similarities with other TRPV1 agonists, e.g. the hydroperoxyeicosatetraenoic acid 12-HPETE, and we hypothesized that it may be an endogenous ligand for TRPV1 in sensory neurons innervating the vasculature. Here, we demonstrate that 20-HETE both activates and sensitizes mouse and human TRPV1, in a kinase-dependent manner, involving the residue Ser(502) in heterologously expressed hTRPV1, at physiologically relevant concentrations. PMID:22389490

  19. Comparison of behavioral effects of the NMDA receptor channel blockers memantine and ketamine in rats.

    PubMed

    Kotermanski, Shawn E; Johnson, Jon W; Thiels, Edda

    2013-08-01

    Memantine and ketamine block N-methyl-D-aspartate (NMDA) receptors with similar affinity and kinetics, yet their behavioral consequences differ: e.g., memantine is used to alleviate symptoms of Alzheimer's disease, whereas ketamine reproduces symptoms of schizophrenia. The two drugs exhibit different pharmacokinetics, which may play a principal role in their differential behavioral effects. To gain insight into the drugs' behavioral consequences, we treated adult male rats acutely with varying doses (0-40 mg/kg i.p.) of memantine or ketamine and assessed exploratory behavior and spatial working memory. To examine the importance of pharmacokinetics, we assessed behavior either 15 or 45 min after drug administration. Both drugs decreased ambulation, fine movements, and rearing at the beginning of the exploratory activity test; however, at the end of the test, high doses of only memantine increased ambulation and fine movements. High doses of both drugs disrupted spontaneous alternation, a measure of working memory, but high doses of only memantine elicited perseverative behavior. Surprisingly, ketamine's effects were influenced by the delay between drug administration and testing no more frequently than were memantine's. Our findings show that, regardless of test delay, memantine and ketamine evoke similar behavioral effects at lower doses, consistent with NMDA receptors being both drugs' principal site of action, but can have divergent effects at higher doses. Our results suggest that the divergence of memantine's and ketamine's behavioral consequences is likely to result from differences in mechanisms of NMDA receptor antagonism or actions at other targets.

  20. The Transient Receptor Potential (TRP) Channel Family in Colletotrichum graminicola: A Molecular and Physiological Analysis

    PubMed Central

    Lange, Mario; Weihmann, Fabian; Schliebner, Ivo; Horbach, Ralf; Deising, Holger B.; Wirsel, Stefan G. R.

    2016-01-01

    Calcium (Ca2+) is a universal second messenger in all higher organisms and centrally involved in the launch of responses to environmental stimuli. Ca2+ signals in the cytosol are initiated by the activation of Ca2+ channels in the plasma membrane and/or in endomembranes. Yeast (Saccharomyces cerevisiae) contains a Ca2+-permeable channel of the TRP family, TRPY1, which is localized in the vacuolar membrane and contributes to cytosolic free Ca2+ ([Ca2+]cyt) elevations, for example in response to osmotic upshock. A TRPY1 homologue in the rice blast fungus is known to be important for growth and pathogenicity. To determine the role of the TRP channel family in the maize pathogen Colletotrichum graminicola, proteins homologous to TRPY1 were searched. This identified not one, but four genes in the C. graminicola genome, which had putative orthologs in other fungi, and which we named CgTRPF1 through 4. The topology of the CgTRPF proteins resembled that of TRPY1, albeit with a variable number of transmembrane (TM) domains additional to the six-TM-domain core and a diverse arrangement of putatively Ca2+-binding acidic motifs. All CgTRPF genes were expressed in axenic culture and throughout the infection of maize. Like TRPY1, all TRPF proteins of C. graminicola were localized intracellularly, albeit three of them were found not in large vacuoles, but co-localized in vesicular structures. Deletion strains for the CgTRPF genes were not altered in processes thought to involve Ca2+ release from internal stores, i.e. spore germination, the utilization of complex carbon sources, and the generation of tip-focussed [Ca2+]cyt spikes. Heterologous expression of CgTRPF1 through 4 in a tryp1Δ yeast mutant revealed that none of the channels mediated the release of Ca2+ in response to osmotic upshock. Accordingly, aequorin-based [Ca2+]cyt measurements of C. graminicola showed that in this fungus, osmotic upshock-triggered [Ca2+]cyt elevations were generated entirely by influx of Ca2

  1. The Transient Receptor Potential (TRP) Channel Family in Colletotrichum graminicola: A Molecular and Physiological Analysis.

    PubMed

    Lange, Mario; Weihmann, Fabian; Schliebner, Ivo; Horbach, Ralf; Deising, Holger B; Wirsel, Stefan G R; Peiter, Edgar

    2016-01-01

    Calcium (Ca2+) is a universal second messenger in all higher organisms and centrally involved in the launch of responses to environmental stimuli. Ca2+ signals in the cytosol are initiated by the activation of Ca2+ channels in the plasma membrane and/or in endomembranes. Yeast (Saccharomyces cerevisiae) contains a Ca2+-permeable channel of the TRP family, TRPY1, which is localized in the vacuolar membrane and contributes to cytosolic free Ca2+ ([Ca2+]cyt) elevations, for example in response to osmotic upshock. A TRPY1 homologue in the rice blast fungus is known to be important for growth and pathogenicity. To determine the role of the TRP channel family in the maize pathogen Colletotrichum graminicola, proteins homologous to TRPY1 were searched. This identified not one, but four genes in the C. graminicola genome, which had putative orthologs in other fungi, and which we named CgTRPF1 through 4. The topology of the CgTRPF proteins resembled that of TRPY1, albeit with a variable number of transmembrane (TM) domains additional to the six-TM-domain core and a diverse arrangement of putatively Ca2+-binding acidic motifs. All CgTRPF genes were expressed in axenic culture and throughout the infection of maize. Like TRPY1, all TRPF proteins of C. graminicola were localized intracellularly, albeit three of them were found not in large vacuoles, but co-localized in vesicular structures. Deletion strains for the CgTRPF genes were not altered in processes thought to involve Ca2+ release from internal stores, i.e. spore germination, the utilization of complex carbon sources, and the generation of tip-focussed [Ca2+]cyt spikes. Heterologous expression of CgTRPF1 through 4 in a tryp1Δ yeast mutant revealed that none of the channels mediated the release of Ca2+ in response to osmotic upshock. Accordingly, aequorin-based [Ca2+]cyt measurements of C. graminicola showed that in this fungus, osmotic upshock-triggered [Ca2+]cyt elevations were generated entirely by influx of Ca2

  2. Eudistomin D and penaresin derivatives as modulators of ryanodine receptor channels and sarcoplasmic reticulum Ca2+ ATPase in striated muscle.

    PubMed

    Diaz-Sylvester, Paula L; Porta, Maura; Juettner, Vanessa V; Lv, Yuanzhao; Fleischer, Sidney; Copello, Julio A

    2014-04-01

    Eudistomin D (EuD) and penaresin (Pen) derivatives are bioactive alkaloids from marine sponges found to induce Ca(2+) release from striated muscle sarcoplasmic reticulum (SR). Although these alkaloids are believed to affect ryanodine receptor (RyR) gating in a "caffeine-like" manner, no single-channel study confirmed this assumption. Here, EuD and MBED (9-methyl-7-bromoeudistomin D) were contrasted against caffeine on their ability to modulate the SR Ca(2+) loading/leak from cardiac and skeletal muscle SR microsomes as well as the function of RyRs in planar bilayers. The effects of these alkaloids on [(3)H]ryanodine binding and SR Ca(2+) ATPase (SERCA) activity were also tested. MBED (1-5 μM) fully mimicked maximal activating effects of caffeine (20 mM) on SR Ca(2+) leak. At the single-channel level, MBED mimicked the agonistic action of caffeine on cardiac RyR gating (i.e., stabilized long openings characteristic of "high-open-probability" mode). EuD was a partial agonist at the maximal doses tested. The tested Pen derivatives displayed mild to no agonism on RyRs, SR Ca(2+) leak, or [(3)H]ryanodine binding studies. Unlike caffeine, EuD and some Pen derivatives significantly inhibited SERCA at concentrations required to modulate RyRs. Instead, MBED's affinity for RyRs (EC50 ∼ 0.5 μM) was much larger than for SERCA (IC50 > 285 μM). In conclusion, MBED is a potent RyR agonist and, potentially, a better choice than caffeine for microsomal and cell studies due to its reported lack of effects on adenosine receptors and phosphodiesterases. As a high-affinity caffeine-like probe, MBED could also help identify the caffeine-binding site in RyRs. PMID:24423447

  3. A Leukocyte Immune-Type Receptor Subset Is a Marker of Antiviral Cytotoxic Cells in Channel Catfish, Ictalurus punctatus.

    PubMed

    Taylor, Erin B; Moulana, Mohadetheh; Stuge, Tor B; Quiniou, Sylvie M A; Bengten, Eva; Wilson, Melanie

    2016-03-15

    Channel catfish, Ictalurus punctatus, leukocyte immune type receptors (LITRs) represent a multigene family that encodes Ig superfamily proteins that mediate activating or inhibitory signaling. In this study, we demonstrate the use of mAb CC41 to monitor viral cytotoxic responses in catfish and determine that CC41 binds to a subset of LITRs on the surface of catfish clonal CTLs. Homozygous gynogenetic catfish were immunized with channel catfish virus (CCV)-infected MHC-matched clonal T cells (G14D-CCV), and PBL were collected at various times after immunization for flow cytometric analyses. The percentage of CC41(+) cells was significantly increased 5 d after primary immunization with G14D-CCV and at 3 d after a booster immunization as compared with control fish only injected with G14D. Moreover, CC41(+) cells magnetically isolated from the PBL specifically killed CCV-infected targets as measured by (51)Cr release assays and expressed messages for CD3γδ, perforin, and at least one of the CD4-like receptors as analyzed by RNA flow cytometry. When MLC effector cells derived from a G14D-CCV-immunized fish were preincubated with CC41 mAb, killing of G14D-CCV targets was reduced by ∼40%, suggesting that at least some LITRs have a role in target cell recognition and/or cytotoxicity. The availability of a LITR-specific mAb has allowed, to our knowledge for the first time, functional characterization of LITRs in an autologous system. In addition, the identification of an LITR subset as a cytotoxic cell marker will allow for more effective monitoring of catfish immune responses to pathogens. PMID:26856701

  4. Phosphatidylinositol (4,5)-Bisphosphate Regulation of N-Methyl-d-aspartate Receptor Channels in Cortical Neurons

    PubMed Central

    Mandal, Madhuchhanda

    2009-01-01

    The membrane phospholipid phosphatidylinositol (4,5)-bisphosphate (PIP2) has been implicated in the regulation of several ion channels and transporters. In this study, we examined the impact of PIP2 on N-methyl-d-aspartate receptors (NMDARs) in cortical neurons. Blocking PIP2 synthesis by inhibiting phosphoinositide-4 kinase, or stimulating PIP2 hydrolysis via activation of phospholipase C (PLC), or blocking PIP2 function with an antibody caused a significant reduction of NMDAR-mediated currents. On the other hand, inhibition of PLC or application of PIP2 caused an enhancement of NMDAR currents. These electrophysiological effects were accompanied by changes in NMDAR surface clusters induced by agents that manipulate PIP2 levels. The PIP2 regulation of NMDAR currents was abolished by the dynamin inhibitory peptide, which blocks receptor internalization. Agents perturbing actin stability prevented PIP2 regulation of NMDAR currents, suggesting the actin-dependence of this effect of PIP2. Cofilin, a major actin depolymerizing factor, which has a common binding sequence for actin and PIP2, was required for PIP2 regulation of NMDAR currents. It is noteworthy that the PIP2 regulation of NMDAR channels was impaired in a transgenic mouse model of Alzheimer's disease, probably because of the amyloid-β disruption of PIP2 metabolism. Taken together, our data suggest that continuous synthesis of PIP2 at the membrane might be important for the maintenance of NMDARs at the cell surface. When PIP2 is lost, cofilin is released from the PIP2 complex and is rendered free to depolymerize actin. With the actin cytoskeleton no longer intact, NMDARs are internalized via a dynamin/clathrin-dependent mechanism, leading to reduced NMDAR currents. PMID:19770351

  5. The endocannabinoid 2-AG controls skeletal muscle cell differentiation via CB1 receptor-dependent inhibition of Kv7 channels

    PubMed Central

    Iannotti, Fabio A.; Silvestri, Cristoforo; Mazzarella, Enrico; Martella, Andrea; Calvigioni, Daniela; Piscitelli, Fabiana; Ambrosino, Paolo; Petrosino, Stefania; Czifra, Gabriella; Bíró, Tamás; Harkany, Tibor; Taglialatela, Maurizio; Di Marzo, Vincenzo

    2014-01-01

    Little is known of the involvement of endocannabinoids and cannabinoid receptors in skeletal muscle cell differentiation. We report that, due to changes in the expression of genes involved in its metabolism, the levels of the endocannabinoid 2-arachidonoylglycerol (2-AG) are decreased both during myotube formation in vitro from murine C2C12 myoblasts and during mouse muscle growth in vivo. The endocannabinoid, as well as the CB1 agonist arachidonoyl-2-chloroethylamide, prevent myotube formation in a manner antagonized by CB1 knockdown and by CB1 antagonists, which, per se, instead stimulate differentiation. Importantly, 2-AG also inhibits differentiation of primary human satellite cells. Muscle fascicles from CB1 knockout embryos contain more muscle fibers, and postnatal mice show muscle fibers of an increased diameter relative to wild-type littermates. Inhibition of Kv7.4 channel activity, which plays a permissive role in myogenesis and depends on phosphatidylinositol 4,5-bisphosphate (PIP2), underlies the effects of 2-AG. We find that CB1 stimulation reduces both total and Kv7.4-bound PIP2 levels in C2C12 cells and inhibits Kv7.4 currents in transfected CHO cells. We suggest that 2-AG is an endogenous repressor of myoblast differentiation via CB1-mediated inhibition of Kv7.4 channels. PMID:24927567

  6. Simultaneous measurement of Ca2+ influx and reversal potentials in recombinant N-methyl-D-aspartate receptor channels.

    PubMed Central

    Schneggenburger, R

    1996-01-01

    The Ca(2+) permeability of N-methyl-D-aspartate receptor (NMDA-R) channels was studied in human embryonic kidney cells transfected with the NR1-NR2A subunit combination. To determine the fractional Ca(2+) current (P(f)), measurements of fura-2-based Ca(2+) influx and whole-cell currents were made in symmetrical monovalent ion concentrations at membrane potentials between -50 mV and the reversal potential. The ratios of Ca(2+) flux over net whole-cell charge at 2, 5, and 10 mM external Ca(2+) concentrations ([Ca](o)) were identical at a membrane potential close to the reversal potential of the monovalent current component. Assuming unity of P(f) at this potential, the percentage of current carried by Ca(2+) was found to be 18.5 +/- 1.3% at 2 mM [Ca](o) and -50 mV. This value, which is higher than the ones reported previously, was confirmed in independent experiments in which a pure flux of Ca(2+) through NMDA-R channels was used to calibrate the Ca(2+) influx signals. The measured values of fractional Ca(2+) currents, which agree with the predictions of the Goldman-Hodgkin-Katz equations, are also compatible with a two-barrier model for ion permeation, in which the differences between the energy barriers for Ca(2+) and monovalent ions are similar on the external and internal membrane sides. Images FIGURE 5 PMID:9172740

  7. Expression and functionality of transient receptor potential melastatin 4 (TRPM4)-like channels during development of the zebrafish.

