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Sample records for channel sulfonylurea sensitivity

  1. ATP-modulated K+ channels sensitive to antidiabetic sulfonylureas are present in adenohypophysis and are involved in growth hormone release.

    PubMed Central

    Bernardi, H; De Weille, J R; Epelbaum, J; Mourre, C; Amoroso, S; Slama, A; Fosset, M; Lazdunski, M

    1993-01-01

    The adenohypophysis contains high-affinity binding sites for antidiabetic sulfonylureas that are specific blockers of ATP-sensitive K+ channels. The binding protein has a M(r) of 145,000 +/- 5000. The presence of ATP-sensitive K+ channels (26 pS) has been demonstrated by electrophysiological techniques. Intracellular perfusion of adenohypophysis cells with an ATP-free medium to activate ATP-sensitive K+ channels induces a large hyperpolarization (approximately 30 mV) that is antagonized by antidiabetic sulfonylureas. Diazoxide opens ATP-sensitive K+ channels in adenohypophysis cells as it does in pancreatic beta cells and also induces a hyperpolarization (approximately 30 mV) that is also suppressed by antidiabetic sulfonylureas. As in pancreatic beta cells, glucose and antidiabetic sulfonylureas depolarize the adenohypophysis cells and thereby indirectly increase Ca2+ influx through L-type Ca2+ channels. The K+ channel opener diazoxide has an opposite effect. Opening ATP-sensitive K+ channels inhibits growth hormone secretion and this inhibition is eliminated by antidiabetic sulfonylureas. Images PMID:8433992

  2. A role of the sulfonylurea receptor 1 in endocytic trafficking of ATP-sensitive potassium channels

    PubMed Central

    Bruederle, Cathrin E.; Gay, Joel; Shyng, Show-Ling

    2011-01-01

    The ATP-sensitive potassium (KATP) channel consisting of sulfonylurea receptor 1 (SUR1) and inward rectifier potassium channel 6.2 (Kir6.2) has a well-established role in insulin secretion. Mutations in either subunit can lead to disease due to aberrant channel gating, altered channel density at the cell surface or a combination of both. Endocytic trafficking of channels at the plasma membrane is one way to influence surface channel numbers. It has been previously reported that channel endocytosis is dependent on a tyrosine-based motif in Kir6.2 while SUR1 alone is unable to internalize. In this study, we followed endocytic trafficking of surface channels in real time by live cell imaging of channel subunits tagged with an extracellular minimal α-bungarotoxin binding peptide labeled with a fluorescent dye. We demonstrate that SUR1 undergoes endocytosis independent of Kir6.2. Moreover, mutations in the putative endocytosis motif of Kir6.2, Y330C, Y330A and F333I are unable to prevent channel endocytosis. These findings challenge the notion that Kir6.2 bears the sole endocytic signal for KATP channels and support a role of SUR1 in this trafficking process. PMID:21649805

  3. 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

  4. Phosphatidylinositol 4,5-biphosphate (PIP2) modulates syntaxin-1A binding to sulfonylurea receptor 2A to regulate cardiac ATP-sensitive potassium (KATP) channels.

    PubMed

    Xie, Li; Liang, Tao; Kang, Youhou; Lin, Xianguang; Sobbi, Roozbeh; Xie, Huanli; Chao, Christin; Backx, Peter; Feng, Zhong-Ping; Shyng, Show-Ling; Gaisano, Herbert Y

    2014-10-01

    Cardiac sarcolemmal syntaxin (Syn)-1A interacts with sulfonylurea receptor (SUR) 2A to inhibit ATP-sensitive potassium (KATP) channels. Phosphatidylinositol 4,5-bisphosphate (PIP2), a ubiquitous endogenous inositol phospholipid, known to bind Kir6.2 subunit to open KATP channels, has recently been shown to directly bind Syn-1A in plasma membrane to form Syn-1A clusters. Here, we sought to determine whether the interaction between Syn-1A and PIP2 interferes with the ability of Syn-1A to bind SUR2A and inhibit KATP channel activity. We found that PIP2 dose-dependently reduced SUR2A binding to GST-Syn-1A by in vitro pulldown assays. FRET studies in intact cells using TIRFM revealed that increasing endogenous PIP2 levels led to increased Syn-1A (-EGFP) cluster formation and a severe reduction in availability of Syn-1A molecules to interact with SUR2A (-mCherry) molecules outside the Syn-1A clusters. Correspondingly, electrophysiological studies employing SUR2A/Kir6.2-expressing HEK cells showed that increasing endogenous or exogenous PIP2 diminished the inhibitory effect of Syn-1A on KATP currents. The physiological relevance of these findings was confirmed by ability of exogenous PIP2 to block exogenous Syn-1A inhibition of cardiac KATP currents in inside-out patches of mouse ventricular myocytes. The effect of PIP2 on physical and functional interactions between Syn-1A and KATP channels is specific and not observed with physiologic concentrations of other phospholipids. To unequivocally demonstrate the specificity of PIP2 interaction with Syn-1A and its impact on KATP channel modulation by Syn-1A, we employed a PIP2-insensitive Syn-1A-5RK/A mutant. The Syn-1A-5RK/A mutant retains the ability to interact with SUR2A in both in vitro binding and in vivo FRET assays, although as expected the interaction is no longer disrupted by PIP2. Interestingly, at physiological PIP2 concentrations, Syn-1A-5RK/A inhibited KATP currents to a greater extent than Syn-1A-WT, indicating

  5. Sensitivity of a renal K+ channel (ROMK2) to the inhibitory sulfonylurea compound glibenclamide is enhanced by coexpression with the ATP-binding cassette transporter cystic fibrosis transmembrane regulator.

    PubMed Central

    McNicholas, C M; Guggino, W B; Schwiebert, E M; Hebert, S C; Giebisch, G; Egan, M E

    1996-01-01

    We demonstrate here that coexpression of ROMK2, an inwardly rectifying ATP-sensitive renal K+ channel (IKATP) with cystic fibrosis transmembrane regulator (CFTR) significantly enhances the sensitivity of ROMK2 to the sulfonylurea compound glibenclamide. When expressed alone, ROMK2 is relatively insensitive to glibenclamide. The interaction between ROMK2, CFTR, and glibenclamide is modulated by altering the phosphorylation state of either ROMK2, CFTR, or an associated protein, as exogenous MgATP and the catalytic subunit of protein kinase A significantly attenuate the inhibitory effect of glibenclamide on ROMK2. Thus CFTR, which has been demonstrated to interact with both Na+ and Cl- channels in airway epithelium, modulates the function of renal ROMK2 K+ channels. PMID:8755607

  6. ATP-sensitive K/sup +/ channels that are blocked by hypoglycemia-inducing sulfonylureas in insulin-secreting cells are activated by galanin, a hyperglycemia-inducing hormone

    SciTech Connect

    de Weille, J.; Schmid-Antomarchi, H.; Fosset, M.; Lazdunski, M.

    1988-02-01

    The action of the hyperglycemia-inducing hormone galanin, a 29-amino acid peptide names from its N-terminal glycine and C-terminal amidated alanine, was studied in rat insulinoma (RINm5F) cells using electrophysiological and /sup 86/Rb/sup +/ flux techniques. Galanin hyperpolarizes and reduces spontaneous electrical activity by activating a population of APT-sensitive K/sup +/ channels with a single-channel conductance of 30 pS (at -60 mV). Galanin-induced hyperpolarization and reduction of spike activity are reversed by the hypoglycemia-inducing sulfonylurea glibenclamine. Glibenclamide blocks the galanin-activated ATP-sensitive K/sup +/ channel. /sup 86/Rb/sup +/ efflux from insulinoma cells is stimulated by galanin in a dose-dependent manner. The half-maximum value of activation is found at 1.6 nM. Galanin-induced /sup 86/Rb/sup +/ efflux is abolished by glibenclamide. The half-maximum value of inhibition is found at 0.3 nM, which is close to the half-maximum value of inhibition of the ATP-dependent K/sup +/ channel reported earlier. /sup 86/Rb/sup +/ efflux studies confirm the electrophysiological demonstration that galanin activates and ATP-dependent K/sup +/ channel.

  7. [Sulfonylurea].

    PubMed

    Usuda, Rika

    2015-03-01

    SU drug promotes insulin secretion by acting on pancreatic β cell. The hypoglycemic effect is the most powerful among oral diabetic drugs with high cost-effectiveness. Particularly for the Japanese with type 2 diabetes caused by a decrease in insulin secretion as a main pathological condition, it was widely used until the present since 1957 and largely contributed for the sickness. On the other hand, it is true that a number of issues such as a prolonged hypoglycemic coma or obesity due to a neglect of proper usage, patient education, and a possibility of second failure have been discussed so far. After a structure of K(ATP) channel on pancreatic β cell as playing an important role with insulin secretion is clarified recently and neonatal diabetes mellitus by the genetic defect is reported, a new possibility for SU drug receives attention. Furthermore, a receptor of SU drug on β cell membrane was solely known as a target molecule for SU drug, but since a binding to Epac2 as a protein to detect a part of SU drug's cAMP signal within β cell is determined, a relation with an enhancing mechanism of insulin secretion by incretin is being clarified at present. As understanding a new potential for SU drug, we consider a positioning and proper usage for SU drug again. PMID:25812366

  8. Cerebrovascular Safety of Sulfonylureas: The Role of KATP Channels in Neuroprotection and the Risk of Stroke in Patients With Type 2 Diabetes.

    PubMed

    Liu, Rui; Wang, Haitao; Xu, Baofeng; Chen, Wenliang; Turlova, Ekaterina; Dong, Nan; Sun, Christopher L F; Lu, Yangqingqin; Fu, Hanhui; Shi, Ranran; Barszczyk, Andrew; Yang, Dongzi; Jin, Tianru; Mannucci, Edoardo; Feng, Zhong-Ping; Sun, Hong-Shuo

    2016-09-01

    Sulfonylureas are ATP-sensitive potassium (KATP) channel blockers commonly used in the treatment of type 2 diabetes mellitus (T2DM). Activation of KATP channels plays a neuroprotective role in ischemia; thus, whether sulfonylureas affect the outcomes of stroke in patients with T2DM needs to be further studied. In our study, streptozotocin (STZ)-induced diabetic mice subjected to transient middle cerebral artery occlusion (MCAO) showed larger areas of brain damage and poorer behavioral outcomes. Blocking the KATP channel by tolbutamide increased neuronal injury induced by oxygen-glucose deprivation (OGD) in vitro and permanent MCAO (pMCAO) in vivo. Activating the KATP channel by diazoxide reduced the effects of both the OGD and pMCAO. Western blot analysis in STZ mouse brains indicated an early increase in protein levels of N-methyl-d-aspartate receptor 2B and postsynaptic density protein-95, followed by a decrease in phosphorylation of glycogen synthase kinase 3β. Our systematic meta-analysis indicated that patients with T2DM treated with sulfonylureas had a higher odds ratio for stroke morbidity than those who received comparator drugs. Taken together, these results suggest that sulfonylurea treatment in patients with T2DM may inhibit the neuroprotective effects of KATP channels and increase the risk of stroke. PMID:27207539

  9. Hormone-regulated K+ channels in follicle-enclosed oocytes are activated by vasorelaxing K+ channel openers and blocked by antidiabetic sulfonylureas.

    PubMed Central

    Honoré, E; Lazdunski, M

    1991-01-01

    Follicular oocytes from Xenopus laevis contain K+ channels activated by members of the recently recognized class of vasorelaxants that include cromakalim and pinacidil and blocked by antidiabetic sulfonylureas, such as glibenclamide. These channels are situated on the adherent follicular cells and are not present in denuded oocytes. Cromakalim-activated K+ channels are also activated by increases in intracellular cAMP, and cAMP-activated K+ channels are blocked by glibenclamide. Although cromakalim and cAMP effects are synergistic, cromakalim activation of K+ channels is drastically reduced or abolished by treatments that stimulate protein kinase C (e.g., muscarinic effectors, phorbol esters). Gonadotropins, known to play an essential role in ovarian physiology, also activate cromakalim and sulfonylurea-sensitive K+ channels. Follicular oocytes constitute an excellent system for studying regulation of cromakalim-sensitive K+ channels that are important in relation to a variety of disease processes, such as cardiovascular dysfunction and asthma, as well as brain function. PMID:1647032

  10. Evidence for the existence of a sulfonylurea-receptor-like protein in plants: Modulation of stomatal movements and guard cell potassium channels by sulfonylureas and potassium channel openers

    PubMed Central

    Leonhardt, Nathalie; Marin, Elena; Vavasseur, Alain; Forestier, Cyrille

    1997-01-01

    Limitation of water loss and control of gas exchange is accomplished in plant leaves via stomatal guard cells. Stomata open in response to light when an increase in guard cell turgor is triggered by ions and water influx across the plasma membrane. Recent evidence demonstrating the existence of ATP-binding cassette proteins in plants led us to analyze the effect of compounds known for their ability to modulate ATP-sensitive potassium channels (K-ATP) in animal cells. By using epidermal strip bioassays and whole-cell patch-clamp experiments with Vicia faba guard cell protoplasts, we describe a pharmacological profile that is specific for the outward K+ channel and very similar to the one described for ATP-sensitive potassium channels in mammalian cells. Tolbutamide and glibenclamide induced stomatal opening in bioassays and in patch-clamp experiments, a specific inhibition of the outward K+ channel by these compounds was observed. Conversely, application of potassium channel openers such as cromakalim or RP49356 triggered stomatal closure. An apparent competition between sulfonylureas and potassium channel openers occurred in bioassays, and outward potassium currents, previously inhibited by glibenclamide, were partially recovered after application of cromakalim. By using an expressed sequence tag clone from an Arabidopsis thaliana homologue of the sulfonylurea receptor, a 7-kb transcript was detected by Northern blot analysis in guard cells and other tissues. Beside the molecular evidence recently obtained for the expression of ATP-binding cassette protein transcripts in plants, these results give pharmacological support to the presence of a sulfonylurea-receptor-like protein in the guard-cell plasma membrane tightly involved in the outward potassium channel regulation during stomatal movements. PMID:9391169

  11. Sulfonylureas suppress the stimulatory action of Mg-nucleotides on Kir6.2/SUR1 but not Kir6.2/SUR2A KATP channels: a mechanistic study.

    PubMed

    Proks, Peter; de Wet, Heidi; Ashcroft, Frances M

    2014-11-01

    Sulfonylureas, which stimulate insulin secretion from pancreatic β-cells, are widely used to treat both type 2 diabetes and neonatal diabetes. These drugs mediate their effects by binding to the sulfonylurea receptor subunit (SUR) of the ATP-sensitive K(+) (KATP) channel and inducing channel closure. The mechanism of channel inhibition is unusually complex. First, sulfonylureas act as partial antagonists of channel activity, and second, their effect is modulated by MgADP. We analyzed the molecular basis of the interactions between the sulfonylurea gliclazide and Mg-nucleotides on β-cell and cardiac types of KATP channel (Kir6.2/SUR1 and Kir6.2/SUR2A, respectively) heterologously expressed in Xenopus laevis oocytes. The SUR2A-Y1206S mutation was used to confer gliclazide sensitivity on SUR2A. We found that both MgATP and MgADP increased gliclazide inhibition of Kir6.2/SUR1 channels and reduced inhibition of Kir6.2/SUR2A-Y1206S. The latter effect can be attributed to stabilization of the cardiac channel open state by Mg-nucleotides. Using a Kir6.2 mutation that renders the KATP channel insensitive to nucleotide inhibition (Kir6.2-G334D), we showed that gliclazide abolishes the stimulatory effects of MgADP and MgATP on β-cell KATP channels. Detailed analysis suggests that the drug both reduces nucleotide binding to SUR1 and impairs the efficacy with which nucleotide binding is translated into pore opening. Mutation of one (or both) of the Walker A lysines in the catalytic site of the nucleotide-binding domains of SUR1 may have a similar effect to gliclazide on MgADP binding and transduction, but it does not appear to impair MgATP binding. Our results have implications for the therapeutic use of sulfonylureas. PMID:25348414

  12. Optical control of insulin release using a photoswitchable sulfonylurea

    PubMed Central

    Broichhagen, Johannes; Schönberger, Matthias; Cork, Simon C.; Frank, James A.; Marchetti, Piero; Bugliani, Marco; Shapiro, A. M. James; Trapp, Stefan; Rutter, Guy A.; Hodson, David J.; Trauner, Dirk

    2014-01-01

    Sulfonylureas are widely prescribed for the treatment of type 2 diabetes mellitus (T2DM). Through their actions on ATP-sensitive potassium (KATP) channels, sulfonylureas boost insulin release from the pancreatic beta cell mass to restore glucose homeostasis. A limitation of these compounds is the elevated risk of developing hypoglycemia and cardiovascular disease, both potentially fatal complications. Here, we describe the design and development of a photoswitchable sulfonylurea, JB253, which reversibly and repeatedly blocks KATP channel activity following exposure to violet-blue light. Using in situ imaging and hormone assays, we further show that JB253 bestows light sensitivity upon rodent and human pancreatic beta cell function. Thus, JB253 enables the optical control of insulin release and may offer a valuable research tool for the interrogation of KATP channel function in health and T2DM. PMID:25311795

  13. CARDIAC SULFONYLUREA RECEPTOR SHORT FORM-BASED CHANNELS CONFER A GLIBENCLAMIDE-INSENSITIVE KATP ACTIVITY

    PubMed Central

    Pu, Jie-Lin,; Ye, Bin; Kroboth, Stacie L.; McNally, Elizabeth M.; Makielski, Jonathan C.; Shi, Nian-Qing

    2008-01-01

    The cardiac sarcolemmal ATP-sensitive potassium channel (KATP) consists of a Kir6.2 pore and a SUR2 regulatory subunit, which is an ATP-binding cassette (ABC) transporter. KATP channels have been proposed to play protective roles during ischemic preconditioning. A SUR2 mutant mouse was previously generated by disrupting the first nucleotide-binding domain (NBD1), where a glibenclamide action site was located. In the mutant ventricular myocytes, a non-conventional glibenclamide-insensitive (10 μM), ATP-sensitive current (IKATPn) was detected in 33% of single-channel recordings with an average amplitude of 12.3±5.4 pA per patch, an IC50 to ATP inhibition at 10 μM, and a mean burst duration at 20.6±1.8 ms. Newly designed SUR2-isoform or variant-specific antibodies identified novel SUR2 short forms in the sizes of 28 and 68 kDa in addition to a 150-kDa long form in the sarcolemmal membrane of wild-type (WT) heart. We hypothesized that channels constituted by these short forms that lack NBD1, confer IKATPn. The absence of the long form in the mutant corresponded to loss of the conventional glibenclamide-sensitive KATP currents (IKATP) in isolated cardiomyocytes and vascular smooth muscle cells but the SUR2 short forms remained intact. Nested exonic RT-PCR in the mutant indicated that the short forms lacked NBD1 but contained NBD2. The SUR2 short forms co-immunoprecipitated with Kir6.1 or Kir6.2 suggesting that the short forms may function as hemi-transporters reported in other eukaryotic ABC transporter subgroups. Our results indicate that different KATP compositions may co-exist in cardiac sarcolemmal membrane. PMID:18001767

  14. Role of ATP-sensitive K+ channels in cardiac arrhythmias.

    PubMed

    Nakaya, Haruaki

    2014-05-01

    The sarcolemmal adenosine triphosphate (ATP)-sensitive K(+) (sarcKATP) channel in the heart is a hetero-octamer comprising the pore-forming subunit Kir6.2 and the regulatory subunit sulfonylurea receptor SUR2A. By functional analysis of genetically engineered mice lacking sarcKATP channels, the pathophysiological roles of the K(+) channel in the heart have been extensively evaluated. Although mitochondrial KATP (mitoKATP) channel is proposed to be an important effector for the protection of ischemic myocardium and the inhibition of ischemia/reperfusion-induced ventricular arrhythmias, the molecular identity of mitoKATP channel has not been established. Although selective sarcKATP-channel blockers can prevent ischemia/reperfusion-induced ventricular arrhythmias by inhibiting the action potential shortening in the acute phase, the drugs may aggravate the ischemic damages due to intracellular Ca(2+) overload. The sarcKATP channel is also mandatory for optimal adaptation to hemodynamic stress such as sympathetic activation. Dysfunction of mutated sarcKATP channels in atrial cells may lead to electrical instability and atrial fibrillation. Recently, it has been proposed that the gain-of-function mutation of cardiac Kir6.1 channel can be a pathogenic substrate for J wave syndromes, a cause of idiopathic ventricular fibrillation as early repolarization syndrome or Brugada syndrome, whereas loss of function of the channel mutations can underlie sudden infant death syndrome. However, precise role of Kir6.1 channels in cardiac cells remains to be defined and further study may be needed to clarify the role of Kir6.1 channel in the heart. PMID:24367007

  15. BLX‐1002 restores glucose sensitivity and enhances insulin secretion stimulated by GLP‐1 and sulfonylurea in type 2 diabetic pancreatic islets

    PubMed Central

    Zhang, Qimin; Zhang, Fan; Sjöholm, Åke

    2014-01-01

    Abstract BLX‐1002 is a novel thiazolidinedione with no peroxisome proliferator‐activated receptor (PPAR) activity that has been shown to improve glycemia in type 2 diabetes without weight gain. We previously found that BLX‐1002 selectively augments glucose‐sensitive (but not basal) insulin secretion in normal mouse β‐cells. We have now extended these observations to other insulin secretagogues and to diabetic rat islets. To this end, dynamics of insulin secretion stimulated by glucose, GLP‐1, and the sulfonylurea tolbutamide were examined in pancreatic islets from nondiabetic Wistar and type 2 diabetic Goto‐Kakizaki rats ex vivo. BLX‐1002 restored normal glucose‐sensitive insulin secretion in otherwise “glucose‐blind” islets from GK rats, but did not affect basal or glucose‐stimulated secretion in normal Wistar rat islets. The stimulatory effect of BLX‐1002 on insulin secretion at high glucose required Ca2+ and involved phosphatidylinositol 3‐kinase (PI3K) activity. Consistent with its effects on insulin secretion, BLX‐1002 also augmented insulin secretion and cytoplasmic‐free Ca2+ concentrations ([Ca2+]i) stimulated by high glucose, GLP‐1, and tolbutamide in islets from GK, but not Wistar, rats. The inactive analog BLX‐1237 had no effects. In conclusion, our findings suggest that BLX‐1002 potentiates insulin secretion by different stimuli in diabetic β‐cells only, in a Ca2+‐dependent manner and involving PI3K. PMID:24872354

  16. ATP-sensitive K(+) channels in rat colonic epithelium.

    PubMed

    Pouokam, Ervice; Bader, Sandra; Brück, Brigitta; Schmidt, Bärbel; Diener, Martin

    2013-06-01

    ATP-sensitive K(+) (KATP) channels couple the metabolic state of a cell to its electrical activity. They consist of a hetero-octameric complex with pore-forming Kir6.x (Kir6.1, Kir6.2) and regulatory sulfonylurea receptor (SUR) subunits. Functional data indicate that KATP channels contribute to epithelial K(+) currents at colonic epithelia. However, their molecular identity and their properties are largely unknown. Therefore, changes in short-circuit current (I sc) induced by the KATP channel opener pinacidil (5 10(-4) mol l(-1)) were measured in Ussing chambers under control conditions and in the presence of different blockers of KATP channels. The channel subunits expressed by the colonic epithelium were identified by immunohistochemistry and by RT-PCR. The K(+) channel opener, when administered at the serosal side, induced an increase in I sc consistent with the induction of transepithelial Cl(-) secretion after activation of basolateral K(+) channels, whereas mucosal administration of pinacidil resulted in a negative I sc. The increase in I sc evoked by serosal pinacidil was inhibited by serosal administration of glibenclamide (5 10(-4) mol l(-1)) and gliclazide (10(-6) mol l(-1)), but was resistant even against a high concentration (10(-2) mol l(-1)) of tolbutamide. In contrast, none of these inhibitors (administered at the mucosal side) reduced significantly the negative I sc induced by mucosal pinacidil. Instead, pinacidil inhibited Cl(-) currents across apical Cl(-) channels in basolaterally depolarized epithelia indicating that the negative I sc induced by mucosal pinacidil is due to a transient inhibition of Cl(-) secretion. In mRNA prepared from isolated colonic crypts, messenger RNA for both pore-forming subunits, Kir6.1 and Kir6.2, and two regulatory subunits (SUR1 and SUR2B) was found. Expression within the colonic epithelium was confirmed for these subunits by immunohistochemistry. In consequence, KATP channels are present in the basolateral membrane

  17. Acid-sensitive ion channels and receptors.

    PubMed

    Holzer, Peter

    2009-01-01

    Acidosis is a noxious condition associated with inflammation, ischaemia or defective acid containment. As a consequence, acid sensing has evolved as an important property of afferent neurons with unmyelinated and thinly myelinated nerve fibres. Protons evoke multiple currents in primary afferent neurons, which are carried by several acid-sensitive ion channels. Among these, acid-sensing ion channels (ASICs) and transient receptor potential (TRP) vanilloid-1 (TRPV1) ion channels have been most thoroughly studied. ASICs survey moderate decreases in extracellular pH, whereas TRPV1 is activated only by severe acidosis resulting in pH values below 6. Two-pore-domain K(+) (K(2P)) channels are differentially regulated by small deviations of extra- or intracellular pH from physiological levels. Other acid-sensitive channels include TRPV4, TRPC4, TRPC5, TRPP2 (PKD2L1), ionotropic purinoceptors (P2X), inward rectifier K(+) channels, voltage-activated K(+) channels, L-type Ca(2+) channels, hyperpolarization-activated cyclic nucleotide gated channels, gap junction channels, and Cl(-) channels. In addition, acid-sensitive G protein coupled receptors have also been identified. Most of these molecular acid sensors are expressed by primary sensory neurons, although to different degrees and in various combinations. Emerging evidence indicates that many of the acid-sensitive ion channels and receptors play a role in acid sensing, acid-induced pain and acid-evoked feedback regulation of homeostatic reactions. The existence and apparent redundancy of multiple pH surveillance systems attests to the concept that acid-base regulation is a vital issue for cell and tissue homeostasis. Since upregulation and overactivity of acid sensors appear to contribute to various forms of chronic pain, acid-sensitive ion channels and receptors are considered as targets for novel analgesic drugs. This approach will only be successful if the pathological implications of acid sensors can be differentiated

  18. Acid-sensitive ion channels and receptors

    PubMed Central

    Holzer, Peter

    2015-01-01

    Acidosis is a noxious condition associated with inflammation, ischaemia or defective acid containment. As a consequence, acid sensing has evolved as an important property of afferent neurons with unmyelinated and thinly myelinated nerve fibres. Protons evoke multiple currents in primary afferent neurons, which are carried by several acid-sensitive ion channels. Among these, acid-sensing ion channels (ASICs) and transient receptor potential (TRP) vanilloid-1 (TRPV1) ion channels have been most thoroughly studied. ASICs survey moderate decreases in extracellular pH whereas TRPV1 is activated only by severe acidosis resulting in pH values below 6. Two-pore domain K+ (K2P) channels are differentially regulated by small deviations of extra- or intracellular pH from physiological levels. Other acid-sensitive channels comprise TRPV4, TRPC4, TRPC5, TRPP2 (PKD2L1), ionotropic purinoceptors (P2X), inward rectifier K+ channels, voltage-activated K+ channels, L-type Ca2+ channels, hyperpolarization-activated cyclic nucleotide-gated channels, gap junction channels, and Cl− channels. In addition, acid-sensitive G protein-coupled receptors have also been identified. Most of these molecular acid sensors are expressed by primary sensory neurons, although to different degrees and in various combinations. Emerging evidence indicates that many of the acid-sensitive ion channels and receptors play a role in acid sensing, acid-induced pain and acid-evoked feedback regulation of homeostatic reactions. The existence and apparent redundancy of multiple pH surveillance systems attests to the concept that acid-base regulation is a vital issue for cell and tissue homeostasis. Since upregulation and overactivity of acid sensors appear to contribute to various forms of chronic pain, acid-sensitive ion channels and receptors are considered as targets for novel analgesic drugs. This approach will only be successful if the pathological implications of acid sensors can be differentiated

  19. Essential role of adenosine, adenosine A1 receptors, and ATP-sensitive K+ channels in cerebral ischemic preconditioning.

    PubMed Central

    Heurteaux, C; Lauritzen, I; Widmann, C; Lazdunski, M

    1995-01-01

    Preconditioning with sublethal ischemia protects against neuronal damage after subsequent lethal ischemic insults in hippocampal neurons. A pharmacological approach using agonists and antagonists at the adenosine A1 receptor as well as openers and blockers of ATP-sensitive K+ channels has been combined with an analysis of neuronal death and gene expression of subunits of glutamate and gamma-aminobutyric acid receptors, HSP70, c-fos, c-jun, and growth factors. It indicates that the mechanism of ischemic tolerance involves a cascade of events including liberation of adenosine, stimulation of adenosine A1 receptors, and, via these receptors, opening of sulfonylurea-sensitive ATP-sensitive K+ channels. Images Fig. 2 Fig. 3 PMID:7753861

  20. Molecular determinants of ATP-sensitive potassium channel MgATPase activity: diabetes risk variants and diazoxide sensitivity

    PubMed Central

    Fatehi, Mohammad; Carter, Chris R.J.; Youssef, Nermeen; Hunter, Beth E.; Holt, Andrew; Light, Peter E.

    2015-01-01

    ATP-sensitive K+ (KATP) channels play an important role in insulin secretion. KATP channels possess intrinsic MgATPase activity that is important in regulating channel activity in response to metabolic changes, although the precise structural determinants are not clearly understood. Furthermore, the sulfonylurea receptor 1 (SUR1) S1369A diabetes risk variant increases MgATPase activity, but the molecular mechanisms remain to be determined. Therefore, we hypothesized that residue–residue interactions between 1369 and 1372, predicted from in silico modelling, influence MgATPase activity, as well as sensitivity to the clinically used drug diazoxide that is known to increase MgATPase activity. We employed a point mutagenic approach with patch-clamp and direct biochemical assays to determine interaction between residues 1369 and 1372. Mutations in residues 1369 and 1372 predicted to decrease the residue interaction elicited a significant increase in MgATPase activity, whereas mutations predicted to possess similar residue interactions to wild-type (WT) channels elicited no alterations in MgATPase activity. In contrast, mutations that were predicted to increase residue interactions resulted in significant decreases in MgATPase activity. We also determined that a single S1369K substitution in SUR1 caused MgATPase activity and diazoxide pharmacological profiles to resemble those of channels containing the SUR2A subunit isoform. Our results provide evidence, at the single residue level, for a molecular mechanism that may underlie the association of the S1369A variant with type 2 diabetes. We also show a single amino acid difference can account for the markedly different diazoxide sensitivities between channels containing either the SUR1 or SUR2A subunit isoforms. PMID:26181369

  1. Molecular determinants of ATP-sensitive potassium channel MgATPase activity: diabetes risk variants and diazoxide sensitivity.

    PubMed

    Fatehi, Mohammad; Carter, Chris R J; Youssef, Nermeen; Hunter, Beth E; Holt, Andrew; Light, Peter E

    2015-01-01

    ATP-sensitive K(+) (KATP) channels play an important role in insulin secretion. KATP channels possess intrinsic MgATPase activity that is important in regulating channel activity in response to metabolic changes, although the precise structural determinants are not clearly understood. Furthermore, the sulfonylurea receptor 1 (SUR1) S1369A diabetes risk variant increases MgATPase activity, but the molecular mechanisms remain to be determined. Therefore, we hypothesized that residue-residue interactions between 1369 and 1372, predicted from in silico modelling, influence MgATPase activity, as well as sensitivity to the clinically used drug diazoxide that is known to increase MgATPase activity. We employed a point mutagenic approach with patch-clamp and direct biochemical assays to determine interaction between residues 1369 and 1372. Mutations in residues 1369 and 1372 predicted to decrease the residue interaction elicited a significant increase in MgATPase activity, whereas mutations predicted to possess similar residue interactions to wild-type (WT) channels elicited no alterations in MgATPase activity. In contrast, mutations that were predicted to increase residue interactions resulted in significant decreases in MgATPase activity. We also determined that a single S1369K substitution in SUR1 caused MgATPase activity and diazoxide pharmacological profiles to resemble those of channels containing the SUR2A subunit isoform. Our results provide evidence, at the single residue level, for a molecular mechanism that may underlie the association of the S1369A variant with type 2 diabetes. We also show a single amino acid difference can account for the markedly different diazoxide sensitivities between channels containing either the SUR1 or SUR2A subunit isoforms. PMID:26181369

  2. Mechanism of sulfonylurea herbicide resistance in the broadleaf weed, Kochia scoparia

    SciTech Connect

    Saari, L.L.; Cotterman, J.C.; Primiani, M.M. )

    1990-05-01

    Selection of kochia (Kochia scoparia) biotypes resistant to the sulfonylurea herbicide chlorsulfuron has occurred through the continued use of this herbicide in monoculture cereal-growing areas in the United States. The apparent sulfonylurea resistance observed in kochia was confirmed in greenhouse tests. Fresh and dry weight accumulation in the resistance kochia was 2- to >350-fold higher in the presence of four sulfonylurea herbicides as compared to the susceptible biotype. Acetolactate synthase (ALS) activity isolated from sulfonylurea-resistant kochia was less sensitive to inhibition by three classes of ALS-inhibiting herbicides, sulfonylureas, imidazolinones, and sulfonanilides. The decrease in ALS sensitivity to inhibition (as measured by the ratio of resistant I{sub 50} to susceptible I{sub 50}) was 5- to 28-fold, 2- to 6-fold, and 20-fold for sulfonylurea herbicides, imidazolinone herbicides, and a sulfonanilide herbicide, respectively. No differences were observed in the ALS-specific activities or the rates of ({sup 14}C)chlorsulfuron uptake, translocation, and metabolism between susceptible and resistant kochia biotypes. The K{sub m} values for pyruvate using ALS from susceptible and resistant kochia were 2.13 and 1.74 mM, respectively. Based on these results, the mechanism of sulfonylurea resistance in this kochia biotype is due solely to a less sulfonylurea-sensitive ALS enzyme.