    PubMed

    Cheng, Henrique; Ellis, Jayne; Kleinow, Kevin M

    2015-12-01

    Calcium signaling, from localized spikes to coordinated waves, are linked to cleavage, patterning, differentiation, and growth during embryonic development. The basis for control of these Ca(2+) signals is poorly defined. In this study, the expression and functionality of the transient receptor potential melastatin 4 protein (TRPM4), an ion channel that controls Ca(2+) entry into cells, was examined in the zebrafish embryo and adult. Originating with the human TRPM4 gene, Ensembl ortholog, NCBI BLAST, and Homologene searches identified a zebrafish TRPM4 "like" gene encoding a predicted protein of 1199 amino acids and sharing a 42-43% sequence identity with the mouse, rat, and human. Custom-designed zebrafish primers identified TRPM4 transcripts throughout the 0-123h period of embryonic development with greatest and lowest relative expression at 12 and 123h post-fertilization, respectively. Perforated patch clamp recordings in 27h embryonic cells revealed Ca(2+)-activated currents with the characteristics of those described for mammalian TRPM4. Similarly, TRPM4-like expression and functionality was observed in brain and liver cells from adult fish. These findings suggest that a TRPM4-like channel is available for Ca(2+) regulation during early development of the zebrafish. PMID:26432160

  8. Characterization of a ligand-gated cation channel based on an inositol receptor in the silkworm, Bombyx mori.

    PubMed

    Kikuta, Shingo; Endo, Haruka; Tomita, Natsuo; Takada, Tomoyuki; Morita, Chiharu; Asaoka, Kiyoshi; Sato, Ryoichi

    2016-07-01

    Insect herbivores recognize non-volatile compounds in plants to direct their feeding behavior. Gustatory receptors (Gr) appear to be required for nutrient recognition by gustatory organs in the mouthparts of insects. Gr10 is expressed in Bombyx mori (BmGr10) mouthparts such as maxillary galea, maxillary palp, and labrum. BmGr10 is predicted to function in sugar recognition; however, the precise biochemical function remains obscure. Larvae of B. mori are monophagous feeders able to find and feed on mulberry leaves. Soluble mulberry leaf extract contains sucrose, glucose, fructose, and myo-inositol. In this study, we identified BmGr10 as an inositol receptor using electrophysiological analysis with the Xenopus oocyte expression system and Ca(2+) imaging techniques using mammalian cells. These results demonstrated that Xenopus oocytes or HEK293T cells expressing BmGr10 specifically respond to myo-inositol and epi-inositol but do not respond to any mono-, di-, or tri-saccharides or to some sugar alcohols. These inositols caused Ca(2+) and Na(+) influxes into the cytoplasm independently of a G protein-mediated signaling cascade, indicating that BmGr10 is a ligand-gated cation channel. Overall, BmGr10 plays an important role in the myo-inositol recognition required for B. mori larval feeding behavior. PMID:27132146

  9. Pharmacological characterization of muscarinic receptor-activated cation channels in guinea-pig ileum.

    PubMed Central

    Chen, S.; Inoue, R.; Ito, Y.

    1993-01-01

    1. The pharmacological properties of cationic currents activated by acetylcholine (ACh) (Icat) in guinea-pig ileal smooth muscle cells were investigated, with conventional single patch electrode or nystatin-perforated whole-cell recording. Cs-aspartate was used as the internal solution to allow selective measurement of Icat. 2. Well-known K channel blockers, tetraethylammonium (TEA), 4-aminopyridine (4-AP), procaine and quinine as well as a Ca releasing agent, caffeine, all produced concentration-dependent inhibition of Icat with rapid onset (time constant approximately 100 ms), when applied externally. The recovery from the inhibition on washout also occurred rapidly in the order of 100 ms except in the case of quinine. Approximate values of the half inhibitory concentrations (IC50) were 10 nM for TEA and caffeine, 1-5 mM for 4-AP and procaine, and 1 microM for quinine. The mode of inhibition was voltage-dependent, i.e., depolarization relieved the inhibition with no change in reversal potential. 3. Externally applied diphenylamine-2-carboxylate (DPC) derivatives, DCDPC and flufenamic acid, produced potent inhibition of Icat at micromolar concentrations (IC50s were < 30 microM for DCDPC and 32 microM for flufenamic acid). The onset of and recovery from inhibition occurred slowly and the degree of inhibition depended on the membrane potential only weakly, without any discernible change in the reversal potential. 4. All of the above-tested drugs exhibited comparable inhibitory actions on the voltage-dependent Ca current in the concentration ranges effective at inhibiting Icat. However, amongst them, quinine and flufenamic acid seemed to have several-fold better selectivity for the Icat channel than for the voltage-dependent Ca channel.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:7689404

  10. Metabotropic glutamate receptors activate dendritic calcium waves and TRPM channels which drive rhythmic respiratory patterns in mice

    PubMed Central

    Mironov, S L

    2008-01-01

    Respiration in vertebrates is generated by a compact network which is located in the lower brainstem but cellular mechanisms which underlie persistent oscillatory activity of the respiratory network are yet unknown. Using two-photon imaging and patch-clamp recordings in functional brainstem preparations of mice containing pre-Bötzinger complex (preBötC), we examined the actions of metabotropic glutamate receptors (mGluR1/5) on the respiratory patterns. The agonist DHPG potentiated and antagonist LY367385 depressed respiration-related activities. In the inspiratory neurons, we observed rhythmic activation of non-selective channels which had a conductance of 24 pS. Their activity was enhanced with membrane depolarization and after elevation of calcium from the cytoplasmic side of the membrane. They were activated by a non-hydrolysable PIP2 analogue and blocked by flufenamate, ATP4− and Gd3+. All these properties correspond well to those of TRPM4 channels. Calcium imaging of functional slices revealed rhythmic transients in small clusters of neurons present in a network. Calcium transients in the soma were preceded by the waves in dendrites which were dependent on mGluR activation. Initiation and propagation of waves required calcium influx and calcium release from internal stores. Calcium waves activated TPRM4-like channels in the soma and promoted generation of inspiratory bursts. Simulations of activity of neurons communicated via dendritic calcium waves showed emerging activity within neuronal clusters and its synchronization between the clusters. The experimental and theoretical data provide a subcellular basis for a recently proposed group-pacemaker hypothesis and describe a novel mechanism of rhythm generation in neuronal networks. PMID:18308826

  11. Intercellular Odontoblast Communication via ATP Mediated by Pannexin-1 Channel and Phospholipase C-coupled Receptor Activation

    PubMed Central

    Sato, Masaki; Furuya, Tadashi; Kimura, Maki; Kojima, Yuki; Tazaki, Masakazu; Sato, Toru; Shibukawa, Yoshiyuki

    2015-01-01

    Extracellular ATP released via pannexin-1 channels, in response to the activation of mechanosensitive-TRP channels during odontoblast mechanical stimulation, mediates intercellular communication among odontoblasts in dental pulp slice preparation dissected from rat incisor. Recently, odontoblast cell lines, such as mouse odontoblast lineage cells, have been widely used to investigate physiological/pathological cellular functions. To clarify whether the odontoblast cell lines also communicate with each other by diffusible chemical substance(s), we investigated the chemical intercellular communication among cells from mouse odontoblast cell lines following mechanical stimulation. A single cell was stimulated using a glass pipette filled with standard extracellular solution. We measured intracellular free Ca2+ concentration ([Ca2+]i) by fura-2 in stimulated cells, as well as in cells located nearby. Direct mechanical stimulation to a single odontoblast increased [Ca2+]i, which showed sensitivity to capsazepine. In addition, we observed increases in [Ca2+]i not only in the mechanically stimulated odontoblast, but also in nearby odontoblasts. We could observe mechanical stimulation-induced increase in [Ca2+]i in a stimulated human embryo kidney (HEK) 293 cell, but not in nearby HEK293 cells. The increase in [Ca2+]i in nearby odontoblasts, but not in the stimulated odontoblast, was inhibited by adenosine triphosphate (ATP) release channel (pannexin-1) inhibitor in a concentration- and spatial-dependent manner. Moreover, in the presence of phospholipase C (PLC) inhibitor, the increase in [Ca2+]i in nearby odontoblasts, following mechanical stimulation of a single odontoblast, was abolished. We could record some inward currents evoked from odontoblasts near the stimulated odontoblast, but the currents were observed in only 4.8% of the recorded odontoblasts. The results of this study showed that ATP is released via pannexin-1, from a mechanically stimulated odontoblast

  12. Regulation of cyclic nucleotide-gated channels and membrane excitability in olfactory receptor cells by carbon monoxide

    NASA Technical Reports Server (NTRS)

    Leinders-Zufall, T.; Shepherd, G. M.; Zufall, F.

    1995-01-01

    1. The effect of the putative neural messenger carbon monoxide (CO) and the role of the cGMP second-messenger system for olfactory signal generation was examined in isolated olfactory receptor neurons (ORNs) of the tiger salamander. 2. With the use of whole cell voltage-clamp recordings in combination with a series of ionic and pharmological tests, it is demonstrated that exogenously applied CO is a potent activator (K1/2 = 2.9 microM) of cyclic nucleotide-gated (CNG) channels previously described to mediate odor transduction. 3. Several lines of evidence suggest that CO mediates its effect through stimulation of a soluble guanylyl cyclase (sGC) leading to formation of the second-messenger cGMP. This conclusion is based on the findings that CO responses show an absolute requirement for guanosine 5'-triphosphate (GTP) in the internal solution, that no direct effect of CO on CNG currents in the absence of GTP is detectable, and that a blocker of sGC activation, LY85383 (10 microM), completely inhibits the CO response. 4. The dose-response curve for cGMP at CNG channels is used as a calibration to provide a quantitative estimate of the CO-stimulated cGMP formation. This analysis implies that CO is a potent activator of olfactory sGC. 5. Perforated patch recordings using amphotericin B demonstrate that low micromolar doses of CO effectively depolarize the membrane potential of ORNs through tonic activation of CNG channels. This effect in turn regulates excitable and adaptive properties of ORNs and modulates neuronal responsiveness. 6. These data argue for an important role of the cGMP pathway in olfactory signaling and support the idea that CO may function as a diffusible messenger in the olfactory system.

  13. Graded recruitment and inactivation of single InsP3 receptor Ca2+-release channels: implications for quantal [corrected] Ca2+release.

    PubMed

    Ionescu, Lucian; Cheung, King-Ho; Vais, Horia; Mak, Don-On Daniel; White, Carl; Foskett, J Kevin

    2006-06-15

    Modulation of cytoplasmic free Ca2+ concentration ([Ca2+]i) by receptor-mediated generation of inositol 1,4,5-trisphosphate (InsP3) and activation of its receptor (InsP3R), a Ca2+-release channel in the endoplasmic reticulum, is a ubiquitous signalling mechanism. A fundamental aspect of InsP3-mediated signalling is the graded release of Ca2+ in response to incremental levels of stimuli. Ca2+ release has a transient fast phase, whose rate is proportional to [InsP3], followed by a much slower one even in constant [InsP3]. Many schemes have been proposed to account for quantal Ca2+ release, including the presence of heterogeneous channels and Ca2+ stores with various mechanisms of release termination. Here, we demonstrate that mechanisms intrinsic to the single InsP3R channel can account for quantal Ca2+ release. Patch-clamp electrophysiology of isolated insect Sf9 cell nuclei revealed a consistent and high probability of detecting functional endogenous InsP3R channels, enabling InsP3-induced channel inactivation to be identified as an inevitable consequence of activation, and allowing the average number of activated channels in the membrane patch (N(A)) to be accurately quantified. InsP3-activated channels invariably inactivated, with average duration of channel activity reduced by high [Ca2+]i and suboptimal [InsP3]. Unexpectedly, N(A) was found to be a graded function of both [Ca2+]i and [InsP3]. A qualitative model involving Ca2+-induced InsP3R sequestration and inactivation can account for these observations. These results suggest that apparent heterogeneous ligand sensitivity can be generated in a homogeneous population of InsP3R channels, providing a mechanism for graded Ca2+ release that is intrinsic to the InsP3R Ca2+ release channel itself.

  14. Granzyme B-Induced Neurotoxicity Is Mediated via Activation of PAR-1 Receptor and Kv1.3 Channel

    PubMed Central

    Wang, Tongguang; Lee, Myoung-Hwa; Choi, Elliot; Pardo-Villamizar, Carlos A.; Lee, Sung Bin; Yang, In Hong; Calabresi, Peter A.; Nath, Avindra

    2012-01-01

    Increasing evidence supports a critical role of T cells in neurodegeneration associated with acute and subacute brain inflammatory disorders. Granzyme B (GrB), released by activated T cells, is a cytotoxic proteinase which may induce perforin-independent neurotoxicity. Here, we studied the mechanism of perforin-independent GrB toxicity by treating primary cultured human neuronal cells with recombinant GrB. GrBactivated the protease-activated receptor (PAR)-1 receptor on the neuronal cell surface leading to decreased intracellular cyclic AMP levels. This was followed by increased expression and translocation of the voltage gated potassium channel, Kv1.3 to the neuronal cell membrane. Similar expression of Kv1.3 was also seen in neurons of the cerebral cortex adjacent to active inflammatory lesions in patients with multiple sclerosis. Kv1.3 expression was followed by activation of Notch-1 resulting in neurotoxicity. Blocking PAR-1, Kv1.3 or Notch-1 activation using specific pharmacological inhibitors or siRNAs prevented GrB-induced neurotoxicity. Furthermore, clofazimine protected against GrB-induced neurotoxicity in rat hippocampus, in vivo. These observations indicate that GrB released from T cells induced neurotoxicity by interacting with the membrane bound Gi-coupled PAR-1 receptor and subsequently activated Kv1.3 and Notch-1. These pathways provide novel targets to treat T cell-mediated neuroinflammatory disorders. Kv1.3 is of particular interest since it is expressed on the cell surface, only under pathological circumstances, and early in the cascade of events making it an attractive therapeutic target. PMID:22952817

  15. Suppression of aberrant transient receptor potential cation channel, subfamily V, member 6 expression in hyperproliferative colonic crypts by dietary calcium.