  3. Regulation of ATP-sensitive K sup + channels in insulinoma cells: Activation by somatostatin and protein kinase C and the role of cAMP

    SciTech Connect

    De Weille, J.R.; Schmid-Antomarchi, H.; Fosset, M.; Lazdunski, M. )

    1989-04-01

    The actions of somatostatin and of the phorbol ester 4{beta}-phorbol 12-myristate 13-acetate (PMA) were studied in rat insulinoma (RINm5F) cells by electrophysiological and {sup 86}Rb{sup +} flux techniques. Both PMA and somatostatin hyperpolarize insulinoma cells by activating ATP-sensitive K{sup +} channels. The presence of intracellular GTP is required for the somatostatin effects. PMA- and somatostatin-induced hyperpolarization and channel activity are inhibited by the sulfonylurea glibenclamide. Glibenclamide-sensitive {sup 86}Rb{sup +} efflux from insulinoma cells is stimulated by somatostatin in a dose-dependent manner (half maximal effect at 0.7 nM) and abolished by pertussis toxin pretreatment. Mutual roles of a GTP-binding protein, of protein kinase C, and of cAMP in the regulation of ATP-sensitive K{sup +} channels are discussed.

  4. A novel sulfonylurea receptor family member expressed in the embryonic Drosophila dorsal vessel and tracheal system.

    PubMed

    Nasonkin, I; Alikasifoglu, A; Ambrose, C; Cahill, P; Cheng, M; Sarniak, A; Egan, M; Thomas, P M

    1999-10-01

    Sulfonylurea receptors (SURx) are required subunits of the ATP-sensitive potassium channel. SURx alone is electrophysiologically inert. However, when SURx is combined with an inward rectifier Kir6.2 subunit, ATP-sensitive potassium channel activity is generated. We report the identification, characterization, and localization of Dsur, a novel Drosophila gene that is highly related to the vertebrate SUR family. The Dsur coding sequence contains structural features characteristic of the ABC transporter family and, in addition, harbors 1.7 kilobases of a distinctive sequence that does not share homology with any known gene. When Dsur alone is expressed in Xenopus oocytes glibenclamide-sensitive potassium channel activity occurs. During Drosophila embryogenesis, the Dsur gene is specifically expressed in the developing tracheal system and dorsal vessel. Studies of the Drosophila genome support that only a single Dsur gene is present. Our data reveal conservation of glibenclamide-sensitive potassium channels in Drosophila and suggest that Dsur may play an important role during Drosophila embryogenesis. The lack of gene duplication in the Drosophila system provides a unique opportunity for functional studies of SUR using a genetic approach. PMID:10506204

  5. Sulfonylureas and their use in clinical practice

    PubMed Central

    Sola, Daniele; Schianca, Gian Piero Carnevale; Maffioli, Pamela; Bigliocca, Marcello; Mella, Roberto; Corlianò, Francesca; Fra, Gian Paolo; Bartoli, Ettore; Derosa, Giuseppe

    2015-01-01

    Many anti-diabetic drugs with different mechanisms of action are now available for treatment of type 2 diabetes mellitus. Sulfonylureas have been extensively used for treatment of type 2 diabetes for nearly 50 years and, even in our times, are widely used for treatment of this devastating chronic illness. Here, we review some of the available data on sulfonylureas, evaluating their mechanism of action and their effects on glycemic control. We can conclude that sulfonylureas are still the most used anti-diabetic agents: maybe this is due to their lower cost, to the possibility of mono-dosing and to the presence of an association with metformin in the same tablet. However, sulfonylureas, especially the older ones, are linked to a greater prevalence of hypoglycemia, and cardiovascular risk; newer prolonged-release preparations of sulfonylureas are undoubtedly safer, mainly due to reducing hypoglycemia, and for this reason should be preferred. PMID:26322096

  6. HYPOGLYCEMIA INDUCED BY ANTIDIABETIC SULFONYLUREAS.

    PubMed

    Confederat, Luminiţa; Constantin, Sandra; Lupaşcu, Florentina; Pânzariu, Andreea; Hăncianu, Monica; Profire, Lenuţa

    2015-01-01

    Diabetes mellitus is a major health problem due to its increasing prevalence and life-threatening complications. Antidiabetic sulfonylureas represent the first-line drugs in type 2 diabetes even though the most common associated risk is pharmacologically-induced hypoglycemia. In the development of this side effect are involved several factors including the pharmacokinetic and pharmacodynamic profile of the drug, patient age and behavior, hepatic or renal dysfunctions, or other drugs associated with a high risk of interactions. If all these are controlled, the risk-benefit balance can be equal to other oral antidiabetic drugs. PMID:26204670

  7. Database search of spontaneous reports and pharmacological investigations on the sulfonylureas and glinides-induced atrophy in skeletal muscle

    PubMed Central

    Mele, Antonietta; Calzolaro, Sara; Cannone, Gianluigi; Cetrone, Michela; Conte, Diana; Tricarico, Domenico

    2014-01-01

    The ATP-sensitive K+ (KATP) channel is an emerging pathway in the skeletal muscle atrophy which is a comorbidity condition in diabetes. The “in vitro” effects of the sulfonylureas and glinides were evaluated on the protein content/muscle weight, fibers viability, mitochondrial succinic dehydrogenases (SDH) activity, and channel currents in oxidative soleus (SOL), glycolitic/oxidative flexor digitorum brevis (FDB), and glycolitic extensor digitorum longus (EDL) muscle fibers of mice using biochemical and cell-counting Kit-8 assay, image analysis, and patch-clamp techniques. The sulfonylureas were: tolbutamide, glibenclamide, and glimepiride; the glinides were: repaglinide and nateglinide. Food and Drug Administration-Adverse Effects Reporting System (FDA-AERS) database searching of atrophy-related signals associated with the use of these drugs in humans has been performed. The drugs after 24 h of incubation time reduced the protein content/muscle weight and fibers viability more effectively in FDB and SOL than in the EDL. The order of efficacy of the drugs in reducing the protein content in FDB was: repaglinide (EC50 = 5.21 × 10−6) ≥ glibenclamide(EC50 = 8.84 × 10−6) > glimepiride(EC50 = 2.93 × 10−5) > tolbutamide(EC50 = 1.07 × 10−4) > nateglinide(EC50 = 1.61 × 10−4) and it was: repaglinide(7.15 × 10−5) ≥ glibenclamide(EC50 = 9.10 × 10−5) > nateglinide(EC50 = 1.80 × 10−4) ≥ tolbutamide(EC50 = 2.19 × 10−4) > glimepiride(EC50=–) in SOL. The drug-induced atrophy can be explained by the KATP channel block and by the enhancement of the mitochondrial SDH activity. In an 8-month period, muscle atrophy was found in 0.27% of the glibenclamide reports in humans and in 0.022% of the other not sulfonylureas and glinides drugs. No reports of atrophy were found for the other sulfonylureas and glinides in the FDA-AERS. Glibenclamide induces atrophy in animal experiments and in human patients. Glimepiride shows less potential for inducing

  8. [Risk and benefit of sulfonylureas--their role in view of new treatment options for type 2 diabetes].

    PubMed

    Rustenbeck, Ingo

    2016-02-01

    Currently, the therapy with oral antidiabetic drugs undergoes major changes. The use of sulfonylureas is in marked decline. The major argument in favor of newer oral antidiabetic drugs is the lower risk of hypoglycemia. At the present time however, it is unclear whether DDP4 inhibitors or SGLT2 inhibitors lead to better outcomes with respect to cardiovascular events and overall mortality. Most evidence on the therapeutic use of sulfonylureas has been gained with glibenclamide and to some degree sulfonylureas and glibenclamide have become synonymous. Since sulfonylureas vary considerably in their affinity for the K(ATP) channel subtypes and in their pharmacokinetic properties, the epidemiological evidence that outcomes tend to be less favorable with glibenclamide than with glimepiride or gliclazide has gained some attention. Beyond debate is the efficacy of metformin to diminish cardiovascular events in type 2 diabetes, probably due to effects beyond the lowering of blood glucose. PMID:26983335

  9. Hysteresis of gating underlines sensitization of TRPV3 channels

    PubMed Central

    Liu, Beiying; Yao, Jing; Zhu, Michael X.

    2011-01-01

    Vanilloid receptors of the transient receptor potential family have functions in thermal sensation and nociception. Among them, transient receptor potential vanilloid (TRPV)3 displays a unique property by which the repeated stimulation causes successive increases in its activity. The property has been known as sensitization and is observed in both native cells and cells heterologously expressing TRPV3. Transient increases in intracellular calcium levels have been implicated to play a key role in this process by mediating interaction of calmodulin with the channel. In support of the mechanism, BAPTA, a fast calcium chelator, accelerates the sensitization, whereas the slow chelator EGTA is ineffectual. Here, we show that the sensitization of TRPV3 also occurred independently of Ca2+. It was observed in both inside-out and outside-out membrane patches. BAPTA, but not EGTA, has a direct potentiation effect on channel activation. Analogues of BAPTA lacking Ca2+-buffering capability were similarly effective. The stimulation-induced sensitization and the potentiation by BAPTA are distinguishable in reversibility. We conclude that the sensitization of TRPV3 is intrinsic to the channel itself and occurs as a result of hysteresis of channel gating. BAPTA accelerates the sensitization process by potentiating the gating of the channel. PMID:22006988

  10. Sensitivity analysis of channel-bend hydraulics influenced by vegetation

    NASA Astrophysics Data System (ADS)

    Bywater-Reyes, S.; Manners, R.; McDonald, R.; Wilcox, A. C.

    2015-12-01

    Alternating bars influence hydraulics by changing the force balance of channels as part of a morphodynamic feedback loop that dictates channel geometry. Pioneer woody riparian trees recruit on river bars and may steer flow, alter cross-stream and downstream force balances, and ultimately change channel morphology. Quantifying the influence of vegetation on stream hydraulics is difficult, and researchers increasingly rely on two-dimensional hydraulic models. In many cases, channel characteristics (channel drag and lateral eddy viscosity) and vegetation characteristics (density, frontal area, and drag coefficient) are uncertain. This study uses a beta version of FaSTMECH that models vegetation explicitly as a drag force to test the sensitivity of channel-bend hydraulics to riparian vegetation. We use a simplified, scale model of a meandering river with bars and conduct a global sensitivity analysis that ranks the influence of specified channel characteristics (channel drag and lateral eddy viscosity) against vegetation characteristics (density, frontal area, and drag coefficient) on cross-stream hydraulics. The primary influence on cross-stream velocity and shear stress is channel drag (i.e., bed roughness), followed by the near-equal influence of all vegetation parameters and lateral eddy viscosity. To test the implication of the sensitivity indices on bend hydraulics, we hold calibrated channel characteristics constant for a wandering gravel-bed river with bars (Bitterroot River, MT), and vary vegetation parameters on a bar. For a dense vegetation scenario, we find flow to be steered away from the bar, and velocity and shear stress to be reduced within the thalweg. This provides insight into how the morphodynamic evolution of vegetated bars differs from unvegetated bars.

  11. BK Channels Reveal Novel Phosphate Sensitivity in SNr Neurons

    PubMed Central

    Ji, Juan Juan; Chen, Lianwan; Duan, Xuezhi; Song, Xueqin; Su, Wenting; Zhang, Peng; Li, Li; Bai, Shuyun; Sun, Yingchun; Inagaki, Nobuya

    2012-01-01

    Whether large conductance Ca2+-activated potassium (BK) channels are present in the substantia nigra pars reticulata (SNr) is a matter of debate. Using the patch-clamp technique, we examined the functional expression of BK channels in neurons of the SNr and showed that the channels were activated or inhibited by internal high-energy phosphates (IHEPs) at positive and negative membrane potentials, respectively. SNr neurons showed membrane potential hyperpolarization under glucose-deprivation conditions which was attenuated by paxilline, a specific BK channel blocker. In addition, Fluo-3 fluorescence recording detected an increase in the level of internal free calcium ([Ca2+]i) during ischemic hyperpolarization. These results confirm that BK channels are present in SNr neurons and indicate that their unique IHEP sensitivity is requisite in neuronal ischemic responses. Bearing in mind that the KATP channel blocker tolbutamide also attenuated the hyperpolarization, we suggest that BK channels may play a protective role in the basal ganglia by modulating the excitability of SNr neurons along with KATP channels under ischemic stresses. PMID:23284908

  12. Progressing From Metformin to Sulfonylureas or Meglitinides.

    PubMed

    Grant, Joan S; Graven, Lucinda J

    2016-09-01

    The article provides an overview of sulfonylureas and meglitinides as second-line agents for treating type 2 diabetes mellitus (T2DM). Implications for occupational health clinicians who work with these individuals when they take either of these medications to achieve target glycemic indices are emphasized. PMID:27621259

  13. Running out of time: the decline of channel activity and nucleotide activation in adenosine triphosphate-sensitive K-channels.

    PubMed

    Proks, Peter; Puljung, Michael C; Vedovato, Natascia; Sachse, Gregor; Mulvaney, Rachel; Ashcroft, Frances M

    2016-08-01

    KATP channels act as key regulators of electrical excitability by coupling metabolic cues-mainly intracellular adenine nucleotide concentrations-to cellular potassium ion efflux. However, their study has been hindered by their rapid loss of activity in excised membrane patches (rundown), and by a second phenomenon, the decline of activation by Mg-nucleotides (DAMN). Degradation of PI(4,5)P2 and other phosphoinositides is the strongest candidate for the molecular cause of rundown. Broad evidence indicates that most other determinants of rundown (e.g. phosphorylation, intracellular calcium, channel mutations that affect rundown) also act by influencing KATP channel regulation by phosphoinositides. Unfortunately, experimental conditions that reproducibly prevent rundown have remained elusive, necessitating post hoc data compensation. Rundown is clearly distinct from DAMN. While the former is associated with pore-forming Kir6.2 subunits, DAMN is generally a slower process involving the regulatory sulfonylurea receptor (SUR) subunits. We speculate that it arises when SUR subunits enter non-physiological conformational states associated with the loss of SUR nucleotide-binding domain dimerization following prolonged exposure to nucleotide-free conditions. This review presents new information on both rundown and DAMN, summarizes our current understanding of these processes and considers their physiological roles.This article is part of the themed issue 'Evolution brings Ca(2+) and ATP together to control life and death'. PMID:27377720

  14. Running out of time: the decline of channel activity and nucleotide activation in adenosine triphosphate-sensitive K-channels

    PubMed Central

    Proks, Peter; Puljung, Michael C.; Vedovato, Natascia; Sachse, Gregor; Mulvaney, Rachel; Ashcroft, Frances M.

    2016-01-01

    KATP channels act as key regulators of electrical excitability by coupling metabolic cues—mainly intracellular adenine nucleotide concentrations—to cellular potassium ion efflux. However, their study has been hindered by their rapid loss of activity in excised membrane patches (rundown), and by a second phenomenon, the decline of activation by Mg-nucleotides (DAMN). Degradation of PI(4,5)P2 and other phosphoinositides is the strongest candidate for the molecular cause of rundown. Broad evidence indicates that most other determinants of rundown (e.g. phosphorylation, intracellular calcium, channel mutations that affect rundown) also act by influencing KATP channel regulation by phosphoinositides. Unfortunately, experimental conditions that reproducibly prevent rundown have remained elusive, necessitating post hoc data compensation. Rundown is clearly distinct from DAMN. While the former is associated with pore-forming Kir6.2 subunits, DAMN is generally a slower process involving the regulatory sulfonylurea receptor (SUR) subunits. We speculate that it arises when SUR subunits enter non-physiological conformational states associated with the loss of SUR nucleotide-binding domain dimerization following prolonged exposure to nucleotide-free conditions. This review presents new information on both rundown and DAMN, summarizes our current understanding of these processes and considers their physiological roles. This article is part of the themed issue ‘Evolution brings Ca2+ and ATP together to control life and death’. PMID:27377720

  15. Investigation of parameter sensitivity of short channel mosfets

    NASA Astrophysics Data System (ADS)

    Selberherr, S.; Schütz, A.; Pötzl, H.

    1982-02-01

    A strategy to examine the sensitivity of electrical device parameters on geometrical and technological tolerances is described. An approach is offered to determine the limit of device miniaturzation for a given fabrication process and a desired operating condition. As a didactic example of practical relevance the minimum channel length for a modern silicon gate, double implant process due to threshold uncertainty is estimated. A method to calculate global sensitivity numbers for the reproducability of miniaturized devices is suggested. As an experimental determination of sensitivities is extremely difficult and expensive, numerical simulations are ideally suited for this purpose.

  16. Differential polyamine sensitivity in inwardly rectifying Kir2 potassium channels.

    PubMed

    Panama, Brian K; Lopatin, Anatoli N

    2006-03-01

    Recent studies have shown that Kir2 channels display differential sensitivity to intracellular polyamines, and have raised a number of questions about several properties of inward rectification important to the understanding of their physiological roles. In this study, we have carried out a detailed characterization of steady-state and kinetic properties of block of Kir2.1-3 channels by spermine. High-resolution recordings from outside-out patches showed that in all Kir2 channels current-voltage relationships display a 'crossover' effect upon change in extracellular K+. Experiments at different concentrations of spermine allowed for the characterization of two distinct shallow components of rectification, with the voltages for half-block negative (V1(1/2)) and positive (V2(1/2)) to the voltage of half-block for the major steep component of rectification (V0(1/2)). While V1(1/2) and V2(1/2) voltages differ significantly between Kir2 channels, they were coupled to each other according to the equation V1(1/2)-V2(1/2) = constant, strongly suggesting that similar structures may underlie both components. In Kir2.3 channels, the V2(1/2) was approximately 50 mV positive to V0(1/2), leading to a pattern of outward currents distinct from that of Kir2.1 and Kir2.2 channels. The effective valency of spermine block (Z0) was highest in Kir2.2 channels while the valencies in Kir2.1 and Kir2.3 channels were not significantly different. The voltage dependence of spermine unblock was similar in all Kir2 channels, but the rates of unblock were approximately 7-fold and approximately 16-fold slower in Kir2.3 channels than those in Kir2.1 and Kir2.2 when measured at high and physiological extracellular K+, respectively. In all Kir2 channels, the instantaneous phase of activation was present. The instantaneous phase was difficult to resolve at high extracellular K+ but it became evident and accounted for nearly 30-50% of the total current when recorded at physiological extracellular K

  17. Differential sensitivity of rat voltage-sensitive sodium channel isoforms to pyrazoline-type insecticides

    SciTech Connect

    Silver, Kristopher S.; Soderlund, David M. . E-mail: dms6@cornell.edu

    2006-07-15

    Pyrazoline-type insecticides are potent inhibitors of insect and mammalian voltage-sensitive sodium channels. In mammals, there are nine sodium channel {alpha} subunit isoforms that have unique distributions and pharmacological properties, but no published data exist that compare the relative sensitivity of these different mammalian sodium channel isoforms to inhibition by pyrazoline-type insecticides. This study employed the Xenopus oocyte expression system to examine the relative sensitivity of rat Na{sub v}1.2a, Na{sub v}1.4, Na{sub v}1.5, and Na{sub v}1.8 sodium channel {alpha} subunit isoforms to the pyrazoline-type insecticides indoxacarb, DCJW, and RH 3421. Additionally, we assessed the effect of coexpression with the rat {beta}1 auxiliary subunit on the sensitivity of the Na{sub v}1.2a and Na{sub v}1.4 isoforms to these compounds. The relative sensitivity of the four sodium channel {alpha} subunits differed for each of the three compounds we examined. With DCJW, the order of sensitivity was Na{sub v}1.4 > Na{sub v}1.2a > Na{sub v}1.5 > Na{sub v}1.8. In contrast, the relative sensitivity of these isoforms to indoxacarb differed from that to DCJW: the Na{sub v}1.8 isoform was most sensitive, the Na{sub v}1.4 isoform was completely insensitive, and the sensitivities of the Na{sub v}1.5 and Na{sub v}1.2a isoforms were intermediate between these two extremes. Moreover, the pattern of sensitivity to RH 3421 among these four isoforms was different from that for either indoxacarb or DCJW: the Na{sub v}1.4 isoform was most sensitive to RH 3421, whereas the sensitivities of the remaining three isoforms were substantially less than that of the Na{sub v}1.4 isoform and were approximately equivalent. The only statistically significant effect of coexpression of either the Na{sub v}1.2a or Na{sub v}1.4 isoforms with the {beta}1 subunit was the modest reduction in the sensitivity of the Na{sub v}1.2a isoform to RH 3421. These results demonstrate that mammalian sodium

  18. Sensitivity analysis of vegetation-induced flow steering in channels

    NASA Astrophysics Data System (ADS)

    Bywater-Reyes, S.; Wilcox, A. C.; Lightbody, A.; Stella, J. C.

    2014-12-01

    Morphodynamic feedbacks result in alternating bars within channels, and the resulting convective accelerations dictate the cross-stream force balance of channels and in turn influence morphology. Pioneer woody riparian trees recruit on river bars and may steer flow and alter this force balance. This study uses two-dimensional hydraulic modeling to test the sensitivity of the flow field to riparian vegetation at the reach scale. We use two test systems with different width-to-depth ratios, substrate sizes, and vegetation structure: the gravel-bed Bitterroot River, MT and the sand-bed Santa Maria River, AZ. We model vegetation explicitly as a drag force by spatially specifying vegetation density, height, and drag coefficient, across varying hydraulic (e.g., discharge, eddy viscosity) conditions and compare velocity vectors between runs. We test variations in vegetation configurations, including the present-day configuration of vegetation in our field systems (extracted from LiDAR), removal of vegetation (e.g., from floods or management actions), and expansion of vegetation. Preliminary model runs suggest that the sensitivity of convective accelerations to vegetation reflects a balance between the extent and density of vegetation inundated and other sources of channel roughness. This research quantifies how vegetation alters hydraulics at the reach scale, a fundamental step to understanding vegetation-morphodynamic interactions.

  19. Cytomembrane ATP-sensitive K+ channels in neurovascular unit targets of ischemic stroke in the recovery period

    PubMed Central

    Zhang, Yang; Pan, Sipei; Zheng, Xiaolu; Wan, Qi

    2016-01-01

    The present study was to analyze the mechanism of cytomembrane ATP-sensitive K+ channels (KATP) in the neurovascular unit treatment of ischemic stroke in the recovery period. A total of 24 healthy adult male Wistar rats of 5–8 weeks age, weighing 160–200 g were randomly divided into the control (sham-operation group), model, KATP blocker and KATP opener groups (n=6 rats per group). Nylon cerebral artery occlusion was conducted using nylon monofilament coated with Poly-L-lysine, which was used to produce a cerebral infarction model. After feeding normally for 3 days, 5-hydroxydecanoate (40 mg/Kg), and diazoxide (40 mg/Kg) were injected to the abdominal cavity in the blocker, and opener groups, respectively. The control received an equivalent normal saline that was injected into the sham-operation and model groups. The animals were mutilated and samples were collected after 3 days. RT-PCR was used to detect the expression levels of the three subunits of KATP, i.e., kir6.1, and sulfonylurea receptor (SUR) 1 and SUR2 mRNA, as well as to calculate infarct size in tetrazolium chloride staining. The expression level of mRNA in the opener group were significantly higher, followed by the model and blocker groups, with the control group being the lowest (P<0.05). Infarct size in the opener group was markedly smaller than the model and blocker groups, and infarct size in the blocker group was significantly larger (P<0.05). Thus, the target treatment on KATP may improve the prognosis of ischemic stroke during the recovery period. PMID:27446320

  20. Potentiation of sulfonylurea action by an EPAC-selective cAMP analog in INS-1 cells: comparison of tolbutamide and gliclazide and a potential role for EPAC activation of a 2-APB-sensitive Ca2+ influx.

    PubMed

    Jarrard, Rachel E; Wang, Yuchen; Salyer, Amy E; Pratt, Evan P S; Soderling, Ian M; Guerra, Marcy L; Lange, Allison M; Broderick, Hilary J; Hockerman, Gregory H

    2013-01-01

    Tolbutamide and gliclazide block the K(ATP) channel K(ir)6.2/Sur1, causing membrane depolarization and stimulating insulin secretion in pancreatic beta cells. We examined the ability of the EPAC-selective cAMP analog 8-pCPT-2'-O-Me-cAMP-AM to potentiate the action of these drugs and the mechanism that might account for it. Insulin secretion stimulated by both 200 μM tolbutamide and 20 μM gliclazide, concentrations that had equivalent effects on membrane potential, was inhibited by thapsigargin (1 μM) or the L-type Ca(2+) channel blocker nicardipine (2 μM) and was potentiated by 8-pCPT-2'-O-Me-cAMP-AM at concentrations ≥2 μM in INS-1 cells. Ca(2+) transients stimulated by either tolbutamide or gliclazide were inhibited by thapsigargin or nicardipine and were significantly potentiated by 8-pCPT-2'-O-Me-cAMP-AM at 5 μM but not 1 μM. Both tolbutamide and gliclazide stimulated phospholipase C activity; however, only gliclazide did so independently of its activity at K(ATP) channels, and this activity was partially inhibited by pertussis toxin. 8-pCPT-2'-O-Me-cAMP-AM alone (5 μM) did not stimulate insulin secretion, but did increase intracellular Ca(2+) concentration significantly, and this activity was inhibited by 25 μM 2-aminoethoxydiphenylborate (2-APB) or the removal of extracellular Ca(2+). 8-pCPT-2'-O-Me-cAMP-AM potentiation of insulin secretion stimulated by tolbutamide was markedly inhibited by 2-APB (25 μM) and enhanced by the PKC inhibitor bisindolylmaleimide I (1 μM). Our data demonstrate that the actions of both tolbutamide and gliclazide are strongly potentiated by 8-pCPT-2'-O-Me-cAMP-AM, that gliclazide can stimulate phospholipase C activity via a partially pertussis toxin-sensitive mechanism, and that 8-pCPT-2'-O-Me-cAMP-AM potentiation of tolbutamide action may involve activation of a 2-APB-sensitive Ca(2+) influx. PMID:23071106

  1. Voltage-sensitive potassium channels in Limulus ventral photoreceptors

    PubMed Central

    1978-01-01

    The steady-state slope conductance of Limulus ventral photoreceptors increases markedly when the membrane is depolarized from rest. The ionic basis of this rectification has been examined with a voltage- clamp technique. Tail currents that occur when membrane potential is repolarized after having been depolarized have been identified. The tail currents reverse direction at a voltage that becomes more positive when Ko is increased. Rectification is reduced by extracellular 4- aminopyridine and by intracellular injection of tetra-ethyl-ammonium (TEA). These results indicate that the membrane rectification around resting potential is due primarily to voltage-sensitive K+ channels. The increase in gK caused by depolarization is not mediated by a voltage-dependent rise in in Cai++, since intracellular injection of Ca++ causes a decrease rather than an increase in slope conductance. TEA can be used to examine the functional role of the K+ channels because it blocks them without substantially affecting the light- activated Na+ conductance. The effect of TEA on response-intensity curves shows that the K+ channels serve to compress the voltage range of receptor potentials. PMID:621492

  2. Engineering vanilloid-sensitivity into the rat TRPV2 channel

    PubMed Central

    Zhang, Feng; Hanson, Sonya M; Jara-Oseguera, Andres; Krepkiy, Dmitriy; Bae, Chanhyung; Pearce, Larry V; Blumberg, Peter M; Newstead, Simon; Swartz, Kenton J

    2016-01-01

    The TRPV1 channel is a detector of noxious stimuli, including heat, acidosis, vanilloid compounds and lipids. The gating mechanisms of the related TRPV2 channel are poorly understood because selective high affinity ligands are not available, and the threshold for heat activation is extremely high (>50°C). Cryo-EM structures of TRPV1 and TRPV2 reveal that they adopt similar structures, and identify a putative vanilloid binding pocket near the internal side of TRPV1. Here we use biochemical and electrophysiological approaches to investigate the resiniferatoxin(RTx) binding site in TRPV1 and to explore the functional relationships between TRPV1 and TRPV2. Collectively, our results support the interaction of vanilloids with the proposed RTx binding pocket, and demonstrate an allosteric influence of a tarantula toxin on vanilloid binding. Moreover, we show that sensitivity to RTx can be engineered into TRPV2, demonstrating that the gating and permeation properties of this channel are similar to TRPV1. DOI: http://dx.doi.org/10.7554/eLife.16409.001 PMID:27177419

  3. Engineering vanilloid-sensitivity into the rat TRPV2 channel.

    PubMed

    Zhang, Feng; Hanson, Sonya M; Jara-Oseguera, Andres; Krepkiy, Dmitriy; Bae, Chanhyung; Pearce, Larry V; Blumberg, Peter M; Newstead, Simon; Swartz, Kenton J

    2016-01-01

    The TRPV1 channel is a detector of noxious stimuli, including heat, acidosis, vanilloid compounds and lipids. The gating mechanisms of the related TRPV2 channel are poorly understood because selective high affinity ligands are not available, and the threshold for heat activation is extremely high (>50°C). Cryo-EM structures of TRPV1 and TRPV2 reveal that they adopt similar structures, and identify a putative vanilloid binding pocket near the internal side of TRPV1. Here we use biochemical and electrophysiological approaches to investigate the resiniferatoxin(RTx) binding site in TRPV1 and to explore the functional relationships between TRPV1 and TRPV2. Collectively, our results support the interaction of vanilloids with the proposed RTx binding pocket, and demonstrate an allosteric influence of a tarantula toxin on vanilloid binding. Moreover, we show that sensitivity to RTx can be engineered into TRPV2, demonstrating that the gating and permeation properties of this channel are similar to TRPV1. PMID:27177419

  4. Age at the time of sulfonylurea initiation influences treatment outcomes in KCNJ11-related neonatal diabetes

    PubMed Central

    Thurber, Brian W.; Carmody, David; Tadie, Elizabeth C.; Pastore, Ashley N.; Dickens, Jazzmyne T.; Wroblewski, Kristen E.; Naylor, Rochelle N.; Philipson, Louis H.; Greeley, Siri Atma W.

    2015-01-01

    Aims/hypothesis Individuals with heterozygous activating mutations of the KCNJ11 gene encoding a subunit of the ATP-sensitive potassium channel (KATP) can usually be treated with oral sulfonylurea (SU) pills in lieu of insulin injections. The aim of this study was to test our hypothesis that younger age at the time of initiation of SU therapy is correlated with lower required doses of SU therapy, shorter transition time and decreased likelihood of requiring additional diabetes medications. Methods We performed a retrospective cohort study using data on 58 individuals with neonatal diabetes due to KCNJ11mutations identified through the University of Chicago Monogenic Diabetes Registry (http://monogenicdiabetes.uchicago.edu/registry). We assessed the influence of age at initiation of SU therapy on treatment outcomes. Results HbA1c fell from an average of 8.5% (69 mmol/mol) before transition to 6.2% (44 mmol/mol) after SU therapy (p < 0.001). Age of initiation of SU correlated with the dose (mg kg−1 day−1) of SU required at follow-up (r = 0.80, p < 0.001). Similar associations were observed across mutation subtypes. Ten participants required additional glucose-lowering medications and all had initiated SU at age 13 years or older. No serious adverse events were reported. Conclusions/interpretation Earlier age at initiation of SU treatment is associated with improved response to SU therapy. Declining sensitivity to SU may be due to loss of beta cell mass over time in those treated with insulin. Our data support the need for early genetic diagnosis and appropriate personalised treatment in all cases of neonatal diabetes. PMID:25877689

  5. Kir6.2 activation by sulfonylurea receptors: a different mechanism of action for SUR1 and SUR2A subunits via the same residues

    PubMed Central

    Principalli, Maria A; Dupuis, Julien P; Moreau, Christophe J; Vivaudou, Michel; Revilloud, Jean

    2015-01-01

    ATP-sensitive potassium channels (K-ATP channels) play a key role in adjusting the membrane potential to the metabolic state of cells. They result from the unique combination of two proteins: the sulfonylurea receptor (SUR), an ATP-binding cassette (ABC) protein, and the inward rectifier K+ channel Kir6.2. Both subunits associate to form a heterooctamer (4 SUR/4 Kir6.2). SUR modulates channel gating in response to the binding of nucleotides or drugs and Kir6.2 conducts potassium ions. The activity of K-ATP channels varies with their localization. In pancreatic β-cells, SUR1/Kir6.2 channels are partly active at rest while in cardiomyocytes SUR2A/Kir6.2 channels are mostly closed. This divergence of function could be related to differences in the interaction of SUR1 and SUR2A with Kir6.2. Three residues (E1305, I1310, L1313) located in the linker region between transmembrane domain 2 and nucleotide-binding domain 2 of SUR2A were previously found to be involved in the activation pathway linking binding of openers onto SUR2A and channel opening. To determine the role of the equivalent residues in the SUR1 isoform, we designed chimeras between SUR1 and the ABC transporter multidrug resistance-associated protein 1 (MRP1), and used patch clamp recordings on Xenopus oocytes to assess the functionality of SUR1/MRP1 chimeric K-ATP channels. Our results reveal that the same residues in SUR1 and SUR2A are involved in the functional association with Kir6.2, but they display unexpected side-chain specificities which could account for the contrasted properties of pancreatic and cardiac K-ATP channels. PMID:26416970

  6. Diverse roles of K(ATP) channels learned from Kir6.2 genetically engineered mice.

    PubMed

    Seino, S; Iwanaga, T; Nagashima, K; Miki, T

    2000-03-01

    The regulation of insulin secretion from pancreatic beta-cells depends critically on the activities of their plasma membrane ion channels. ATP-sensitive K+ channels (K(ATP) channels) are present in many cells and regulate a variety of cellular functions by coupling cell metabolism with membrane potential. The activity of the K(ATP) channels in pancreatic beta-cells is regulated by changes in the ATP and ADP concentrations (ATP/ADP ratio) caused by glucose metabolism. Thus, the K(ATP) channels are the ATP and ADP sensors in the regulation of glucose-induced insulin secretion. K(ATP) channels are also the target of sulfonylureas, which are widely used in the treatment of type 2 diabetes. Molecular cloning of the two subunits of the pancreatic beta-cell K(ATP) channel, Kir6.2 (an inward rectifier K+ channel member) and SUR1 (a receptor for sulfonylureas), has provided great insight into its structure and function. Kir6.2 subunits form the K+ ion-permeable pore and primarily confer inhibition of the channels by ATP, while SUR1 subunits confer activation of the channels by MgADP and K+ channel openers, such as diazoxide, as well as inhibition by sulfonylureas. The SUR1 subunits also enhance the sensitivity of the channels to ATP. To determine the physiological roles of K(ATP) channels directly, we have generated two kinds of genetically engineered mice: mice expressing a dominant-negative form of Kir6.2 specifically in the pancreatic beta-cells (Kir6.2G132S Tg mice) and mice lacking Kir6.2 (Kir6.2 knockout mice). Studies of these mice elucidated various roles of the K(ATP) channels in endocrine pancreatic function: 1) the K(ATP) channels are the major determinant of the resting membrane potential of pancreatic beta-cells, 2) both glucose- and sulfonylurea-induced membrane depolarization of beta-cells require closure of the K(ATP) channels, 3) both glucose- and sulfonylurea-induced rises in intracellular calcium concentration in beta-cells require closure of the K

  7. The 3-dimensional, 4-channel model of human visual sensitivity to grayscale scrambles.

    PubMed

    Silva, Andrew E; Chubb, Charles

    2014-08-01

    Previous research supports the claim that human vision has three dimensions of sensitivity to grayscale scrambles (textures composed of randomly scrambled mixtures of different grayscales). However, the preattentive mechanisms (called here "field-capture channels") that confer this sensitivity remain obscure. The current experiments sought to characterize the specific field-capture channels that confer this sensitivity using a task in which the participant is required to detect the location of a small patch of one type of grayscale scramble in an extended background of another type. Analysis of the results supports the existence of four field-capture channels: (1) the (previously characterized) "blackshot" channel, sharply tuned to the blackest grayscales; (2) a (previously unknown) "gray-tuned" field-capture channel whose sensitivity is zero for black rising sharply to maximum sensitivity for grayscales slightly darker than mid-gray then decreasing to half-height for brighter grayscales; (3) an "up-ramped" channel whose sensitivity is zero for black, increases linearly with increasing grayscale reaching a maximum near white; (4) a (complementary) "down-ramped" channel whose sensitivity is maximal for black, decreases linearly reaching a minimum near white. The sensitivity functions of field-capture channels (3) and (4) are linearly dependent; thus, these four field-capture channels collectively confer sensitivity to a 3-dimensional space of histogram variations. PMID:24932891

  8. Severe hypoglycemia in users of sulfonylurea antidiabetic agents and antihyperlipidemics.

    PubMed

    Leonard, C E; Bilker, W B; Brensinger, C M; Han, X; Flory, J H; Flockhart, D A; Gagne, J J; Cardillo, S; Hennessy, S

    2016-05-01

    Drug-drug interactions causing severe hypoglycemia due to antidiabetic drugs is a major clinical and public health problem. We assessed whether sulfonylurea use with a statin or fibrate was associated with severe hypoglycemia. We conducted cohort studies of users of glyburide, glipizide, and glimepiride plus a statin or fibrate within a Medicaid population. The outcome was a validated, diagnosis-based algorithm for severe hypoglycemia. Among 592,872 persons newly exposed to a sulfonylurea+antihyperlipidemic, the incidence of severe hypoglycemia was 5.8/100 person-years. Adjusted hazard ratios (HRs) for sulfonylurea+statins were consistent with no association. Most overall HRs for sulfonylurea+fibrate were elevated, with sulfonylurea-specific adjusted HRs as large as 1.50 (95% confidence interval (CI): 1.24-1.81) for glyburide+gemfibrozil, 1.37 (95% CI: 1.11-1.69) for glipizide+gemfibrozil, and 1.63 (95% CI: 1.29-2.06) for glimepiride+fenofibrate. Concomitant therapy with a sulfonylurea and fibrate is associated with an often delayed increased rate of severe hypoglycemia. PMID:26566262

  9. The evolutionary divergence of TRPA1 channel: heat-sensitive, cold-sensitive and temperature-insensitive

    PubMed Central

    Chen, Jun

    2015-01-01

    The Transient Receptor Potential Ankyrin 1 ion channel is heat-sensitive in invertebrate and ancestral vertebrates, cold-sensitive in rodents, and temperature-insensitive in primates. This remarkable divergence in temperature sensitivity is in contrast to its role in sensing electrophilic compounds, which is conserved during animal evolution.

  10. A new pH-sensitive rectifying potassium channel in mitochondria from the embryonic rat hippocampus.

    PubMed

    Kajma, Anna; Szewczyk, Adam

    2012-10-01

    Patch-clamp single-channel studies on mitochondria isolated from embryonic rat hippocampus revealed the presence of two different potassium ion channels: a large-conductance (288±4pS) calcium-activated potassium channel and second potassium channel with outwardly rectifying activity under symmetric conditions (150/150mM KCl). At positive voltages, this channel displayed a conductance of 67.84pS and a strong voltage dependence at holding potentials from -80mV to +80mV. The open probability was higher at positive than at negative voltages. Patch-clamp studies at the mitoplast-attached mode showed that the channel was not sensitive to activators and inhibitors of mitochondrial potassium channels but was regulated by pH. Moreover, we demonstrated that the channel activity was not affected by the application of lidocaine, an inhibitor of two-pore domain potassium channels, or by tertiapin, an inhibitor of inwardly rectifying potassium channels. In summary, based on the single-channel recordings, we characterised for the first time mitochondrial pH-sensitive ion channel that is selective for cations, permeable to potassium ions, displays voltage sensitivity and does not correspond to any previously described potassium ion channels in the inner mitochondrial membrane. This article is part of a Special Issue entitled: 17th European Bioenergetics Conference (EBEC 2012). PMID:22406520

  11. SODIUM CHANNELS (NAV1.2/B1) EXPRESSED IN XENOPUS OOCYTES DEMONSTRATE SENSITIVITY TO PYRETHROIDS.

    EPA Science Inventory

    Voltage-sensitive sodium channels (VSSCs) are hypothesized to be a primary target of pyrethroid insecticides. However, multiple isoforms of VSSCs exist and the sensitivity of different isoforms to pyrethroids has not been well characterized. The Nav1.2/1 channel predominates in a...

  12. Regulation of Shaker-type potassium channels by hypoxia. Oxygen-sensitive K+ channels in PC12 cells.

    PubMed

    Conforti, L; Millhorn, D E

    2000-01-01

    Little is known about the molecular composition of the O2-sensitive K+ (Ko2) channels. The possibility that these channels belong to the Shaker subfamily (Kv1) of voltage-dependent K+ (Kv) channels has been raised in pulmonary artery (PA) smooth muscle cells. Numerous findings suggest that the Ko2 channel in PC12 cells is a Kv1 channel, formed by the Kv1.2 alpha subunit. The Ko2 channel in PC12 cells is a slow-inactivating voltage-dependent K+ channel of 20 pS conductance. Other Kv channels, also expressed in PC12 cells, are not inhibited by hypoxia. Selective up-regulation by chronic hypoxia of the Kv1.2 alpha subunit expression correlates with an increase O2-sensitivity of the K+ current. Other Kv1 alpha subunit genes encoding slow-inactivating Kv channels, such as Kv1.3, Kv2.1, Kv3.1 and Kv3.2 are not modulated by chronic hypoxia. The Ko2 current in PC12 cells is blocked by 5 mM externally applied tetraethylammonium chloride (TEA) and by charydbotoxin (CTX). The responses of the Kv1.2 K+ channel to hypoxia have been studied in the Xenopus oocytes and compared to those of Kv2.1, also proposed as Ko2 channel in PA smooth muscle cells. Two-electrode voltage clamp experiments show that hypoxia induces inhibition of K+ current amplitude only in oocytes injected with Kv1.2 cRNA. These data indicate that Kv1.2 K+ channels are inhibited by hypoxia. PMID:10849667

  13. High temperature sensitivity is intrinsic to voltage-gated potassium channels.

    PubMed

    Yang, Fan; Zheng, Jie

    2014-01-01

    Temperature-sensitive transient receptor potential (TRP) ion channels are members of the large tetrameric cation channels superfamily but are considered to be uniquely sensitive to heat, which has been presumed to be due to the existence of an unidentified temperature-sensing domain. Here we report that the homologous voltage-gated potassium (Kv) channels also exhibit high temperature sensitivity comparable to that of TRPV1, which is detectable under specific conditions when the voltage sensor is functionally decoupled from the activation gate through either intrinsic mechanisms or mutations. Interestingly, mutations could tune Shaker channel to be either heat-activated or heat-deactivated. Therefore, high temperature sensitivity is intrinsic to both TRP and Kv channels. Our findings suggest important physiological roles of heat-induced variation in Kv channel activities. Mechanistically our findings indicate that temperature-sensing TRP channels may not contain a specialized heat-sensor domain; instead, non-obligatory allosteric gating permits the intrinsic heat sensitivity to drive channel activation, allowing temperature-sensitive TRP channels to function as polymodal nociceptors. PMID:25030910

  14. High temperature sensitivity is intrinsic to voltage-gated potassium channels

    PubMed Central

    Yang, Fan; Zheng, Jie

    2014-01-01

    Temperature-sensitive transient receptor potential (TRP) ion channels are members of the large tetrameric cation channels superfamily but are considered to be uniquely sensitive to heat, which has been presumed to be due to the existence of an unidentified temperature-sensing domain. Here we report that the homologous voltage-gated potassium (Kv) channels also exhibit high temperature sensitivity comparable to that of TRPV1, which is detectable under specific conditions when the voltage sensor is functionally decoupled from the activation gate through either intrinsic mechanisms or mutations. Interestingly, mutations could tune Shaker channel to be either heat-activated or heat-deactivated. Therefore, high temperature sensitivity is intrinsic to both TRP and Kv channels. Our findings suggest important physiological roles of heat-induced variation in Kv channel activities. Mechanistically our findings indicate that temperature-sensing TRP channels may not contain a specialized heat-sensor domain; instead, non-obligatory allosteric gating permits the intrinsic heat sensitivity to drive channel activation, allowing temperature-sensitive TRP channels to function as polymodal nociceptors. DOI: http://dx.doi.org/10.7554/eLife.03255.001 PMID:25030910

  15. Inactivation of the first nucleotide-binding fold of the sulfonylurea receptor, and familial persistent hyperinsulinemic hypoglycemia of infancy

    SciTech Connect

    Thomas, P.M.; Wohllk, N.; Huang, E.

    1996-09-01

    Familial persistent hyperinsulinemic hypoglycemia of infancy is a disorder of glucose homeostasis and is characterized by unregulated insulin secretion and profound hypoglycemia. Loss-of-function mutations in the second nucleotide-binding fold of the sulfonylurea receptor, a subunit of the pancreatic-islet {beta}-cell ATP-dependent potassium channel, has been demonstrated to be causative for persistent hyperinsulinemic hypoglycemia of infancy. We now describe three additional mutations in the first nucleotide-binding fold of the sulfonylurea-receptor gene. One point mutation disrupts the highly conserved Walker A motif of the first nucleotide-binding-fold region. The other two mutations occur in noncoding sequences required for RNA processing and are predicted to disrupt the normal splicing pathway of the sulfonylurea-receptor mRNA precursor. These data suggest that both nucleotide-binding-fold regions of the sulfortylurea receptor are required for normal regulation of {beta}-cell ATP-dependent potassium channel activity and insulin secretion. 32 refs., 4 figs., 1 tab.

  16. Phosphorylation-dependent changes in nucleotide binding, conformation, and dynamics of the first nucleotide binding domain (NBD1) of the sulfonylurea receptor 2B (SUR2B).

    PubMed

    de Araujo, Elvin D; Alvarez, Claudia P; López-Alonso, Jorge P; Sooklal, Clarissa R; Stagljar, Marijana; Kanelis, Voula

    2015-09-11

    The sulfonylurea receptor 2B (SUR2B) forms the regulatory subunit of ATP-sensitive potassium (KATP) channels in vascular smooth muscle. Phosphorylation of the SUR2B nucleotide binding domains (NBD1 and NBD2) by protein kinase A results in increased channel open probability. Here, we investigate the effects of phosphorylation on the structure and nucleotide binding properties of NBD1. Phosphorylation sites in SUR2B NBD1 are located in an N-terminal tail that is disordered. Nuclear magnetic resonance (NMR) data indicate that phosphorylation of the N-terminal tail affects multiple residues in NBD1, including residues in the NBD2-binding site, and results in altered conformation and dynamics of NBD1. NMR spectra of NBD1 lacking the N-terminal tail, NBD1-ΔN, suggest that phosphorylation disrupts interactions of the N-terminal tail with the core of NBD1, a model supported by dynamic light scattering. Increased nucleotide binding of phosphorylated NBD1 and NBD1-ΔN, compared with non-phosphorylated NBD1, suggests that by disrupting the interaction of the NBD core with the N-terminal tail, phosphorylation also exposes the MgATP-binding site on NBD1. These data provide insights into the molecular basis by which phosphorylation of SUR2B NBD1 activates KATP channels. PMID:26198630

  17. Molecular physiology of pH-sensitive background K(2P) channels.

    PubMed

    Lesage, Florian; Barhanin, Jacques

    2011-12-01

    Background K(2P) channels are tightly regulated by different stimuli including variations of external and internal pH. pH sensitivity relies on proton-sensing residues that influence channel gating and activity. Gene inactivation in the mouse is a revealing implication of K(2P) channels in many physiological functions ranging from hormone secretion to central respiratory adaptation. Surprisingly, only a few phenotypic traits of these mice have yet been directly related to the pH sensitivity of K(2P) channels. PMID:22170960

  18. Distinct pharmacological and molecular properties of the acid-sensitive outwardly rectifying (ASOR) anion channel from those of the volume-sensitive outwardly rectifying (VSOR) anion channel.

    PubMed

    Sato-Numata, Kaori; Numata, Tomohiro; Inoue, Ryuji; Okada, Yasunobu

    2016-05-01

    Expressed by many cell types, acid-sensitive outwardly rectifying (ASOR) anion channels are known to be activated by extracellular acidification and involved in acidotoxic necrotic cell death. In contrast, ubiquitously expressed volume-sensitive outwardly rectifying (VSOR) anion channels are activated by osmotic cell swelling and involved in cell volume regulation and apoptotic cell death. Distinct inhibitors to distinguish ASOR from VSOR anion channels have not been identified. Although leucine-rich repeats containing 8A (LRRC8A) was recently found to be an essential component of VSOR anion channels, the possibility of an LRRC8 family member serving as a component of ASOR anion channels has not been examined. In this study, we explored the effects of 12 known VSOR channel inhibitors and small interfering RNA (siRNA)-mediated knockdown of LRRC8 family members on ASOR and VSOR currents in HeLa cells. Among these inhibitors, eight putative VSOR blockers, including 4-(2-butyl-6,7-dichlor-2-cyclopentylindan-1-on-5-yl) oxobutyric acid (DCPIB) and 5-nitro-2-(3-phenylpropylamino)-benzoic acid (NPPB), were totally ineffective at blocking ASOR channel activity, whereas suramin, R-(+)-[(2-n-butyl-6,7-dichloro-2-cyclopentyl-2,3-dihydro-1-oxo-1H-inden-5-yl)oxy] acetic acid (DIOA), arachidonic acid, and niflumic acid were found to be effective ASOR anion channel antagonists. In addition, gene-silencing studies showed that no LRRC8 family members are essentially involved in ASOR anion channel activity, whereas LRRC8A is involved in VSOR anion channel activity in HeLa cells. PMID:26743872

  19. Heteromeric Heat-sensitive Transient Receptor Potential Channels Exhibit Distinct Temperature and Chemical Response*

    PubMed Central

    Cheng, Wei; Yang, Fan; Liu, Shuang; Colton, Craig K.; Wang, Chunbo; Cui, Yuanyuan; Cao, Xu; Zhu, Michael X.; Sun, Changsen; Wang, KeWei; Zheng, Jie

    2012-01-01

    TRPV1 and TRPV3 are two heat-sensitive ion channels activated at distinct temperature ranges perceived by human as hot and warm, respectively. Compounds eliciting human sensations of heat or warmth can also potently activate these channels. In rodents, TRPV3 is expressed predominantly in skin keratinocytes, whereas in humans TRPV1 and TRPV3 are co-expressed in sensory neurons of dorsal root ganglia and trigeminal ganglion and are known to form heteromeric channels with distinct single channel conductances as well as sensitivities to TRPV1 activator capsaicin and inhibitor capsazepine. However, how heteromeric TRPV1/TRPV3 channels respond to heat and other stimuli remains unknown. In this study, we examined the behavior of heteromeric TRPV1/TRPV3 channels activated by heat, capsaicin, and voltage. Our results demonstrate that the heteromeric channels exhibit distinct temperature sensitivity, activation threshold, and heat-induced sensitization. Changes in gating properties apparently originate from interactions between TRPV1 and TRPV3 subunits. Our results suggest that heteromeric TRPV1/TRPV3 channels are unique heat sensors that may contribute to the fine-tuning of sensitivity to sensory inputs. PMID:22184123

  20. A Heat-Sensitive TRP Channel Expressed in Keratinocytes

    NASA Astrophysics Data System (ADS)

    Peier, Andrea M.; Reeve, Alison J.; Andersson, David A.; Moqrich, Aziz; Earley, Taryn J.; Hergarden, Anne C.; Story, Gina M.; Colley, Sian; Hogenesch, John B.; McIntyre, Peter; Bevan, Stuart; Patapoutian, Ardem

    2002-06-01

    Mechanical and thermal cues stimulate a specialized group of sensory neurons that terminate in the skin. Three members of the transient receptor potential (TRP) family of channels are expressed in subsets of these neurons and are activated at distinct physiological temperatures. Here, we describe the cloning and characterization of a novel thermosensitive TRP channel. TRPV3 has a unique threshold: It is activated at innocuous (warm) temperatures and shows an increased response at noxious temperatures. TRPV3 is specifically expressed in keratinocytes; hence, skin cells are capable of detecting heat via molecules similar to those in heat-sensing neurons.

  1. Potential Roles of Amiloride-Sensitive Sodium Channels in Cancer Development

    PubMed Central

    Xu, Siguang; Liu, Cui; Ma, Yana; Ji, Hong-Long; Li, Xiumin

    2016-01-01

    The ENaC/degenerin ion channel superfamily includes the amiloride-sensitive epithelial sodium channel (ENaC) and acid sensitive ionic channel (ASIC). ENaC is a multimeric ion channel formed by heteromultimeric membrane glycoproteins, which participate in a multitude of biological processes by mediating the transport of sodium (Na+) across epithelial tissues such as the kidney, lungs, bladder, and gut. Aberrant ENaC functions contribute to several human disease states including pseudohypoaldosteronism, Liddle syndrome, cystic fibrosis, and salt-sensitive hypertension. Increasing evidence suggests that ion channels not only regulate ion homeostasis and electric signaling in excitable cells but also play important roles in cancer cell behaviors such as proliferation, apoptosis, invasion, and migration. Indeed, ENaCs/ASICs had been reported to be associated with cancer characteristics. Given their cell surface localization and pharmacology, pharmacological strategies to target ENaC/ASIC family members may be promising cancer therapeutics. PMID:27403419

  2. Potential Roles of Amiloride-Sensitive Sodium Channels in Cancer Development.

    PubMed

    Xu, Siguang; Liu, Cui; Ma, Yana; Ji, Hong-Long; Li, Xiumin

    2016-01-01

    The ENaC/degenerin ion channel superfamily includes the amiloride-sensitive epithelial sodium channel (ENaC) and acid sensitive ionic channel (ASIC). ENaC is a multimeric ion channel formed by heteromultimeric membrane glycoproteins, which participate in a multitude of biological processes by mediating the transport of sodium (Na(+)) across epithelial tissues such as the kidney, lungs, bladder, and gut. Aberrant ENaC functions contribute to several human disease states including pseudohypoaldosteronism, Liddle syndrome, cystic fibrosis, and salt-sensitive hypertension. Increasing evidence suggests that ion channels not only regulate ion homeostasis and electric signaling in excitable cells but also play important roles in cancer cell behaviors such as proliferation, apoptosis, invasion, and migration. Indeed, ENaCs/ASICs had been reported to be associated with cancer characteristics. Given their cell surface localization and pharmacology, pharmacological strategies to target ENaC/ASIC family members may be promising cancer therapeutics. PMID:27403419

  3. Tactile Sensitivity of Children: Effects of Frequency, Masking, and the Non-Pacinian I Psychophysical Channel

    ERIC Educational Resources Information Center

    Guclu, Burak; Oztek, Cigdem

    2007-01-01

    Tactile perception depends on the contributions of four psychophysical tactile channels mediated by four corresponding receptor systems. The sensitivity of the tactile channels is determined by detection thresholds that vary as a function of the stimulus frequency. It has been widely reported that tactile thresholds increase (i.e., sensitivity…

  4. NEGATIVE-ION MASS SPECTROMETRY OF SULFONYLUREA HERBICIDES

    EPA Science Inventory

    Sulfonylurea herbicides have been studied using neg-ion desorption chem.-ionization (DCI) mass spectrometry (MS) and DCI-MS/MS techniques. Both {M-H]- and M.- ions were obsd. in the DCI mass spectra. The collisonally activated dissocn. (CAD) spectra were characteristic of the str...

  5. POTENTIAL ENVIRONMENTAL RISKS ASSOCIATED WITH THE NEW SULFONYLUREA HERBICIDES

    EPA Science Inventory

    The first sulfonylurea herbicide, chlorsulfuron was introduced in 1982. embers of this new herbicide class are known for their high toxicity toward plant growth, low application rate, extremely low toxicity to humans and other animals, and phytotoxicity reported due to the inhibi...

  6. TOLERANCE OF MEADOW FOXTAIL (ALOPECURUS PRATENSIS) TO TWO SULFONYLUREA HERBICIDES

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Meadow foxtail is a rhizomatous grass widely grown for hay and pasture in wet meadows of the western United States and Canada. I compared the influence various rates of these two sulfonylurea herbicides on meadow foxtail biomass. Treatments of 0.035, 0.070, 0.105, and 0.140 kg/ai ha of chlorsulfur...

  7. Angular Sensitivity of Gated Micro-Channel Plate Framing Cameras

    SciTech Connect

    Landen, O L; Lobban, A; Tutt, T; Bell, P M; Costa, R; Ze, F

    2000-07-24

    Gated, microchannel-plate-based (MCP) framing cameras have been deployed worldwide for 0.2 - 9 keV x-ray imaging and spectroscopy of transient plasma phenomena. For a variety of spectroscopic and imaging applications, the angular sensitivity of MCPs must be known for correctly interpreting the data. We present systematic measurements of angular sensitivity at discrete relevant photon energies and arbitrary MCP gain. The results can been accurately predicted by using a simple 2D approximation to the 3D MCP geometry and by averaging over all possible photon ray paths.

  8. Gold nanoparticles embedded silicon channel biosensor for improved sensitivity

    NASA Astrophysics Data System (ADS)

    Chang, H. Y.; Arshad, M. K. Md.; M. Nuzaihan M., N.; Fathil, M. F. M.; Hashim, U.

    2016-07-01

    This project discusses the fabrication steps of a biosensor device on silicon-on-insulator (SOI) wafer. Conventional photolithography technique is used to fabricate the device. The gold nanoparticles (GNPs) are then used to enhance the sensitivity of the device. By incorporating the GNPs, it is expected to get higher current compared with the device without GNPs due to better conductivity of gold and higher volume-to-ratio. Hence, with the addition of GNPs, it may boost up the signal and enhance the sensitivity of the device.

  9. Mechano-sensitivity of epithelial sodium channels (ENaCs): laminar shear stress increases ion channel open probability.

    PubMed

    Althaus, Mike; Bogdan, Roman; Clauss, Wolfgang G; Fronius, Martin

    2007-08-01

    Epithelial cells are exposed to a variety of mechanical forces, but little is known about the impact of these forces on epithelial ion channels. Here we show that mechanical activation of epithelial sodium channels (ENaCs), which are essential for electrolyte and water balance, occurs via an increased ion channel open probability. ENaC activity of heterologously expressed rat (rENaC) and Xenopus (xENaC) orthologs was measured by whole-cell as well as single-channel recordings. Laminar shear stress (LSS), producing shear forces in physiologically relevant ranges, was used to mechanically stimulate ENaCs and was able to activate ENaC currents in whole-cell recordings. Preceding pharmacological activation of rENaC with Zn2+ and xENaC with gadolinium and glibenclamide largely prevented LSS-activated currents. In contrast, proteolytic cleavage with trypsin potentiated the LSS effect on rENaC whereas the LSS effect on xENaC was reversed (inhibition of xENaC current). Further, we found that exposure of excised outside-out patches to LSS led to an increased ion channel open probability without affecting the number of active channels. We suggest that mechano-sensitivity of ENaC may represent a ubiquitous feature for the physiology of epithelia, providing a putative mechanism for coupling transepithelial Na+ reabsorption to luminal transport. PMID:17426066

  10. Pharmacological characterization of volume-sensitive, taurine permeable anion channels in rat supraoptic glial cells

    PubMed Central

    Brès, Vanessa; Hurbin, Amandine; Duvoid, Anne; Orcel, Hélène; Moos, Françoise C; Rabié, Alain; Hussy, Nicolas

    2000-01-01

    To characterize the volume-sensitive, osmolyte permeable anion channels responsible for the osmodependent release of taurine from supraoptic nucleus (SON) astrocytes, we investigated the pharmacological properties of the [3H]-taurine efflux from acutely isolated SON.Taurine release induced by hypotonic stimulus (250 mosmol l−1) was not antagonized by the taurine transporter blocker guanidinoethyl sulphonate, confirming the lack of implication of the transporter.The osmodependent release of taurine was blocked by a variety of Cl− channel inhibitors with the order of potency: NPPB>niflumic acid>DPC>DIDS>ATP. On the other hand, release of taurine was only weakly affected by other compounds (dideoxyforskolin, 4-bromophenacyl bromide, mibefradil) known to block volume-activated anion channels in other cell preparations, and was completely insensitive to tamoxifen, a broad inhibitor of these channels.Although the molecular identity of volume-sensitive anion channels is not firmly established, a few genes have been postulated as potential candidates to encode such channels. We checked the expression in the SON of three of them, ClC3, phospholemman and VDAC1, and found that the transcripts of these genes are found in SON neurons, but not in astrocytes. Similar observation was previously reported for ClC2.In conclusion, the osmodependent taurine permeable channels of SON astrocytes display a particular pharmacological profile, suggesting the expression of a particular type or subtype of volume-sensitive anion channel, which is likely to be formed by yet unidentified proteins. PMID:10952690

  11. Pharmacological characterization of volume-sensitive, taurine permeable anion channels in rat supraoptic glial cells.

    PubMed

    Brès, V; Hurbin, A; Duvoid, A; Orcel, H; Moos, F C; Rabié, A; Hussy, N

    2000-08-01

    To characterize the volume-sensitive, osmolyte permeable anion channels responsible for the osmodependent release of taurine from supraoptic nucleus (SON) astrocytes, we investigated the pharmacological properties of the [(3)H]-taurine efflux from acutely isolated SON. Taurine release induced by hypotonic stimulus (250 mosmol l(-1)) was not antagonized by the taurine transporter blocker guanidinoethyl sulphonate, confirming the lack of implication of the transporter. The osmodependent release of taurine was blocked by a variety of Cl(-) channel inhibitors with the order of potency: NPPB>niflumic acid>DPC>DIDS>ATP. On the other hand, release of taurine was only weakly affected by other compounds (dideoxyforskolin, 4-bromophenacyl bromide, mibefradil) known to block volume-activated anion channels in other cell preparations, and was completely insensitive to tamoxifen, a broad inhibitor of these channels. Although the molecular identity of volume-sensitive anion channels is not firmly established, a few genes have been postulated as potential candidates to encode such channels. We checked the expression in the SON of three of them, ClC(3), phospholemman and VDAC(1), and found that the transcripts of these genes are found in SON neurons, but not in astrocytes. Similar observation was previously reported for ClC(2). In conclusion, the osmodependent taurine permeable channels of SON astrocytes display a particular pharmacological profile, suggesting the expression of a particular type or subtype of volume-sensitive anion channel, which is likely to be formed by yet unidentified proteins. PMID:10952690

  12. Sulfonylurea receptor 1 in central nervous system injury: a focused review

    PubMed Central

    Simard, J Marc; Woo, S Kyoon; Schwartzbauer, Gary T; Gerzanich, Volodymyr

    2012-01-01

    The sulfonylurea receptor 1 (Sur1)-regulated NCCa-ATP channel is a nonselective cation channel that is regulated by intracellular calcium and adenosine triphosphate. The channel is not constitutively expressed, but is transcriptionally upregulated de novo in all cells of the neurovascular unit, in many forms of central nervous system (CNS) injury, including cerebral ischemia, traumatic brain injury (TBI), spinal cord injury (SCI), and subarachnoid hemorrhage (SAH). The channel is linked to microvascular dysfunction that manifests as edema formation and delayed secondary hemorrhage. Also implicated in oncotic cell swelling and oncotic (necrotic) cell death, the channel is a major molecular mechanism of ‘accidental necrotic cell death' in the CNS. In animal models of SCI, pharmacological inhibition of Sur1 by glibenclamide, as well as gene suppression of Abcc8, prevents delayed capillary fragmentation and tissue necrosis. In models of stroke and TBI, glibenclamide ameliorates edema, secondary hemorrhage, and tissue damage. In a model of SAH, glibenclamide attenuates the inflammatory response due to extravasated blood. Clinical trials of an intravenous formulation of glibenclamide in TBI and stroke underscore the importance of recent advances in understanding the role of the Sur1-regulated NCCa-ATP channel in acute ischemic, traumatic, and inflammatory injury to the CNS. PMID:22714048

  13. Integrated cooling channels in position-sensitive silicon detectors

    NASA Astrophysics Data System (ADS)

    Andricek, L.; Boronat, M.; Fuster, J.; Garcia, I.; Gomis, P.; Marinas, C.; Ninkovic, J.; Perelló, M.; Villarejo, M. A.; Vos, M.