    PubMed

    Peleg, Sara; Sellin, Joseph H; Wang, Yu; Freeman, Michael R; Umar, Shahid

    2010-09-01

    Dietary calcium is believed to reduce colon cancer risk, but the mechanism by which this occurs is poorly understood. Employing the Citrobacter rodentium-induced transmissible murine colonic hyperplasia (TMCH) model, we previously showed that a high-calcium diet (hCa) significantly abrogated hyperplasia in the distal colons of NIH-Swiss mice. Here, we explored the mechanism of dietary protection by hCa by analyzing the expression of genes involved in the regulation of Ca uptake/flux in the intestinal epithelium, including the Ca-sensing receptor, vitamin D receptor, Ca binding protein, and transient receptor potential cation channels, subfamily V, members 5 and 6 (TRPV5/6). Interestingly, while TRPV6 expression increased significantly during TMCH, the expression of the other gene products was unchanged. This elevated TRPV6 expression was significantly abrogated by a hCa diet. Immunofluorescence revealed apical membrane localization of TRPV6 in the normal colon, whereas during TMCH we observed intense apical pole and cytoplasmic staining along the entire longitudinal crypt axis, including the expanded proliferating zone. The hCa diet reversed this effect. In humans, overexpression of TRPV6 was associated with early-stage colon cancer, and in colon carcinoma cells, inhibition of TRPV6 expression by small interfering RNA inhibited their proliferation and induced apoptosis. TRPV6 small interfering RNA also diminished the transcriptional activity of the calcium-dependent nuclear factors in activated T cells. Thus the aberrant overexpression of TRPV6 contributes to colonic crypt hyperplasia in mice and to colon cancer cell proliferation in humans. Therefore, it is likely that suppression of TRPV6 by a hCa diet is required for its protective effects in the colon.

  16. G-protein coupled receptor 18 (GPR18) in channel catfish: Expression analysis and efficacy as immunostimulant against Aeromonas hydrophila infection

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The objectives of this study were: 1) to determine the transcriptional profiles of G-protein coupled receptor 18 (GPR18) in channel catfish after infection with A. hydrophila compared to that in healthy catfish; 2) to determine whether over-expression of GPR18 in catfish gill cells will offer protec...

  17. Rapid and Contrasting Effects of Rosiglitazone on Transient Receptor Potential TRPM3 and TRPC5 ChannelsS⃞

    PubMed Central

    Majeed, Yasser; Bahnasi, Yahya; Seymour, Victoria A. L.; Wilson, Lesley A.; Milligan, Carol J.; Agarwal, Anil K.; Sukumar, Piruthivi; Naylor, Jacqueline

    2011-01-01

    The aim of this study was to generate new insight into chemical regulation of transient receptor potential (TRP) channels with relevance to glucose homeostasis and the metabolic syndrome. Human TRP melastatin 2 (TRPM2), TRPM3, and TRP canonical 5 (TRPC5) were conditionally overexpressed in human embryonic kidney 293 cells and studied by using calcium-measurement and patch-clamp techniques. Rosiglitazone and other peroxisome proliferator-activated receptor-γ (PPAR-γ) agonists were investigated. TRPM2 was unaffected by rosiglitazone at concentrations up to 10 μM but was inhibited completely at higher concentrations (IC50, ∼22.5 μM). TRPM3 was more potently inhibited, with effects occurring in a biphasic concentration-dependent manner such that there was approximately 20% inhibition at low concentrations (0.1–1 μM) and full inhibition at higher concentrations (IC50, 5–10 μM). PPAR-γ antagonism by 2-chloro-5-nitrobenzanilide (GW9662) did not prevent inhibition of TRPM3 by rosiglitazone. TRPC5 was strongly stimulated by rosiglitazone at concentrations of ≥10 μM (EC50, ∼30 μM). Effects on TRPM3 and TRPC5 occurred rapidly and reversibly. Troglitazone and pioglitazone inhibited TRPM3 (IC50, 12 μM) but lacked effect on TRPC5, suggesting no relevance of PPAR-γ or the thiazolidinedione moiety to rosiglitazone stimulation of TRPC5. A rosiglitazone-related but nonthiazolidinedione PPAR-γ agonist, N-(2-benzoylphenyl)-O-[2-(methyl-2-pyridinylamino)ethyl]-l-tyrosine (GW1929), was a weak stimulator of TRPM3 and TRPC5. The natural PPAR-γ agonist 15-deoxy prostaglandin J2, had no effect on TRPM3 or TRPC5. The data suggest that rosiglitazone contains chemical moieties that rapidly, strongly, and differentially modulate TRP channels independently of PPAR-γ, potentially contributing to biological consequences of the agent and providing the basis for novel TRP channel pharmacology. PMID:21406603

  18. Patch clamp combined with voltage/concentration clamp to determine the kinetics and voltage dependency of N-methyl-D-aspartate (NMDA) receptor open channel blockers.

    PubMed

    Parsons, Chris G; Gilling, Kate E

    2014-01-01

    Electrophysiological techniques can be used to great effect to help determine the mechanism of action of a compound. However, many factors can compromise the resulting data and their analysis, such as the speed of solution exchange, expression of additional ion channel populations including other ligand-gated receptors and voltage-gated channels, compounds having multiple binding sites, and current desensitization and rundown. In this chapter, such problems and their solutions are discussed and illustrated using data from experiments involving the uncompetitive NMDA receptor antagonist memantine. Memantine differs from many other NMDA receptor channel blockers in that it is well tolerated and does not cause psychotomimetic effects at therapeutic doses. Various electrophysiological parameters of NMDA-induced current blockade by memantine have been proposed to be important in determining therapeutic tolerability; potency, onset and offset kinetics, and voltage dependency. These were all measured using whole cell patch clamp techniques using hippocampal neurons. Full results are shown here for memantine, and these are summarized and compared to those from similar experiments with other NMDA channel blockers. The interpretation of these results is discussed, as are theories concerning the tolerability of NMDA channel blockers, with the aim of illustrating how electrophysiological data can be used to form and support a physiological hypothesis.

  19. Lipopolysaccharide Inhibits the Channel Activity of the P2X7 Receptor

    PubMed Central

    Leiva-Salcedo, Elias; Coddou, Claudio; Rodríguez, Felipe E.; Penna, Antonello; Lopez, Ximena; Neira, Tanya; Fernández, Ricardo; Imarai, Mónica; Rios, Miguel; Escobar, Jorge; Montoya, Margarita; Huidobro-Toro, J. Pablo; Escobar, Alejandro; Acuña-Castillo, Claudio

    2011-01-01

    The purinergic P2X7 receptor (P2X7R) plays an important role during the immune response, participating in several events such as cytokine release, apoptosis, and necrosis. The bacterial endotoxin lipopolysaccharide (LPS) is one of the strongest stimuli of the immune response, and it has been shown that P2X7R activation can modulate LPS-induced responses. Moreover, a C-terminal binding site for LPS has been proposed. In order to evaluate if LPS can directly modulate the activity of the P2X7R, we tested several signaling pathways associated with P2X7R activation in HEK293 cells that do not express the TLR-4 receptor. We found that LPS alone was unable to induce any P2X7R-related activity, suggesting that the P2X7R is not directly activated by the endotoxin. On the other hand, preapplication of LPS inhibited ATP-induced currents, intracellular calcium increase, and ethidium bromide uptake and had no effect on ERK activation in HEK293 cells. In splenocytes-derived T-regulatory cells, in which ATP-induced apoptosis is driven by the P2X7R, LPS inhibited ATP-induced apoptosis. Altogether, these results demonstrate that LPS modulates the activity of the P2X7R and suggest that this effect could be of physiological relevance. PMID:21941410

  20. Lipopolysaccharide inhibits the channel activity of the P2X7 receptor.

    PubMed

    Leiva-Salcedo, Elias; Coddou, Claudio; Rodríguez, Felipe E; Penna, Antonello; Lopez, Ximena; Neira, Tanya; Fernández, Ricardo; Imarai, Mónica; Rios, Miguel; Escobar, Jorge; Montoya, Margarita; Huidobro-Toro, J Pablo; Escobar, Alejandro; Acuña-Castillo, Claudio

    2011-01-01

    The purinergic P2X7 receptor (P2X7R) plays an important role during the immune response, participating in several events such as cytokine release, apoptosis, and necrosis. The bacterial endotoxin lipopolysaccharide (LPS) is one of the strongest stimuli of the immune response, and it has been shown that P2X7R activation can modulate LPS-induced responses. Moreover, a C-terminal binding site for LPS has been proposed. In order to evaluate if LPS can directly modulate the activity of the P2X7R, we tested several signaling pathways associated with P2X7R activation in HEK293 cells that do not express the TLR-4 receptor. We found that LPS alone was unable to induce any P2X7R-related activity, suggesting that the P2X7R is not directly activated by the endotoxin. On the other hand, preapplication of LPS inhibited ATP-induced currents, intracellular calcium increase, and ethidium bromide uptake and had no effect on ERK activation in HEK293 cells. In splenocytes-derived T-regulatory cells, in which ATP-induced apoptosis is driven by the P2X7R, LPS inhibited ATP-induced apoptosis. Altogether, these results demonstrate that LPS modulates the activity of the P2X7R and suggest that this effect could be of physiological relevance. PMID:21941410

  1. Functional modulation of cerebral gamma-aminobutyric acidA receptor/benzodiazepine receptor/chloride ion channel complex with ethyl beta-carboline-3-carboxylate: Presence of independent binding site for ethyl beta-carboline-3-carboxylate

    SciTech Connect

    Taguchi, J.; Kuriyama, K. )

    1990-05-01

    Effect of ethyl beta-carboline-3-carboxylate (beta-CCE) on the function of gamma-aminobutyric acid (GABA)A receptor/benzodiazepine receptor/chloride ion channel complex was studied. Beta-CCE noncompetitively and competitively inhibited (3H)flunitrazepam binding to benzodiazepine receptor, but not (3H)muscimol binding to GABAA receptor as well as t-(3H)butylbicycloorthobenzoate (( 3H) TBOB) binding to chloride ion channel, in particulate fraction of the mouse brain. Ro15-1788 also inhibited competitively (3H) flunitrazepam binding. On the other hand, the binding of beta-(3H)CCE was inhibited noncompetitively and competitively by clonazepam and competitively by Ro15-1788. In agreement with these results, benzodiazepines-stimulated (3H)muscimol binding was antagonized by beta-CCE and Ro15-1788. Gel column chromatography for the solubilized fraction from cerebral particulate fraction by 0.2% sodium deoxycholate (DOC-Na) in the presence of 1 M KCl indicated that beta-(3H)CCE binding site was eluted in the same fraction (molecular weight, 250,000) as the binding sites for (3H)flunitrazepam, (3H)muscimol and (3H)TBOB. GABA-stimulated 36Cl- influx into membrane vesicles prepared from the bovine cerebral cortex was stimulated and attenuated by flunitrazepam and beta-CCE, respectively. These effects of flunitrazepam and beta-CCE on the GABA-stimulated 36Cl- influx were antagonized by Ro15-1788. The present results suggest that the binding site for beta-CCE, which resides on GABAA receptor/benzodiazepine receptor/chloride ion channel complex, may be different from that for benzodiazepine. Possible roles of beta-CCE binding site in the allosteric inhibitions on benzodiazepine binding site as well as on the functional coupling between chloride ion channel and GABAA receptor are also suggested.

  2. A data-driven model of a modal gated ion channel: the inositol 1,4,5-trisphosphate receptor in insect Sf9 cells.

    PubMed

    Ullah, Ghanim; Mak, Don-On Daniel; Pearson, John E

    2012-08-01

    The inositol 1,4,5-trisphosphate (IP(3)) receptor (IP(3)R) channel is crucial for the generation and modulation of intracellular Ca(2+) signals in animal cells. To gain insight into the complicated ligand regulation of this ubiquitous channel, we constructed a simple quantitative continuous-time Markov-chain model from the data. Our model accounts for most experimentally observed gating behaviors of single native IP(3)R channels from insect Sf9 cells. Ligand (Ca(2+) and IP(3)) dependencies of channel activity established six main ligand-bound channel complexes, where a complex consists of one or more states with the same ligand stoichiometry and open or closed conformation. Channel gating in three distinct modes added one complex and indicated that three complexes gate in multiple modes. This also restricted the connectivity between channel complexes. Finally, latencies of channel responses to abrupt ligand concentration changes defined a model with specific network topology between 9 closed and 3 open states. The model with 28 parameters can closely reproduce the equilibrium gating statistics for all three gating modes over a broad range of ligand concentrations. It also captures the major features of channel response latency distributions. The model can generate falsifiable predictions of IP(3)R channel gating behaviors and provide insights to both guide future experiment development and improve IP(3)R channel gating analysis. Maximum likelihood estimates of the model parameters and of the parameters in the De Young-Keizer model yield strong statistical evidence in favor of our model. Our method is simple and easily applicable to the dynamics of other ion channels and molecules.

  3. A single-channel method for evaluation of very magnitudes of Ca2+ ion fluxes through epsilon4/zeta1 N-methyl-D-aspartate receptor channels in bilayer lipid membranes.

    PubMed

    Wakabayashi, M; Hirano, A; Sugawara, M; Uchino, S; Nakajima-Iijima, S

    2001-01-01

    A single-channel method for evaluating agonist selectivity in terms of the very number of Ca2+ ions passed through the epsilon4/zeta1 N-methyl-D-aspartate (NMDA) receptor ion channel in bilayer lipid membranes (BLMs) is described. The number of Ca2+ passed through the single-channel was obtained from single-channel recordings in a medium where the primary permeant ion is Ca2+. The recombinant epsilon4/zeta1 NMDA channel was partially purified from Chinese hamster ovary cells expressing the channel and incorporated in BLMs formed by the tip-dip method. It was found that the epsilon4/zeta1 channel in BLMs is permeable to Ca2+ and Na+, but the number of Ca2+ passed through the channel is much fewer than that of Na+. The integrated Ca2+ currents induced by three typical agonists NMDA, L-glutamate and L-CCG-IV were obtained at concentration of 50 microM, where the integrated currents for all the agonists reached their saturated values. The integrated Ca2+ currents obtained are (3.1+/-0.21) x 10(-13) C/s for NMDA, (4.6+/-0.31) x 10(-13) C/s for L-glutamate and (5.7+/-0.25) x 10(-13) C/s for L-CCG-IV, respectively, suggesting that the three kinds of agonists have different efficacies to induce permeation of Ca2+. The range of the agonist selectivity thus obtained is much narrower than that of binding affinities for the NMDA receptors from rat brain. The present method is able to detect Ca2+ permeation with a detection limit of approximately 10(5) Ca2+ ions/s.

  4. Different Contribution of Redox-Sensitive Transient Receptor Potential Channels to Acetaminophen-Induced Death of Human Hepatoma Cell Line

    PubMed Central

    Badr, Heba; Kozai, Daisuke; Sakaguchi, Reiko; Numata, Tomohiro; Mori, Yasuo

    2016-01-01

    Acetaminophen (APAP) is a safe analgesic antipyretic drug at prescribed doses. Its overdose, however, can cause life-threatening liver damage. Though, involvement of oxidative stress is widely acknowledged in APAP-induced hepatocellular death, the mechanism of this increased oxidative stress and the associated alterations in Ca2+ homeostasis are still unclear. Among members of transient receptor potential (TRP) channels activated in response to oxidative stress, we here identify that redox-sensitive TRPV1, TRPC1, TRPM2, and TRPM7 channels underlie Ca2+ entry and downstream cellular damages induced by APAP in human hepatoma (HepG2) cells. Our data indicate that APAP treatment of HepG2 cells resulted in increased reactive oxygen species (ROS) production, glutathione (GSH) depletion, and Ca2+ entry leading to increased apoptotic cell death. These responses were significantly suppressed by pretreatment with the ROS scavengers N-acetyl-L-cysteine (NAC) and 4,5-dihydroxy-1,3-benzene disulfonic acid disodium salt monohydrate (Tiron), and also by preincubation of cells with the glutathione inducer Dimethylfumarate (DMF). TRP subtype-targeted pharmacological blockers and siRNAs strategy revealed that suppression of either TRPV1, TRPC1, TRPM2, or TRPM7 reduced APAP-induced ROS formation, Ca2+ influx, and cell death; the effects of suppression of TRPV1 or TRPC1, known to be activated by oxidative cysteine modifications, were stronger than those of TRPM2 or TRPM7. Interestingly, TRPV1 and TRPC1 were labeled by the cysteine-selective modification reagent, 5,5′-dithiobis (2-nitrobenzoic acid)-2biotin (DTNB-2Bio), and this was attenuated by pretreatment with APAP, suggesting that APAP and/or its oxidized metabolites act directly on the modification target cysteine residues of TRPV1 and TRPC1 proteins. In human liver tissue, TRPV1, TRPC1, TRPM2, and TRPM7 channels transcripts were localized mainly to hepatocytes and Kupffer cells. Our findings strongly suggest that APAP

  5. Role of amino-terminal half of the S4-S5 linker in type 1 ryanodine receptor (RyR1) channel gating.

    PubMed

    Murayama, Takashi; Kurebayashi, Nagomi; Oba, Toshiharu; Oyamada, Hideto; Oguchi, Katsuji; Sakurai, Takashi; Ogawa, Yasuo

    2011-10-14

    The type 1 ryanodine receptor (RyR1) is a Ca(2+) release channel found in the sarcoplasmic reticulum of skeletal muscle and plays a pivotal role in excitation-contraction coupling. The RyR1 channel is activated by a conformational change of the dihydropyridine receptor upon depolarization of the transverse tubule, or by Ca(2+) itself, i.e. Ca(2+)-induced Ca(2+) release (CICR). The molecular events transmitting such signals to the ion gate of the channel are unknown. The S4-S5 linker, a cytosolic loop connecting the S4 and S5 transmembrane segments in six-transmembrane type channels, forms an α-helical structure and mediates signal transmission in a wide variety of channels. To address the role of the S4-S5 linker in RyR1 channel gating, we performed alanine substitution scan of N-terminal half of the putative S4-S5 linker (Thr(4825)-Ser(4829)) that exhibits high helix probability. The mutant RyR1 was expressed in HEK cells, and CICR activity was investigated by caffeine-induced Ca(2+) release, single-channel current recordings, and [(3)H]ryanodine binding. Four mutants (T4825A, I4826A, S4828A, and S4829A) had reduced CICR activity without changing Ca(2+) sensitivity, whereas the L4827A mutant formed a constitutive active channel. T4825I, a disease-associated mutation for malignant hyperthermia, exhibited enhanced CICR activity. An α-helical wheel representation of the N-terminal S4-S5 linker provides a rational explanation to the observed activities of the mutants. These results suggest that N-terminal half of the S4-S5 linker may form an α-helical structure and play an important role in RyR1 channel gating.

  6. Mechanism of muscarinic receptor-induced K+ channel activation as revealed by hydrolysis-resistant GTP analogues

    PubMed Central

    1988-01-01

    The role of a guanine nucleotide-binding protein (Gk) in the coupling between muscarinic receptor activation and opening of an inwardly rectifying K+ channel [IK(M)] was examined in cardiac atrial myocytes, using hydrolysis-resistant GTP analogues. In the absence of muscarinic agonist, GTP analogues produced a membrane current characteristic of IK(M). The initial rate of appearance of this receptor-independent IK(M) was measured for the various analogues in order to explore the kinetic properties of IK(M) activation. We found that IK(M) activation is controlled solely by the intracellular analogue/GTP ratio and not by the absolute concentrations of the nucleotides. Analogues competed with GTP for binding to Gk with the following relative affinities: GTP gamma S greater than GTP greater than GppNHp greater than GppCH2p. At sufficiently high intracellular concentrations, however, all GTP analogues produced the same rate of IK(M) activation. This analogue- independent limiting rate is likely to correspond to the rate of GDP release from inactive, GDP-bound Gk. Muscarinic receptor stimulation by nanomolar concentrations of acetylcholine (ACh), which do not elicit IK(M) under control conditions, catalyzed IK(M) activation in the presence of GTP analogues. The rate of Gk activation by ACh (kACh) was found to be described by the simple relationship kACh = 8.4 X 10(8) min- 1 M-1.[ACh] + 0.44 min-1, the first term of which presumably reflects the agonist-catalyzed rate of GDP release from the Gk.GDP complex, while the second term corresponds to the basal rate of receptor- independent GDP release. Combined with the estimated K0.5 of the IK(M)- [ACh] dose-effect relationship, 160 nM, this result also allowed us to estimate the rate of Gk.GTP hydrolysis, kcat, to be near 135 min-1. These results provide, for the first time, a quantitative description of the salient features of G-protein function in vivo. PMID:2455765

  7. 3-Iodothyronamine increases transient receptor potential melastatin channel 8 (TRPM8) activity in immortalized human corneal epithelial cells.

    PubMed

    Lucius, Alexander; Khajavi, Noushafarin; Reinach, Peter S; Köhrle, Josef; Dhandapani, Priyavathi; Huimann, Philipp; Ljubojevic, Nina; Grötzinger, Carsten; Mergler, Stefan

    2016-03-01

    3-Iodothyronamine (3T1AM) is an endogenous thyroid hormone metabolite that interacts with the human trace amine-associated receptor 1 (hTAAR1), a G-protein-coupled receptor, to induce numerous physiological responses including dose-dependent body temperature lowering in rodents. 3T1AM also directly activates cold-sensitive transient receptor potential melastatin 8 (TRPM8) channels in human conjunctival epithelial cells (HCjEC) at constant temperature as well as reducing rises in IL-6 release induced by transient receptor potential vanilloid 1 (TRPV1) activation by capsaicin (CAP). Here, we describe that 3T1AM-induced TRPM8 activation suppresses through crosstalk TRPV1 activation in immortalized human corneal epithelial cells (HCEC). RT-PCR and immunofluorescent staining identified TRPM8 gene and protein expression. Increases in Ca(2+) influx induced by the TRPM8 agonists either 3T1AM (0.1-10 μM), menthol (500 μM), icilin (15-60 μM) or temperature lowering (either <17°C or >17°C) were all blocked by 10-20 μM BCTC, a mixed TRPV1/TRPM8 antagonist. BCTC blocked 3T1AM-induced recombinant TRPM8 activation of Ca(2+) transients in an osteosarcoma heterologous expression system. The effects of BCTC in HCEC were attributable to selective TRPM8 inhibition since whole-cell patch-clamp currents underlying Ca(2+) rises induced by 20 μM CAP were BCTC insensitive. On the other hand, Ca(2+) transients induced by activating TRPV1 with either CAP or a hyperosmolar medium were suppressed during exposure to either 1 μM 3T1AM or 15 μM icilin. All of these modulatory effects on intracellular Ca(2+) regulation induced by the aforementioned agents were attributable to changes in underlying inward and outward current. Taken together, TRPM8 activation by 3T1AM markedly attenuates and even eliminates hyperosmolar and CAP induced TRPV1 activation through crosstalk.

  8. Opposing roles of smooth muscle BK channels and ryanodine receptors in the regulation of nerve-evoked constriction of mesenteric resistance arteries.

    PubMed

    Krishnamoorthy, Gayathri; Sonkusare, Swapnil K; Heppner, Thomas J; Nelson, Mark T

    2014-04-01

    In depolarized smooth muscle cells of pressurized cerebral arteries, ryanodine receptors (RyRs) generate "Ca2+ sparks" that activate large-conductance, Ca2+ -, and voltage-sensitive potassium (BK) channels to oppose pressure-induced (myogenic) constriction. Here, we show that BK channels and RyRs have opposing roles in the regulation of arterial tone in response to sympathetic nerve activation by electrical field stimulation. Inhibition of BK channels with paxilline increased both myogenic and nerve-induced constrictions of pressurized, resistance-sized mesenteric arteries from mice. Inhibition of RyRs with ryanodine increased myogenic constriction, but it decreased nerve-evoked constriction along with a reduction in the amplitude of nerve-evoked increases in global intracellular Ca2+. In the presence of L-type voltage-dependent Ca2+ channel (VDCC) antagonists, nerve stimulation failed to evoke a change in arterial diameter, and BK channel and RyR inhibitors were without effect, suggesting that nerve- induced constriction is dependent on activation of VDCCs. Collectively, these results indicate that BK channels and RyRs have different roles in the regulation of myogenic versus neurogenic tone: whereas BK channels and RyRs act in concert to oppose myogenic vasoconstriction, BK channels oppose neurogenic vasoconstriction and RyRs augment it. A scheme for neurogenic vasoregulation is proposed in which RyRs act in conjunction with VDCCs to regulate nerve-evoked constriction in mesenteric resistance arteries.

  9. Mapping the Interaction Sites between AMPA Receptors and TARPs Reveals a Role for the Receptor N-Terminal Domain in Channel Gating

    PubMed Central

    Cais, Ondrej; Herguedas, Beatriz; Krol, Karolina; Cull-Candy, Stuart G.; Farrant, Mark; Greger, Ingo H.

    2014-01-01

    Summary AMPA-type glutamate receptors (AMPARs) mediate fast neurotransmission at excitatory synapses. The extent and fidelity of postsynaptic depolarization triggered by AMPAR activation are shaped by AMPAR auxiliary subunits, including the transmembrane AMPAR regulatory proteins (TARPs). TARPs profoundly influence gating, an effect thought to be mediated by an interaction with the AMPAR ion channel and ligand binding domain (LBD). Here, we show that the distal N-terminal domain (NTD) contributes to TARP modulation. Alterations in the NTD-LBD linker result in TARP-dependent and TARP-selective changes in AMPAR gating. Using peptide arrays, we identify a TARP interaction region on the NTD and define the path of TARP contacts along the LBD surface. Moreover, we map key binding sites on the TARP itself and show that mutation of these residues mediates gating modulation. Our data reveal a TARP-dependent allosteric role for the AMPAR NTD and suggest that TARP binding triggers a drastic reorganization of the AMPAR complex. PMID:25373908

  10. γ-Aminobutyric acid type B (GABAB) receptor expression is needed for inhibition of N-type (Cav2.2) calcium channels by analgesic α-conotoxins.

    PubMed

    Cuny, Hartmut; de Faoite, Andrew; Huynh, Thuan G; Yasuda, Takahiro; Berecki, Géza; Adams, David J

    2012-07-01

    α-Conotoxins Vc1.1 and RgIA are small peptides isolated from the venom of marine cone snails. They have effective anti-nociceptive actions in rat models of neuropathic pain. Pharmacological studies in rodent dorsal root ganglion (DRG) show their analgesic effect is mediated by inhibition of N-type (Ca(v)2.2) calcium channels via a pathway involving γ-aminobutyric acid type B (GABA(B)) receptor. However, there is no direct demonstration that functional GABA(B) receptors are needed for inhibition of the Ca(v)2.2 channel by analgesic α-conotoxins. This study examined the effect of the GABA(B) agonist baclofen and α-conotoxins Vc1.1 and RgIA on calcium channel currents after transient knockdown of the GABA(B) receptor using RNA interference. Isolated rat DRG neurons were transfected with small interfering RNAs (siRNA) targeting GABA(B) subunits R1 and R2. Efficient knockdown of GABA(B) receptor expression at mRNA and protein levels was confirmed by quantitative real time PCR (qRT-PCR) and immunocytochemical analysis, respectively. Whole-cell patch clamp recordings conducted 2-4 days after transfection showed that inhibition of N-type calcium channels in response to baclofen, Vc1.1 and RgIA was significantly reduced in GABA(B) receptor knockdown DRG neurons. In contrast, neurons transfected with a scrambled nontargeting siRNA were indistinguishable from untransfected neurons. In the HEK 293 cell heterologous expression system, Vc1.1 and RgIA inhibition of Ca(v)2.2 channels needed functional expression of both human GABA(B) receptor subunits. Together, these results confirm that GABA(B) receptors must be activated for the modulation of N-type (Ca(v)2.2) calcium channels by analgesic α-conotoxins Vc1.1 and RgIA.

  11. Expression of serotonin receptor genes in cranial ganglia.

    PubMed

    Maeda, Naohiro; Ohmoto, Makoto; Yamamoto, Kurumi; Kurokawa, Azusa; Narukawa, Masataka; Ishimaru, Yoshiro; Misaka, Takumi; Matsumoto, Ichiro; Abe, Keiko

    2016-03-23

    Taste cells release neurotransmitters to gustatory neurons to transmit chemical information they received. Sweet, umami, and bitter taste cells use ATP as a neurotransmitter. However, ATP release from sour taste cells has not been observed so far. Instead, they release serotonin when they are activated by sour/acid stimuli. Thus it is still controversial whether sour taste cells use ATP, serotonin, or both. By reverse transcription-polymerase chain reaction and subsequent in situ hybridization (ISH) analyses, we revealed that of 14 serotonin receptor genes only 5-HT3A and 5-HT3B showed significant/clear signals in a subset of neurons of cranial sensory ganglia in which gustatory neurons reside. Double-fluorescent labeling analyses of ISH for serotonin receptor genes with wheat germ agglutinin (WGA) in cranial sensory ganglia of pkd1l3-WGA mice whose sour neural pathway is visualized by the distribution of WGA originating from sour taste cells in the posterior region of the tongue revealed that WGA-positive cranial sensory neurons rarely express either of serotonin receptor gene. These results suggest that serotonin receptors expressed in cranial sensory neurons do not play any role as neurotransmitter receptor from sour taste cells. PMID:26854841

  12. Cell-attached single-channel recordings in intact prefrontal cortex pyramidal neurons reveal compartmentalized D1/D5 receptor modulation of the persistent sodium current

    PubMed Central

    Gorelova, Natalia; Seamans, Jeremy K.

    2015-01-01

    The persistent Na+ current (INap) is believed to be an important target of dopamine modulation in prefrontal cortex (PFC) neurons. While past studies have tested the effects of dopamine on INap, the results have been contradictory largely because of difficulties in measuring INap using somatic whole-cell recordings. To circumvent these confounds we used the cell-attached patch-clamp technique to record single Na+ channels from the soma, proximal dendrite (PD) or proximal axon (PA) of intact prefrontal layer V pyramidal neurons. Under baseline conditions, numerous well resolved Na+ channel openings were recorded that exhibited an extrapolated reversal potential of 73 mV, a slope conductance of 14–19 pS and were blocked by tetrodotoxin (TTX). While similar in most respects, the propensity to exhibit prolonged bursts lasting >40 ms was many fold greater in the axon than the soma or dendrite. Bath application of the D1/D5 receptor agonist SKF81297 shifted the ensemble current activation curve leftward and increased the number of late events recorded from the PD but not the soma or PA. However, the greatest effect was on prolonged bursting where the D1/D5 receptor agonist increased their occurrence 3 fold in the PD and nearly 7 fold in the soma, but not at all in the PA. As a result, D1/D5 receptor activation equalized the probability of prolonged burst occurrence across the proximal axosomatodendritic region. Therefore, D1/D5 receptor modulation appears to be targeted mainly to Na+ channels in the PD/soma and not the PA. By circumventing the pitfalls of previous attempts to study the D1/D5 receptor modulation of INap, we demonstrate conclusively that D1/D5 receptor activation can increase the INap generated proximally, however questions still remain as to how D1/D5 receptor modulates Na+ currents in the more distal initial segment where most of the INap is normally generated. PMID:25729354

  13. Activation of ATP-sensitive potassium channels in rat pancreatic beta-cells by linoleic acid through both intracellular metabolites and membrane receptor signalling pathway.