    2016-06-01

    We present an approach to construct position-sensitive silicon detectors with an integrated cooling circuit. Tests on samples demonstrate that a very modest liquid flow very effectively cool the devices up to a power dissipation of over 10 W/cm2. The liquid flow is found to have a negligible impact on the mechanical stability. A finite-element simulation predicts the cooling performance to an accuracy of approximately 10%.

  14. Transcainide causes two modes of open-channel block with different voltage sensitivities in batrachotoxin-activated sodium channels.

    PubMed Central

    Zamponi, G W; French, R J

    1994-01-01

    Transcainide, a complex derivative of lidocaine, blocks the open state of BTX-activated sodium channels from bovine heart and rat skeletal muscle in two distinct ways. When applied to either side of the membrane, transcainide caused discrete blocking events a few hundred milliseconds in duration (slow block), and a concomitant reduction in apparent single-channel amplitude, presumably because of rapid block beyond the temporal resolution of our recordings (fast block). We quantitatively analyzed block from the cytoplasmic side. Both modes of block occurred via binding of the drug to the open channel, approximately followed 1:1 stoichiometry, and were similar for both channel subtypes. For slow block, the blocking rate increased, and the unblocking rate decreased with depolarization, yielding an overall enhancement of block at positive potentials, and suggesting a blocking site at an apparent electrical distance about 45% of the way from the cytoplasmic end of the channel (z delta approximately 0.45). In contrast, the fast blocking mode was only slightly enhanced by depolarization (z delta approximately 0.15). Phenomenologically, the bulky and complex transcainide molecule combines the almost voltage-insensitive blocking action of phenylhydrazine (Zamponi and French, 1994a (companion paper)) with a slow open-channel blocking action that shows a voltage dependence typical of simpler amines. Only the slower blocking mode was sensitive to the removal of external sodium ions, suggesting that the two types of block occur at distinct sites. Dose-response relations were also consistent with independent binding of transcainide to two separate sites on the channel. PMID:7811913

  15. Hypoglycemia After Antimicrobial Drug Prescription for Older Patients Using Sulfonylureas

    PubMed Central

    Parekh, Trisha M.; Raji, Mukaila; Lin, Yu-Li; Tan, Alai; Kuo, Yong-Fang; Goodwin, James S.

    2016-01-01

    IMPORTANCE Certain antimicrobial drugs interact with sulfonylureas to increase the risk of hypoglycemia. OBJECTIVE To determine the risk of hypoglycemia and associated costs in older patients prescribed glipizide or glyburide who fill a prescription for an antimicrobial drug. DESIGN, SETTING, AND PARTICIPANTS This was a retrospective cohort study of Texas Medicare claims from 2006 to 2009 for patients 66 years or older who were prescribed glipizide or glyburide and who also filled a prescription for 1 of the 16 antimicrobials most commonly prescribed for this population. METHODS We assessed hypoglycemia events and associated Medicare costs in patients prescribed 1 of 7 antimicrobial agents thought to interact with sulfonylureas, using noninteracting antimicrobials as a comparison. We used a repeated measure logistic regression, controlling for age, sex, ethnicity, Medicaid eligibility, comorbidity, prior emergency department visits for hypoglycemia, prior hospitalizations for any cause, nursing home residence, and indication for the antimicrobial. We estimated odds of hypoglycemia, number needed to harm, deaths during hospitalization for hypoglycemia, and Medicare costs for hypoglycemia treatment. MAIN OUTCOMES AND MEASURES Any hospitalization or emergency department visit owing to hypoglycemia within 14 days of antimicrobial exposure. RESULTS In multivariable analyses controlling for patient characteristics and indication for antimicrobial drug use, clarithromycin (odds ratio [OR], 3.96 [95% CI, 2.42–6.49]), levofloxacin (OR, 2.60 [95% CI, 2.18–3.10]), sulfamethoxazole-trimethoprim (OR, 2.56 [95% CI, 2.12–3.10]), metronidazole (OR, 2.11 [95% CI, 1.28–3.47]), and ciprofloxacin (OR, 1.62 [95% CI, 1.33–1.97]) were associated with higher rates of hypoglycemia compared with a panel of noninteracting antimicrobials. The number needed to harm ranged from 71 for clarithromycin to 334 for ciprofloxacin. Patient factors associated with hypoglycemia included older

  16. Polyanions decelerate the kinetics of positively charged gramicidin channels as shown by sensitized photoinactivation.

    PubMed Central

    Antonenko, Yuri N; Borisenko, Vitali; Melik-Nubarov, Nikolay S; Kotova, Elena A; Woolley, G Andrew

    2002-01-01

    The effects of different anionic polymers on the kinetic properties of ionic channels formed by neutral gramicidin A (gA) and its positively charged analogs gramicidin-tris(2-aminoethyl)amine (gram-TAEA) and gramicidin-ethylenediamine (gram-EDA) in a bilayer lipid membrane were studied using a method of sensitized photoinactivation. The addition of Konig's polyanion caused substantial deceleration of the photoinactivation kinetics of gram-TAEA channels, which expose three positive charges to the aqueous phase at both sides of the membrane. In contrast, channels formed of gram-EDA, which exposes one positive charge, and neutral gA channels were insensitive to Konig's polyanion. The effect strongly depended on the nature of the polyanion added, namely: DNA, RNA, polyacrylic acid, and polyglutamic acid were inactive, whereas modified polyacrylic acid induced deceleration of the channel kinetics at high concentrations. In addition, DNA was able to prevent the action of Konig's polyanion. In single-channel experiments, the addition of Konig's polyanion resulted in the appearance of long-lived gram-TAEA channels. The deceleration of the gram-TAEA channel kinetics was ascribed to electrostatic interaction of the polyanion with gram-TAEA that reduces the mobility of gram-TAEA monomers and dimers in the membrane via clustering of channels. PMID:11867447

  17. Direct Activation of Epac by Sulfonylurea is Isoform Selective

    PubMed Central

    Herbst, Katie J.; Coltharp, Carla; Amzel, L. Mario; Zhang, Jin

    2011-01-01

    Summary Commonly used as a treatment for Type II diabetes, sulfonylureas (SUs) stimulate insulin secretion from pancreatic β cells by binding to sulfonylurea receptors. Recently, SUs have been shown to also activate exchange protein directly activated by cAMP 2 (Epac2), however little is known about this molecular action. Using biosensor imaging and biochemical analysis, we show that SUs activate Epac2 and the downstream signaling via direct binding to Epac2. We further identify R447 of Epac2 to be critically involved in SU binding. This distinct binding site from cAMP points to a new mode of allosteric activation of Epac2. We also show that SUs selectively activate Epac2 isoform, but not the closely related Epac1, further establishing SUs as a new class of isoform-selective enzyme activators. PMID:21338921

  18. Magnesium Sensitizes Slow Vacuolar Channels to Physiological Cytosolic Calcium and Inhibits Fast Vacuolar Channels in Fava Bean Guard Cell Vacuoles.

    PubMed

    Pei; Ward; Schroeder

    1999-11-01

    Vacuolar ion channels in guard cells play important roles during stomatal movement and are regulated by many factors including Ca(2+), calmodulin, protein kinases, and phosphatases. We report that physiological cytosolic and luminal Mg(2+) levels strongly regulate vacuolar ion channels in fava bean (Vicia faba) guard cells. Luminal Mg(2+) inhibited fast vacuolar (FV) currents with a K(i) of approximately 0.23 mM in a voltage-dependent manner at positive potentials on the cytoplasmic side. Cytosolic Mg(2+) at 1 mM also inhibited FV currents. Furthermore, in the absence of cytosolic Mg(2+), cytosolic Ca(2+) at less than 10 µM did not activate slow vacuolar (SV) currents. However, when cytosolic Mg(2+) was present, submicromolar concentrations of cytosolic Ca(2+) activated SV currents with a K(d) of approximately 227 nM, suggesting a synergistic Mg(2+)-Ca(2+) effect. The activation potential of SV currents was shifted toward physiological potentials in the presence of cytosolic Mg(2+) concentrations. The direction of SV currents could also be changed from outward to both outward and inward currents. Our data predict a model for SV channel regulation, including a cytosolic binding site for Ca(2+) with an affinity in the submicromolar range and a cytosolic low-affinity Mg(2+)-Ca(2+) binding site. SV channels are predicted to contain a third binding site on the vacuolar luminal side, which binds Ca(2+) and is inhibitory. In conclusion, cytosolic Mg(2+) sensitizes SV channels to physiological cytosolic Ca(2+) elevations. Furthermore, we propose that cytosolic and vacuolar Mg(2+) concentrations ensure that FV channels do not function as a continuous vacuolar K(+) leak, which would prohibit stomatal opening. PMID:10557247

  19. Sensitivity of the plant vacuolar malate channel to pH, Ca2+ and anion-channel blockers.

    PubMed

    Pantoja, O; Smith, J A C

    2002-03-01

    The organic anion malate is accumulated in the central vacuole of most plant cells. Malate has several important roles in plant vacuoles, such as the maintenance of charge balance and pH regulation, as an osmolyte involved in the generation of cell turgor, and as a storage form of CO2. Transport of malate across the vacuolar membrane is important for the regulation of cytoplasmic pH and the control of cellular metabolism, particularly in plants showing crassulacean acid metabolism (CAM), in which large fluxes of malate occur during the day/night cycle. By applying the patch-clamp technique, in the whole-vacuole configuration, to isolated vacuoles from leaf mesophyll cells of the CAM plant Kalanchoë daigremontiana, we studied the regulation of the vacuolar malate channel by pH and Ca2+, as well as its sensitivity to anion-channel blockers. Malate currents were found to be insensitive to Ca2+ on the cytoplasmic side of the membrane over a range from approximately 10(-8) M to 10(-4) M. In contrast, decreasing cytoplasmic pH below 7.5 had a significant modulatory effect on channel activity, reducing malate currents by 40%, whereas increasing cytoplasmic pH above 7.5 resulted in no change in current. Several known Cl?-channel blockers inhibited the vacuolar malate currents: niflumic acid and indanoyloxyacetic acid (IAA-94) proved to be the most effective inhibitors, exerting half-maximal effects at concentrations of approximately 20 mM, suggesting that the plant vacuolar malate channel may share certain similarities with other classes of known anion channels. PMID:11891587

  20. 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

  1. Growth Inhibition of Pathogenic Bacteria by Sulfonylurea Herbicides

    PubMed Central

    Kreisberg, Jason F.; Ong, Nicholas T.; Krishna, Aishwarya; Joseph, Thomas L.; Wang, Jing; Ong, Catherine; Ooi, Hui Ann; Sung, Julie C.; Siew, Chern Chiang; Chang, Grace C.; Biot, Fabrice; Cuccui, Jon; Wren, Brendan W.; Chan, Joey; Sivalingam, Suppiah P.; Zhang, Lian-Hui; Verma, Chandra

    2013-01-01

    Emerging resistance to current antibiotics raises the need for new microbial drug targets. We show that targeting branched-chain amino acid (BCAA) biosynthesis using sulfonylurea herbicides, which inhibit the BCAA biosynthetic enzyme acetohydroxyacid synthase (AHAS), can exert bacteriostatic effects on several pathogenic bacteria, including Burkholderia pseudomallei, Pseudomonas aeruginosa, and Acinetobacter baumannii. Our results suggest that targeting biosynthetic enzymes like AHAS, which are lacking in humans, could represent a promising antimicrobial drug strategy. PMID:23263008

  2. [The use of gliclazide in individualized sulfonylurea therapy].

    PubMed

    Winkler, Gábor

    2014-04-01

    In addition to the common blood glucose lowering effect, sulfonylurea compounds are different in many aspects from each other. Based on earlier findings the second generation gliclazide has special advantages within this group. Although the number of experimental and clinical observations on gliclazide is continuously increasing, these novel findings are not in the focus anymore due to the appearance of new antidiabetics. This article reviews recent experimental (effect on receptors, the absence of Epac2 activation, antioxidant properties, possible incentive of factors participating in beta-cell differentiation) and pharmacogenomic data, and compares them with clinical observations obtained from gliclazide treatment (hypoglycemias, parameters of cardiovascular outcome). The data underline the advantages of gliclazide, the highly pancreas-selective nature, preservation of the ischemic precondition, favourable hemodynamic properties and potential reduction of the beta-cell loss as compared to other compounds of the group. However, gliclazide is not free from disadvantages characteristic to sulfonylureas in general (blood glucose independent insulin stimulation, beta-cell depletion). Comparing gliclazide with other derivatives of the group, the above data indicate individual benefits for the application when sulfonylurea compound is the drug of choice. PMID:24681676

  3. Mechanical sensitivity of Piezo1 ion channels can be tuned by cellular membrane tension

    PubMed Central

    Lewis, Amanda H; Grandl, Jörg

    2015-01-01

    Piezo1 ion channels mediate the conversion of mechanical forces into electrical signals and are critical for responsiveness to touch in metazoans. The apparent mechanical sensitivity of Piezo1 varies substantially across cellular environments, stimulating methods and protocols, raising the fundamental questions of what precise physical stimulus activates the channel and how its stimulus sensitivity is regulated. Here, we measured Piezo1 currents evoked by membrane stretch in three patch configurations, while simultaneously visualizing and measuring membrane geometry. Building on this approach, we developed protocols to minimize resting membrane curvature and tension prior to probing Piezo1 activity. We find that Piezo1 responds to lateral membrane tension with exquisite sensitivity as compared to other mechanically activated channels and that resting tension can drive channel inactivation, thereby tuning overall mechanical sensitivity of Piezo1. Our results explain how Piezo1 can function efficiently and with adaptable sensitivity as a sensor of mechanical stimulation in diverse cellular contexts. DOI: http://dx.doi.org/10.7554/eLife.12088.001 PMID:26646186

  4. Developmentally-regulated sodium channel subunits are differentially sensitive to {alpha}-cyano containing pyrethroids

    SciTech Connect

    Meacham, Connie A.; Brodfuehrer, Peter D.; Watkins, Jennifer A.; Shafer, Timothy J.

    2008-09-15

    Juvenile rats have been reported to be more sensitive to the acute neurotoxic effects of the pyrethroid deltamethrin than adults. While toxicokinetic differences between juveniles and adults are documented, toxicodynamic differences have not been examined. Voltage-gated sodium channels, the primary targets of pyrethroids, are comprised of {alpha} and {beta} subunits, each of which have multiple isoforms that are expressed in a developmentally-regulated manner. To begin to test whether toxicodynamic differences could contribute to age-dependent deltamethrin toxicity, deltamethrin effects were examined on sodium currents in Xenopus laevis oocytes injected with different combinations of rat {alpha} (Na{sub v}1.2 or Na{sub v}1.3) and {beta} ({beta}{sub 1} or {beta}{sub 3}) subunits. Deltamethrin induced tail currents in all isoform combinations and increased the percent of modified channels in a concentration-dependent manner. Effects of deltamethrin were dependent on subunit combination; Na{sub v}1.3-containing channels were modified to a greater extent than were Na{sub v}1.2-containing channels. In the presence of a {beta} subunit, deltamethrin effects were significantly greater, an effect most pronounced for Na{sub v}1.3 channels; Na{sub v}1.3/{beta}{sub 3} channels were more sensitive to deltamethrin than Na{sub v}1.2/{beta}{sub 1} channels. Na{sub v}1.3/{beta}{sub 3} channels are expressed embryonically, while the Na{sub v}1.2 and {beta}{sub 1} subunits predominate in adults, supporting the hypothesis for age-dependent toxicodynamic differences. Structure-activity relationships for sensitivity of these subunit combinations were examined for other pyrethroids. Permethrin and tetramethrin did not modify currents mediated by either subunit combination. Cypermethrin, {beta}-cyfluthrin, esfenvalerate and fenpropathrin all modified sodium channel function; effects were significantly greater on Na{sub v}1.3/{beta}{sub 3} than on Na{sub v}1.2/{beta}{sub 1} channels. These

  5. Sarcolemmal ATP-sensitive potassium channels modulate skeletal muscle function under low-intensity workloads

    PubMed Central

    Zhu, Zhiyong; Sierra, Ana; Burnett, Colin M.-L.; Chen, Biyi; Subbotina, Ekaterina; Koganti, Siva Rama Krishna; Gao, Zhan; Wu, Yuejin; Anderson, Mark E.; Song, Long-Sheng; Goldhamer, David J.; Coetzee, William A.; Hodgson-Zingman, Denice M.

    2014-01-01

    ATP-sensitive potassium (KATP) channels have the unique ability to adjust membrane excitability and functions in accordance with the metabolic status of the cell. Skeletal muscles are primary sites of activity-related energy consumption and have KATP channels expressed in very high density. Previously, we demonstrated that transgenic mice with skeletal muscle–specific disruption of KATP channel function consume more energy than wild-type littermates. However, how KATP channel activation modulates skeletal muscle resting and action potentials under physiological conditions, particularly low-intensity workloads, and how this can be translated to muscle energy expenditure are yet to be determined. Here, we developed a technique that allows evaluation of skeletal muscle excitability in situ, with minimal disruption of the physiological environment. Isometric twitching of the tibialis anterior muscle at 1 Hz was used as a model of low-intensity physical activity in mice with normal and genetically disrupted KATP channel function. This workload was sufficient to induce KATP channel opening, resulting in membrane hyperpolarization as well as reduction in action potential overshoot and duration. Loss of KATP channel function resulted in increased calcium release and aggravated activity-induced heat production. Thus, this study identifies low-intensity workload as a trigger for opening skeletal muscle KATP channels and establishes that this coupling is important for regulation of myocyte function and thermogenesis. These mechanisms may provide a foundation for novel strategies to combat metabolic derangements when energy conservation or dissipation is required. PMID:24344248

  6. Sarcolemmal ATP-sensitive potassium channel protects cardiac myocytes against lipopolysaccharide-induced apoptosis.

    PubMed

    Zhang, Xiaohui; Zhang, Xiaohua; Xiong, Yiqun; Xu, Chaoying; Liu, Xinliang; Lin, Jian; Mu, Guiping; Xu, Shaogang; Liu, Wenhe

    2016-09-01

    The sarcolemmal ATP-sensitive K+ (sarcKATP) channel plays a cardioprotective role during stress. However, the role of the sarcKATP channel in the apoptosis of cardiomyocytes and association with mitochondrial calcium remains unclear. For this purpose, we developed a model of LPS-induced sepsis in neonatal rat cardiomyocytes (NRCs). The TUNEL assay was performed in order to detect the apoptosis of cardiac myocytes and the MTT assay was performed to determine cellular viability. Exposure to LPS significantly decreased the viability of the NRCs as well as the expression of Bcl-2, whereas it enhanced the activity and expression of the apoptosis-related proteins caspase-3 and Bax, respectively. The sarcKATP channel blocker, HMR-1098, increased the apoptosis of NRCs, whereas the specific sarcKATP channel opener, P-1075, reduced the apoptosis of NRCs. The mitochondrial calcium uniporter inhibitor ruthenium red (RR) partially inhibited the pro-apoptotic effect of HMR-1098. In order to confirm the role of the sarcKATP channel, we constructed a recombinant adenovirus vector carrying the sarcKATP channel mutant subunit Kir6.2AAA to inhibit the channel activity. Kir6.2AAA adenovirus infection in NRCs significantly aggravated the apoptosis of myocytes induced by LPS. Elucidating the regulatory mechanisms of the sarcKATP channel in apoptosis may facilitate the development of novel therapeutic targets and strategies for the management of sepsis and cardiac dysfunction. PMID:27430376

  7. EXAMINATION OF THE ANTICONVULSANT PROPERTIES OF VOLTAGE-SENSITIVE CALCIUM CHANNEL INHIBITORS IN AMYGDALA KINDLED SEIZURES

    EPA Science Inventory

    Representatives from three different classes of voltage-sensitive calcium (VSC) channel inhibitors were assessed for their protection against amygdala kindled seizures. dult male long Evans rats (n=12) were implanted with electrodes in the amygdala and were stimulated once daily ...

  8. A KINETIC STUDY OF THE METHANOLYSIS OF THE SULFONYLUREAS BENSULFURON METHYL AND SULFOMETURON METHYL USING CAPILLARY ELECTROPHORESIS

    EPA Science Inventory

    The instability of sulfonylureas in solution in methanol has led us to a kinetic study of methanolysis of two sulfonylureas using capillary electrophoresis. In a preliminary experiment solutions of the seven compounds, bensulfuron methyl, sulfometuron methyl, nicosulfuron, chlori...

  9. Identification and localization of an arachidonic acid-sensitive potassium channel in the cochlea.

    PubMed

    Sokolowski, Bernd H A; Sakai, Yoshihisa; Harvey, Margaret C; Duzhyy, Dmytro E

    2004-07-14

    Receptor cells of the auditory and vestibular end organs of vertebrates acquire various types of potassium channels during development. Their expression and kinetics can differ along the tonotopic axis as well as in different cell types of the sensory epithelium. These variations can play a crucial role in modulating sensory transduction and cochlear tuning. Whole-cell tight-seal recordings of isolated hair cells revealed the presence of an arachidonic acid-sensitive A-type channel in the short (outer) hair cells of the chicken cochlea. This polyunsaturated fatty acid blocked the A-current, thereby increasing the amplitude and duration of the voltage response in these cells. We identified the gene encoding this channel as belonging to a member of the Shal subfamily, Kv4.2. Expression of the recombinant channel shows half-activation and inactivation potentials shifted to more positive values relative to native channels, suggesting that the native channel is coexpressed with an accessory subunit. RT-PCR revealed that transcription begins early in development, whereas in situ hybridization showed mRNA expression limited to the intermediate and short hair cells located in specific regions of the adult cochlea. Additional localization, using immunofluorescent staining, revealed clustering in apical-lateral regions of the receptor cell as well as in the cochlear ganglion. These experiments provide evidence that in addition to membrane proteins modulating excitation in these receptor cells, fatty acids contribute to the coding of auditory stimuli via these channels. PMID:15254081

  10. Inhibition of diacylglycerol-sensitive TRPC channels by synthetic and natural steroids.

    PubMed

    Miehe, Susanne; Crause, Peter; Schmidt, Thorsten; Löhn, Matthias; Kleemann, Heinz-Werner; Licher, Thomas; Dittrich, Werner; Rütten, Hartmut; Strübing, Carsten

    2012-01-01

    TRPC channels are a family of nonselective cation channels that regulate ion homeostasis and intracellular Ca(2+) signaling in numerous cell types. Important physiological functions such as vasoregulation, neuronal growth, and pheromone recognition have been assigned to this class of ion channels. Despite their physiological relevance, few selective pharmacological tools are available to study TRPC channel function. We, therefore, screened a selection of pharmacologically active compounds for TRPC modulating activity. We found that the synthetic gestagen norgestimate inhibited diacylglycerol-sensitive TRPC3 and TRPC6 with IC(50)s of 3-5 µM, while half-maximal inhibition of TRPC5 required significantly higher compound concentrations (>10 µM). Norgestimate blocked TRPC-mediated vasopressin-induced cation currents in A7r5 smooth muscle cells and caused vasorelaxation of isolated rat aorta, indicating that norgestimate could be an interesting tool for the investigation of TRP channel function in native cells and tissues. The steroid hormone progesterone, which is structurally related to norgestimate, also inhibited TRPC channel activity with IC(50)s ranging from 6 to 18 µM but showed little subtype selectivity. Thus, TRPC channel inhibition by high gestational levels of progesterone may contribute to the physiological decrease of uterine contractility and immunosuppression during pregnancy. PMID:22530015

  11. Cell volume-sensitive chloride channels: phenotypic properties and molecular identity.

    PubMed

    Okada, Yasunobu

    2006-01-01

    Cell volume regulation is essential for the survival of cells. After osmotic swelling, animal cells show a regulatory volume decrease by releasing intracellular K(+), Cl (-)and water. In most cell types, volume-regulatory Cl(-) efflux is induced by activation of electroconductive anion pathways. Among these volume-activated Cl(-) channels, the most important and specific is a volume-sensitive outwardly rectifying (VSOR) Cl(-) channel. The phonotypical properties have been well described. Extracellular application of anionic forms of ATP and glibenclamide give rise to voltage-dependent open-channel block of this channel, the fact suggesting that its outer vestibule and pore are larger and smaller, respectively, than the sizes of ATP and glibenclamide. Consistent with this prediction, the pore radius of VSOR Cl(-) channel (0.63 nm) which has been recently determined is slightly smaller than the radii of ATP and glibenclamide. The activities of VSOR Cl(-) channels are implicated not only in regulatory volume decrease but also in many other physiological or pathophysiological cell events including cell death induction. Despite their ubiquitous expression and physiological/ pathophysiological significance, there is still a paucity of the molecular information of the VSOR Cl(-) channel. PMID:17065805

  12. Insecticide sensitivity of native chloride and sodium channels in a mosquito cell line.

    PubMed

    Jenson, Lacey J; Anderson, Troy D; Bloomquist, Jeffrey R

    2016-06-01

    The aim of this study was to investigate the utility of cultured Anopheles gambiae Sua1B cells for insecticide screening applications without genetic engineering or other treatments. Sua1B cells were exposed to the known insecticidal compounds lindane and DIDS, which inhibited cell growth at micromolar concentrations. In patch clamp studies, DIDS produced partial inhibition (69%) of chloride current amplitudes, and an IC50 of 5.1μM was determined for Sua1B cells. A sub-set of chloride currents showed no response to DIDS; however, inhibition (64%) of these currents was achieved using a low chloride saline solution, confirming their identity as chloride channels. In contrast, lindane increased chloride current amplitude (EC50=116nM), which was reversed when cells were bathed in calcium-free extracellular solution. Voltage-sensitive chloride channels were also inhibited by the presence of fenvalerate, a type 2 pyrethroid, but not significantly blocked by type 1 allethrin, an effect not previously shown in insects. Although no evidence of fast inward currents typical of sodium channels was observed, studies with fenvalerate in combination with veratridine, a sodium channel activator, revealed complete inhibition of cell growth that was best fit by a two-site binding model. The high potency effect was completely inhibited in the presence of tetrodotoxin, a specific sodium channel blocker, suggesting the presence of some type of sodium channel. Thus, Sua1B cells express native insect ion channels with potential utility for insecticide screening. PMID:27155485

  13. Inhibition of the ATP-sensitive potassium channel from mouse pancreatic β-cells by surfactants

    PubMed Central

    Smith, Paul A; Proks, Peter

    1998-01-01

    We have used patch-clamp methods to study the effects of the detergents, Cremophor, Tween 80 and Triton X100 on the KATP channel in the pancreatic β-cell from mouse.All three detergents blocked KATP channel activity with the following order of potency: Tween 80 (Ki<∼83 nM)>Triton X100 (Ki=350 nM)>Cremophor. In all cases the block was poorly reversible.Single-channel studies suggested that at low doses, the detergents act as slow blockers of the KATP channel.Unlike the block produced by tolbutamide, that produced by detergent was not affected by intracellular Mg2+-nucleotide, diazoxide or trypsin treatment, nor did it involve an acceleration of rundown or increase in ATP sensitivity of the chanel.The detergents could block the pore-forming subunit, Kir6.2ΔC26, which can be expressed independently of SUR1 (the regulatory subunit of the KATP channel). These data suggest that the detergents act on Kir6.2 and not SUR1.The detergents had no effect on another member of the inward rectifier family: Kir1.1a (ROMK1).Voltage-dependent K-currents in the β-cell were reversibly blocked by the detergents with a far lower potency than that found for the KATP channel.Like other insulin secretagogues that act by blocking the KATP channel, Cremophor elevated intracellular Ca2+ in single β-cells to levels that would be expected to elicit insulin secretion.Given the role of the KATP channel in many physiological processes, we conclude that plasma borne detergent may have pharmacological actions mediated through blockage of the KATP channel PMID:9647478

  14. Characterization of apamin-sensitive Ca(2+)-activated potassium channels in human leukaemic T lymphocytes.

    PubMed

    Hanselmann, C; Grissmer, S

    1996-11-01

    1. The whole-cell recording mode of the patch-clamp technique was used to study the effect of extracellularly applied ions, toxins and drugs on voltage-independent, apamin-sensitive Ca(2+)-activated K+ channels, K(Ca), expressed in the Jurkat human leukaemic T cell line. 2. Extracellular Ba2+ and Sr+ produced a voltage-dependent block. The equilibrium dissociation constant of the Ba2+/K(Ca) channel complex increased e-fold for a 20 mV change of potential. Ba2+ block of Jurkat K(Ca) channels is therefore as steep as expected from the movement of a single divalent cation about half-way into the electric field of the membrane from the outside. 3. We determined the ion selectivity as well as the conductance of these channels. Calculated permeability ratios, PX/PK, for these K(Ca) channels were 1.0, 0.96, 0.26 and 0.53 for K+, Rb+, Cs+ and NH4+, respectively. Conductance ratios, gX/gK, for the same ions were 1.0, 1.0, 0.67 and 0.11, respectively. Most strikingly this channel can also carry significant current with Cs+ as current carrier. 4. Scyllatoxin (ScTX), a thirty-one amino acid peptide toxin, reduced current through these K(Ca) channels with a half-blocking concentration of approximately 0.3 nM independent of the pH. Other drugs that were able to reduce current through these channels include the classical calcium antagonists diltiazem and verapamil. In contrast, nifedipine, clotrimazole and kaliotoxin (100 nM) were unable to block current through these channels in Jurkat T cells. PMID:8930831

  15. Chronic ethanol-induced changes in cardiac and neuronal ATP-sensitive potassium channels

    SciTech Connect

    Bangalore, R.; Hawthorn, M.; Triggle, D.J. )

    1992-02-26

    The present study was designed to investigate the effect of chronic ethanol consumption on cardiac and neuronal ATP-sensitive potassium channels. These channels have been shown to be regulated under diseased conditions such as congestive heart failure. Rats were chronically fed with a liquid diet containing ethanol or equicaloric amount of dextrin for the three weeks. This diet induced tolerance to ethanol as assessed by the longer time the ethanol fed rats could stay on a rotorod compared to control rats when challenged with an i.p. injection of ethanol, ATP-sensitive potassium channels were characterized using ({sup 3}H)glibenclamide binding to membrane preparations from heart, olfactory bulb, hippocampus, striatum, cerebellum, cortex, brain stem and spinal cord. Chronic ethanol consumption caused a significant increase in the K{sub D} value in the hippocampus and cerebellum, and a significant decrease in the K{sub D} value in the cortex. The K{sub D} value did not change in other brain areas and heart with chronic ethanol consumption. In contrast, chronic ethanol caused a significant decrease in the B{sub max} value in the heart, and a slight but significant increase in the B{sub max} value in the spinal cord. Chronic ethanol did not affect the B{sub max} value in other brain areas. ATP-sensitive potassium channels are differently regulated by ethanol in cardiac and neuronal preparations.

  16. The pH sensitivity of Aqp0 channels in tetraploid and diploid teleosts

    PubMed Central

    Chauvigné, François; Zapater, Cinta; Stavang, Jon Anders; Taranger, Geir Lasse; Cerdà, Joan; Finn, Roderick Nigel

    2015-01-01

    Water homeostasis and the structural integrity of the vertebrate lens is partially mediated by AQP0 channels. Emerging evidence indicates that external pH may be involved in channel gating. Here we show that a tetraploid teleost, the Atlantic salmon, retains 4 aqp0 genes (aqp0a1, -0a2, -0b1, and -0b2), which are highly, but not exclusively, expressed in the lens. Functional characterization reveals that, although each paralog permeates water efficiently, the permeability is respectively shifted to the neutral, alkaline, or acidic pH in Aqp0a1, -0a2, and -0b1, whereas that of Aqp0b2 is not regulated by external pH. Mutagenesis studies demonstrate that Ser38, His39, and His40 residues in the extracellular transmembrane domain of α-helix 2 facing the water pore are critical for the pH modulation of water transport. To validate these findings, we show that both zebrafish Aqp0a and -0b are functional water channels with respective pH sensitivities toward alkaline or acid pH ranges and that an N-terminal allelic variant (Ser19) of Aqp0b exists that abolishes water transport in Xenopus laevis oocytes. The data suggest that the alkaline pH sensitivity is a conserved trait in teleost Aqp0 a-type channels, whereas mammalian AQP0 and some teleost Aqp0 b-type channels display an acidic pH permeation preference.—Chauvigné, F., Zapater, C., Stavang, J. A., Taranger, G. L., Cerdà, J., Finn, R. N. The pH sensitivity of Aqp0 channels in tetraploid and diploid teleosts. PMID:25667219

  17. Cost-effectiveness of Canagliflozin versus Sitagliptin When Added to Metformin and Sulfonylurea in Type 2 Diabetes in Canada.