    PubMed

    Zhao, Yu-Feng; Pei, Jianming; Chen, Chen

    2008-09-01

    ATP-sensitive potassium channels (K(ATP) channels) determine the excitability of pancreatic beta-cells and importantly regulate glucose-stimulated insulin secretion (GSIS). Long-chain free fatty acids (FFAs) decrease GSIS after long-term exposure to beta-cells, but the effects of exogenous FFAs on K(ATP) channels are not yet well clarified. In this study, the effects of linoleic acid (LA) on membrane potential (MP) and K(ATP) channels were observed in primary cultured rat pancreatic beta-cells. LA (20 microM) induced hyperpolarization of MP and opening of K(ATP) channels, which was totally reversed and inhibited by tolbutamide, a K(ATP) channel blocker. Inhibition of LA metabolism by acyl-CoA synthetase inhibitor, triacsin C (10 microM), partially inhibited LA-induced opening of K(ATP) channels by 64%. The non-FFA G protein-coupled receptor (GPR) 40 agonist, GW9508 (40 microM), induced an opening of K(ATP) channels, which was similar to that induced by LA under triacsin C treatment. Blockade of protein kinases A and C did not influence the opening of K(ATP) channels induced by LA and GW9508, indicating that these two protein kinase pathways are not involved in the action of LA on K(ATP) channels. The present study demonstrates that LA induces hyperpolarization of MP by activating K(ATP) channels via both intracellular metabolites and activation of GPR40. It indicates that not only intracellular metabolites of FFAs but also GPR40-mediated pathways take part in the inhibition of GSIS and beta-cell dysfunction induced by FFAs.

  14. Identification and in vitro pharmacological characterization of a novel and selective α7 nicotinic acetylcholine receptor agonist, Br-IQ17B

    PubMed Central

    Tang, Jing-shu; Xie, Bing-xue; Bian, Xi-ling; Xue, Yu; Wei, Ning-ning; Zhou, Jing-heng; Hao, Yu-chen; Li, Gang; Zhang, Liang-ren; Wang, Ke-wei

    2015-01-01

    Aim: Alpha7-nicotinic acetylcholine receptor (α7 nAChR) is a ligand-gated Ca2+-permeable ion channel implicated in cognition and neuropsychiatric disorders. Activation of α7 nAChR improves learning, memory, and sensory gating in animal models. To identify novel α7 nAChR agonists, we synthesized a series of small molecules and characterized a representative compound, Br-IQ17B, N-[(3R)-1-azabicyclo[2,2,2]oct-3-yl]-5-bromoindolizine-2-carboxamide, which specifically activates α7 nAChR. Methods: Two-electrode voltage clamp (TEVC) recordings were primarily used for screening in Xenopus oocytes expressing human α7 nAChR. Assays, including radioisotope ligand binding, Western blots, whole-cell recordings of hippocampal culture neurons, and spontaneous IPSC recordings of brain slices, were also utilized to evaluate and confirm the specific activation of α7 nAChR by Br-IQ17B. Results: Br-IQ17B potently activates α7 nAChR with an EC50 of 1.8±0.2 μmol/L. Br-IQ17B is selective over other subtypes such as α4β2 and α3β4, but it blocks 5-HT3A receptors. Br-IQ17B displaced binding of the α7 blocker [3H]-MLA to hippocampal crude membranes with a Ki of 14.9±3.2 nmol/L. In hippocampal neurons, Br-IQ17B evoked α7-like currents that were inhibited by MLA and enhanced in the presence of the α7 PAM PNU-120596. In brain slice recordings, Br-IQ17B enhanced GABAergic synaptic transmission in CA1 neurons. Mechanistically, Br-IQ17B increased ERK1/2 phosphorylation that was MLA-sensitive. Conclusion: We identified the novel, potent, and selective α7 agonist Br-IQ17B, which enhances synaptic transmission. Br-IQ17B may be a helpful tool to understand new aspects of α7 nAChR function, and it also has potential for being developed as therapy for schizophrenia and cognitive deficits. PMID:25948478

  15. Abnormal interactions of calsequestrin with the ryanodine receptor calcium release channel complex linked to exercise-induced sudden cardiac death.

    PubMed

    Terentyev, Dmitry; Nori, Alessandra; Santoro, Massimo; Viatchenko-Karpinski, Serge; Kubalova, Zuzana; Gyorke, Inna; Terentyeva, Radmila; Vedamoorthyrao, Srikanth; Blom, Nico A; Valle, Giorgia; Napolitano, Carlo; Williams, Simon C; Volpe, Pompeo; Priori, Silvia G; Gyorke, Sandor

    2006-05-12

    Catecholaminergic polymorphic ventricular tachycardia (CPVT) is a familial arrhythmogenic disorder associated with mutations in the cardiac ryanodine receptor (RyR2) and cardiac calsequestrin (CASQ2) genes. Previous in vitro studies suggested that RyR2 and CASQ2 interact as parts of a multimolecular Ca(2+)-signaling complex; however, direct evidence for such interactions and their potential significance to myocardial function remain to be determined. We identified a novel CASQ2 mutation in a young female with a structurally normal heart and unexplained syncopal episodes. This mutation results in the nonconservative substitution of glutamine for arginine at amino acid 33 of CASQ2 (R33Q). Adenoviral-mediated expression of CASQ2(R33Q) in adult rat myocytes led to an increase in excitation-contraction coupling gain and to more frequent occurrences of spontaneous propagating (Ca2+ waves) and local Ca2+ signals (sparks) with respect to control cells expressing wild-type CASQ2 (CASQ2WT). As revealed by a Ca2+ indicator entrapped inside the sarcoplasmic reticulum (SR) of permeabilized myocytes, the increased occurrence of spontaneous Ca2+ sparks and waves was associated with a dramatic decrease in intra-SR [Ca2+]. Recombinant CASQ2WT and CASQ2R33Q exhibited similar Ca(2+)-binding capacities in vitro; however, the mutant protein lacked the ability of its WT counterpart to inhibit RyR2 activity at low luminal [Ca2+] in planar lipid bilayers. We conclude that the R33Q mutation disrupts interactions of CASQ2 with the RyR2 channel complex and impairs regulation of RyR2 by luminal Ca2+. These results show that intracellular Ca2+ cycling in normal heart relies on an intricate interplay of CASQ2 with the proteins of the RyR2 channel complex and that disruption of these interactions can lead to cardiac arrhythmia. PMID:16601229

  16. Inhibition of the transient receptor potential melastatin-2 channel causes increased DNA damage and decreased proliferation in breast adenocarcinoma cells

    PubMed Central

    HOPKINS, MANDI M.; FENG, XIAOXING; LIU, MENGWEI; PARKER, LAUREN P.; KOH, DAVID W.

    2015-01-01

    Transient receptor potential, melastatin-2 (TRPM2) is a plasma membrane cation channel with important roles in sensory functions and promoting cell death. However, we demonstrated here that TRPM2 was present in the nuclei of MCF-7 and MDA-MB-231 human breast adenocarcinoma cells, and its pharmacologic inhibition or RNAi silencing caused decreased cell proliferation. Neither an effect on proliferation nor a localization of TRPM2 in the nucleus was observed in noncancerous HMEC and MCF-10A human mammary epithelial cells. Investigation of possible effects of TRPM2 function in the nucleus demonstrated that pharmacologic inhibition or RNAi silencing of TRPM2 in MCF-7 and MDA-MB-231 human breast adenocarcinoma cells caused up to 4-fold increases in DNA damage levels, as compared to noncancerous breast cells after equivalent treatments. These results indicate that TRPM2 has a novel nuclear function in human breast adenocarcinoma cells that facilitates the integrity of genomic DNA, a finding that is distinct from its previously reported role as a plasma membrane cation channel in noncancerous cells. In summary, we report here a novel effect promoted by TRPM2, where it functions to minimize DNA damage and thus may have a role in the protection of genomic DNA in breast cancer cells. Our study therefore provides compelling evidence that TRPM2 has a unique role in breast adenocarcinoma cells. Accordingly, these studies suggest that TRPM2 is a potential therapeutic target, where its pharmacologic inhibition may provide an innovative strategy to selectively increase DNA damage levels in breast cancer cells. PMID:25760245

  17. Opioid receptors from a lower vertebrate (Catostomus commersoni): Sequence, pharmacology, coupling to a G-protein-gated inward-rectifying potassium channel (GIRK1), and evolution

    PubMed Central

    Darlison, Mark G.; Greten, Florian R.; Harvey, Robert J.; Kreienkamp, Hans-Jürgen; Stühmer, Thorsten; Zwiers, Henk; Lederis, Karl; Richter, Dietmar

    1997-01-01

    The molecular evolution of the opioid receptor family has been studied by isolating cDNAs that encode six distinct opioid receptor-like proteins from a lower vertebrate, the teleost fish Catostomus commersoni. One of these, which has been obtained in full-length form, encodes a 383-amino acid protein that exhibits greatest sequence similarity to mammalian μ-opioid receptors; the corresponding gene is expressed predominantly in brain and pituitary. Transfection of the teleost cDNA into HEK 293 cells resulted in the appearance of a receptor having high affinity for the μ-selective agonist [d-Ala2, MePhe4-Gly-ol5]enkephalin (DAMGO) (Kd = 0.63 ± 0.15 nM) and for the nonselective antagonist naloxone (Kd = 3.1 ± 1.3 nM). The receptor had negligible affinity for U50488 and [d-Pen2, d-Pen5]enkephalin (DPDPE), which are κ- and δ-opioid receptor selective agonists, respectively. Stimulation of transfected cells with 1 μM DAMGO lowered forskolin-induced cAMP levels, an effect that could be reversed by naloxone. Experiments in Xenopus oocytes have demonstrated that the fish opioid receptor can, in an agonist-dependent fashion, activate a coexpressed mouse G-protein-gated inward-rectifying potassium channel (GIRK1). The identification of six distinct fish opioid receptor-like proteins suggests that additional mammalian opioid receptors remain to be identified at the molecular level. Furthermore, our data indicate that the μ-opioid receptor arose very early in evolution, perhaps before the appearance of vertebrates, and that the pharmacological and functional properties of this receptor have been conserved over a period of ≈400 million years implying that it fulfills an important physiological role. PMID:9223341

  18. Cisapride, a selective serotonin 5-HT4-receptor agonist, inhibits voltage-dependent K(+) channels in rabbit coronary arterial smooth muscle cells.

    PubMed

    Kim, Hye Won; Li, Hongliang; Kim, Han Sol; Shin, Sung Eun; Jung, Won-Kyo; Ha, Kwon-Soo; Han, Eun-Taek; Hong, Seok-Ho; Choi, Il-Whan; Park, Won Sun

    2016-09-23

    We investigated the effect of cisapride, a selective serotonin 5-HT4-receptor agonist, on voltage-dependent K(+) (Kv) channels using freshly isolated smooth muscle cells from the coronary arteries of rabbits. The amplitude of Kv currents was reduced by cisapride in a concentration-dependent manner, with an IC50 value of 6.77 ± 6.01 μM and a Hill coefficient of 0.51 ± 0.18. The application of cisapride shifted the steady-state inactivation curve toward a more negative potential, but had no significant effect on the steady-state activation curve. This suggested that cisapride inhibited the Kv channel in a closed state by changing the voltage sensitivity of Kv channels. The application of another selective serotonin 5-HT4-receptor agonist, prucalopride, did not affect the basal Kv current and did not alter the inhibitory effect of cisapride on Kv channels. From these results, we concluded that cisapride inhibited vascular Kv current in a concentration-dependent manner by shifting the steady-state inactivation curve, independent of its own function as a selective serotonin 5-HT4-receptor agonist. PMID:27569285

  19. 20-Hydroxyeicosatetraenoic Acid (20-HETE) Modulates Canonical Transient Receptor Potential-6 (TRPC6) Channels in Podocytes

    PubMed Central

    Roshanravan, Hila; Kim, Eun Y.; Dryer, Stuart E.

    2016-01-01

    The arachidonic acid metabolite 20-hydroxyeicosatetraenoic acid (20-HETE) regulates renal function, including changes in glomerular function evoked during tubuloglomerular feedback (TGF). This study describes the cellular actions of 20-HETE on cultured podocytes, assessed by whole-cell recordings from cultured podocytes combined with pharmacological and cell-biological manipulations of cells. Bath superfusion of 20-HETE activates cationic currents that are blocked by the pan-TRP blocker SKF-96365 and by 50 μM La3+, and which are attenuated after siRNA knockdown of TRPC6 subunits. Similar currents are evoked by a membrane-permeable analog of diacylgycerol (OAG), but OAG does not occlude responses to maximally-activating concentrations of 20-HETE (20 μM). Exposure to 20-HETE also increased steady-state surface abundance of TRPC6 subunits in podocytes as assessed by cell-surface biotinylation assays, and increased cytosolic concentrations of reactive oxygen species (ROS). TRPC6 activation by 20-HETE was eliminated in cells pretreated with TEMPOL, a membrane-permeable superoxide dismutase mimic. Activation of TRPC6 by 20-HETE was also blocked when whole-cell recording pipettes contained GDP-βS, indicating a role for either small or heterotrimeric G proteins in the transduction cascade. Responses to 20-HETE were eliminated by siRNA knockdown of podocin, a protein that organizes NADPH oxidase complexes with TRPC6 subunits in this cell type. In summary, modulation of ionic channels in podocytes may contribute to glomerular actions of 20-HETE.

  20. 20-Hydroxyeicosatetraenoic Acid (20-HETE) Modulates Canonical Transient Receptor Potential-6 (TRPC6) Channels in Podocytes

    PubMed Central

    Roshanravan, Hila; Kim, Eun Y.; Dryer, Stuart E.

    2016-01-01

    The arachidonic acid metabolite 20-hydroxyeicosatetraenoic acid (20-HETE) regulates renal function, including changes in glomerular function evoked during tubuloglomerular feedback (TGF). This study describes the cellular actions of 20-HETE on cultured podocytes, assessed by whole-cell recordings from cultured podocytes combined with pharmacological and cell-biological manipulations of cells. Bath superfusion of 20-HETE activates cationic currents that are blocked by the pan-TRP blocker SKF-96365 and by 50 μM La3+, and which are attenuated after siRNA knockdown of TRPC6 subunits. Similar currents are evoked by a membrane-permeable analog of diacylgycerol (OAG), but OAG does not occlude responses to maximally-activating concentrations of 20-HETE (20 μM). Exposure to 20-HETE also increased steady-state surface abundance of TRPC6 subunits in podocytes as assessed by cell-surface biotinylation assays, and increased cytosolic concentrations of reactive oxygen species (ROS). TRPC6 activation by 20-HETE was eliminated in cells pretreated with TEMPOL, a membrane-permeable superoxide dismutase mimic. Activation of TRPC6 by 20-HETE was also blocked when whole-cell recording pipettes contained GDP-βS, indicating a role for either small or heterotrimeric G proteins in the transduction cascade. Responses to 20-HETE were eliminated by siRNA knockdown of podocin, a protein that organizes NADPH oxidase complexes with TRPC6 subunits in this cell type. In summary, modulation of ionic channels in podocytes may contribute to glomerular actions of 20-HETE. PMID:27630573

  1. Open-channel block of N-methyl-D-aspartate (NMDA) responses by memantine: therapeutic advantage against NMDA receptor-mediated neurotoxicity.

    PubMed

    Chen, H S; Pellegrini, J W; Aggarwal, S K; Lei, S Z; Warach, S; Jensen, F E; Lipton, S A

    1992-11-01

    Excessive activation of NMDA receptors is thought to mediate the calcium-dependent neurotoxicity associated with hypoxic-ischemic brain injury, trauma, epilepsy, and several neurodegenerative diseases. For this reason, various NMDA antagonists have been investigated for their therapeutic potential in these diseases, but heretofore none have proven to be both effective and safe. In the present study, memantine, an adamantane derivative similar to the antiviral drug amantadine, is shown to block the channels activated by NMDA receptor stimulation. From whole-cell and single-channel recording experiments, the mechanism of action of memantine is deduced to be open-channel block, similar to MK-801; however, unlike MK-801, memantine is well tolerated clinically. Compared to MK-801, memantine's safety may be related to its faster kinetics of action with rapid blocking and unblocking rates at low micromolar concentrations. Furthermore, at these levels memantine is an uncompetitive antagonist and should theoretically allow near-normal physiological NMDA activity throughout the brain even in the face of pathologically high focal concentrations of glutamate. These pharmacological properties confer upon memantine a therapeutic advantage against NMDA receptor-mediated neurotoxicity with few side effects compared with other organic NMDA open-channel blockers. Moreover, memantine is increasingly effective against escalating levels of glutamate, such as those observed during a stroke. Low micromolar concentrations of memantine, levels known to be tolerated by patients receiving the drug for the treatment of Parkinson's disease, prevent NMDA receptor-mediated neurotoxicity in cultures of rat cortical and retinal ganglion cell neurons; memantine also appears to be both safe and effective in a rat stroke model. These results suggest that memantine has considerable therapeutic potential for the myriad of clinical entities associated with NMDA receptor-mediated neurotoxicity.

  2. Transient receptor potential vanilloid 1 channels modulate the anxiolytic effect of diazepam.

    PubMed

    Manna, Shyamshree S S; Umathe, Sudhir N

    2011-11-24

    The present study investigated the interaction between the vanilloid and GABAergic systems on anxiety. Swiss mice were subjected to social interaction test, an animal model for assessing anxiety-related behavior, after intracerebroventricular administration of capsaicin, (TRPV1 agonist) or capsazepine, (TRPV1 antagonist) either alone or in combination with traditional anxiolytic drug, diazepam. Results showed that capsaicin (1, 10, and 100 μg/mouse) decreased the interaction time exhibiting an anxiogenic-like response, while capsazepine (10, and 100 μg/mouse) produced anxiolytic-like response similar to that of diazepam (0.25-4 mg/kg, i.p). Prior administration of capsaicin at a dose, inactive per se (0.1 μg/mouse) attenuated the anxiolytic effect of diazepam, whereas, co-administration of capsazepine and diazepam both in their sub-effective as well as effective doses exhibited significant anxiolytic-like effect. Interestingly, the combined treatment of diazepam (2mg/kg) and capsazepine (100μg/mouse) produced no sedative or locomotor deficit effects. On the contrary, a higher dose of diazepam (>2mg/kg) alone was found to be a sedative or locomotor depressant, indicating that the anxiolytic effect of diazepam, at least in part involve TRPV1 receptor. Morever, capsazepine pretreatment blocked the anxiogenic effect of capsaicin (1, and 100 μg/mouse). Taken together, these findings suggest that blockade of TRPV1 might be a functional tool to prevent the risks associated with the long-term use of benzodiazepines.

  3. Overexpression of Transient Receptor Protein Cation Channel Subfamily A Member 1, Confers an Independent Prognostic Indicator in Nasopharyngeal Carcinoma

    PubMed Central

    Wu, You-Ting; Yen, Shao-Lun; Li, Chien-Feng; Chan, Ti-Chun; Chen, Tzu-Ju; Lee, Sung-Wei; He, Hong-Lin; Chang, I-Wei; Hsing, Chung-Hsi; Shiue, Yow-Ling

    2016-01-01

    Background: Detection of oncogenes provides chances to understand tumor development and progression. Transient receptor protein cation channel subfamily A, member 1 (TRPA1) transcript was significantly upregulated in nasopharyngeal carcinoma (NPC) with a stepwise upregulation from low- to high-stage NPCs from a preliminary data analysis in the Gene Expression Omnibus database. The TRPA1 gene is a member of the TRP channel family, encoding integral membrane proteins that functions as cation channels. Loss of calcium homeostasis takes place in cancer cells. Methods: Immunostaining of TRPA1 was analyzed on 124 biopsies from NPC patients retrospectively. The H-score method was used to evaluate the immunoexpression of TRPA1. The correlations between H-score of TRPA1 protein level and clinicopathological factors, as well as the significances of TRPA1 protein level for disease-specific, distal-metastasis-free and local recurrence-free survivals were assessed. Results: These patients were characterized to be no initial metastasis and medicated with the traditional procedure. The TRPA1 score was found to be associated with clinicopathological parameters and patient survivals. Along with the guideline of 7th edition of the American Joint Committee on Cancer, we found that TRPA1 upregulation (50%) was associated with advanced primary tumor (P = 0.009) and overall clinical stage (P = 0.019). In univariate log-rank testing, primary tumor, nodal status, stage and TRPA1 protein level significantly contributed to worse disease-specific survival, distal metastasis-free survival and local recurrence-free survival. In multivariate analysis, high TRPA1 protein level and tumor stage emerged as independent prognostic indicators for inferior disease-specific survival (P = 0.014; P = 0.003), distal metastasis-free survival (P = 0.004; P = 0.034) and recurrence-free survival (P = 0.017; P = 0.015). Conclusions: The upregulation of TRPA1 protein level is frequently correlated to unfavorable

  4. Sphingosine and FTY720 are potent inhibitors of the transient receptor potential melastatin 7 (TRPM7) channels

    PubMed Central

    Qin, Xin; Yue, Zhichao; Sun, Baonan; Yang, Wenzhong; Xie, Jia; Ni, Eric; Feng, Yi; Mahmood, Rafat; Zhang, Yanhui; Yue, Lixia

    2013-01-01

    Background and Purpose Transient receptor potential melastatin 7 (TRPM7) is a unique channel kinase which is crucial for various physiological functions. However, the mechanism by which TRPM7 is gated and modulated is not fully understood. To better understand how modulation of TRPM7 may impact biological processes, we investigated if TRPM7 can be regulated by the phospholipids sphingosine (SPH) and sphingosine-1-phosphate (S1P), two potent bioactive sphingolipids that mediate a variety of physiological functions. Moreover, we also tested the effects of the structural analogues of SPH, N,N-dimethyl-D-erythro-sphingosine (DMS), ceramides and FTY720 on TRPM7. Experimental Approach HEK293 cells stably expressing TRPM7 were used for whole-cell, single-channel and macropatch current recordings. Cardiac fibroblasts were used for native TRPM7 current recording. Key Results SPH potently inhibited TRPM7 in a concentration-dependent manner, whereas S1P and other ceramides did not produce noticeable effects. DMS also markedly inhibited TRPM7. Moreover, FTY720, an immunosuppressant and the first oral drug for treatment of multiple sclerosis, inhibited TRPM7 with a similar potency to that of SPH. In contrast, FTY720-P has no effect on TRPM7. It appears that SPH and FTY720 inhibit TRPM7 by reducing channel open probability. Furthermore, endogenous TRPM7 in cardiac fibroblasts was markedly inhibited by SPH, DMS and FTY720. Conclusions and Implications This is the first study demonstrating that SPH and FTY720 are potent inhibitors of TRPM7. Our results not only provide a new modulation mechanism of TRPM7, but also suggest that TRPM7 may serve as a direct target of SPH and FTY720, thereby mediating S1P-independent physiological/pathological functions of SPH and FTY720. Linked Article This article is commented on by Rohacs, pp. 1291–1293 of this issue. To view this commentary visit http://dx.doi.org/10.1111/bph.12070 PMID:23145923

  5. Increases in reactive oxygen species enhance vascular endothelial cell migration through a mechanism dependent on the transient receptor potential melastatin 4 ion channel.

    PubMed

    Sarmiento, Daniela; Montorfano, Ignacio; Cerda, Oscar; Cáceres, Mónica; Becerra, Alvaro; Cabello-Verrugio, Claudio; Elorza, Alvaro A; Riedel, Claudia; Tapia, Pablo; Velásquez, Luis A; Varela, Diego; Simon, Felipe

    2015-03-01

    A hallmark of severe inflammation is reactive oxygen species (ROS) overproduction induced by increased inflammatory mediators secretion. During systemic inflammation, inflammation mediators circulating in the bloodstream interact with endothelial cells (ECs) raising intracellular oxidative stress at the endothelial monolayer. Oxidative stress mediates several pathological functions, including an exacerbated EC migration. Because cell migration critically depends on calcium channel-mediated Ca(2+) influx, the molecular identification of the calcium channel involved in oxidative stress-modulated EC migration has been the subject of intense investigation. The transient receptor potential melastatin 4 (TRPM4) protein is a ROS-modulated non-selective cationic channel that performs several cell functions, including regulating intracellular Ca(2+) overload and Ca(2+) oscillation. This channel is expressed in multiple tissues, including ECs, and contributes to the migration of certain immune cells. However, whether the TRPM4 ion channel participates in oxidative stress-mediated EC migration is not known. Herein, we investigate whether oxidative stress initiates or enhances EC migration and study the role played by the ROS-modulated TRPM4 ion channel in oxidative stress-mediated EC migration. We demonstrate that oxidative stress enhances, but does not initiate, EC migration in a dose-dependent manner. Notably, we demonstrate that the TRPM4 ion channel is critical in promoting H2O2-enhanced EC migration. These results show that TRPM4 is a novel pharmacological target for the possible treatment of severe inflammation and other oxidative stress-mediated inflammatory diseases.

  6. Biphasic regulation of InsP3 receptor gating by dual Ca2+ release channel BH3-like domains mediates Bcl-xL control of cell viability

    PubMed Central

    Yang, Jun; Vais, Horia; Gu, Wenen; Foskett, J. Kevin

    2016-01-01

    Antiapoptotic Bcl-2 family members interact with inositol trisphosphate receptor (InsP3R) Ca2+ release channels in the endoplasmic reticulum to modulate Ca2+ signals that affect cell viability. However, the molecular details and consequences of their interactions are unclear. Here, we found that Bcl-xL activates single InsP3R channels with a biphasic concentration dependence. The Bcl-xL Bcl-2 homology 3 (BH3) domain-binding pocket mediates both high-affinity channel activation and low-affinity inhibition. Bcl-xL activates channel gating by binding to two BH3 domain-like helices in the channel carboxyl terminus, whereas inhibition requires binding to one of them and to a previously identified Bcl-2 interaction site in the channel-coupling domain. Disruption of these interactions diminishes cell viability and sensitizes cells to apoptotic stimuli. Our results identify BH3-like domains in an ion channel and they provide a unifying model of the effects of antiapoptotic Bcl-2 proteins on the InsP3R that play critical roles in Ca2+ signaling and cell viability. PMID:26976600

  7. STIM1 converts TRPC1 from a receptor-operated to a store-operated channel: moving TRPC1 in and out of lipid rafts.

    PubMed

    Alicia, Sampieri; Angélica, Zepeda; Carlos, Saldaña; Alfonso, Salgado; Vaca, Luis

    2008-11-01

    While the role of members from the TRPC family of channels as receptor-operated channels (ROC) is well established and supported by numerous studies, the role of this family of channels as store-operated channels (SOC) has been the focus of a heated controversy over the last few years. In the present study, we have explored the modulation of STIM1 on human TRPC1 channel. We show that the association of STIM1 to TRPC1 favors the insertion of TRPC1 into lipid rafts, where TRPC1 functions as a SOC. In the absence of STIM1, TRPC1 associates to other members from the TRPC family of channels to form ROCs. A novel TIRFM-FRET method illustrates the relevance of the dynamic association between STIM1 and TRPC1 for the activation of SOC and the lipid raft localization of the STIM1-TRPC1 complex. This study provides new evidence about the dual activity of TRPC1 (forming ROC or SOC) and the partners needed to determine TRPC1 functional fate. It highlights also the role of plasma membrane microdomains and ER-PM junctions in modulating TRPC1 channel function and its association to STIM1.

  8. Increases in reactive oxygen species enhance vascular endothelial cell migration through a mechanism dependent on the transient receptor potential melastatin 4 ion channel.

    PubMed

    Sarmiento, Daniela; Montorfano, Ignacio; Cerda, Oscar; Cáceres, Mónica; Becerra, Alvaro; Cabello-Verrugio, Claudio; Elorza, Alvaro A; Riedel, Claudia; Tapia, Pablo; Velásquez, Luis A; Varela, Diego; Simon, Felipe

    2015-03-01

    A hallmark of severe inflammation is reactive oxygen species (ROS) overproduction induced by increased inflammatory mediators secretion. During systemic inflammation, inflammation mediators circulating in the bloodstream interact with endothelial cells (ECs) raising intracellular oxidative stress at the endothelial monolayer. Oxidative stress mediates several pathological functions, including an exacerbated EC migration. Because cell migration critically depends on calcium channel-mediated Ca(2+) influx, the molecular identification of the calcium channel involved in oxidative stress-modulated EC migration has been the subject of intense investigation. The transient receptor potential melastatin 4 (TRPM4) protein is a ROS-modulated non-selective cationic channel that performs several cell functions, including regulating intracellular Ca(2+) overload and Ca(2+) oscillation. This channel is expressed in multiple tissues, including ECs, and contributes to the migration of certain immune cells. However, whether the TRPM4 ion channel participates in oxidative stress-mediated EC migration is not known. Herein, we investigate whether oxidative stress initiates or enhances EC migration and study the role played by the ROS-modulated TRPM4 ion channel in oxidative stress-mediated EC migration. We demonstrate that oxidative stress enhances, but does not initiate, EC migration in a dose-dependent manner. Notably, we demonstrate that the TRPM4 ion channel is critical in promoting H2O2-enhanced EC migration. These results show that TRPM4 is a novel pharmacological target for the possible treatment of severe inflammation and other oxidative stress-mediated inflammatory diseases. PMID:24518820

  9. Spinal transient receptor potential ankyrin 1 channel induces mechanical hypersensitivity, increases cutaneous blood flow, and mediates the pronociceptive action of dynorphin A.