    PubMed

    Sabapathy, Suthakar; Neslusan, Cheryl; Yoong, Kim; Teschemaker, Anna; Johansen, Pierre; Willis, Michael

    2016-01-01

    BackgroundCanagliflozin, an agent that inhibits sodium glucose co-transporter 2, is approved as add-on to metformin plus sulfonylurea for the treatment of type 2 diabetes in Canada. Canagliflozin offers greater glycemic control, as well as important additional benefits such as weight loss and blood pressure reductions, versus dipeptidyl peptidase-4 inhibitors such as sitagliptin.  ObjectiveThis analysis evaluated the cost-effectiveness of canagliflozin 300 mg and canagliflozin 100 mg versus sitagliptin 100 mg in patients with type 2 diabetes inadequately controlled on metformin plus sulfonylurea from the perspective of the Canadian Agency for Drugs and Technologies in Health. MethodsA 40-year cost-effectiveness analysis was performed using the validated Economic and Health Outcomes Model of Type 2 Diabetes Mellitus (ECHO-T2DM). Patient characteristics, treatment effects, and rates of hypoglycemia and adverse events were sourced from the canagliflozin clinical program. Canada-specific costs and utilities were applied. Sensitivity analyses were conducted using alternative values for key model inputs. ResultsBoth canagliflozin 300 and 100 mg dominated sitagliptin 100 mg over 40 years, providing quality-adjusted life-year gains of 0.31 and 0.28, and cost offsets of $2,217 and $2,560, respectively. Both canagliflozin doses dominated sitagliptin in each of the sensitivity analyses. ConclusionsSimulation results suggested that canagliflozin 300 and 100 mg provided better health outcomes and lower costs than sitagliptin 100 mg as a third-line therapy added-on to metformin and sulfonylurea in patients with type 2 diabetes in Canada. PMID:27463416

  18. ATP-sensitive potassium channels: uncovering novel targets for treating depression.

    PubMed

    Fan, Yi; Kong, Hui; Ye, Xinhai; Ding, Jianhua; Hu, Gang

    2016-07-01

    ATP-sensitive potassium (K-ATP) channels have been shown to couple membrane electrical activity to energy metabolism in a variety of cells and are important in several physiological systems. In the brain, K-ATP channels are strongly expressed in the neuronal circuitry. The distributional profile and functional significance of K-ATP channels suggest that they may be involved in stress-induced depression. First, we showed that chronic mild stress (CMS) significantly increased the expression of hippocampal Kir6.2 and Kir6.1 subunits of K-ATP channels. Next, using Kir6.2 knockout (Kir6.2(-/-)) mice, we presented that Kir6.2 deficiency resulted in antidepressant-like behaviors under non-stress conditions, but aggravated depressive behaviors accompanied by the loss of CA3 neuron and the reduction of brain-derived neurotrophic factor in hippocampus under chronic stress. Finally, we demonstrated that the K-ATP channel opener iptakalim, as well as a classical antidepressant fluoxetine, can reverse CMS-induced depression-related behaviors and counteract the deleterious effects of stress on hippocampus in wild-type mice, but only partially alleviate these symptoms in Kir6.2(-/-) mice. Collectively, our findings demonstrate that K-ATP channels are involved in the pathogenesis of depression and may be a promising target for the therapy of depression. PMID:26289962

  19. Sulfonylureas and Glinides as New PPARγ Agonists:. Virtual Screening and Biological Assays

    NASA Astrophysics Data System (ADS)

    Scarsi, Marco; Podvinec, Michael; Roth, Adrian; Hug, Hubert; Kersten, Sander; Albrecht, Hugo; Schwede, Torsten; Meyer, Urs A.; Rücker, Christoph

    2007-12-01

    This work combines the predictive power of computational drug discovery with experimental validation by means of biological assays. In this way, a new mode of action for type 2 diabetes drugs has been unvealed. Most drugs currently employed in the treatment of type 2 diabetes either target the sulfonylurea receptor stimulating insulin release (sulfonylureas, glinides), or target PPARγ improving insulin resistance (thiazolidinediones). Our work shows that sulfonylureas and glinides bind to PPARγ and exhibit PPARγ agonistic activity. This result was predicted in silico by virtual screening and confirmed in vitro by three biological assays. This dual mode of action of sulfonylureas and glinides may open new perspectives for the molecular pharmacology of antidiabetic drugs, since it provides evidence that drugs can be designed which target both the sulfonylurea receptor and PPARγ. Targeting both receptors could in principle allow to increase pancreatic insulin secretion, as well as to improve insulin resistance.

  20. Review of safety considerations in the elderly using sulfonylureas.

    PubMed

    Tirmizi, Samad; Mazzola, Nissa

    2015-02-01

    Sulfonylureas (SUs) are a class of medications used to treat type 2 diabetes mellitus that work by increasing insulin secretion in the pancreas. Second-generation SUs are most often used today because of adverse effects with those in the first generation. However, there is still a risk of hypoglycemia even with the newer medications in the class. The American Diabetes Association encourages a patient-centered approach when initiating pharmacologic therapy. Understanding the pharmacokinetic differences among these medications can allow the consultant pharmacist to make a decision on which medication may be best suited for the patient. PMID:25695418

  1. Myometrial relaxation of mice via expression of two pore domain acid sensitive K(+) (TASK-2) channels.

    PubMed

    Kyeong, Kyu-Sang; Hong, Seung Hwa; Kim, Young Chul; Cho, Woong; Myung, Sun Chul; Lee, Moo Yeol; You, Ra Young; Kim, Chan Hyung; Kwon, So Yeon; Suzuki, Hikaru; Park, Yeon Jin; Jeong, Eun-Hwan; Kim, Hak Soon; Kim, Heon; Lim, Seung Woon; Xu, Wen-Xie; Lee, Sang Jin; Ji, Il Woon

    2016-09-01

    Myometrial relaxation of mouse via expression of two-pore domain acid sensitive (TASK) channels was studied. In our previous report, we suggested that two-pore domain acid-sensing K(+) channels (TASK-2) might be one of the candidates for the regulation of uterine circular smooth muscles in mice. In this study, we tried to show the mechanisms of relaxation via TASK-2 channels in marine myometrium. Isometric contraction measurements and patch clamp technique were used to verify TASK conductance in murine myometrium. Western blot and immunehistochemical study under confocal microscopy were used to investigate molecular identity of TASK channel. In this study, we showed that TEA and 4-AP insensitive non-inactivating outward K(+) current (NIOK) may be responsible for the quiescence of murine pregnant longitudinal myometrium. The characteristics of NIOK coincided with two-pore domain acid-sensing K(+) channels (TASK-2). NIOK in the presence of K(+) channel blockers was inhibited further by TASK inhibitors such as quinidine, bupivacaine, lidocaine, and extracellular acidosis. Furthermore, oxytocin and estrogen inhibited NIOK in pregnant myometrium. When compared to non-pregnant myometrium, pregnant myometrium showed stronger inhibition of NIOK by quinidine and increased immunohistochemical expression of TASK-2. Finally, TASK-2 inhibitors induced strong myometrial contraction even in the presence of L-methionine, a known inhibitor of stretch-activated channels in the longitudinal myometrium of mouse. Activation of TASK-2 channels seems to play an essential role for relaxing uterus during pregnancy and it might be one of the alternatives for preventing preterm delivery. PMID:27610042

  2. Myometrial relaxation of mice via expression of two pore domain acid sensitive K+ (TASK-2) channels

    PubMed Central

    Kyeong, Kyu-Sang; Hong, Seung Hwa; Cho, Woong; Myung, Sun Chul; Lee, Moo Yeol; You, Ra Young; Kim, Chan Hyung; Kwon, So Yeon; Suzuki, Hikaru; Park, Yeon Jin; Jeong, Eun-Hwan; Kim, Hak Soon; Kim, Heon; Lim, Seung Woon; Xu, Wen-Xie; Lee, Sang Jin

    2016-01-01

    Myometrial relaxation of mouse via expression of two-pore domain acid sensitive (TASK) channels was studied. In our previous report, we suggested that two-pore domain acid-sensing K+ channels (TASK-2) might be one of the candidates for the regulation of uterine circular smooth muscles in mice. In this study, we tried to show the mechanisms of relaxation via TASK-2 channels in marine myometrium. Isometric contraction measurements and patch clamp technique were used to verify TASK conductance in murine myometrium. Western blot and immunehistochemical study under confocal microscopy were used to investigate molecular identity of TASK channel. In this study, we showed that TEA and 4-AP insensitive non-inactivating outward K+ current (NIOK) may be responsible for the quiescence of murine pregnant longitudinal myometrium. The characteristics of NIOK coincided with two-pore domain acid-sensing K+ channels (TASK-2). NIOK in the presence of K+ channel blockers was inhibited further by TASK inhibitors such as quinidine, bupivacaine, lidocaine, and extracellular acidosis. Furthermore, oxytocin and estrogen inhibited NIOK in pregnant myometrium. When compared to non-pregnant myometrium, pregnant myometrium showed stronger inhibition of NIOK by quinidine and increased immunohistochemical expression of TASK-2. Finally, TASK-2 inhibitors induced strong myometrial contraction even in the presence of L-methionine, a known inhibitor of stretch-activated channels in the longitudinal myometrium of mouse. Activation of TASK-2 channels seems to play an essential role for relaxing uterus during pregnancy and it might be one of the alternatives for preventing preterm delivery. PMID:27610042

  3. Molecular identification of O2 sensors and O2-sensitive potassium channels in the pulmonary circulation.

    PubMed

    Archer, S L; Weir, E K; Reeve, H L; Michelakis, E

    2000-01-01

    Small, muscular pulmonary arteries (PAs) constrict within seconds of the onset of alveolar hypoxia, diverting blood flow to better-ventilated lobes, thereby matching ventilation to perfusion and optimizing systemic PO2. This hypoxic pulmonary vasoconstriction (HPV) is enhanced by endothelial derived vasoconstrictors, such as endothelin, and inhibited by endothelial derived nitric oxide. However, the essence of the response is intrinsic to PA smooth muscle cells in resistance arteries (PASMCs). HPV is initiated by inhibition of the Kv channels in PASMCs which set the membrane potential (EM). It is currently uncertain whether this reflects an initial inhibitory effect of hypoxia on the K+ channels or an initial release of intracellular Ca2+, which then inhibits K+ channels. In either scenario, the resulting depolarization activates L-type, voltage gated Ca2+ channels, which raises cytosolic calcium levels [Ca2+]i and causes vasoconstriction. Nine families of Kv channels are recognized from cloning studies (Kv1-Kv9), each with subtypes (i.e. Kv1.1-1.6). The contribution of an individual Kv channel to the whole-cell current (IK) is difficult to determine pharmacologically because Kv channel inhibitors are nonspecific. Furthermore, the PASMC's IK is an ensemble, reflecting activity of several channels. The K+ channels which set EM, and inhibition of which initiates HPV, conduct an outward current which is slowly inactivating, and which is blocked by the Kv inhibitor 4-aminopyridine (4-AP) but not by inhibitors of Ca(2+)- or ATP-sensitive K+ channels. Using anti-Kv antibodies to immunolocalize and inhibit Kv channels, we showed that the PASMC contains numerous types of Kv channels from the Kv1 and Kv2 families., Furthermore Kv1.5 and Kv2.1 may be important in determining the EM and play a role as effectors of HPV in PASMCs. While the Kv channels in PASMCs are the "effectors" of HPV, it is uncertain whether they are intrinsically O2-sensitive or are under the control of

  4. Neurotoxin-sensitive sodium channels in neurons developing in vivo and in vitro.

    PubMed

    Couraud, F; Martin-Moutot, N; Koulakoff, A; Berwald-Netter, Y

    1986-01-01

    Fetal mouse brain cells were investigated by 22Na+ flux assays with the aim to determine the ontogenetic time course of appearance of functional voltage-sensitive sodium channels. Their pharmacological properties were assessed by measurement of the response to known neurotoxins, acting at site 1, 2, or 3 of the Na+ channel. Brain cell suspensions, prepared at 11-19 d of prenatal development in vivo, and fetal brain neurons in culture were explored. In vivo neurotoxin-sensitive Na+ influx becomes detectable at 12 d of gestation, in concordance with the time of appearance of saturable binding sites for alpha-scorpion toxin (alpha-ScTx) and saxitoxin. Progression in fetal age or in time in vitro is accompanied by an increase in the initial rate and in the amplitude of Na+ uptake stimulated by batrachotoxin or veratridine. The general pharmacological properties of developing Na+ channels are very similar to the known properties of voltage-dependent Na+ channels in adult nerve: Batrachotoxin acts as a full channel agonist and veratridine as a partial agonist. Their respective apparent affinities are increased in presence of alpha-ScTx, in agreement with the known positive cooperativity of toxins acting at sites 2 and 3 of the Na+ channel. alpha-ScTx alone induces a small increase in Na+ permeability; its effect is greatly amplified in the presence of batrachotoxin or veratridine. The apparent affinity of alpha-ScTx is reduced by cell depolarization. Tetrodotoxin and saxitoxin block the increase in Na+ permeability induced by batrachotoxin, veratridine, and alpha-ScTx.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:2418173

  5. Mutants in Drosophila TRPC Channels Reduce Olfactory Sensitivity to Carbon Dioxide

    PubMed Central

    Badsha, Farhath; Kain, Pinky; Prabhakar, Sunil; Sundaram, Susinder; Padinjat, Raghu; Hasan, Gaiti

    2012-01-01

    Background Members of the canonical Transient Receptor Potential (TRPC) class of cationic channels function downstream of Gαq and PLCβ in Drosophila photoreceptors for transducing visual stimuli. Gαq has recently been implicated in olfactory sensing of carbon dioxide (CO2) and other odorants. Here we investigated the role of PLCβ and TRPC channels for sensing CO2 in Drosophila. Methodology/Principal Findings Through behavioral assays it was demonstrated that Drosophila mutants for plc21c, trp and trpl have a reduced sensitivity for CO2. Immuno-histochemical staining for TRP, TRPL and TRPγ indicates that all three channels are expressed in Drosophila antennae including the sensory neurons that express CO2 receptors. Electrophysiological recordings obtained from the antennae of protein null alleles of TRP (trp343) and TRPL (trpl302), showed that the sensory response to multiple concentrations of CO2 was reduced. However, trpl302; trp343 double mutants still have a residual response to CO2. Down-regulation of TRPC channels specifically in CO2 sensing olfactory neurons reduced the response to CO2 and this reduction was obtained even upon down-regulation of the TRPCs in adult olfactory sensory neurons. Thus the reduced response to CO2 obtained from the antennae of TRPC RNAi strains is not due to a developmental defect. Conclusion These observations show that reduction in TRPC channel function significantly reduces the sensitivity of the olfactory response to CO2 concentrations of 5% or less in adult Drosophila. It is possible that the CO2 receptors Gr63a and Gr21a activate the TRPC channels through Gαq and PLC21C. PMID:23185459

  6. Sensitivity of the sea circulation to the atmospheric forcing in the Sicily Channel

    NASA Astrophysics Data System (ADS)

    Omrani, Hiba; Arsouze, Thomas; Béranger, Karine; Boukthir, Moncef; Drobinski, Philippe; Lebeaupin-Brossier, Cindy; Mairech, Hanen

    2016-01-01

    We investigate the sensitivity of the sea surface circulation in the Sicily Channel to surface winds, using a 15-year long (1994-2008) air-sea coupled numerical simulation. Analysis is based on the clustering of six main wind regimes over the Sicily Channel domain. The analysis of the corresponding sea current clusters shows that sea circulation in this area is sensitive to surface wind patterns. This wind modulates the strength of the two main branches of the sea circulation in the Sicily Channel (i.e. the Atlantic Tunisian Current and the Atlantic Ionian Stream). The modulation of these two currents depends on the wind regime, and displays a strong seasonal variability. It is also shown that the sea circulation in the Sicily Channel is strongly controlled by the thermohaline circulation and the bathymetry (geostrophic current). However, the contribution to the total current of its ageostrophic component forced by the surface winds is significant, with a correlation coefficient varying from 0.3 to 0.7.

  7. TRPM2 Channel-Mediated ROS-Sensitive Ca2+ Signaling Mechanisms in Immune Cells

    PubMed Central

    Syed Mortadza, Sharifah Alawieyah; Wang, Lu; Li, Dongliang; Jiang, Lin-Hua

    2015-01-01

    Transient receptor potential melastatin 2 (TRPM2) proteins form Ca2+-permeable cationic channels that are potently activated by reactive oxygen species (ROS). ROS are produced during immune responses as signaling molecules as well as anti-microbial agents. ROS-sensitive TRPM2 channels are widely expressed in cells of the immune system and located on the cell surface as a Ca2+ influx pathway in macrophages, monocytes, neutrophils, lymphocytes, and microglia but preferentially within the lysosomal membranes as a Ca2+ release mechanism in dendritic cells; ROS activation of the TRPM2 channels, regardless of the subcellular location, results in an increase in the intracellular Ca2+ concentrations. Recent studies have revealed that TRPM2-mediated ROS-sensitive Ca2+ signaling mechanisms play a crucial role in a number of processes and functions in immune cells. This mini-review discusses the recent advances in revelation of the various roles the TRPM2 channels have in immune cell functions and the implications in inflammatory diseases. PMID:26300888

  8. The TRPM2 ion channel is required for sensitivity to warmth.

    PubMed

    Tan, Chun-Hsiang; McNaughton, Peter A

    2016-08-25

    Thermally activated ion channels are known to detect the entire thermal range from extreme heat (TRPV2), painful heat (TRPV1, TRPM3 and ANO1), non-painful warmth (TRPV3 and TRPV4) and non-painful coolness (TRPM8) through to painful cold (TRPA1). Genetic deletion of each of these ion channels, however, has only modest effects on thermal behaviour in mice, with the exception of TRPM8, the deletion of which has marked effects on the perception of moderate coolness in the range 10-25 °C. The molecular mechanism responsible for detecting non-painful warmth, in particular, is unresolved. Here we used calcium imaging to identify a population of thermally sensitive somatosensory neurons which do not express any of the known thermally activated TRP channels. We then used a combination of calcium imaging, electrophysiology and RNA sequencing to show that the ion channel generating heat sensitivity in these neurons is TRPM2. Autonomic neurons, usually thought of as exclusively motor, also express TRPM2 and respond directly to heat. Mice in which TRPM2 had been genetically deleted showed a striking deficit in their sensation of non-noxious warm temperatures, consistent with the idea that TRPM2 initiates a 'warm' signal which drives cool-seeking behaviour. PMID:27533035

  9. Magnification of low-sensitivity channels in seismographs with standard characteristics

    NASA Astrophysics Data System (ADS)

    Broucek, I.; Tobyas, V.

    The accuracy of an approximate method of computing the magnification of the low-sensitivity channel is analyzed on an analog computer for the parameters of seismographs with standard characteristics used in the network of seismic stations of CAPG (Commission of the Academies of Sciences of the Socialist Countries for Planetary Geophysical Research). The errors of the method of computation depend on the magnitude of the coupling coefficient of the basic channel of the seismograph: for short periods of motion they approach zero and they are usually largest outside the passband in the range of long periods. The error in the magnification of the low-sensitivity channel of a short-period seismograph with an A IV amplitude characteristic and a maximum magnification of the basic channel of 100000 is 65% at the most, of an intermediate-period seismograph with a magnification of 1000 as much as 24%, and of a long-period seismograph with a maximum magnification of 900 up to 19%.

  10. Apamin-sensitive K+ channels mediate an endothelium-dependent hyperpolarization in rabbit mesenteric arteries.

    PubMed Central

    Murphy, M E; Brayden, J E

    1995-01-01

    1. Vascular endothelial cells release a variety of substances which affect the membrane potential and tone of underlying vascular smooth muscle. In the presence of N omega-nitro-L-arginine to inhibit nitric oxide synthase and indomethacin to inhibit cyclo-oxygenase, acetylcholine (ACh; EC50 approximately 1 microM) elicited the release of an endothelium-derived hyperpolarizing factor (EDHF) in rabbit mesenteric arteries. 2. The hyperpolarization due to EDHF was blocked by apamin (IC50 approximately 0.3 nM), and by other inhibitors of the apamin-sensitive K+ channel (10 nM scyllatoxin, 100 microM d-tubocurarine, 300 microM gallamine) in the presence of indomethacin and N omega-nitro-L-arginine. The hyperpolarization was not blocked by glibenclamide (5 microM), iberiotoxin (10 nM), tetraethylammonium (1 mM), barium (500 microM), 4-aminopyridine (500 microM), ouabain (10 microM), bumetanide (10 microM), or nimodipine (100 nM). 3. In the presence of apamin and N omega-nitro-L-arginine, but the absence of indomethacin, ACh triggered a hyperpolarization that was blocked by glibenclamide, an inhibitor of ATP-sensitive K+ (KATP) channels. A similar glibenclamide-sensitive hyperpolarization was caused by Iloprost, a stable analogue of prostacyclin. 4. In experiments which distinguished the effects of EDHF, prostanoids and nitric oxide, hyperpolarizations and/or relaxations triggered by ACh were antagonized by muscarinic antagonists, the relative potencies (atropine approximately 4-DAMP > pirenzepine) of which indicated that the release of all three endothelium-derived factors was mediated by M3 receptors. 5. Our results suggest that ACh stimulates M3 receptors on endothelial cells, triggering the release of nitric oxide and prostanoids, which hyperpolarize underlying smooth muscle by activation of KATP channels, and the release of an EDHF, which hyperpolarizes smooth muscle through the activation of apamin-sensitive K+ (KAS) channels. Images Figure 4 PMID:8788937

  11. Temperature sensitivity of surface channel effects on high-purity germanium detectors

    NASA Astrophysics Data System (ADS)

    Hull, E. L.; Pehl, R. H.; Madden, N. W.; Luke, P. N.; Cork, C. P.; Malone, D. L.; Xing, J. S.; Komisarcik, K.; Vanderwerp, J. D.; Friesel, D. L.

    1995-02-01

    The temperature sensitivity of surface channel effects on planar high-purity germanium detectors was measured using 60-keV gamma-ray scanning techniques, as part of a radiation damage study. When measured in this manner, the surface effects on most detectors showed extreme temperature sensitivity in the 72-95 K region. The effect of the surface channel increased with increasing temperature to such an extent that the efficiency, as measured by the count rate in the 1332-keV peak from a 60Co source, decreased by a factor of over two in some cases. Since the peak efficiency for the 1332-keV gamma ray decreased as the temperature increased throughout the operating range (72-120 K) the effect of the surface channel must continue to increase beyond the temperature (95 K) at which the 60-keV scan loses its sensitivity because of the strong attenuation of these much lower energy gamma rays. Radiation damage had no measurable effect on the surface characteristics. No correlation between the surface effects and the resolution changes of the 1332-keV peak was observed.

  12. 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.

  13. Integration of spatio-temporal contrast sensitivity with a multi-slice channelized Hotelling observer

    NASA Astrophysics Data System (ADS)

    Avanaki, Ali N.; Espig, Kathryn S.; Marchessoux, Cedric; Krupinski, Elizabeth A.; Bakic, Predrag R.; Kimpe, Tom R. L.; Maidment, Andrew D. A.

    2013-03-01

    Barten's model of spatio-temporal contrast sensitivity function of human visual system is embedded in a multi-slice channelized Hotelling observer. This is done by 3D filtering of the stack of images with the spatio-temporal contrast sensitivity function and feeding the result (i.e., the perceived image stack) to the multi-slice channelized Hotelling observer. The proposed procedure of considering spatio-temporal contrast sensitivity function is generic in the sense that it can be used with observers other than multi-slice channelized Hotelling observer. Detection performance of the new observer in digital breast tomosynthesis is measured in a variety of browsing speeds, at two spatial sampling rates, using computer simulations. Our results show a peak in detection performance in mid browsing speeds. We compare our results to those of a human observer study reported earlier (I. Diaz et al. SPIE MI 2011). The effects of display luminance, contrast and spatial sampling rate, with and without considering foveal vision, are also studied. Reported simulations are conducted with real digital breast tomosynthesis image stacks, as well as stacks from an anthropomorphic software breast phantom (P. Bakic et al. Med Phys. 2011). Lesion cases are simulated by inserting single micro-calcifications or masses. Limitations of our methods and ways to improve them are discussed.

  14. Action of insecticidal N-alkylamides at site 2 of the voltage-sensitive sodium channel

    SciTech Connect

    Ottea, J.A.; Payne, G.T.; Soderlund, D.M. )

    1990-08-01

    Nine synthetic N-alkylamides were examined as inhibitors of the specific binding of ({sup 3}H)batrachotoxinin A 20{alpha}-benzoate (({sup 3}H)BTX-B) to sodium channels and as activators of sodium uptake in mouse brain synaptoneurosomes. In the presence of scorpion (Leiurus quinquestriatus) venom, the six insecticidal analogues were active as both inhibitors of ({sup 3}H)BTX-B binding and stimulators of sodium uptake. These findings are consistent with an action of these compounds at the alkaloid activator recognition site (site 2) of the voltage-sensitive sodium channel. The three noninsecticidal N-alkylamides also inhibited ({sup 3}H)BTX-B binding but were ineffective as activators of sodium uptake. Concentration-response studies revealed that some of the insecticidal amides also enhanced sodium uptake through a second, high-affinity interaction that does not involve site 2, but this secondary effect does not appear to be correlated with insecticidal activity. The activities of N-alkylamides as sodium channel activators were influenced by the length of the alkenyl chain and the location of unsaturation within the molecule. These results further define the actions of N-alkylamides on sodium channels and illustrate the significance of the multiple binding domains of the sodium channel as target sites for insect control agents.

  15. Iptakalim protects against hypoxic brain injury through multiple pathways associated with ATP-sensitive potassium channels.

    PubMed

    Zhu, H-L; Luo, W-Q; Wang, H

    2008-12-10

    The rapid and irreversible brain injury produced by anoxia when stroke occurs is well known. Cumulative evidence suggests that the activation of neuronal ATP-sensitive potassium (KATP) channels may have inherent protective effects during cerebral hypoxia, yet little information regarding the therapeutic effects of KATP channel openers is available. We hypothesized that pretreatment with a KATP channel opener might protect against brain injury induced by cerebral hypoxia. In this study, adult Wistar rats were treated with iptakalim, a new KATP channel opener, which is selective for SUR2 type KATP channels, by intragastric administration at doses of 2, 4, or 8 mg/kg/day for 7 days before being exposed to simulated high altitude equivalent to 8000 m in a decompression chamber for 8 h leading to hypoxic brain injury. By light and electron microscopic images, we observed that hypobaric hypoxia-induced brain injury could be prevented by pretreatment with iptakalim. It was also observed that the permeability of the blood-brain barrier, water content, Na+ and Ca2+ concentration, and activities of Na+,K+-ATPase, Ca2+-ATPase and Mg2+-ATPase in rat cerebral cortex were increased and the gene expression of the occludin or aquaporin-4 was down- or upregulated respectively, which could also be prevented by the pretreatment with iptakalim at doses of 2, 4, or 8 mg/kg in a dose-dependent manner. Furthermore, we found that in an oxygen-and-glucose-deprived model in ECV304 cells and rat cortical astrocytes, pretreatment with iptakalim significantly increased survived cell rates and decreased lactate dehydrogenate release, which were significantly antagonized by glibenclamide, a K(ATP) channel blocker. We conclude that iptakalim is a promising drug that may protect against brain injury induced by acute hypobaric hypoxia through multiple pathways associated with SUR2-type K(ATP) channels, suggesting a new therapeutic strategy for stroke treatment. PMID:18951957

  16. ATP-Sensitive Potassium (KATP) Channel Openers Diazoxide and Nicorandil Lower Intraocular Pressure In Vivo

    PubMed Central

    Chowdhury, Uttio Roy; Holman, Bradley H.; Fautsch, Michael P.

    2013-01-01

    Purpose. To evaluate the expression of ATP-sensitive potassium (KATP) channel subunits and study the effect of KATP channel openers diazoxide and nicorandil on intraocular pressure (IOP) in an in vivo mouse model. Methods. Expression of KATP channel subunits in normal C57BL/6 mouse eyes was studied by immunohistochemistry and confocal microscopy. Wild-type C57BL/6 mice were treated with KATP channel openers diazoxide (n = 10) and nicorandil (n = 10) for 14 days. Similar treatments with diazoxide were performed on Kir6.2(−/−) mice (n = 10). IOP was recorded with a handheld tonometer 1 hour, 4 hours, and 23 hours following daily treatment. Posttreatment histology was examined by light and transmission electron microscopy. Results. The KATP channel subunits SUR2B, Kir6.1, and Kir6.2 were identified in all tissues within mouse eyes. Treatment with diazoxide in wild-type mice decreased IOP by 21.5 ± 3.2% with an absolute IOP reduction of 3.9 ± 0.6 mm Hg (P = 0.002). Nicorandil also decreased IOP (18.9 ± 1.8%) with an absolute IOP reduction of 3.4 ± 0.4 mm Hg (P = 0.002). Treatment with diazoxide in Kir6.2(−/−) mice had no effect on IOP. No morphological abnormalities were observed in diazoxide- or nicorandil-treated eyes. Conclusions. KATP channel openers diazoxide and nicorandil are effective regulators of IOP in mouse eyes. Kir6.2 appears to be a major KATP channel subunit through which IOP is lowered following treatment with diazoxide. PMID:23778875

  17. Sulfonylurea receptor 1 expression in human cerebral infarcts.

    PubMed

    Mehta, Rupal I; Ivanova, Svetlana; Tosun, Cigdem; Castellani, Rudy J; Gerzanich, Volodymyr; Simard, J Marc

    2013-09-01

    In animal models of stroke, sulfonylurea receptor 1 (Sur1), a member of the adenosine triphosphate binding cassette transporter gene family, is transcriptionally upregulated in neural and vascular cells in which it plays a leading role in edema formation and necrotic cell death. To date, expression of Sur1 in the brains of humans with cerebral infarcts has not been systematically evaluated. We examined Sur1 expression in postmortem specimens obtained from 13 patients within the first 31 days after focal infarcts, 5 patients with lacunar infarcts, and 6 normal control brains using immunohistochemistry. Elevated immunoreactivity for Sur1 was detected in all cases of focal infarcts, with 3 distinct temporal patterns of expression: 1) neurons and endothelium showed the greatest elevation during the first week, after which levels declined; 2) astrocytes and microglia/macrophages showed progressive increases during the first 31 days; and 3) neutrophils near the infarct showed prominent immunoreactivity that did not change over time. Upregulation of Sur1 was corroborated using in situ hybridization for Abcc8 mRNA. Sulfonylurea receptor 1 immunoreactivity in lacunar infarcts was less prominent and more sporadic than in nonlacunar infarcts. In conjunction with previous studies, these data suggest that Sur1 may be a promising treatment target in patients with acute cerebral infarction. PMID:23965746

  18. Sulfonylurea Receptor 1 Expression in Human Cerebral Infarcts

    PubMed Central

    Mehta, Rupal I.; Ivanova, Svetlana; Tosun, Cigdem; Castellani, Rudy J.; Gerzanich, Volodymyr

    2013-01-01

    Abstract In animal models of stroke, sulfonylurea receptor 1 (Sur1), a member of the adenosine triphosphate binding cassette transporter gene family, is transcriptionally upregulated in neural and vascular cells in which it plays a leading role in edema formation and necrotic cell death. To date, expression of Sur1 in the brains of humans with cerebral infarcts has not been systematically evaluated. We examined Sur1 expression in postmortem specimens obtained from 13 patients within the first 31 days after focal infarcts, 5 patients with lacunar infarcts, and 6 normal control brains using immunohistochemistry. Elevated immunoreactivity for Sur1 was detected in all cases of focal infarcts, with 3 distinct temporal patterns of expression: 1) neurons and endothelium showed the greatest elevation during the first week, after which levels declined; 2) astrocytes and microglia/macrophages showed progressive increases during the first 31 days; and 3) neutrophils near the infarct showed prominent immunoreactivity that did not change over time. Upregulation of Sur1 was corroborated using in situ hybridization for Abcc8 mRNA. Sulfonylurea receptor 1 immunoreactivity in lacunar infarcts was less prominent and more sporadic than in nonlacunar infarcts. In conjunction with previous studies, these data suggest that Sur1 may be a promising treatment target in patients with acute cerebral infarction. PMID:23965746

  19. Crystal structure of the human K2P TRAAK, a lipid- and mechano-sensitive K+ ion channel

    PubMed Central

    Brohawn, Stephen G.; del Mármol, Josefina; MacKinnon, Roderick

    2012-01-01

    TRAAK channels, members of the two pore domain K+ channel family, are expressed almost exclusively in the nervous system and control the resting membrane potential. Their gating is sensitive to polyunsaturated fatty acids, mechanical deformation of the membrane, and temperature changes. Physiologically these channels appear to control the noxious input threshold for temperature and pressure sensitivity in dorsal root ganglia neurons. We present the crystal structure of human TRAAK at 3.8 Å resolution. The channel comprises two protomers, each containing two distinct pore domains, which create a two-fold symmetric K+ channel. The extracellular surface features a 35 Å tall helical cap that creates a bifurcated pore entryway and accounts for the insensitivity of two pore K+ channels to inhibitory toxins. Two diagonally opposed gate-forming inner helices form membrane interacting structures that may underlie this channel’s sensitivity to chemical and mechanical properties of the cell membrane. PMID:22282805

  20. Localization of sulfonylurea receptor subunits, SUR2A and SUR2B, in rat heart.

    PubMed

    Zhou, Ming; He, Hui-Jing; Suzuki, Ryoji; Liu, Ke-Xiang; Tanaka, Osamu; Sekiguchi, Masaki; Itoh, Hideaki; Kawahara, Katsumasa; Abe, Hiroshi

    2007-08-01

    To understand the possible functions and subcellular localizations of sulfonylurea receptors (SURs) in cardiac muscle, polyclonal anti-SUR2A and anti-SUR2B antisera were raised. Immunoblots revealed both SUR2A and SUR2B expression in mitochondrial fractions of rat heart and other cellular fractions such as microsomes and cell membranes. Immunostaining detected ubiquitous expression of both SUR2A and SUR2B in rat heart in the atria, ventricles, interatrial and interventricular septa, and smooth muscles and endothelia of the coronary arteries. Electron microscopy revealed SUR2A immunoreactivity in the cell membrane, endoplasmic reticulum (ER), and mitochondria. SUR2B immunoreactivity was mainly localized in the mitochondria as well as in the ER and cell membrane. Thus, SUR2A and SUR2B are not only the regulatory subunits of sarcolemmal K(ATP) channels but may also function as regulatory subunits in mitochondrial K(ATP) channels and play important roles in cardioprotection. PMID:17438353

  1. Apamin-sensitive, small-conductance, calcium-activated potassium channels mediate cholinergic inhibition of chick auditory hair cells.