    PubMed

    Wei, H; Saarnilehto, M; Falck, L; Viisanen, H; Lasierra, M; Koivisto, A; Pertovaara, A

    2013-06-01

    We characterized pain behavior and cutaneous blood flow response induced by activation of the spinal transient receptor potential ankyrin 1 (TRPA1) channel using intrathecal drug administrations in the rat. Additionally, we assessed whether the pronociceptive actions induced by intrathecally administered dynorphin A, cholecystokinin or prostaglandin F(2α) are mediated by the spinal TRPA1 channel. Cinnamaldehyde, a TRPA1 agonist, produced a dose-related (3-10 μg) cutaneous blood flow increase and mechanical hypersensitivity effect. These effects at the currently used doses were of short duration and attenuated, although not completely, by pretreatment with A-967079, a TRPA1 antagonist. The cinnamaldehyde-induced hypersensitivity was also reduced by pretreatment with minocycline (an inhibitor of microglial activation), but not by carbenoxolone (a gap junction decoupler). In vitro study, however, indicated that minocycline only poorly blocks the TRPA1 channel. The mechanical hypersensitivity effect induced by dynorphin A, but not that by cholecystokinin or prostaglandin F(2α), was attenuated by a TRPA1 antagonist Chembridge-5861528 as well as A-967079. The cinnamaldehyde-induced cutaneous blood flow increase was not suppressed by MK-801, an NMDA receptor antagonist, or bicuculline, a GABA(A) receptor antagonist. The results indicate that spinal TRPA1 channels promote mechanical pain hypersensitivity and due to antidromic activation of nociceptive nerve fibers increase cutaneous blood flow. The attenuation of the cinnamaldehyde-induced hypersensitivity effect by minocycline may be explained by action other than block of the TRPA1 channel. Moreover, the spinal TRPA1 channel is involved in mediating the pronociceptive action of dynorphin A, but not that of the spinal cholecystokinin or prostaglandin F(2α).

  10. Sequence analysis, characterization and mRNA distribution of channel catfish (Ictalurus punctatus Rafinesque, 1818) chemokine (C-X-C motif) receptor 4 (CXCR4) cDNA.

    PubMed

    Yeh, Hung-Yueh; Klesius, Phillip H

    2010-04-15

    Chemokine receptor CXCR4, a member of the G protein-coupled receptor superfamily, binds selectively CXCL12. This protein plays many important roles in immunological as well as pathophysiological functions. In this study, we identified and characterized the channel catfish CXCR4 transcript. The full-length nucleic acid sequence of channel catfish CXCR4 cDNA comprised of 1994 nucleotides, including an open reading frame, which appears to encode a putative peptide of 357 amino acid residues with a calculated molecular mass of 40.1kDa. By comparison with the human counterpart, the channel catfish CXCR4 peptide can be divided into domains, including seven transmembrane domains, four cytoplasmic domains, and four extracellular domains. The CXCR4 transcript was detected in spleen, anterior kidney, liver, intestine, skin and gill of all catfish examined in this study. Because four CXCL of channel catfish have been identified, the result provides valuable information for further exploring the channel catfish chemokine signalling pathways and their roles in immune responses to infection. PMID:19853928

  11. On the Roles of the Transient Receptor Potential Canonical 3 (TRPC3) Channel in Endothelium and Macrophages: Implications in Atherosclerosis.

    PubMed

    Vazquez, Guillermo; Solanki, Sumeet; Dube, Prabhatachandra; Smedlund, Kathryn; Ampem, Prince

    2016-01-01

    In the cardiovascular and hematopoietic systems the Transient Receptor Potential Canonical 3 (TRPC3) channel has a well-recognized role in a number of signaling mechanisms that impact the function of diverse cells and tissues in physiology and disease. The latter includes, but is not limited to, molecular and cellular mechanisms associated to the pathogenesis of cardiac hypertrophy, hypertension and endothelial dysfunction. Despite several of these functions being closely related to atherorelevant mechanisms, the potential roles of TRPC3 in atherosclerosis, the major cause of coronary artery disease, have remained largely unexplored. Over recent years, a series of studies from the authors' laboratory revealed novel functions of TRPC3 in mechanisms related to endothelial inflammation, monocyte adhesion to endothelium and survival and apoptosis of macrophages. The relevance of these new TRPC3 functions to atherogenesis has recently began to receive validation through studies in mouse models of atherosclerosis with conditional gain or loss of TRPC3 function. This chapter summarizes these novel findings and provides a discussion of their impact in the context of atherosclerosis, in an attempt to delineate a framework for further exploration of this terra incognita in the TRPC field. PMID:27161230

  12. Effect of adenosine and adenosine receptor antagonist on Müller cell potassium channel in Rat chronic ocular hypertension models.

    PubMed

    Yang, Zijian; Huang, Ping; Liu, Xiaohong; Huang, Shouyue; Deng, Lianfu; Jin, Zhe; Xu, Shuo; Shen, Xi; Luo, Xunda; Zhong, Yisheng

    2015-01-01

    Müller cells are principal glial cells in rat retina and have attracted much attention in glaucoma studies. However, it is not clear whether adenosine and adenosine receptor (AR) antagonists play any roles in the regulation of potassium channels in Müller cells and subsequently in the promotion of glutamine synthetase (GS) and L-Glutamate/L-Aspartate Transporter (GLAST) functions. We found that chronic ocular hypertension (COH) in rat down-regulated Müller cells Kir2.1, Kir4.1, TASK-1, GS and GLAST expressions and attenuated the peak of inward potassium current. Retinal ganglion cells (RGC) count was lower in the COH rats than that in the sham operation animals. Intravitreal injection of selective A2A AR antagonist SCH442416 up-regulated Müller cell Kir4.1, TASK-1, GS and GLAST expressions and enhanced inward potassium currents compared with those in the COH rats with vehicle control. Meanwhile, the RGC count was higher following intravitreal injection of SCH442416 in the COH rats than that after vehicle injection. The fact that PKA inhibitor H-89 blocked these SCH442416 effects suggested that the PKA signaling pathway was involved in the observed ocular responses following the intravitreal SCH442416 injection. PMID:26063641

  13. Blockade of transient receptor potential cation channel subfamily V member 1 promotes regeneration after sciatic nerve injury

    PubMed Central

    Ren, Fei; Zhang, Hong; Qi, Chao; Gao, Mei-ling; Wang, Hong; Li, Xia-qing

    2015-01-01

    The transient receptor potential cation channel subfamily V member 1 (TRPV1) provides the sensation of pain (nociception). However, it remains unknown whether TRPV1 is activated after peripheral nerve injury, or whether activation of TRPV1 affects neural regeneration. In the present study, we established rat models of unilateral sciatic nerve crush injury, with or without pretreatment with AMG517 (300 mg/kg), a TRPV1 antagonist, injected subcutaneously into the ipsilateral paw 60 minutes before injury. At 1 and 2 weeks after injury, we performed immunofluorescence staining of the sciatic nerve at the center of injury, at 0.3 cm proximal and distal to the injury site, and in the dorsal root ganglia. Our results showed that Wallerian degeneration occurred distal to the injury site, and neurite outgrowth and Schwann cell regeneration occurred proximal to the injury. The number of regenerating myelinated and unmyelinated nerve clusters was greater in the AMG517-pretreated rats than in the vehicle-treated group, most notably 2 weeks after injury. TRPV1 expression in the injured sciatic nerve and ipsilateral dorsal root ganglia was markedly greater than on the contralateral side. Pretreatment with AMG517 blocked this effect. These data indicate that TRPV1 is activated or overexpressed after sciatic nerve crush injury, and that blockade of TRPV1 may accelerate regeneration of the injured sciatic nerve. PMID:26487864

  14. Stress-induced switch in Numb isoforms enhances Notch-dependent expression of subtype-specific transient receptor potential channel.

    PubMed

    Kyriazis, George A; Belal, Cherine; Madan, Meenu; Taylor, David G; Wang, Jang; Wei, Zelan; Pattisapu, Jogi V; Chan, Sic L

    2010-02-26

    The Notch signaling pathway plays an essential role in the regulation of cell specification by controlling differentiation, proliferation, and apoptosis. Numb is an intrinsic regulator of the Notch pathway and exists in four alternative splice variants that differ in the length of their phosphotyrosine-binding domain (PTB) and proline-rich region domains. The physiological relevance of the existence of the Numb splice variants and their exact regulation are still poorly understood. We previously reported that Numb switches from isoforms containing the insertion in PTB to isoforms lacking this insertion in neuronal cells subjected to trophic factor withdrawal (TFW). The functional relevance of the TFW-induced switch in Numb isoforms is not known. Here we provide evidence that the TFW-induced switch in Numb isoforms regulates Notch signaling strength and Notch target gene expression. PC12 cells stably overexpressing Numb isoforms lacking the PTB insertion exhibited higher basal Notch activity and Notch-dependent transcription of the transient receptor potential channel 6 (TRPC6) when compared with those overexpressing Numb isoforms with the PTB insertion. The differential regulation of TRPC6 expression is correlated with perturbed calcium signaling and increased neuronal vulnerability to TFW-induced death. Pharmacological inhibition of the Notch pathway or knockdown of TRPC6 function ameliorates the adverse effects caused by the TFW-induced switch in Numb isoforms. Taken together, our results indicate that Notch and Numb interaction may influence the sensitivity of neuronal cells to injurious stimuli by modulating calcium-dependent apoptotic signaling cascades.

  15. Mineralocorticoid receptor stimulation induces urinary storage dysfunction via upregulation of epithelial sodium channel expression in the rat urinary bladder epithelium.

    PubMed

    Yamamoto, Seiji; Hotta, Yuji; Maeda, Kotomi; Kataoka, Tomoya; Maeda, Yasuhiro; Hamakawa, Takashi; Sasaki, Shoichi; Yasui, Takahiro; Asai, Kiyofumi; Kimura, Kazunori

    2016-04-01

    We aimed to evaluate mineralocorticoid receptor (MR) expression in rat bladder and the physiological role of the MR-epithelial sodium channel (ENaC) pathway in controlling bladder function in 10-12-week-old, male Sprague-Dawley rats. First, we examined the mRNA expression of MR and localization of MR and ENaC-α proteins in the urinary bladder. MR mRNA expression was observed in untreated-rat urinary bladders, and MR and ENaC-α proteins were localized in the epithelium. Next, rats were treated with vehicle (controls) or fludrocortisone (an MR agonist) for 3 days, and ENaC-α protein expression levels and bladder function were evaluated on day 4. ENaC-α protein expression was significantly higher in fludrocortisone-treated rats than in controls. In addition, cystometry was performed during intravesical infusion of saline and amiloride (an ENaC inhibitor). While intercontraction intervals (ICIs) during saline infusion were significantly shorter in the fludrocortisone group than in the controls, infusion of amiloride normalized the ICIs in the fludrocortisone group. However, no intra- or inter-group differences in maximum intravesical pressure were observed. Taken together, MR protein is localized in the rat urinary bladder epithelium, and may regulate ENaC expression and bladder afferent input. The MR-ENaC pathway may be a therapeutic target for ameliorating storage symptoms. PMID:26976493

  16. Evodiamine suppresses capsaicin-induced thermal hyperalgesia through activation and subsequent desensitization of the transient receptor potential V1 channels.

    PubMed

    Iwaoka, Emiko; Wang, Shenglan; Matsuyoshi, Nobuyuki; Kogure, Yoko; Aoki, Shunji; Yamamoto, Satoshi; Noguchi, Koichi; Dai, Yi

    2016-01-01

    Evodiae fructus (EF), a fruit of Evodia rutaecarpa Bentham, has long been used as an analgesic drug in traditional Chinese and Japanese medicine. However, the underlying molecular mechanism of its pharmacological action is unclear. Here, using calcium imaging, whole-cell patch-clamp recording, and behavioral analysis, we investigated the pharmacological action of EF and its principal compound, evodiamine, on the transient receptor potential (TRP) V1 channels. Dorsal root ganglion (DRG) neurons and TRPV1- or TRPA1-transfected human embryonic kidney-derived (HEK) 293 cells were used for calcium imaging or whole-cell patch-clamp recording. Twenty male adult Sprague-Dawley rats were used for the capsaicin-induced thermal hyperalgesia behavioral analyses. We found that evodiamine induced significant increases in intracellular calcium and robust inward currents in a subpopulation of isolated rat DRG neurons, most of which were also sensitive to capsaicin. The effect of evodiamine was completely blocked by capsazepine, a competitive antagonist of TRPV1. Evodiamine induced significant inward currents in TRPV1-, but not TRPA1-transfected HEK293 cells. Pretreatment with evodiamine reduced capsaicin-induced currents significantly. Furthermore, the in vivo pre-treatment of evodiamine suppressed thermal hyperalgesia induced by intraplantar injection of capsaicin in rats. These results identify that the analgesic effect of EF and evodiamine may be due to the activation and subsequent desensitization of TRPV1 in sensory neurons.

  17. Alteration of the mu opioid receptor: Ca2+ channel signaling pathway in a subset of rat sensory neurons following chronic femoral artery occlusion.

    PubMed

    Hassan, Bassil; Kim, Joyce S; Farrag, Mohamed; Kaufman, Marc P; Ruiz-Velasco, Victor

    2014-12-15

    The exercise pressor reflex, a crucial component of the cardiovascular response under physiological and pathophysiological states, is activated via metabolic and mechanical mediators that originate from contracting muscles and stimulate group III and IV afferents. We reported previously that stimulation of mu opioid receptors (MOR), expressed in both afferents, led to a significant attenuation of the reflex in rats whose femoral arteries had been occluded for 72 h. The present study examined the effect of arterial occlusion on the signaling components involved in the opioid-mediated modulation of Ca(2+) channels in rat dorsal root ganglion neurons innervating the triceps surae muscles. We focused on neurons that were transfected with cDNA coding for enhanced green fluorescent protein whose expression is driven by the voltage-gated Na(+) channel 1.8 (Na(V)1.8) promoter region, a channel expressed primarily in nociceptive neurons. With the use of a small interference RNA approach, our results show that the pertussis toxin-sensitive Gα(i3) subunit couples MOR with Ca(2+) channels. We observed a significant leftward shift of the MOR agonist [D-Ala2-N-Me-Phe4-Glycol5]-enkephalin concentration-response relationship in neurons isolated from rats with occluded arteries compared with those that were perfused freely. Femoral occlusion did not affect Ca(2+) channel density or the fraction of the main Ca(2+) channel subtype. Furthermore, Western blotting analysis indicated that the leftward shift did not result from either increased Gα(i3) or MOR expression. Finally, all neurons from both groups exhibited an inward current following exposure of the transient potential receptor vanilloid 1 (TRPV1) agonist, 8-methyl-N-vanillyl-6-nonenamide. These findings suggest that sensory neurons mediating the exercise pressor reflex express Na(V)1.8 and TRPV1 channels, and femoral occlusion alters the MOR pharmacological profile. PMID:25231620

  18. Phosphorylation of the Drosophila Transient Receptor Potential Ion Channel Is Regulated by the Phototransduction Cascade and Involves Several Protein Kinases and Phosphatases

    PubMed Central

    Voolstra, Olaf; Bartels, Jonas-Peter; Oberegelsbacher, Claudia; Pfannstiel, Jens; Huber, Armin

    2013-01-01

    Protein phosphorylation plays a cardinal role in regulating cellular processes in eukaryotes. Phosphorylation of proteins is controlled by protein kinases and phosphatases. We previously reported the light-dependent phosphorylation of the Drosophila transient receptor potential (TRP) ion channel at multiple sites. TRP generates the receptor potential upon stimulation of the photoreceptor cell by light. An eye-enriched protein kinase C (eye-PKC) has been implicated in the phosphorylation of TRP by in vitro studies. Other kinases and phosphatases of TRP are elusive. Using phosphospecific antibodies and mass spectrometry, we here show that phosphorylation of most TRP sites depends on the phototransduction cascade and the activity of the TRP ion channel. A candidate screen to identify kinases and phosphatases provided in vivo evidence for an involvement of eye-PKC as well as other kinases and phosphatases in TRP phosphorylation. PMID:24040070

  19. Light at the end of the Ca(2+)-release channel tunnel: structures and mechanisms involved in ion translocation in ryanodine receptor channels.

    PubMed

    Williams, A J; West, D J; Sitsapesan, R

    2001-02-01

    RyR and InsP3R are Ca(2+)-release channels. When induced to open by the appropriate stimulus, these channels allow Ca2+ to leave intracellular storage organelles at an astonishing rate. Investigations of the ion-handling properties of isolated RyR channels have demonstrated that, at least in comparison to voltage-gated channels of surface membranes, these channels display limited powers of discrimination between physiologically relevant cations and this relative lack of selectivity is likely to contribute to the ability of Ca(2+)-release channels to maintain high rates of cation translocation without compromising function. A range of ion-handling properties in RyR are consistent with the proposal that this channel functions as a single-ion channel and theoretical considerations indicate that the high rates of ion translocation monitored for RyR would require the pore of such a structure to be short and possess a large capture radius. Measurements of the dimensions of regions of RyR involved in ion conduction and discrimination indicate that this is likely to be the case. In each monomer of RyR/InsP3R, residues making up the last two trans-membrane spanning domains and a luminal loop linking these two helices contribute to the formation of the channel pore. The luminal loops of both RyR and InsP3R contain amino acid sequences similar to those known to form the selectivity filter of K+ channels. In addition the luminal loops of both Ca(2+)-release channels contain sequences that are likely to form helices that may be analogous to the pore helix visualised in KcsA. The correlation in structural elements of the luminal loops of RyR/InsP3R and KcsA has prompted us to speculate on the tertiary arrangement for this region of the Ca(2+)-release channels using the established structure of KcsA as a framework.

  20. Novel regulatory mechanism in human urinary bladder: central role of transient receptor potential melastatin 4 channels in detrusor smooth muscle function.

    PubMed

    Hristov, Kiril L; Smith, Amy C; Parajuli, Shankar P; Malysz, John; Rovner, Eric S; Petkov, Georgi V

    2016-04-01

    Transient receptor potential melastatin 4 (TRPM4) channels are Ca(2+)-activated nonselective cation channels that have been recently identified as regulators of detrusor smooth muscle (DSM) function in rodents. However, their expression and function in human DSM remain unexplored. We provide insights into the functional role of TRPM4 channels in human DSM under physiological conditions. We used a multidisciplinary experimental approach, including RT-PCR, Western blotting, immunohistochemistry and immunocytochemistry, patch-clamp electrophysiology, and functional studies of DSM contractility. DSM samples were obtained from patients without preoperative overactive bladder symptoms. RT-PCR detected mRNA transcripts for TRPM4 channels in human DSM whole tissue and freshly isolated single cells. Western blotting and immunohistochemistry with confocal microscopy revealed TRPM4 protein expression in human DSM. Immunocytochemistry further detected TRPM4 protein expression in DSM single cells. Patch-clamp experiments showed that 9-phenanthrol, a selective TRPM4 channel inhibitor, significantly decreased the transient inward cation currents and voltage step-induced whole cell currents in freshly isolated human DSM cells. In current-clamp mode, 9-phenanthrol hyperpolarized the human DSM cell membrane potential. Furthermore, 9-phenanthrol attenuated the spontaneous phasic, carbachol-induced and nerve-evoked contractions in human DSM isolated strips. Significant species-related differences in TRPM4 channel activity between human, rat, and guinea pig DSM were revealed, suggesting a more prominent physiological role for the TRPM4 channel in the regulation of DSM function in humans than in rodents. In conclusion, TRPM4 channels regulate human DSM excitability and contractility and are critical determinants of human urinary bladder function. Thus, TRPM4 channels could represent promising novel targets for the pharmacological or genetic control of overactive bladder.

  1. Neonatal Diabetes Caused by Mutations in Sulfonylurea Receptor 1: Interplay between Expression and Mg-Nucleotide Gating Defects of ATP-Sensitive Potassium Channels

    PubMed Central

    Zhou, Qing; Garin, Intza; Castaño, Luis; Argente, Jesús; Muñoz-Calvo, Ma. Teresa; Perez de Nanclares, Guiomar; Shyng, Show-Ling

    2010-01-01

    Context: ATP-sensitive potassium (KATP) channels regulate insulin secretion by coupling glucose metabolism to β-cell membrane potential. Gain-of-function mutations in the sulfonylurea receptor 1 (SUR1) or Kir6.2 channel subunit underlie neonatal diabetes. Objective: The objective of the study was to determine the mechanisms by which two SUR1 mutations, E208K and V324M, associated with transient neonatal diabetes affect KATP channel function. Design: E208K or V324M mutant SUR1 was coexpressed with Kir6.2 in COS cells, and expression and gating properties of the resulting channels were assessed biochemically and electrophysiologically. Results: Both E208K and V324M augment channel response to MgADP stimulation without altering sensitivity to ATP4− or sulfonylureas. Surprisingly, whereas E208K causes only a small increase in MgADP response consistent with the mild transient diabetes phenotype, V324M causes a severe activating gating defect. Unlike E208K, V324M also impairs channel expression at the cell surface, which is expected to dampen its functional impact on β-cells. When either mutation was combined with a mutation in the second nucleotide binding domain of SUR1 previously shown to abolish Mg-nucleotide response, the activating effect of E208K and V324M was also abolished. Moreover, combination of E208K and V324M results in channels with Mg-nucleotide sensitivity greater than that seen in individual mutations alone. Conclusion: The results demonstrate that E208K and V324M, located in distinct domains of SUR1, enhance transduction of Mg-nucleotide stimulation from the SUR1 nucleotide binding folds to Kir6.2. Furthermore, they suggest that diabetes severity is determined by interplay between effects of a mutation on channel expression and channel gating. PMID:20810569

  2. Palmitoylethanolamide normalizes intestinal motility in a model of post-inflammatory accelerated transit: involvement of CB1 receptors and TRPV1 channels

    PubMed Central

    Capasso, Raffaele; Orlando, Pierangelo; Pagano, Ester; Aveta, Teresa; Buono, Lorena; Borrelli, Francesca; Di Marzo, Vincenzo; Izzo, Angelo A

    2014-01-01

    Background and Purpose Palmitoylethanolamide (PEA), a naturally occurring acylethanolamide chemically related to the endocannabinoid anandamide, interacts with targets that have been identified in peripheral nerves controlling gastrointestinal motility, such as cannabinoid CB1 and CB2 receptors, TRPV1 channels and PPARα. Here, we investigated the effect of PEA in a mouse model of functional accelerated transit which persists after the resolution of colonic inflammation (post-inflammatory irritable bowel syndrome). Experimental Approach Intestinal inflammation was induced by intracolonic administration of oil of mustard (OM). Mice were tested for motility and biochemical and molecular biology changes 4 weeks later. PEA, oleoylethanolamide and endocannabinoid levels were measured by liquid chromatography-mass spectrometry and receptor and enzyme mRNA expression by qRT-PCR. Key Results OM induced transient colitis and a functional post-inflammatory increase in upper gastrointestinal transit, associated with increased intestinal anandamide (but not 2-arachidonoylglycerol, PEA or oleoylethanolamide) levels and down-regulation of mRNA for TRPV1 channels. Exogenous PEA inhibited the OM-induced increase in transit and tended to increase anandamide levels. Palmitic acid had a weaker effect on transit. Inhibition of transit by PEA was blocked by rimonabant (CB1 receptor antagonist), further increased by 5′-iodoresiniferatoxin (TRPV1 antagonist) and not significantly modified by the PPARα antagonist GW6471. Conclusions and Implications Intestinal endocannabinoids and TRPV1 channel were dysregulated in a functional model of accelerated transit exhibiting aspects of post-inflammatory irritable bowel syndrome. PEA counteracted the accelerated transit, the effect being mediated by CB1 receptors (possibly via increased anandamide levels) and modulated by TRPV1 channels. PMID:24818658

  3. Activation of muscarinic M3 receptors inhibits large-conductance voltage- and Ca2+-activated K+ channels in rat urinary bladder smooth muscle cells

    PubMed Central

    Parajuli, Shankar P.

    2013-01-01

    Large conductance voltage- and Ca2+-activated K+ (BK) channels are key regulators of detrusor smooth muscle (DSM) contraction and relaxation during urine voiding and storage. Here, we explored whether BK channels are regulated by muscarinic receptors (M-Rs) in native freshly isolated rat DSM cells under physiological conditions using the perforated whole cell patch-clamp technique and pharmacological inhibitors. M-R activation with carbachol (1 μM) initially evoked large transient outward BK currents, followed by inhibition of the spontaneous transient outward BK currents (STBKCs) in DSM cells. Carbachol (1 μM) also inhibited the amplitude and frequency of spontaneous transient hyperpolarizations (STHs) and depolarized the DSM cell membrane potential. Selective inhibition of the muscarinic M3 receptors (M3-Rs) with 4-diphenylacetoxy-N-methylpiperidine (4-DAMP; 0.1 μM), but not muscarinic M2 receptors with methoctramine (1 μM), blocked the carbachol inhibitory effects on STBKCs. Furthermore, blocking the inositol 1,4,5-triphosphate (IP3) receptors with xestospongin-C (1 μM) inhibited the carbachol-induced large transient outward BK currents without affecting carbachol inhibitory effects on STBKCs. Upon pharmacological inhibition of all known cellular sources of Ca2+ for BK channel activation, carbachol (1 μM) did not affect the voltage-step-induced steady-state BK currents, suggesting that the muscarinic effects in DSM cells are mediated by mobilization of intracellular Ca2+. In conclusion, our findings provide strong evidence that activation of M3-Rs leads to inhibition of the STBKCs, STHs, and depolarization of DSM cells. Collectively, the data suggest the existence of functional interactions between BK channels and M3-Rs at a cellular level in DSM. PMID:23703523

  4. [The role of adenosine Al receptors and mitochondrial K+ATP channels in the mechanism of increasing the resistance to acute hypoxia in the combined effects of hypoxia and hypercapnia].

    PubMed

    Tregub, P P; Kulikov, V P; Stepanova, L A; Zabrodina, A S; Nagibaeva, M E

    2014-01-01

    We studied the role of the role of mitoK+ATp channels and Al-adenosine receptor in the mechanism of increasing the resistance to acute hypoxia after hypoxic, hypercapnic and hypercapnic-hypoxic preconditioning. It is shown that mitochondrial ATP-sensitive potassium channels and Al-adenosine receptors, an important mechanism of preconditioning have a high value to increase the resistance to acute hypoxia/ischemia in the combined effect of hypoxia and hypercapnia. However, with regard to the adenosine receptor, this mechanism is realized without the participation hypercapnic component, which apparently starts neuroprotection without activation of the adenosine Al receptors. PMID:25980226

  5. A Specific Subset of Transient Receptor Potential Vanilloid-Type Channel Subunits in Caenorhabditis elegans Endocrine Cells Function as Mixed Heteromers to Promote Neurotransmitter Release

    PubMed Central

    Jose, Antony M.; Bany, I. Amy; Chase, Daniel L.; Koelle, Michael R.

    2007-01-01

    Transient receptor potential (TRP) channel subunits form homotetramers that function in sensory transduction. Heteromeric channels also form, but their physiological subunit compositions and functions are largely unknown. We found a dominant-negative mutant of the C. elegans TRPV (vanilloid-type) subunit OCR-2 that apparently incorporates into and inactivates OCR-2 homomers as well as heteromers with the TRPV subunits OCR-1 and -4, resulting in a premature egg-laying defect. This defect is reproduced by knocking out all three OCR genes, but not by any single knockout. Thus a mixture of redundant heteromeric channels prevents premature egg laying. These channels, as well as the G-protein Gαo, function in neuroendocrine cells to promote release of neurotransmitters that block egg laying until eggs filling the uterus deform the neuroendocrine cells. The TRPV channel OSM-9, previously suggested to be an obligate heteromeric partner of OCR-2 in sensory neurons, is expressed in the neuroendocrine cells but has no detectable role in egg laying. Our results identify a specific set of heteromeric TRPV channels that redundantly regulate neuroendocrine function and show that a subunit combination that functions in sensory neurons is also present in neuroendocrine cells but has no detectable function in these cells. PMID:17057248

  6. Simultaneous optical recording in multiple cells by digital holographic microscopy of chloride current associated to activation of the ligand-gated chloride channel GABA(A) receptor.

    PubMed

    Jourdain, Pascal; Boss, Daniel; Rappaz, Benjamin; Moratal, Corinne; Hernandez, Maria-Clemencia; Depeursinge, Christian; Magistretti, Pierre Julius; Marquet, Pierre

    2012-01-01

    Chloride channels represent a group of targets for major clinical indications. However, molecular screening for chloride channel modulators has proven to be difficult and time-consuming as approaches essentially rely on the use of fluorescent dyes or invasive patch-clamp techniques which do not lend themselves to the screening of large sets of compounds. To address this problem, we have developed a non-invasive optical method, based on digital holographic microcopy (DHM), allowing monitoring of ion channel activity without using any electrode or fluorescent dye. To illustrate this approach, GABA(A) mediated chloride currents have been monitored with DHM. Practically, we show that DHM can non-invasively provide the quantitative determination of transmembrane chloride fluxes mediated by the activation of chloride channels associated with GABA(A) receptors. Indeed through an original algorithm, chloride currents elicited by application of appropriate agonists of the GABA(A) receptor can be derived from the quantitative phase signal recorded with DHM. Finally, chloride currents can be determined and pharmacologically characterized non-invasively simultaneously on a large cellular sampling by DHM.

  7. Activation of protein kinase C and inositol 1,4,5-triphosphate receptors antagonistically modulate voltage-gated sodium channels in striatal neurons.

    PubMed

    Hourez, Raphaël; Azdad, Karima; Vanwalleghem, Gilles; Roussel, Céline; Gall, David; Schiffmann, Serge N

    2005-10-19

    Regulation of voltage-gated sodium channels is crucial to firing patterns that constitute the output of medium spiny neurons (MSN), projecting neurons of the striatum. This modulation is thus critical for the final integration of information processed within the striatum. It has been shown that the adenylate cyclase pathway reduces sodium currents in MSN through channel phosphorylation by cAMP-dependent protein kinase. However, it is unknown whether a phospholipase C (PLC)-mediated signaling cascade could also modulate voltage-gated sodium channels within MSN. Using the whole-cell patch clamp technique, we investigated the effects of activation of two key components in PLC-mediated signaling cascades: protein kinase C (PKC) and inositol-1,4,5-triphosphate (IP(3))