    PubMed

    Yuhas, W A; Fuchs, P A

    1999-11-01

    Acetylcholine released from efferent neurons in the cochlea causes inhibition of mechanosensory hair cells due to the activation of calcium-dependent potassium channels. Hair cells are known to have large-conductance, "BK"-type potassium channels associated with the afferent synapse, but these channels have different properties than those activated by acetylcholine. Whole-cell (tight-seal) and cell-attached patch-clamp recordings were made from short (outer) hair cells isolated from the chicken basilar papilla (cochlea equivalent). The peptides apamin and charybdotoxin were used to distinguish the calcium-activated potassium channels involved in the acetylcholine response from the BK-type channels associated with the afferent synapse. Differential toxin blockade of these potassium currents provides definitive evidence that ACh activates apamin-sensitive, "SK"-type potassium channels, but does not activate carybdotoxin-sensitive BK channels. This conclusion is supported by tentative identification of small-conductance, calcium-sensitive but voltage-insensitive potassium channels in cell-attached patches. The distinction between these channel types is important for understanding the segregation of opposing afferent and efferent synaptic activity in the hair cell, both of which depend on calcium influx. These different calcium-activated potassium channels serve as sensitive indicators for functionally significant calcium influx in the hair cell. PMID:10573868

  2. Control of heart rate by cAMP sensitivity of HCN channels

    PubMed Central

    Alig, Jacqueline; Marger, Laurine; Mesirca, Pietro; Ehmke, Heimo; Mangoni, Matteo E.; Isbrandt, Dirk

    2009-01-01

    “Pacemaker” f-channels mediating the hyperpolarization-activated nonselective cation current If are directly regulated by cAMP. Accordingly, the activity of f-channels increases when cellular cAMP levels are elevated (e.g., during sympathetic stimulation) and decreases when they are reduced (e.g., during vagal stimulation). Although these biophysical properties seem to make f-channels ideal molecular targets for heart rate regulation by the autonomic nervous system, the exact contribution of the major If-mediating cardiac isoforms HCN2 and HCN4 to sinoatrial node (SAN) function remains highly controversial. To directly investigate the role of cAMP-dependent regulation of hyperpolarization activated cyclic nucleotide activated (HCN) channels in SAN activity, we generated mice with heart-specific and inducible expression of a human HCN4 mutation (573X) that abolishes the cAMP-dependent regulation of HCN channels. We found that hHCN4–573X expression causes elimination of the cAMP sensitivity of If and decreases the maximum firing rates of SAN pacemaker cells. In conscious mice, hHCN4–573X expression leads to a marked reduction in heart rate at rest and during exercise. Despite the complete loss of cAMP sensitivity of If, the relative extent of SAN cell frequency and heart rate regulation are preserved. Our data demonstrate that cAMP-mediated regulation of If determines basal and maximal heart rates but does not play an indispensable role in heart rate adaptation during physical activity. Our data also reveal the pathophysiologic mechanism of hHCN4–573X–linked SAN dysfunction in humans. PMID:19570998

  3. Application of ion-sensitive field effect transistors for ion channel screening.

    PubMed

    Walsh, Kenneth B; DeRoller, Nicholas; Zhu, Yihao; Koley, Goutam

    2014-04-15

    Cell-based screening assays are now widely used for identifying compounds that serve as ion channel modulators. However, instrumentation for the automated, real-time analysis of ion flux from clonal and primary cells is lacking. This study describes the initial development of an ion-sensitive field effect transistor (ISFET)-based screening assay for the acquisition of K(+) efflux data from cells cultured in multi-well plates. Silicon-based K(+)-sensitive ISFETs were tested for their electrical response to varying concentrations of KCl and were found to display a linear response relationship to KCl in the range of 10 µM-1 mM. The ISFETs, along with reference electrodes, were inserted into fast-flow chambers containing either human colonic T84 epithelial cells or U251-MG glioma cells. Application of the Ca(2+) ionophore A23187 (1 µM), to activate Ca(2+)-activated non-selective cation (NSC) channels (T84 cells) and large conductance Ca(2+)-activated K(+) (BK) channels (U251 cells), resulted in time-dependent increases in the extracellular K(+) concentration ([K(+)]o) as measured with the ISFETs. Treatment of the cells with blockers of either the NSC or BK channels, caused a strong inhibition of the A23187-induced increase in [K(+)]o. These results were consistent with ion current measurements obtained using the whole-cell arrangement of the patch clamp procedure. In addition, K(+) efflux data could be acquired in parallel from multiple cell chambers using the ISFET sensors. Given the non-invasive properties of the probes, the ISFET-based assay should be adaptable for screening ion channels in various cell types. PMID:24315877

  4. Phosphorylation of the mitochondrial ATP-sensitive potassium channel occurs independently of PKCε in turtle brain.

    PubMed

    Hawrysh, Peter John; Miles, Ashley Rebecca; Buck, Leslie Thomas

    2016-10-01

    Neurons from the western painted turtle (Chrysemys picta bellii) are remarkably resilient to anoxia. This is partly due to a reduction in the permeability of excitatory glutamatergic ion channels, initiated by mitochondrial ATP-sensitive K(+) (mK(+)ATP) channel activation. The aim of this study was to determine if: 1) PKCε, a kinase associated with hypoxic stress tolerance, is more highly expressed in turtle brain than the anoxia-intolerant rat brain; 2) PKCε translocates to the mitochondrial membrane during anoxia; 3) PKCε modulates mK(+)ATP channels at the Thr-224 phosphorylation site on the Kir6.2 subunit; and 4) Thr-224 phosphorylation sensitises mK(+)ATP channels to anoxia. Soluble and mitochondrial-rich particulate fractions of turtle and rat cerebral cortex were isolated and PKCε expression was determined by Western blot, which revealed that turtle cortical PKCε expression was half that of the rat. Following the transition to anoxia, no changes in PKCε expression in either the soluble or particulate fraction of the turtle cortex were observed. Furthermore, incubation of tissue with tat-conjugated activator or inhibitor peptides had no effect on the amount of PKCε in either fraction. However, we observed a 2-fold increase in Thr-224 phosphorylation following 1h of anoxia. The increased Thr-224 phosphorylation was blocked by the general kinase inhibitor staurosporine but this did not affect the latency or magnitude of mK(+)ATP channel-mediated mitochondrial depolarization following anoxia, as indicated by rhodamine-123. We conclude that PKCε does not play a role in the onset of mitochondrial depolarization and therefore glutamatergic channel arrest in turtle cerebral cortex. PMID:27280321

  5. ATP-sensitive K+ channel knockout induces cardiac proteome remodeling predictive of heart disease susceptibility.

    PubMed

    Arrell, D Kent; Zlatkovic, Jelena; Kane, Garvan C; Yamada, Satsuki; Terzic, Andre

    2009-10-01

    Forecasting disease susceptibility requires detection of maladaptive signatures prior to onset of overt symptoms. A case-in-point are cardiac ATP-sensitive K+ (K(ATP)) channelopathies, for which the substrate underlying disease vulnerability remains to be identified. Resolving molecular pathobiology, even for single genetic defects, mandates a systems platform to reliably diagnose disease predisposition. High-throughput proteomic analysis was here integrated with network biology to decode consequences of Kir6.2 K(ATP) channel pore deletion. Differential two-dimensional gel electrophoresis reproducibly resolved >800 protein species from hearts of asymptomatic wild-type and Kir6.2-knockout counterparts. K(ATP) channel ablation remodeled the cardiac proteome, significantly altering 71 protein spots, from which 102 unique identities were assigned following hybrid linear ion trap quadrupole-Orbitrap tandem mass spectrometry. Ontological annotation stratified the K(ATP) channel-dependent protein cohort into a predominant bioenergetic module (63 resolved identities), with additional focused sets representing signaling molecules (6), oxidoreductases (8), chaperones (6), and proteins involved in catabolism (6), cytostructure (8), and transcription and translation (5). Protein interaction mapping, in conjunction with expression level changes, localized a K(ATP) channel-associated subproteome within a nonstochastic scale-free network. Global assessment of the K(ATP) channel deficient environment verified the primary impact on metabolic pathways and revealed overrepresentation of markers associated with cardiovascular disease. Experimental imposition of graded stress precipitated exaggerated structural and functional myocardial defects in the Kir6.2-knockout, decreasing survivorship and validating the forecast of disease susceptibility. Proteomic cartography thus provides an integral view of molecular remodeling in the heart induced by K(ATP) channel deletion, establishing a

  6. Lack of manifestations of diazoxide/5-hydroxydecanoate-sensitive KATP channel in rat brain nonsynaptosomal mitochondria

    PubMed Central

    Brustovetsky, Tatiana; Shalbuyeva, Natalia; Brustovetsky, Nickolay

    2005-01-01

    Pharmacological modulation of the mitochondrial ATP-sensitive K+ channel (mitoKATP) sensitive to diazoxide and 5-hydroxydecanoate (5-HD) represents an attractive strategy to protect cells against ischaemia/reperfusion- and stroke-related injury. To re-evaluate a functional role for the mitoKATP in brain, we used Percoll-gradient-purified brain nonsynaptosomal mitochondria in a light absorbance assay, in radioisotope measurements of matrix volume, and in measurements of respiration, membrane potential (ΔΨ) and depolarization-induced K+ efflux. The changes in mitochondrial morphology were evaluated by transmission electron microscopy (TEM). Polyclonal antibodies raised against certain fragments of known sulphonylurea receptor subunits, SUR1 and SUR2, and against different epitopes of K+ inward rectifier subunits Kir 6.1 and Kir 6.2 of the ATP-sensitive K+ channel of the plasma membrane (cellKATP), were employed to detect similar subunits in brain mitochondria. A variety of plausible blockers (ATP, 5-hydroxydecanoate, glibenclamide, tetraphenylphosphonium cation) and openers (diazoxide, pinacidil, chromakalim, minoxidil, testosterone) of the putative mitoKATP were applied to show the role of the channel in regulating matrix volume, respiration, and ΔΨ and K+ fluxes across the inner mitochondrial membrane. None of the pharmacological agents applied to brain mitochondria in the various assays pinpointed processes that could be unequivocally associated with mitoKATP activity. In addition, immunoblotting analysis did not provide explicit evidence for the presence of the mitoKATP, similar to the cellKATP, in brain mitochondria. On the other hand, the depolarization-evoked release of K+ suppressed by ATP could be re-activated by carboxyatractyloside, an inhibitor of the adenine nucleotide translocase (ANT). Moreover, bongkrekic acid, another inhibitor of the ANT, inhibited K+ efflux similarly to ATP. These observations implicate the ANT in ATP-sensitive K+ transport in

  7. T-Type Ca2+ Channel Inhibition Sensitizes Ovarian Cancer to Carboplatin.

    PubMed

    Dziegielewska, Barbara; Casarez, Eli V; Yang, Wesley Z; Gray, Lloyd S; Dziegielewski, Jaroslaw; Slack-Davis, Jill K

    2016-03-01

    Ovarian cancer is the deadliest gynecologic cancer, due in large part to the diagnosis of advanced stage disease, the development of platinum resistance, and inadequate treatment alternatives. Recent studies by our group and others have shown that T-type calcium (Ca(2+)) channels play a reinforcing role in cancer cell proliferation, cell-cycle progression, and apoptosis evasion. Therefore, we investigated whether T-type Ca(2+) channels affect ovarian tumor growth and response to platinum agents. Inhibition of T-type Ca(2+) channels with mibefradil or by silencing expression resulted in growth suppression in ovarian cancer cells with a simultaneous increase in apoptosis, which was accompanied by decreased expression of the antiapoptotic gene survivin (BIRC5). Analysis of intracellular signaling revealed mibefradil reduced AKT phosphorylation, increased the levels and nuclear retention of FOXO transcription factors that repress BIRC5 expression, and decreased the expression of FOXM1, which promotes BIRC5 expression. Combining carboplatin with mibefradil synergistically increased apoptosis in vitro. Importantly, mibefradil rendered platinum-resistant ovarian tumors sensitive to carboplatin in a mouse model of peritoneal metastasis. Together, the data provide rationale for future use of T-type channel antagonists together with platinum agents for the treatment of ovarian cancer. Mol Cancer Ther; 15(3); 460-70. ©2016 AACR. PMID:26832797

  8. Evidences for an ATP-sensitive potassium channel (KATP) in muscle and fat body mitochondria of insect.

    PubMed

    Slocinska, Malgorzata; Lubawy, Jan; Jarmuszkiewicz, Wieslawa; Rosinski, Grzegorz

    2013-11-01

    In the present study, we describe the existence of mitochondrial ATP-dependent K(+) channel (mitoKATP) in two different insect tissues, fat body and muscle of cockroach Gromphadorhina coquereliana. We found that pharmacological substances known to modulate potassium channel activity influenced mitochondrial resting respiration. In isolated mitochondria oxygen consumption increased by about 13% in the presence of potassium channel openers (KCOs) such as diazoxide and pinacidil. The opening of mitoKATP was reversed by glibenclamide (potassium channel blocker) and 1 mM ATP. Immunological studies with antibodies raised against the Kir6.1 and SUR1 subunits of the mammalian ATP-sensitive potassium channel, indicated the existence of mitoKATP in insect mitochondria. MitoKATP activation by KCOs resulted in a decrease in superoxide anion production, suggesting that protection against mitochondrial oxidative stress may be a physiological role of mitochondrial ATP-sensitive potassium channel in insects. PMID:23973818

  9. A Theoretical Model for Calculating Voltage Sensitivity of Ion Channels and the Application on Kv1.2 Potassium Channel

    PubMed Central

    Yang, Huaiyu; Gao, Zhaobing; Li, Ping; Yu, Kunqian; Yu, Ye; Xu, Tian-Le; Li, Min; Jiang, Hualiang

    2012-01-01

    Voltage sensing confers conversion of a change in membrane potential to signaling activities underlying the physiological processes. For an ion channel, voltage sensitivity is usually experimentally measured by fitting electrophysiological data to Boltzmann distributions. In our study, a two-state model of the ion channel and equilibrium statistical mechanics principle were used to test the hypothesis of empirically calculating the overall voltage sensitivity of an ion channel on the basis of its closed and open conformations, and determine the contribution of individual residues to the voltage sensing. We examined the theoretical paradigm by performing experimental measurements with Kv1.2 channel and a series of mutants. The correlation between the calculated values and the experimental values is at respective level, R2 = 0.73. Our report therefore provides in silico prediction of key conformations and has identified additional residues critical for voltage sensing. PMID:22768937

  10. S-glutathionylation of an auxiliary subunit confers redox sensitivity to Kv4 channel inactivation.

    PubMed

    Jerng, Henry H; Pfaffinger, Paul J

    2014-01-01

    Reactive oxygen species (ROS) regulate ion channels, modulate neuronal excitability, and contribute to the etiology of neurodegenerative disorders. ROS differentially suppress fast "ball-and-chain" N-type inactivation of cloned Kv1 and Kv3 potassium channels but not of Kv4 channels, likely due to a lack of reactive cysteines in Kv4 N-termini. Recently, we discovered that N-type inactivation of Kv4 channel complexes can be independently conferred by certain N-terminal variants of Kv4 auxiliary subunits (DPP6a, DPP10a). Here, we report that both DPP6a and DPP10a, like Kv subunits with redox-sensitive N-type inactivation, contain a highly conserved cysteine in their N-termini (Cys-13). To test if N-type inactivation mediated by DPP6a or DPP10a is redox sensitive, Xenopus oocyte recordings were performed to examine the effects of two common oxidants, tert-butyl hydroperoxide (tBHP) and diamide. Both oxidants markedly modulate DPP6a- or DPP10a-conferred N-type inactivation of Kv4 channels, slowing the overall inactivation and increasing the peak current. These functional effects are fully reversed by the reducing agent dithiothreitol (DTT) and appear to be due to a selective modulation of the N-type inactivation mediated by these auxiliary subunits. Mutation of DPP6a Cys-13 to serine eliminated the tBHP or diamide effects, confirming the importance of Cys-13 to the oxidative regulation. Biochemical studies designed to elucidate the underlying molecular mechanism show no evidence of protein-protein disulfide linkage formation following cysteine oxidation. Instead, using a biotinylated glutathione (BioGEE) reagent, we discovered that oxidation by tBHP or diamide leads to S-glutathionylation of Cys-13, suggesting that S-glutathionylation underlies the regulation of fast N-type inactivation by redox. In conclusion, our studies suggest that Kv4-based A-type current in neurons may show differential redox sensitivity depending on whether DPP6a or DPP10a is highly expressed

  11. S-Glutathionylation of an Auxiliary Subunit Confers Redox Sensitivity to Kv4 Channel Inactivation

    PubMed Central

    Jerng, Henry H.; Pfaffinger, Paul J.

    2014-01-01

    Reactive oxygen species (ROS) regulate ion channels, modulate neuronal excitability, and contribute to the etiology of neurodegenerative disorders. ROS differentially suppress fast “ball-and-chain” N-type inactivation of cloned Kv1 and Kv3 potassium channels but not of Kv4 channels, likely due to a lack of reactive cysteines in Kv4 N-termini. Recently, we discovered that N-type inactivation of Kv4 channel complexes can be independently conferred by certain N-terminal variants of Kv4 auxiliary subunits (DPP6a, DPP10a). Here, we report that both DPP6a and DPP10a, like Kv subunits with redox-sensitive N-type inactivation, contain a highly conserved cysteine in their N-termini (Cys-13). To test if N-type inactivation mediated by DPP6a or DPP10a is redox sensitive, Xenopus oocyte recordings were performed to examine the effects of two common oxidants, tert-butyl hydroperoxide (tBHP) and diamide. Both oxidants markedly modulate DPP6a- or DPP10a-conferred N-type inactivation of Kv4 channels, slowing the overall inactivation and increasing the peak current. These functional effects are fully reversed by the reducing agent dithiothreitol (DTT) and appear to be due to a selective modulation of the N-type inactivation mediated by these auxiliary subunits. Mutation of DPP6a Cys-13 to serine eliminated the tBHP or diamide effects, confirming the importance of Cys-13 to the oxidative regulation. Biochemical studies designed to elucidate the underlying molecular mechanism show no evidence of protein-protein disulfide linkage formation following cysteine oxidation. Instead, using a biotinylated glutathione (BioGEE) reagent, we discovered that oxidation by tBHP or diamide leads to S-glutathionylation of Cys-13, suggesting that S-glutathionylation underlies the regulation of fast N-type inactivation by redox. In conclusion, our studies suggest that Kv4-based A-type current in neurons may show differential redox sensitivity depending on whether DPP6a or DPP10a is highly expressed

  12. Is gliclazide a sulfonylurea with difference? A review in 2016.

    PubMed

    Singh, Awadhesh Kumar; Singh, Ritu

    2016-06-01

    Sulfonylureas (SUs) remain the most commonly prescribed drug after metformin in the treatment of type 2 diabetes (T2DM), despite the availability of several newer agents. The primary reason of SUs being most popular is their quick glycemic response, time-tested experience and least cost. Although SUs are one amongst the several other second line agents after metformin in all major guidelines, the new Dutch type 2 guidelines specifically advise gliclazide as the preferred second line drug instead of SUs as a class. The World Health Organization (WHO) has also included gliclazide in their Model List of Essential Medicines 2013 motivated by its safety data in elderly patients. Specifically advising gliclazide may have been based on emerging evidence suggesting cardiovascular neutrality of gliclazide over other SUs. This prompted us to do a literature review of gliclazide efficacy and safety data compared to other SUs as well as oral anti-diabetic drugs. PMID:26924475

  13. Intramembrane Aromatic Interactions Influence the Lipid Sensitivities of Pentameric Ligand-gated Ion Channels*

    PubMed Central

    Carswell, Casey L.; Sun, Jiayin; Baenziger, John E.

    2015-01-01

    Although the Torpedo nicotinic acetylcholine receptor (nAChR) reconstituted into phosphatidylcholine (PC) membranes lacking cholesterol and anionic lipids adopts a conformation where agonist binding is uncoupled from channel gating, the underlying mechanism remains to be defined. Here, we examine the mechanism behind lipid-dependent uncoupling by comparing the propensities of two prokaryotic homologs, Gloebacter and Erwinia ligand-gated ion channel (GLIC and ELIC, respectively), to adopt a similar uncoupled conformation. Membrane-reconstituted GLIC and ELIC both exhibit folded structures in the minimal PC membranes that stabilize an uncoupled nAChR. GLIC, with a large number of aromatic interactions at the interface between the outermost transmembrane α-helix, M4, and the adjacent transmembrane α-helices, M1 and M3, retains the ability to flux cations in this uncoupling PC membrane environment. In contrast, ELIC, with a level of aromatic interactions intermediate between that of the nAChR and GLIC, does not undergo agonist-induced channel gating, although it does not exhibit the expected biophysical characteristics of the uncoupled state. Engineering new aromatic interactions at the M4-M1/M3 interface to promote effective M4 interactions with M1/M3, however, increases the stability of the transmembrane domain to restore channel function. Our data provide direct evidence that M4 interactions with M1/M3 are modulated during lipid sensing. Aromatic residues strengthen M4 interactions with M1/M3 to reduce the sensitivities of pentameric ligand-gated ion channels to their surrounding membrane environment. PMID:25519904

  14. Control of channel doping concentration for enhancing the sensitivity of 'top-down' fabricated Si nanochannel FET biosensors

    NASA Astrophysics Data System (ADS)

    Park, Chan Woo; Ahn, Chang-Geun; Yang, Jong-Heon; Baek, In-Bok; Ah, Chil Seong; Kim, Ansoon; Kim, Tae-Youb; Sung, Gun Yong

    2009-11-01

    The sensitivity of 'top-down' fabricated Si nanochannel field effect transistor (FET) biosensors has been analyzed quantitatively, as a function of the channel width and doping concentration. We have fabricated 130-, 150-, and 220 nm-wide Si FET channels with 40 nm-thick p-type silicon-on-insulator (SOI) layers doped at 8 × 1017 and 2 × 1018 cm-3, and characterized their sensitivity in response to the variation of surface charges as hydrogen ion sensors within buffer solutions of various pH levels. Within the range of channel width and doping concentration investigated, the pH sensitivity of Si channels is enhanced much more effectively by decreasing the doping concentration than by reducing the channel width, which suggests a practical strategy for achieving high sensitivity with less effort than to reduce the channel width. Similar behavior has also been confirmed in the immunodetection of prostate specific antigen (PSA). Combined with excellent reproducibility and uniformity of the channel structure, high controllability of the doping concentration can make the 'top-down' fabrication a very useful approach for the massive fabrication of high-sensitivity sensor platforms in a cost-effective way.

  15. O2-sensitive K+ channels: role of the Kv1.2 -subunit in mediating the hypoxic response.

    PubMed

    Conforti, L; Bodi, I; Nisbet, J W; Millhorn, D E

    2000-05-01

    One of the early events in O2 chemoreception is inhibition of O2-sensitive K+ (KO2) channels. Characterization of the molecular composition of the native KO2 channels in chemosensitive cells is important to understand the mechanism(s) that couple O2 to the KO2 channels. The rat phaeochromocytoma PC12 clonal cell line expresses an O2-sensitive voltage-dependent K+ channel similar to that recorded in other chemosensitive cells. Here we examine the possibility that the Kv1.2 alpha-subunit comprises the KO2 channel in PC12 cells. Whole-cell voltage-clamp experiments showed that the KO2 current in PC12 cells is inhibited by charybdotoxin, a blocker of Kv1.2 channels. PC12 cells express the Kv1.2 alpha-subunit of K+ channels: Western blot analysis with affinity-purified anti-Kv1.2 antibody revealed a band at approximately 80 kDa. Specificity of this antibody was established in Western blot and immunohystochemical studies. Anti-Kv1.2 antibody selectively blocked Kv1.2 current expressed in the Xenopus oocyte, but had no effect on Kv2.1 current. Anti-Kv1.2 antibody dialysed through the patch pipette completely blocked the KO2 current, while the anti-Kv2.1 and irrelevant antibodies had no effect. The O2 sensitivity of recombinant Kv1.2 and Kv2.1 channels was studied in Xenopus oocytes. Hypoxia inhibited the Kv1.2 current only. These findings show that the KO2 channel in PC12 cells belongs to the Kv1 subfamily of K+ channels and that the Kv1.2 alpha-subunit is important in conferring O2 sensitivity to this channel. PMID:10790158

  16. O2-sensitive K+ channels: role of the Kv1.2 α-subunit in mediating the hypoxic response

    PubMed Central

    Conforti, Laura; Bodi, Ilona; Nisbet, John W; Millhorn, David E

    2000-01-01

    One of the early events in O2 chemoreception is inhibition of O2-sensitive K+ (KO2) channels. Characterization of the molecular composition of the native KO2 channels in chemosensitive cells is important to understand the mechanism(s) that couple O2 to the KO2 channels. The rat phaeochromocytoma PC12 clonal cell line expresses an O2-sensitive voltage-dependent K+ channel similar to that recorded in other chemosensitive cells. Here we examine the possibility that the Kv1.2 α-subunit comprises the KO2 channel in PC12 cells. Whole-cell voltage-clamp experiments showed that the KO2 current in PC12 cells is inhibited by charybdotoxin, a blocker of Kv1.2 channels. PC12 cells express the Kv1.2 α-subunit of K+ channels: Western blot analysis with affinity-purified anti-Kv1.2 antibody revealed a band at ≈80 kDa. Specificity of this antibody was established in Western blot and immunohystochemical studies. Anti-Kv1.2 antibody selectively blocked Kv1.2 current expressed in the Xenopus oocyte, but had no effect on Kv2.1 current. Anti-Kv1.2 antibody dialysed through the patch pipette completely blocked the KO2 current, while the anti-Kv2.1 and irrelevant antibodies had no effect. The O2 sensitivity of recombinant Kv1.2 and Kv2.1 channels was studied in Xenopus oocytes. Hypoxia inhibited the Kv1.2 current only. These findings show that the KO2 channel in PC12 cells belongs to the Kv1 subfamily of K+ channels and that the Kv1.2 α-subunit is important in conferring O2 sensitivity to this channel. PMID:10790158

  17. 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

  18. Extracellular Spermine Exacerbates Ischemic Neuronal Injury through Sensitization of ASIC1a Channels to Extracellular Acidosis

    PubMed Central

    Duan, Bo; Wang, Yi-Zhi; Yang, Tao; Chu, Xiang-Ping; Yu, Ye; Huang, Yu; Cao, Hui; Hansen, Jillian; Simon, Roger P.; Zhu, Michael X.; Xiong, Zhi-Gang; Xu, Tian-Le

    2011-01-01

    Ischemic brain injury is a major problem associated with stroke. It has been increasingly recognized that acid-sensing ion channels (ASICs) contribute significantly to ischemic neuronal damage, but the underlying mechanism has remained elusive. Here, we show that extracellular spermine, one of the endogenous polyamines, exacerbates ischemic neuronal injury through sensitization of ASIC1a channels to extracellular acidosis. Pharmacological blockade of ASIC1a or deletion of the ASIC1 gene greatly reduces the enhancing effect of spermine in ischemic neuronal damage both in cultures of dissociated neurons and in a mouse model of focal ischemia. Mechanistically, spermine profoundly reduces desensitization of ASIC1a by slowing down desensitization in the open state, shifting steady-state desensitization to more acidic pH, and accelerating recovery between repeated periods of acid stimulation. Spermine-mediated potentiation of ASIC1a activity is occluded by PcTX1 (psalmotoxin 1), a specific ASIC1a inhibitor binding to its extracellular domain. Functionally, the enhanced channel activity is accompanied by increased acid-induced neuronal membrane depolarization and cytoplasmic Ca2+ overload, which may partially explain the exacerbated neuronal damage caused by spermine. More importantly, blocking endogenous spermine synthesis significantly attenuates ischemic brain injury mediated by ASIC1a but not that by NMDA receptors. Thus, extracellular spermine contributes significantly to ischemic neuronal injury through enhancing ASIC1a activity. Our data suggest new neuroprotective strategies for stroke patients via inhibition of polyamine synthesis and subsequent spermine–ASIC interaction. PMID:21307247

  19. Effect of sulfonylureas administered centrally on the blood glucose level in immobilization stress model.

    PubMed

    Sharma, Naveen; Sim, Yun-Beom; Park, Soo-Hyun; Lim, Su-Min; Kim, Sung-Su; Jung, Jun-Sub; Hong, Jae-Seung; Suh, Hong-Won

    2015-05-01

    Sulfonylureas are widely used as an antidiabetic drug. In the present study, the effects of sulfonylurea administered supraspinally on immobilization stress-induced blood glucose level were studied in ICR mice. Mice were once enforced into immobilization stress for 30 min and returned to the cage. The blood glucose level was measured 30, 60, and 120 min after immobilization stress initiation. We found that intracerebroventricular (i.c.v.) injection with 30 µg of glyburide, glipizide, glimepiride or tolazamide attenuated the increased blood glucose level induced by immobilization stress. Immobilization stress causes an elevation of the blood corticosterone and insulin levels. Sulfonylureas pretreated i.c.v. caused a further elevation of the blood corticosterone level when mice were forced into the stress. In addition, sulfonylureas pretreated i.c.v. alone caused an elevation of the plasma insulin level. Furthermore, immobilization stress-induced insulin level was reduced by i.c.v. pretreated sulfonylureas. Our results suggest that lowering effect of sulfonylureas administered supraspinally against immobilization stress-induced increase of the blood glucose level appears to be primarily mediated via elevation of the plasma insulin level. PMID:25954123

  20. Oxygen-Sensitive K+ Channels Modulate Human Chorionic Gonadotropin Secretion from Human Placental Trophoblast.

    PubMed

    Díaz, Paula; Sibley, Colin P; Greenwood, Susan L

    2016-01-01

    Human chorionic gonadotropin (hCG) is a key autocrine/paracrine regulator of placental syncytiotrophoblast, the transport epithelium of the human placenta. Syncytiotrophoblast hCG secretion is modulated by the partial pressure of oxygen (pO2), reactive oxygen species (ROS) and potassium (K+) channels. Here we test the hypothesis that K+ channels mediate the effects of pO2 and ROS on hCG secretion. Placental villous explants from normal term pregnancies were cultured for 6 days at 6% (normoxia), 21% (hyperoxia) or 1% (hypoxia) pO2. On days 3-5, explants were treated with 5mM 4-aminopyridine (4-AP) or tetraethylammonium (TEA), blockers of pO2-sensitive voltage-gated K+ (KV) channels, or ROS (10-1000μM H2O2). hCG secretion and lactate dehydrogenase (LDH) release, a marker of necrosis, were determined daily. At day 6, hCG and LDH were measured in tissue lysate and 86Rb (K+) efflux assessed to estimate syncytiotrophoblast K+ permeability. hCG secretion and 86Rb efflux were significantly greater in explants maintained in 21% pO2 than normoxia. 4-AP/TEA inhibited hCG secretion to a greater extent at 21% than 6% and 1% pO2, and reduced 86Rb efflux at 21% but not 6% pO2. LDH release and tissue LDH/hCG were similar in 6%, 21% and 1% pO2 and unaffected by 4-AP/TEA. H2O2 stimulated 86Rb efflux and hCG secretion at normoxia but decreased 86Rb efflux, without affecting hCG secretion, at 21% pO2. 4-AP/TEA-sensitive K+ channels participate in pO2-sensitive hCG secretion from syncytiotrophoblast. ROS effects on both hCG secretion and 86Rb efflux are pO2-dependent but causal links between the two remain to be established. PMID:26863525

  1. Oxygen-Sensitive K+ Channels Modulate Human Chorionic Gonadotropin Secretion from Human Placental Trophoblast

    PubMed Central

    Díaz, Paula; Sibley, Colin P.; Greenwood, Susan L.

    2016-01-01

    Human chorionic gonadotropin (hCG) is a key autocrine/paracrine regulator of placental syncytiotrophoblast, the transport epithelium of the human placenta. Syncytiotrophoblast hCG secretion is modulated by the partial pressure of oxygen (pO2), reactive oxygen species (ROS) and potassium (K+) channels. Here we test the hypothesis that K+ channels mediate the effects of pO2 and ROS on hCG secretion. Placental villous explants from normal term pregnancies were cultured for 6 days at 6% (normoxia), 21% (hyperoxia) or 1% (hypoxia) pO2. On days 3–5, explants were treated with 5mM 4-aminopyridine (4-AP) or tetraethylammonium (TEA), blockers of pO2-sensitive voltage-gated K+ (KV) channels, or ROS (10–1000μM H2O2). hCG secretion and lactate dehydrogenase (LDH) release, a marker of necrosis, were determined daily. At day 6, hCG and LDH were measured in tissue lysate and 86Rb (K+) efflux assessed to estimate syncytiotrophoblast K+ permeability. hCG secretion and 86Rb efflux were significantly greater in explants maintained in 21% pO2 than normoxia. 4-AP/TEA inhibited hCG secretion to a greater extent at 21% than 6% and 1% pO2, and reduced 86Rb efflux at 21% but not 6% pO2. LDH release and tissue LDH/hCG were similar in 6%, 21% and 1% pO2 and unaffected by 4-AP/TEA. H2O2 stimulated 86Rb efflux and hCG secretion at normoxia but decreased 86Rb efflux, without affecting hCG secretion, at 21% pO2. 4-AP/TEA-sensitive K+ channels participate in pO2-sensitive hCG secretion from syncytiotrophoblast. ROS effects on both hCG secretion and 86Rb efflux are pO2-dependent but causal links between the two remain to be established. PMID:26863525

  2. Yeast K1 killer toxin forms ion channels in sensitive yeast spheroplasts and in artificial liposomes.

    PubMed Central

    Martinac, B; Zhu, H; Kubalski, A; Zhou, X L; Culbertson, M; Bussey, H; Kung, C

    1990-01-01

    The patch-clamp technique was used to examine the plasma membranes of sensitive yeast spheroplasts exposed to partially purified killer toxin preparations. Asolectin liposomes in which the toxin was incorporated were also examined. Excised inside-out patches from these preparations often revealed at 118 pS conductance appearing in pairs. The current through this conductance flickered rapidly among three states: dwelling mostly at the unit-open state, less frequently at the two-unit-open state, and more rarely at the closed state. Membrane voltages from -80 to 80 mV had little influence on the opening probability. The current reversed near the equilibrium potential of K+ in asymmetric KCl solutions and also reversed near O mV at symmetric NaCl vs. KCl solutions. The two levels of the conductance were likely due to the toxin protein, as treatment of spheroplasts or liposomes with extracellular protein preparations from isogenic yeasts deleted for the toxin gene gave no such conductance levels. These results show that in vivo the killer-toxin fraction can form a cation channel that seldom closes regardless of membrane voltage. We suggest that this channel causes the death of sensitive yeast cells. Images PMID:1696721

  3. Macroform and microform-induced change in redox-sensitive chemistries of river channel surface sediments

    NASA Astrophysics Data System (ADS)

    Byrne, P.; Zhang, H.; Heathwaite, A. L.; Binley, A.; Ullah, S.; Kaeser, D.; Heppell, C. M.; Lansdown, K.; Trimmer, M.

    2012-04-01

    In-stream geomorphological features such as riffle-pool sequences (macroforms) can produce steep hydraulic gradients which induce flow in and out of the riverbed - hyporheic exchange flow (HEF). The acceleration of flow over channel obstacles such as large cobbles and boulders (microforms) can create variation in surface-subsurface pressure gradients and generation of HEF. HEF in shallow surface sediments affect the transformation of redox-sensitive chemical forms and, therefore, the attenuation or release of nutrients in river systems. Here, we examine the relationship between stream geomorphological environment (microform and macroform) and concentration profiles of redox-sensitive species (nitrate, sulphate, iron, manganese) in shallow (15cm) subsurface sediments. In-situ passive samplers (diffusive equilibrium in thin films - DET) are used to obtain biogeochemical data from armoured environments at fine scale (cm) depth resolution where there is strong upwelling. The probes were deployed in a 50m reach of the River Eden, Cumbria, UK, during baseflow conditions. The experimental setup allowed for the assessment of differences in redox-sensitive chemistries between a riffle and pool environment and between smooth and rough bed surfaces in the pool. The passive sensing basis of the DET methodology provided a means for investigating how HEF systems generated at two different geomorphological scales influence the concentration and spatial patterns of redox-sensitive species. DET's capability of measuring at high spatial resolution allowed the extent of hyporheic mixing to be targeted, even though it is often limited to the top few centimetres of sediment.

  4. Nordihydroguaiaretic acid depletes ATP and inhibits a swelling-activated, ATP-sensitive taurine channel.

    PubMed

    Ballatori, N; Wang, W

    1997-05-01

    The mechanism by which nordihydroguaiaretic acid (NDGA), a lipoxygenase inhibitor, prevents swelling-activated organic osmolyte efflux was examined in the human hepatoma cell line Hep G2. When swollen in hypotonic medium, Hep G2 cell exhibited a regulatory volume decrease that was associated with the release of intracellular taurine, an amino acid found at a concentrations of 22.0 +/- 2.5 nmol/mg protein (approximately 5 mM) in these cells. Rate coefficients for swelling-activated [3H]taurine uptake and efflux were unaffected when extracellular taurine was increased from 0.1 to 25 mM, indicating that taurine is released via a channel. Taurine efflux was rapidly activated after cell swelling and immediately inactivated when cells were returned to normal size by restoration of isotonicity. Swelling-activated taurine efflux was not altered by replacement of extracellular Na+ with choline+ or K+ but was inhibited when cellular ATP levels were decreased with a variety of chemical agents, consistent with an ATP-regulated channel previously described in other cell types. NDGA inhibited swelling-activated [3H]taurine efflux in Hep G2 cells at concentrations of 50-150 microM; however, these same concentrations of NDGA also lowered cell ATP levels. Likewise, ketoconazole, an inhibitor of cytochrome P-450 monoxygenases, inhibited [3H]taurine efflux only at concentrations at which cell ATP levels were also lowered. In contrast, other inhibitors of cyclooxygenase (indomethacin, 100 microM) or of lipoxygenases (caffeic acid, 100 microM), as well as arachidonic acid itself (100 microM), had no effect on either taurine efflux or cell ATP. The present findings characterize a swelling-activated, ATP-sensitive osmolyte channel in Hep G2 cells and demonstrate that inactivation of the channel by NDGA is related to the ability of this drug to deplete cellular ATP. PMID:9176131

  5. ATP-sensitive K+ channels are functional in expiratory neurones of normoxic cats.

    PubMed Central

    Pierrefiche, O; Bischoff, A M; Richter, D W

    1996-01-01

    1. We analysed spontaneously active expiratory neurones (n = 48) of anaesthetized cats for the presence of ATP-sensitive K+ (KATP) channels. 2. Intracellular injection of ATP reversibly depolarized neurones during all phases of the respiratory cycle. During expiration, membrane potential depolarized by an average of 1.5 +/- 0.1 mV leading to a 25% increase of discharge frequency. During inspiration, ATP induced a 1.8 +/- 0.2 mV depolarization, which was accompanied by a maximum of 20% increase of input resistance (Rn). 3. Extracellular application of diazoxide, an agonist of KATP channels, resulted in reversible membrane hyperpolarization in 68% of neurones (n = 19). This hyperpolarization (2.5 mV during expiration and 3.1 mV during inspiration) was accompanied by a 22% decrease in Rn. 4. Extracellular application of tolbutamide and glibenclamide, two antagonists of KATP channels, evoked reversible depolarizations in 76% of neurones (n = 21). The depolarization was relatively constant throughout the respiratory cycle (1.4 mV during expiration and 2.3 mV during inspiration). Rn increased by 22%. 5. The same sulphonylureas also changed the steepness of membrane depolarization when neurones escaped spontaneous synaptic inhibition during postinspiration. Extracellularly applied tolbutamide and glibenclamide increased the steepness of depolarization by 21%, while diazoxide reduced it by 20%. 6. Antagonism of drugs was verified by simultaneous extra- and intracellular application of diazoxide and glibenclamide, respectively. 7. During voltage clamp at holding potential at -60 to -67 mV, intracellular or extracellular application of tolbutamide and glibenclamide blocked a persistent outward current. 8. We conclude that KATP channels are functional in expiratory neurones of adult cats and contribute to the control of excitability even during normoxia. Images Figure 1 PMID:8842000

  6. GABA transmission via ATP-dependent K+ channels regulates α-synuclein secretion in mouse striatum.

    PubMed

    Emmanouilidou, Evangelia; Minakaki, Georgia; Keramioti, Maria V; Xylaki, Mary; Balafas, Evangelos; Chrysanthou-Piterou, Margarita; Kloukina, Ismini; Vekrellis, Kostas

    2016-03-01

    α-Synuclein is readily released in human and mouse brain parenchyma, even though the normal function of the secreted protein has not been yet elucidated. Under pathological conditions, such as in Parkinson's disease, pathologically relevant species of α-synuclein have been shown to propagate between neurons in a prion-like manner, although the mechanism by which α-synuclein transfer induces degeneration remains to be identified. Due to this evidence extracellular α-synuclein is now considered a critical target to hinder disease progression in Parkinson's disease. Given the importance of extracellular α-synuclein levels, we have now investigated the molecular pathway of α-synuclein secretion in mouse brain. To this end, we have identified a novel synaptic network that regulates α-synuclein release in mouse striatum. In this brain area, the majority of α-synuclein is localized in corticostriatal glutamatergic terminals. Absence of α-synuclein from the lumen of brain-isolated synaptic vesicles suggested that they are unlikely to mediate its release. To dissect the mechanism of α-synuclein release, we have used reverse microdialysis to locally administer reagents that locally target specific cellular pathways. Using this approach, we show that α-synuclein secretion in vivo is a calcium-regulated process that depends on the activation of sulfonylurea receptor 1-sensitive ATP-regulated potassium channels. Sulfonylurea receptor 1 is distributed in the cytoplasm of GABAergic neurons from where the ATP-dependent channel regulates GABA release. Using a combination of specific agonists and antagonists, we were able to show that, in the striatum, modulation of GABA release through the sulfonylurea receptor 1-regulated ATP-dependent potassium channels located on GABAergic neurons controls α-synuclein release from the glutamatergic terminals through activation of the presynaptic GABAB receptors. Considering that sulfonylurea receptors can be selectively targeted, our

  7. Binding of ( sup 125 I)iodipine to parathyroid cell membranes: Evidence of a dihydropyridine-sensitive calcium channel

    SciTech Connect

    Jones, J.I.; Fitzpatrick, L.A. )

    1990-04-01

    The parathyroid cell is unusual, in that an increase in extracellular calcium concentrations inhibits PTH release. Calcium channels are glycoproteins that span cell membranes and allow entry of extracellular calcium into cells. We have demonstrated that the calcium channel agonist (+)202-791, which opens calcium channels, inhibits PTH release and that the antagonist (-)202-791, which closes calcium channels, stimulates PTH release. To identify the calcium channels responsible for these effects, we used a radioligand that specifically binds to calcium channels. Bovine parathyroid cell membranes were prepared and incubated under reduced lighting with (125I) iodipine (SA, 2000 Ci/mmol), which recognizes 1,4-dihydropyridine-sensitive calcium channels. Bound ligand was separated from free ligand by rapid filtration through Whatman GF/B filters. Nonspecific binding was measured by the inclusion of nifedipine at 10 microM. Specific binding represented approximately 40% of the total binding. The optimal temperature for (125I) iodipine binding was 4 C, and binding reached equilibrium by 30 min. The equilibrium dissociation constant (Kd) was approximately 550 pM, and the maximum number of binding sites was 780 fmol/mg protein. Both the calcium channel agonist (+)202-791 and antagonist (-)202-791 competitively inhibited (125I) iodipine binding, with 50% inhibition concentrations of 20 and 300 nM, respectively. These data indicate the presence of dihydropyridine-sensitive calcium channels on parathyroid cell membranes.

  8. Lipid bilayer modification alters the gating properties and pharmacological sensitivity of voltage-gated sodium channel.

    PubMed

    Zhu, Yan; Wu, Bin; Feng, Yi-Jun; Tao, Jie; Ji, Yong-Hua

    2015-06-25

    Voltage-gated sodium channels (VGSCs) are widely distributed in most cells and tissues, performing many physiological functions. As one kind of membrane proteins in the lipid bilayer, whether lipid composition plays a role in the gating and pharmacological sensitivity of VGSCs still remains unknown. Through the application of sphingomyelinase D (SMaseD), the gating and pharmacological sensitivity of the endogenous VGSCs in neuroblastoma ND7-23 cell line to BmK I and BmK AS, two sodium channel-specific modulators from the venom of Buthus martensi Karsch (BmK), were assessed before and after lipid modification. The results showed that, in ND7-23 cells, SMaseD did not change the gating properties of VGSCs. However, SMaseD application altered the slope factor of activation with the treatment of 30 nmol/L BmK I, but caused no significant effects at 100 and 500 nmol/L BmK I. With low concentration of BmK I (30 and 100 nmol/L) treatment, the application of SMaseD exerted hyperpolarizing effects on both slow-inactivation and steady-state inactivation, and increased the recovery time constant, whereas total inactivation and recovery remained unaltered at 500 nmol/L BmK I. Meanwhile, SMaseD modulation hyperpolarized the voltage dependence of slow-inactivation at 0.1 nmol/L BmK AS and altered the slope factor of slow-inactivation at 10 nmol/L BmK AS, whereas other parameters remained unchanged. These results indicated a possibility that the lipid bilayer would disturb the pharmacological sensitivity of VGSCs for the first time, which might open a new way of developing new drugs for treating sodium channelopathies. PMID:26109300

  9. Key Role of Sulfonylurea Receptor 1 in Progressive Secondary Hemorrhage after Brain Contusion

    PubMed Central

    Kilbourne, Michael; Tsymbalyuk, Orest; Tosun, Cigdem; Caridi, John; Ivanova, Svetlana; Keledjian, Kaspar; Bochicchio, Grant; Gerzanich, Volodymyr

    2009-01-01

    Abstract An important but poorly understood feature of traumatic brain injury (TBI) is the clinically serious problem of spatiotemporal progression (“blossoming”) of a hemorrhagic contusion, a phenomenon we term progressive secondary hemorrhage (PSH). Molecular mechanisms of PSH are unknown and efforts to reduce it by promoting coagulation have met with equivocal results. We hypothesized that PSH might be due to upregulation and activation of sulfonylurea receptor 1 (SUR1)-regulated NCCa-ATP channels in capillary endothelial cells, predisposing to oncotic death of endothelial cells and catastrophic failure of capillary integrity. Anesthetized adult male rats underwent left parietal craniectomy for induction of a focal cortical contusion. The regulatory subunit of the channel, SUR1, was prominently upregulated in capillaries of penumbral tissues surrounding the contusion. In untreated rats, PSH was characterized by progressive enlargement of the contusion deep into the site of cortical impact, including corpus callosum, hippocampus, and thalamus, by progressive accumulation of extravasated blood, with a doubling of the volume during the first 12 h after injury, and by capillary fragmentation in penumbral tissues. Block of SUR1 using low-dose (non-hypoglycemogenic) glibenclamide largely eliminated PSH and capillary fragmentation, and was associated with a significant reduction in the size of the necrotic lesion and in preservation of neurobehavioral function. Antisense oligodeoxynucleotide against SUR1, administered after injury, reduced both SUR1 expression and PSH, consistent with a requirement for transcriptional upregulation of SUR1. Our findings provide novel insights into molecular mechanisms responsible for PSH associated with hemorrhagic contusions, and point to SUR1 as a potential therapeutic target in TBI. PMID:19604096

  10. Adenosine Triphosphate-Sensitive Potassium Channel Kir Subunits Implicated in Cardioprotection by Diazoxide

    PubMed Central

    Henn, Matthew C; Janjua, M Burhan; Kanter, Evelyn M; Makepeace, Carol M; Schuessler, Richard B; Nichols, Colin G; Lawton, Jennifer S

    2015-01-01

    Background ATP-sensitive potassium (KATP) channel openers provide cardioprotection in multiple models. Ion flux at an unidentified mitochondrial KATP channel has been proposed as the mechanism. The renal outer medullary kidney potassium channel subunit, potassium inward rectifying (Kir)1.1, has been implicated as a mitochondrial channel pore-forming subunit. We hypothesized that subunit Kir1.1 is involved in cardioprotection (maintenance of volume homeostasis and contractility) of the KATP channel opener diazoxide (DZX) during stress (exposure to hyperkalemic cardioplegia [CPG]) at the myocyte and mitochondrial levels. Methods and Results Kir subunit inhibitor Tertiapin Q (TPN-Q) was utilized to evaluate response to stress. Mouse ventricular mitochondrial volume was measured in the following groups: isolation buffer; 200 μmol/L of ATP; 100 μmol/L of DZX+200 μmol/L of ATP; or 100 μmol/L of DZX+200 μmol/L of ATP+TPN-Q (500 or 100 nmol/L). Myocytes were exposed to Tyrode’s solution (5 minutes), test solution (Tyrode’s, cardioplegia [CPG], CPG+DZX, CPG+DZX+TPN-Q, Tyrode’s+TPN-Q, or CPG+TPN-Q), N=12 for all (10 minutes); followed by Tyrode’s (5 minutes). Volumes were compared. TPN-Q, with or without DZX, did not alter mitochondrial or myocyte volume. Stress (CPG) resulted in myocyte swelling and reduced contractility that was prevented by DZX. TPN-Q prevented the cardioprotection afforded by DZX (volume homeostasis and maintenance of contractility). Conclusions TPN-Q inhibited myocyte cardioprotection provided by DZX during stress; however, it did not alter mitochondrial volume. Because TPN-Q inhibits Kir1.1, Kir3.1, and Kir3.4, these data support that any of these Kir subunits could be involved in the cardioprotection afforded by diazoxide. However, these data suggest that mitochondrial swelling by diazoxide does not involve Kir1.1, 3.1, or 3.4. PMID:26304939

  11. Crystal Structure of the Human K2P TRAAK, a Lipid- and Mechano-Sensitive K[superscript +] Ion Channel

    SciTech Connect

    Brohawn, Stephen G.; del Mármol, Josefina; MacKinnon, Roderick

    2012-03-01

    TRAAK channels, members of the two-pore domain K{sup +} (potassium ion) channel family K2P, are expressed almost exclusively in the nervous system and control the resting membrane potential. Their gating is sensitive to polyunsaturated fatty acids, mechanical deformation of the membrane, and temperature changes. Physiologically, these channels appear to control the noxious input threshold for temperature and pressure sensitivity in dorsal root ganglia neurons. We present the crystal structure of human TRAAK at a resolution of 3.8 angstroms. The channel comprises two protomers, each containing two distinct pore domains, which create a two-fold symmetric K{sup +} channel. The extracellular surface features a helical cap, 35 angstroms tall, that creates a bifurcated pore entry way and accounts for the insensitivity of two-pore domain K{sup +} channels to inhibitory toxins. Two diagonally opposed gate-forming inner helices form membrane-interacting structures that may underlie this channel's sensitivity to chemical and mechanical properties of the cell membrane.

  12. Volume-sensitive outwardly rectifying chloride channels are involved in oxidative stress-induced apoptosis of mesangial cells

    SciTech Connect

    Jiao Jundong; Xu Chaoqian; Yue Peng; Dong Deli; Li Zhe; Du Zhimin; Yang Baofeng . E-mail: yangbf@ems.hrbmu.edu.cn

    2006-02-03

    Volume-sensitive outwardly rectifying (VSOR) Cl{sup -} channels have been electrophysiologically identified in human and mouse mesangial cells, but the functional role of VSOR Cl{sup -} channels in mesangial cell apoptosis is not clear. The aim of the present study was to demonstrate the role of VSOR Cl{sup -} channels in oxidative stress-induced mesangial cell apoptosis. H{sub 2}O{sub 2}-induced Cl{sup -} currents showed phenotypic properties of VSOR Cl{sup -} channels, including outward rectification, voltage-dependent inactivation at more positive potentials, sensitivity to hyperosmolarity, and inhibition by VSOR Cl{sup -} channel blockers. Moreover, blockage of VSOR Cl{sup -} channels by DIDS (100 {mu}M), NPPB (10 {mu}M) or niflumic acid (10 {mu}M) rescued mesangial cell apoptosis induced by H{sub 2}O{sub 2}. Treatment with 150 {mu}M H{sub 2}O{sub 2} for 2 h resulted in significant reduction of cell volume, in contrast, nuclear condensation and/or fragmentation were not observed and the caspase-3 activity was also not increased. The early-phase alterations in cell volume were markedly abolished by pretreatment with VSOR Cl{sup -} channel blockers. We conclude that VSOR Cl{sup -}channels are involved in H{sub 2}O{sub 2}-induced apoptosis in cultured mesangial cells and its mechanism is associated with apoptotic volume decrease processes.

  13. Sulfonylurea Treatment Before Genetic Testing in Neonatal Diabetes: Pros and Cons

    PubMed Central

    Carmody, David; Bell, Charles D.; Hwang, Jessica L.; Dickens, Jazzmyne T.; Sima, Daniela I.; Felipe, Dania L.; Zimmer, Carrie A.; Davis, Ajuah O.; Kotlyarevska, Kateryna; Naylor, Rochelle N.; Philipson, Louis H.

    2014-01-01

    Context: Diabetes in neonates nearly always has a monogenic etiology. Earlier sulfonylurea therapy can improve glycemic control and potential neurodevelopmental outcomes in children with KCNJ11 or ABCC8 mutations, the most common gene causes. Objective: Assess the risks and benefits of initiating sulfonylurea therapy before genetic testing results become available. Design, Setting, and Patients: Observational retrospective study of subjects with neonatal diabetes within the University of Chicago Monogenic Diabetes Registry. Main Outcome Measures: Response to sulfonylurea (determined by whether insulin could be discontinued) and treatment side effects in those treated empirically. Results: A total of 154 subjects were diagnosed with diabetes before 6 months of age. A genetic diagnosis had been determined in 118 (77%), with 73 (47%) having a mutation in KCNJ11 or ABCC8. The median time from clinical diagnosis to genetic diagnosis was 10.4 weeks (range, 1.6 to 58.2 wk). In nine probands, an empiric sulfonylurea trial was initiated within 28 days of diabetes diagnosis. A genetic cause was subsequently found in eight cases, and insulin was discontinued within 14 days of sulfonylurea initiation in all of these cases. Conclusions: Sulfonylurea therapy appears to be safe and often successful in neonatal diabetes patients before genetic testing results are available; however, larger numbers of cases must be studied. Given the potential beneficial effect on neurodevelopmental outcome, glycemic control, and the current barriers to expeditious acquisition of genetic testing, an empiric inpatient trial of sulfonylurea can be considered. However, obtaining a genetic diagnosis remains imperative to inform long-term management and prognosis. PMID:25238204

  14. Voltage-gated and ATP-sensitive K+ channels are associated with cell proliferation and tumorigenesis of human glioma.

    PubMed

    Ru, Qin; Tian, Xiang; Wu, Yu-Xiang; Wu, Ri-Hui; Pi, Ming-Shan; Li, Chao-Ying

    2014-02-01

    Increasing evidence indicates that potassium (K+) channels play important roles in the growth and development of human cancer. In the present study, we investigated the contribution of and the mechanism by which K+ channels control the proliferation and tumor development of U87-MG human glioma cells. A variety of K+ channel blockers and openers were used to differentiate the critical subtype of K+ channels involved. The in vitro data demonstrated that selective blockers of voltage-gated K+ (K(V)) channels or ATP-sensitive K+ (K(ATP)) channels significantly inhibited the proliferation of U87-MG cells, blocked the cell cycle at the G0/G1 phase and induced apoptosis. In the U87-MG xenograft model in nude mice, K(V) or K(ATP) channel blockers markedly suppressed tumor growth in vivo. Furthermore, electrophysiological results showed that KV or KATP channel blockers inhibited K(V)/K(ATP) channel currents as well as cell proliferation and tumor growth over the same concentration range. In contrast, iberiotoxin, a selective blocker of calcium-activated K+ channels, had no apparent effect on the cell proliferation, cell cycle or apoptosis of U87-MG cells. In addition, the results of fluorescence assays indicated that blockers of K(V) or K(ATP) channels attenuated intracellular Ca2+ signaling by blocking Ca2+ influx in U87-MG cells. Taken together, these data suggest that K(V) and K(ATP) channels play important roles in the proliferation of U87-MG cells and that the influence of K(V) and K(ATP) channels may be mediated by a Ca2+-dependent mechanism. PMID:24284968

  15. Contribution of concentration-sensitive sodium channels to the absorption of alveolar fluid in mice.

    PubMed

    Hagiwara, Teruki; Yoshida, Shigeru

    2016-09-01

    The concentration-sensitive sodium channel (Nac) is activated by an increase in the extracellular sodium concentration. Although the expression of Nac in alveolar type II epithelial cells (AEC II) has been reported previously, the physiological role of Nac in the lung has not been established. We characterized Nac expression and examined amiloride-insensitive sodium transport mediated by Nac in mouse lung. Immunofluorescence studies revealed that Nac did not colocalize with either aquaporin 5 or cystic fibrosis transmembrane conductance regulator, but partially colocalized with the epithelial sodium channel γ-subunit. Immunoelectron microscopy studies showed that Nac localized at the basolateral membrane of pulmonary microvascular endothelial cells (PMVECs). Nac mRNA and protein were expressed in PMVECs isolated from the lungs of mice. Image analysis indicated that sodium influx into the alveolar wall was dependent on increases in extracellular sodium concentration. We conclude that Nac expressed in PMVECs and AEC II contributes to the reabsorption of sodium via an amiloride-insensitive pathway during alveolar fluid clearance. PMID:27259686

  16. tmc-1 encodes a sodium-sensitive channel required for salt chemosensation in C. elegans.

    PubMed

    Chatzigeorgiou, Marios; Bang, Sangsu; Hwang, Sun Wook; Schafer, William R

    2013-02-01

    Transmembrane channel-like (TMC) genes encode a broadly conserved family of multipass integral membrane proteins in animals. Human TMC1 and TMC2 genes are linked to human deafness and required for hair-cell mechanotransduction; however, the molecular functions of these and other TMC proteins have not been determined. Here we show that the Caenorhabditis elegans tmc-1 gene encodes a sodium sensor that functions specifically in salt taste chemosensation. tmc-1 is expressed in the ASH polymodal avoidance neurons, where it is required for salt-evoked neuronal activity and behavioural avoidance of high concentrations of NaCl. However, tmc-1 has no effect on responses to other stimuli sensed by the ASH neurons including high osmolarity and chemical repellents, indicating a specific role in salt sensation. When expressed in mammalian cell culture, C. elegans TMC-1 generates a predominantly cationic conductance activated by high extracellular sodium but not by other cations or uncharged small molecules. Thus, TMC-1 is both necessary for salt sensation in vivo and sufficient to generate a sodium-sensitive channel in vitro, identifying it as a probable ionotropic sensory receptor. PMID:23364694

  17. Performance of resistive-charge position sensitive detectors for RBS/Channeling applications

    NASA Astrophysics Data System (ADS)

    Miranda, P. A.; Wahl, U.; Catarino, N.; Ribeiro da Silva, M.; Alves, E.

    2014-10-01

    The performance of two types of 1×1 cm2 photodiode position sensitive detectors (PSDs) based on resistive charge division was evaluated for their use in Rutherford Backscattering/Channeling (RBS/C) experiments in blocking geometry. Their energy resolution was first determined for ~ 5.5 MeV alpha particles from a radioactive sources, and values of full width half maximum (FWHM) of 22 keV and 33 keV were achieved using a shaping time constant of τ = 2.0 μs. Additional tests were performed using backscattered 4He particles from the 2.0 MeV beam of a Van de Graaff accelerator. While the 22 keV FWHM detector failed after exposure to less than 5×106 cm-24He particles, the other did not show any noticeable deterioration due to radiation damage for a fluence of 4×108 cm-2. For this type of PSD position resolution (τ = 0.5 μs) standard deviations of ΔL = 0.072 mm at ~ 5.5 MeV and ΔL = 0.247 mm at 1.1 MeV were achieved. RBS/Channeling experiments using PSD were performed on several crystalline samples, showing that this setup seems suitable for lattice location studies, particularly for heavy ions implantation (D ≳1015 at /cm2) on light substrates like Si, SiC, and AlN.

  18. An epilepsy mutation in the beta1 subunit of the voltage-gated sodium channel results in reduced channel sensitivity to phenytoin.

    PubMed

    Lucas, Paul T; Meadows, Laurence S; Nicholls, Jane; Ragsdale, David S

    2005-05-01

    The antiepileptic drug phenytoin inhibits voltage-gated sodium channels. Phenytoin block is enhanced at depolarized membrane potentials and during high frequency channel activation. These properties, which are important for the clinical efficacy of the drug, depend on voltage-dependent channel gating. In this study, we examined the action of phenytoin on sodium channels, comprising a mutant auxiliary beta1 subunit (mutation C121Wbeta1), which causes the inherited epilepsy syndrome, generalized epilepsy with febrile seizures plus (GEFS+). Whole cell sodium currents in Chinese hamster ovary (CHO) cells coexpressing human Na(v)1.3 sodium channels and C121Wbeta1 exhibited altered gating properties, compared to currents in cells coexpressing Na(v)1.3 and wild type beta1. In addition mutant channels were less sensitive to inhibition by phenytoin, showing reduced tonic block at -70mV (EC(50)=26microM for C121Wbeta1 versus 11microM for wild type beta1) and less frequency-dependent inhibition in response to a 20Hz pulse train ( approximately 40% inhibition for C121Wbeta1 versus approximately 70% inhibition for wild type beta1, with 50microM phenytoin). Mutant and wild type channels did not differ in inactivated state affinity for phenytoin, suggesting that their pharmacological differences were secondary to their differences in voltage-dependent gating, rather than being caused by direct effects of the mutation on the drug receptor. Together, these data show that a sodium channel mutation responsible for epilepsy can also alter channel response to antiepileptic drugs. PMID:15922564

  19. Achromatopsia-associated mutation in the human cone photoreceptor cyclic nucleotide-gated channel CNGB3 subunit alters the ligand sensitivity and pore properties of heteromeric channels.

    PubMed

    Peng, Changhong; Rich, Elizabeth D; Varnum, Michael D

    2003-09-01

    Cone photoreceptor cyclic nucleotide-gated (CNG) channels are thought to form by assembly of two different subunit types, CNGA3 and CNGB3. Recently, mutations in the gene encoding the CNGB3 subunit have been linked to achromatopsia in humans. Here we describe the functional consequences of two achromatopsia-associated mutations in human CNGB3 (hCNGB3). Co-expression in Xenopus oocytes of human CNGA3 (hCNGA3) subunits with hCNGB3 subunits containing an achromatopsia-associated mutation in the S6 transmembrane domain (S435F) generated functional heteromeric channels that exhibited an increase in apparent affinity for both cAMP and cGMP compared with wild type heteromeric channels. In contrast, co-expression of a presumptive null mutation of hCNGB3 (T383f.s.Delta C) with hCNGA3 produced channels with properties indistinguishable from homomeric hCNGA3 channels. The effect of hCNGB3 S435F subunits on cell-surface expression of green fluorescent protein-tagged hCNGA3 subunits and of non-tagged hCNGA3 subunits on surface expression of green fluorescent protein-hCNGB3 S435F subunits were similar to those observed for wild type hCNGB3 subunits, suggesting that the mutation does not grossly disturb subunit assembly or plasma membrane targeting. The S435F mutation was also found to produce changes in the pore properties of the channel, including decreased single channel conductance and decreased sensitivity to block by l-cis-diltiazem. Overall, these results suggest that the functional properties of cone CNG channels may be altered in patients with the S435F mutation, providing evidence supporting the pathogenicity of this mutation in humans. Thus, achromatopsia may arise from a disturbance of cone CNG channel gating and permeation or from the absence of functional CNGB3 subunits. PMID:12815043

  20. Golden channel at a neutrino factory revisited: Improved sensitivities from a magnetized iron neutrino detector

    NASA Astrophysics Data System (ADS)

    Bayes, R.; Laing, A.; Soler, F. J. P.; Cervera Villanueva, A.; Gómez Cadenas, J. J.; Hernández, P.; Martín-Albo, J.; Burguet-Castell, J.

    2012-11-01

    This paper describes the performance and sensitivity to neutrino mixing parameters of a Magnetised Iron Neutrino Detector at a Neutrino Factory with a neutrino beam created from the decay of 10 GeV muons. Specifically, it is concerned with the ability of such a detector to detect muons of the opposite sign to those stored (wrong-sign muons) while suppressing contamination of the signal from the interactions of other neutrino species in the beam. A new, more realistic simulation and analysis, which improves the efficiency of this detector at low energies, has been developed using the GENIE neutrino event generator and the GEANT4 simulation toolkit. Low-energy neutrino events down to 1 GeV were selected, while reducing backgrounds to the 10-4 level. Signal efficiency plateaus of ˜60% for νμ and ˜70% for ν¯μ events were achieved starting at ˜5GeV. Contamination from the νμ→ντ oscillation channel was studied for the first time and was found to be at the level between 1% and 4%. Full response matrices are supplied for all the signal and background channels from 1 GeV to 10 GeV. The sensitivity of an experiment involving a Magnetised Iron Neutrino Detector detector of 100 ktons at 2000 km from the Neutrino Factory is calculated for the case of sin⁡22θ13˜10-1. For this value of θ13, the accuracy in the measurement of the CP-violating phase is estimated to be ΔδCP˜3°-5°, depending on the value of δCP, the CP coverage at 5σ is 85% and the mass hierarchy would be determined with better than 5σ level for all values of δCP.

  1. The structure-activity relationship in herbicidal monosubstituted sulfonylureas

    SciTech Connect

    Li, Zheng-Ming; Ma, Yi; Guddat, Luke; Cheng, Pei-Quan; Wang, Jian-Guo; Pang, Siew S; Dong, Yu-Hui; Lai, Cheng-Ming; Wang, Ling-Xiu; Jia, Guo-Feng; Li, Yong-Hong; Wang, Su-Hua; Liu, Jie; Zhao, Wei-Guang; Wang, Bao-Lei

    2012-05-24

    The herbicide sulfonylurea (SU) belongs to one of the most important class of herbicides worldwide. It is well known for its ecofriendly, extreme low toxicity towards mammals and ultralow dosage application. The original inventor, G Levitt, set out structure-activity relationship (SAR) guidelines for SU structural design to attain superhigh bioactivity. A new approach to SU molecular design has been developed. After the analysis of scores of SU products by X-ray diffraction methodology and after greenhouse herbicidal screening of 900 novel SU structures synthesized in the authors laboratory, it was found that several SU structures containing a monosubstituted pyrimidine moiety retain excellent herbicidal characteristics, which has led to partial revision of the Levitt guidelines. Among the novel SU molecules, monosulfuron and monosulfuron-ester have been developed into two new herbicides that have been officially approved for field application and applied in millet and wheat fields in China. A systematic structural study of the new substrate-target complex and the relative mode of action in comparison with conventional SU has been carried out. A new mode of action has been postulated.

  2. Effect of sulfonylureas on hepatic fatty acid oxidation

    SciTech Connect

    Patel, T.B.

    1986-08-01

    In isolated rat livers perfused with oleic acid (0.1 mM), infusion of tolbutamide or glyburide decreased the rate of ketogenesis in a dose-dependent manner. The inhibition of fatty acid oxidation was maximal at 2.0 mM and 10 M concentrations of tolbutamide and glyburide, respectively. Neither tolbutamide nor glyburide inhibited ketogenesis in livers perfused with octanoate. The inhibition of hepatic ketogenesis by sulfonylureas was independent of perfusate oleic acid concentration. Additionally, in rat livers perfused with oleic acid in the presence of L-(-)-carnitine (10 mM), submaximal concentrations of tolbutamide and glyburide did not inhibit hepatic ketogenesis. Finally, glyburide infusion into livers perfused with (U- $C)oleic acid (0.1 mM) increased the rate of UC label incorporation into hepatic triglycerides by 2.5-fold. These data suggest that both tolbutamide and glyburide inhibit long-chain fatty acid oxidation by inhibition the key regulatory enzyme, carnitine palmitoyltransferase I, most probably by competing with L-(-)-carnitine.

  3. Chiral recognition of pinacidil and its 3-pyridyl isomer by canine cardiac and smooth muscle: Antagonism by sulfonylureas

    SciTech Connect

    Steinberg, M.I.; Wiest, S.A.; Zimmerman, K.M.; Ertel, P.J.; Bemis, K.G.; Robertson, D.W. )

    1991-01-01

    Pinacidil, a potassium channel opener (PCO), relaxes vascular smooth muscle by increasing potassium ion membrane conductance, thereby causing membrane hyperpolarization. PCOs also act on cardiac muscle to decrease action potential duration (APD) selectively. To examine the enantiomeric selectivity of pinacidil, the stereoisomers of pinacidil (a 4-pyridylcyanoguanidine) and its 3-pyridyl isomer (LY222675) were synthesized and studied in canine Purkinje fibers and cephalic veins. The (-)-enantiomers of both pinacidil and LY222675 were more potent in relaxing phenylephrine-contracted cephalic veins and decreasing APD than were their corresponding (+)-enantiomers. The EC50 values for (-)-pinacidil and (-)-LY222675 in relaxing cephalic veins were 0.44 and 0.09 microM, respectively. In decreasing APD, the EC50 values were 3.2 microM for (-)-pinacidil and 0.43 microM for (-)-LY222675. The eudismic ratio was greater for the 3-pyridyl isomer than for pinacidil in both cardiac (71 vs. 22) and vascular (53 vs. 17) tissues. (-)-LY222675 and (-)-pinacidil (0.1-30 microM) also increased 86Rb efflux from cephalic veins to a greater extent than did their respective optical antipodes. The antidiabetic sulfonylurea, glyburide (1-30 microM), shifted the vascular concentration-response curve of (-)-pinacidil to the right by a similar extent at each inhibitor concentration. Glipizide also antagonized the response to (-)-pinacidil, but was about 1/10 as potent with a maximal shift occurring at 10 and 30 microM. Glyburide antagonized the vascular relaxant effects of 0.3 microM (-)-LY222675 (EC50, 2.3 microM) and reversed the decrease in APD caused by 3 microM (-)-LY222675 (EC50, 1.9 microM). Nitroprusside did not alter 86Rb efflux, and vascular relaxation induced by sodium nitroprusside was unaffected by sulfonylureas.

  4. ATP-sensitive potassium channels in capillaries isolated from guinea-pig heart

    PubMed Central

    Schnitzler, Michael Mederos y; Derst, Christian; Daut, Jürgen; Preisig-Müller, Regina

    2000-01-01

    The full-length cDNAs of two different α-subunits (Kir6.1 and Kir6.2) and partial cDNAs of three different β-subunits (SUR1, SUR2A and SUR2B) of ATP-sensitive potassium (KATP) channels of the guinea-pig (gp) were obtained by screening a cDNA library from the ventricle of guinea-pig heart. Cell-specific reverse-transcriptase PCR with gene-specific intron-spanning primers showed that gpKir6.1, gpKir6.2 and gpSUR2B were expressed in a purified fraction of capillary endothelial cells. In cardiomyocytes, gpKir6.1, gpKir6.2, gpSUR1 and gpSUR2A were detected. Patch-clamp measurements were carried out in isolated capillary fragments consisting of 3–15 endothelial cells. The membrane capacitance measured in the whole-cell mode was 19.9 ± 1.0 pF and was independent of the length of the capillary fragment, which suggests that the endothelial cells were not electrically coupled under our experimental conditions. The perforated-patch technique was used to measure the steady-state current-voltage relation of capillary endothelial cells. Application of K+ channel openers (rilmakalim or diazoxide) or metabolic inhibition (250 μm 2,4-dinitrophenol plus 10 mM deoxyglucose) induced a current that reversed near the calculated K+ equilibrium potential. Rilmakalim (1 μm), diazoxide (300 μm) and metabolic inhibition increased the slope conductance measured at −55 mV by a factor of 9.0 (±1.8), 2.5 (±0.2) and 3.9 (±1.7), respectively. The effects were reversed by glibenclamide (1 μm). Our results suggest that capillary endothelial cells from guinea-pig heart express KATP channels composed of SUR2B and Kir6.1 and/or Kir6.2 subunits. The hyperpolarization elicited by the opening of KATP channels may lead to an increase in free cytosolic Ca2+, and thus modulate the synthesis of NO and the permeability of the capillary wall. PMID:10835035

  5. Dopamine midbrain neurons in health and Parkinson's disease: emerging roles of voltage-gated calcium channels and ATP-sensitive potassium channels.

    PubMed

    Dragicevic, E; Schiemann, J; Liss, B

    2015-01-22

    Dopamine (DA) releasing midbrain neurons are essential for multiple brain functions, such as voluntary movement, working memory, emotion and cognition. DA midbrain neurons within the substantia nigra (SN) and the ventral tegmental area (VTA) exhibit a variety of distinct axonal projections and cellular properties, and are differentially affected in diseases like schizophrenia, attention deficit hyperactivity disorder, and Parkinson's disease (PD). Apart from having diverse functions in health and disease states, DA midbrain neurons display distinct electrical activity patterns, crucial for DA release. These activity patterns are generated and modulated by specific sets of ion channels. Recently, two ion channels have been identified, not only contributing to these activity patterns and to functional properties of DA midbrain neurons, but also seem to render SN DA neurons particularly vulnerable to degeneration in PD and its animal models: L-type calcium channels (LTCCs) and ATP-sensitive potassium channels (K-ATPs). In this review, we focus on the emerging physiological and pathophysiological roles of these two ion channels (and their complex interplay with other ion channels), particularly in highly vulnerable SN DA neurons, as selective degeneration of these neurons causes the major motor symptoms of PD. PMID:25450964

  6. Risk of hypoglycemia following intensification of metformin treatment with insulin versus sulfonylurea

    PubMed Central

    Roumie, Christianne L.; Min, Jea Young; Greevy, Robert A.; Grijalva, Carlos G.; Hung, Adriana M.; Liu, Xulei; Elasy, Tom; Griffin, Marie R.

    2016-01-01

    Background: Hypoglycemia remains a common life-threatening event associated with diabetes treatment. We compared the risk of first or recurrent hypoglycemia event among metformin initiators who intensified treatment with insulin versus sulfonylurea. Methods: We assembled a retrospective cohort using databases of the Veterans Health Administration, Medicare and the National Death Index. Metformin initiators who intensified treatment with insulin or sulfonylurea were followed to either their first or recurrent hypoglycemia event using Cox proportional hazard models. Hypoglycemia was defined as hospital admission or an emergency department visit for hypoglycemia, or an outpatient blood glucose value of less than 3.3 mmol/L. We conducted additional analyses for risk of first hypoglycemia event, with death as the competing risk. Results: Among 178 341 metformin initiators, 2948 added insulin and 39 990 added sulfonylurea. Propensity score matching yielded 2436 patients taking metformin plus insulin and 12 180 taking metformin plus sulfonylurea. Patients took metformin for a median of 14 (interquartile range [IQR] 5–30) months, and the median glycated hemoglobin level was 8.1% (IQR 7.2%–9.9%) at intensification. In the group who added insulin, 121 first hypoglycemia events occurred, and 466 first events occurred in the group who added sulfonylurea (30.9 v. 24.6 events per 1000 person-years; adjusted hazard ratio [HR] 1.30, 95% confidence interval [CI] 1.06–1.59). For recurrent hypoglycemia, there were 159 events in the insulin group and 585 events in the sulfonylurea group (39.1 v. 30.0 per 1000 person-years; adjusted HR 1.39, 95% CI 1.12–1.72). In separate competing risk analyses, the adjusted HR for hypoglycemia was 1.28 (95% CI 1.04–1.56). Interpretation: Among patients using metformin who could use either insulin or sulfonylurea, the addition of insulin was associated with a higher risk of hypoglycemia than the addition of sulfonylurea. This finding should

  7. Selective disruption of high sensitivity heat activation but not capsaicin activation of TRPV1 channels by pore turret mutations

    PubMed Central

    Cui, Yuanyuan; Yang, Fan; Cao, Xu; Yarov-Yarovoy, Vladimir

    2012-01-01

    The capsaicin receptor transient receptor potential vanilloid (TRPV)1 is a highly heat-sensitive ion channel. Although chemical activation and heat activation of TRPV1 elicit similar pungent, painful sensation, the molecular mechanism underlying synergistic activation remains mysterious. In particular, where the temperature sensor is located and whether heat and capsaicin share a common activation pathway are debated. To address these fundamental issues, we searched for channel mutations that selectively affected one form of activation. We found that deletion of the first 10 amino acids of the pore turret significantly reduced the heat response amplitude and shifted the heat activation threshold, whereas capsaicin activation remained unchanged. Removing larger portions of the turret disrupted channel function. Introducing an artificial sequence to replace the deleted region restored sensitive capsaicin activation in these nonfunctional channels. The heat activation, however, remained significantly impaired, with the current exhibiting diminishing heat sensitivity to a level indistinguishable from that of a voltage-gated potassium channel, Kv7.4. Our results demonstrate that heat and capsaicin activation of TRPV1 are structurally and mechanistically distinct processes, and the pore turret is an indispensible channel structure involved in the heat activation process but is not part of the capsaicin activation pathway. Synergistic effect of heat and capsaicin on TRPV1 activation may originate from convergence of the two pathways on a common activation gate. PMID:22412190

  8. KCNQ Potassium Channels Modulate Sensitivity of Skin Down-hair (D-hair) Mechanoreceptors.

    PubMed

    Schütze, Sebastian; Orozco, Ian J; Jentsch, Thomas J

    2016-03-11

    M-current-mediating KCNQ (Kv7) channels play an important role in regulating the excitability of neuronal cells, as highlighted by mutations in Kcnq2 and Kcnq3 that underlie certain forms of epilepsy. In addition to their expression in brain, KCNQ2 and -3 are also found in the somatosensory system. We have now detected both KCNQ2 and KCNQ3 in a subset of dorsal root ganglia neurons that correspond to D-hair Aδ-fibers and demonstrate KCNQ3 expression in peripheral nerve endings of cutaneous D-hair follicles. Electrophysiological recordings from single D-hair afferents from Kcnq3(-/-) mice showed increased firing frequencies in response to mechanical ramp-and-hold stimuli. This effect was particularly pronounced at slow indentation velocities. Additional reduction of KCNQ2 expression further increased D-hair sensitivity. Together with previous work on the specific role of KCNQ4 in rapidly adapting skin mechanoreceptors, our results show that different KCNQ isoforms are specifically expressed in particular subsets of mechanosensory neurons and modulate their sensitivity directly in sensory nerve endings. PMID:26733196

  9. Random assembly of SUR subunits in K(ATP) channel complexes.

    PubMed

    Cheng, Wayland W L; Tong, Ailing; Flagg, Thomas P; Nichols, Colin G

    2008-01-01

    Sulfonylurea receptors (SURs) associate with Kir6.x subunits to form tetradimeric K(ATP) channel complexes. SUR1 and SUR2 confer differential channel sensitivities to nucleotides and pharmacological agents, and are expressed in specific, but overlapping, tissues. This raises the question of whether these different SUR subtypes can assemble in the same channel complex and generate channels with hybrid properties. To test this, we engineered dimeric constructs of wild type or N160D mutant Kir6.2 fused to SUR1 or SUR2A. Dimeric fusions formed functional, ATP-sensitive, channels. Coexpression of weakly rectifying SUR1-Kir6.2 (WTF-1) with strongly rectifying SUR1-Kir6.2[N160D] (NDF-1) in COSm6 cells results in mixed subunit complexes that exhibit unique rectification properties. Coexpression of NDF-1 and SUR2A-Kir6.2 (WTF-2) results in similar complex rectification, reflecting the presence of SUR1- and SUR2A-containing dimers in the same channel. The data demonstrate clearly that SUR1 and SUR2A subunits associate randomly, and suggest that heteromeric channels will occur in native tissues. PMID:18690055

  10. Suppression of KATP channel activity protects murine pancreatic β cells against oxidative stress

    PubMed Central

    Gier, Belinda; Krippeit-Drews, Peter; Sheiko, Tatiana; Aguilar-Bryan, Lydia; Bryan, Joseph; Düfer, Martina; Drews, Gisela

    2009-01-01

    The enhanced oxidative stress associated with type 2 diabetes mellitus contributes to disease pathogenesis. We previously identified plasma membrane–associated ATP-sensitive K+ (KATP) channels of pancreatic β cells as targets for oxidants. Here, we examined the effects of genetic and pharmacologic ablation of KATP channels on loss of mouse β cell function and viability following oxidative stress. Using mice lacking the sulfonylurea receptor type 1 (Sur1) subunit of KATP channels, we found that, compared with insulin secretion by WT islets, insulin secretion by Sur1–/– islets was less susceptible to oxidative stress induced by the oxidant H2O2. This was likely, at least in part, a result of the reduced ability of H2O2 to hyperpolarize plasma membrane potential and reduce cytosolic free Ca2+ concentration ([Ca2+]c) in the Sur1–/– β cells. Remarkably, Sur1–/– β cells were less prone to apoptosis induced by H2O2 or an NO donor than WT β cells, despite an enhanced basal rate of apoptosis. This protective effect was attributed to upregulation of the antioxidant enzymes SOD, glutathione peroxidase, and catalase. Upregulation of antioxidant enzymes and reduced sensitivity of Sur1–/– cells to H2O2-induced apoptosis were mimicked by treatment with the sulfonylureas tolbutamide and gliclazide. Enzyme upregulation and protection against oxidant-induced apoptosis were abrogated by agents lowering [Ca2+]c. Sur1–/– mice were less susceptible than WT mice to streptozotocin-induced β cell destruction and subsequent hyperglycemia and death, which suggests that loss of KATP channel activity may protect against streptozotocin-induced diabetes in vivo. PMID:19805912

  11. Trypsin-Sensitive, Rapid Inactivation of a Calcium-Activated Potassium Channel

    NASA Astrophysics Data System (ADS)

    Solaro, Christopher R.; Lingle, Christopher J.

    1992-09-01

    Most calcium-activated potassium channels couple changes in intracellular calcium to membrane excitability by conducting a current with a probability that depends directly on submembrane calcium concentration. In rat adrenal chromaffin cells, however, a large conductance, voltage- and calcium-activated potassium channel (BK) undergoes rapid inactivation, suggesting that this channel has a physiological role different than that of other BK channels. The inactivation of the BK channel, like that of the voltage-gated Shaker B potassium channel, is removed by trypsin digestion and channels are blocked by the Shaker B amino-terminal inactivating domain. Thus, this BK channel shares functional and possibly structural homologies with other inactivating voltage-gated potassium channels.

  12. ATP-Sensitive Potassium (KATP) Channel Activation Decreases Intraocular Pressure in the Anterior Chamber of the Eye

    PubMed Central

    Chowdhury, Uttio Roy; Bahler, Cindy K.; Hann, Cheryl R.; Chang, Minhwang; Resch, Zachary T.; Romero, Michael F.

    2011-01-01

    Purpose. ATP-sensitive potassium channel (KATP) openers target key cellular events, many of which have been implicated in glaucoma. The authors sought to determine whether KATP channel openers influence outflow facility in human anterior segment culture and intraocular pressure (IOP) in vivo. Methods. Anterior segments from human eyes were placed in perfusion organ culture and treated with the KATP channel openers diazoxide, nicorandil, and P1075 or the KATP channel closer glyburide (glibenclamide). The presence, functionality, and specificity of KATP channels were determined by RT-PCR, immunohistochemistry, and inside-out patch clamp in human trabecular meshwork (TM) tissue or primary cultures of normal human trabecular meshwork (NTM) cells. The effect of diazoxide on IOP in anesthetized Brown Norway rats was measured with a rebound tonometer. Results. KATP channel openers increased outflow facility in human anterior segments (0.14 ± 0.02 to 0.26 ± 0.09 μL/min/mm Hg; P < 0.001) compared with fellow control eyes (0.22 ± 0.11 to 0.21 ± 0.11 μL/min/mm Hg; P > 0.5). The effect was reversible, with outflow facility returning to baseline after drug removal. The addition of glyburide inhibited diazoxide from increasing outflow facility. Electrophysiology confirmed the presence and specificity of functional KATP channels. KATP channel subunits Kir6.1, Kir6.2, SUR2A, and SUR2B were expressed in TM and NTM cells. In vivo, diazoxide significantly lowered IOP in Brown Norway rats. Conclusions. Functional KATP channels are present in the trabecular meshwork. When activated by KATP channel openers, these channels increase outflow facility through the trabecular outflow pathway in human anterior segment organ culture and decrease IOP in Brown Norway rat eyes. PMID:21743021

  13. pH-Sensitive K(+) Currents and Properties of K2P Channels in Murine Hippocampal Astrocytes.

    PubMed

    Weller, Johannes; Steinhäuser, Christian; Seifert, Gerald

    2016-01-01

    Based on their intimate spatial association with synapses and the capillary, astrocytes are critically involved in the control of ion, transmitter, and energy homeostasis as well as regulation of the cerebral blood flow. Under pathophysiological conditions, dysfunctional astrocytes can no longer assure homeostatic control although the underlying mechanisms are poorly understood. Specifically, neurological diseases are often accompanied by acidification of the extracellular space, but the properties of astrocytes in such an acidic environment are still a matter of debate. To meet the homeostatic requirements, astrocytes are equipped with intercellular gap junctions, inwardly rectifying K(+) (Kir) channels, and two-pore domain K(+) (K2P) channels. One goal of the present study was to overview current knowledge about astrocyte K(+) channel function during acidosis. In addition, we combined functional and molecular analyses to clarify how low pH affects K(+) channel function in astrocytes freshly isolated from the developing mouse hippocampus. Extracellular acidification led to a decrease of K(+) currents in astrocytes, probably due to modulation of Kir4.1 channels. After blocking Kir4.1 channels, low pH enhanced K(+) current amplitudes. This current activation was mimicked by modulators of TREK-1 channels, which belong to the K2P channels family. We found no evidence for the presence of acid-sensitive ion channels and transient receptor potential vanilloid receptors in hippocampal astrocytes. In conclusion, the assembly of astrocytic K(+) channels allows tolerating short, transient acidification, and glial Kir4.1 and K2P channels can be considered promising new targets in brain diseases accompanied by pH shifts. PMID:26920692

  14. Electrophysiological characterization of spinal neuron sensitization by elevated calcium channel alpha-2-delta-1 subunit protein

    PubMed Central

    Zhou, Chunyi; Luo, Z. David

    2013-01-01

    Background Voltage-gated calcium channel α2δ1 subunit is the binding site for gabapentin, an effective drug in controlling neuropathic pain states including thermal hyperalgesia. Hyperalgesia to noxious thermal stimuli in both spinal-nerve-ligated (SNL) and voltage-gated calcium channel α2δ1 over-expressing transgenic (Tg) mice correlates with higher α2δ1 levels in dorsal root ganglia and dorsal spinal cord. In this study, we investigated whether abnormal synaptic transmission is responsible for thermal hyperalgesia induced by elevated α2δ1 expression in these models. Methods Behavioral sensitivities to thermal stimuli were test in L4 SNL and sham mice, as well as in α2δ1 Tg and wild-type mice. Miniature excitatory (mEPSC) and inhibitory (mIPSC) postsynaptic currents were recorded in superficial dorsal spinal cord neurons from these models using whole-cell patch clamp slice recording techniques. Results The frequency, but not amplitude, of mEPSC in superficial dorsal horn neurons was increased in SNL and α2δ1 Tg mice, which could be attenuated by gabapentin dose dependently. Intrathecal α2δ1 antisense oligodeoxynucleotide treatment diminished increased mEPSC frequency and gabapentin's inhibitory effects in elevated mEPSC frequency in the SNL mice. In contrast, neither the frequency, nor the amplitude, of mIPSC was altered in superficial dorsal horn neurons from the SNL and α2δ1 Tg mice. Conclusions Our findings support a role of peripheral nerve injury-induced α2δ1 in enhancing presynaptic excitatory input onto superficial dorsal spinal cord neurons that contributes to nociception development. PMID:24151064

  15. Overexpressed TRPV3 ion channels in skin keratinocytes modulate pain sensitivity via prostaglandin E2

    PubMed Central

    Huang, Susan M.; Lee, Hyosang; Chung, Man-Kyo; Park, Una; Yu, Yin Yin; Bradshaw, Heather B.; Coulombe, Pierre A.; Walker, J. Michael; Caterina, Michael J.

    2009-01-01

    The ability to sense changes in the environment is essential for survival because it permits responses such as withdrawal from noxious stimuli and regulation of body temperature. Keratinocytes, which occupy much of the skin epidermis, are situated at the interface between the external environment and the body's internal milieu, and have long been appreciated for their barrier function against external insults. The recent discovery of temperature-sensitive TRPV ion channels in keratinocytes has raised the possibility that these cells also actively participate in acute temperature and pain sensation. To address this notion, we generated and characterized transgenic mice that overexpress TRPV3 in epidermal keratinocytes under the control of the keratin 14 promoter. Compared to wild-type controls, keratinocytes overexpressing TRPV3 exhibited larger currents as well as augmented prostaglandin E2 (PGE2) release in response to two TRPV3 agonists, 2-aminoethoxydiphenyl borate (2APB) and heat. Thermal selection behavior and heat-evoked withdrawal behavior of naïve mice overexpressing TRPV3 were not consistently altered. Upon selective pharmacological inhibition of TRPV1 with JNJ-7203212, however, the keratinocyte-specific TRPV3 transgenic mice showed increased escape responses to noxious heat relative to their wild-type littermates. Co-administration of the cyclooxygenase inhibitor, ibuprofen, with the TRPV1 antagonist decreased inflammatory thermal hyperalgesia in transgenic but not wild-type animals. Our results reveal a previously undescribed mechanism for keratinocyte participation in thermal pain transduction through keratinocyte TRPV3 ion channels and the intercellular messenger PGE2. PMID:19091963

  16. Mercury-sensitive water channels as possible sensors of water potentials in pollen

    PubMed Central

    Hill, Adrian E.

    2013-01-01

    The growing pollen tube is central to plant reproduction and is a long-standing model for cellular tip growth in biology. Rapid osmotically driven growth is maintained under variable conditions, which requires osmosensing and regulation. This study explores the mechanism of water entry and the potential role of osmosensory regulation in maintaining pollen growth. The osmotic permeability of the plasmalemma of Lilium pollen tubes was measured from plasmolysis rates to be 1.32±0.31×10–3 cm s–1. Mercuric ions reduce this permeability by 65%. Simulations using an osmotic model of pollen tube growth predict that an osmosensor at the cell membrane controls pectin deposition at the cell tip; inhibiting the sensor is predicted to cause tip bursting due to cell wall thinning. It was found that adding mercury to growing pollen tubes caused such a bursting of the tips. The model indicates that lowering the osmotic permeability per se does not lead to bursting but rather to thickening of the tip. The time course of induced bursting showed no time lag and was independent of mercury concentration, compatible with a surface site of action. The submaximal bursting response to intermediate mercuric ion concentration was independent of the concentration of calcium ions, showing that bursting is not due to a competitive inhibition of calcium binding or entry. Bursting with the same time course was also shown by cells growing on potassium-free media, indicating that potassium channels (implicated in mechanosensing) are not involved in the bursting response. The possible involvement of mercury-sensitive water channels as osmosensors and current knowledge of these in pollen cells are discussed. PMID:24098048

  17. Perturbation of voltage-sensitive Ca2+ channel function by volatile organic solvents.

    PubMed

    Shafer, Timothy J; Bushnell, Philip J; Benignus, Vernon A; Woodward, John J

    2005-12-01

    The mechanisms underlying the acute neurophysiological and behavioral effects of volatile organic compounds (VOCs) remain to be elucidated. However, the function of neuronal ion channels is perturbed by VOCs. The present study examined effects of toluene (TOL), trichloroethylene (TCE), and perchloroethylene (PERC) on whole-cell calcium current (ICa) in nerve growth factor-differentiated pheochromocytoma (PC12) cells. All three VOCs affected ICa in a reversible, concentration-dependent manner. At +10-mV test potentials, VOCs inhibited ICa, whereas at test potentials of -20 and -10 mV, they potentiated it. The order of potency for inhibition (IC50) was PERC (270 microM) > TOL (720 microM) > TCE (1525 microM). VOCs also changed ICa inactivation kinetics from a single- to double-exponential function. Voltage-ramp experiments suggested that VOCs shifted ICa activation in a hyperpolarizing direction; this was confirmed by calculating the half-maximal voltage of activation (V1/2, act) in the absence and presence of VOCs using the Boltzman equation. V(1/2, act) was shifted from approximately -2 mV in control to -11, -12, and -16 mV by TOL, TCE, and PERC, respectively. Similarly, VOCs shifted the half-maximal voltage of steady-state inactivation (V1/2, inact) from approximately -16 mV in control to -32, -35, and -20 mV in the presence of TOL, TCE, and PERC, respectively. Inhibition of ICa by TOL was confirmed in primary cultures of cortical neurons, where 827 microM TOL inhibited current by 61%. These data demonstrate that VOCs perturb voltage-sensitive Ca2+ channel function in neurons, an effect that could contribute to the acute neurotoxicity of these compounds. PMID:16109744

  18. Mercury-sensitive water channels as possible sensors of water potentials in pollen.

    PubMed

    Shachar-Hill, Bruria; Hill, Adrian E; Powell, Janet; Skepper, Jeremy N; Shachar-Hill, Yair

    2013-11-01

    The growing pollen tube is central to plant reproduction and is a long-standing model for cellular tip growth in biology. Rapid osmotically driven growth is maintained under variable conditions, which requires osmosensing and regulation. This study explores the mechanism of water entry and the potential role of osmosensory regulation in maintaining pollen growth. The osmotic permeability of the plasmalemma of Lilium pollen tubes was measured from plasmolysis rates to be 1.32±0.31×10(-3) cm s(-1). Mercuric ions reduce this permeability by 65%. Simulations using an osmotic model of pollen tube growth predict that an osmosensor at the cell membrane controls pectin deposition at the cell tip; inhibiting the sensor is predicted to cause tip bursting due to cell wall thinning. It was found that adding mercury to growing pollen tubes caused such a bursting of the tips. The model indicates that lowering the osmotic permeability per se does not lead to bursting but rather to thickening of the tip. The time course of induced bursting showed no time lag and was independent of mercury concentration, compatible with a surface site of action. The submaximal bursting response to intermediate mercuric ion concentration was independent of the concentration of calcium ions, showing that bursting is not due to a competitive inhibition of calcium binding or entry. Bursting with the same time course was also shown by cells growing on potassium-free media, indicating that potassium channels (implicated in mechanosensing) are not involved in the bursting response. The possible involvement of mercury-sensitive water channels as osmosensors and current knowledge of these in pollen cells are discussed. PMID:24098048

  19. Cross-resistance pattern and alternative herbicides for Cyperus difformis resistant to sulfonylurea herbicides in Korea.

    PubMed

    Kuk, Yong In; Kim, Kyung Hyun; Kwon, Oh Do; Lee, Do Jin; Burgos, Nilda R; Jung, Sunyo; Guh, Ja Ock

    2004-01-01

    A Cyperus difformis L accession from Chonnam province, Korea was tested for resistance to the sulfonylurea herbicide, imazosulfuron. The accession was confirmed to be resistant (R) and was cross-resistant to other sulfonylurea herbicides, bensulfuron-methyl, cyclosulfamuron and pyrazosulfuron-ethyl, the pyrimidinyl thiobenzoate herbicide, bispyribac-sodium, and the imidazolinone herbicide imazapyr, but not to imazaquin. Multiple resistance was tested using twelve herbicides with target sites other than acetolactate synthase (ALS). The R biotype could be controlled by other herbicides with different modes of action such as butachlor, carfentrazone-ethyl, clomeprop, dithiopyr, esprocarb, mefenacet, oxadiazon, pretilachlor, pyrazolate and thiobencarb, applied to soil at recommended rates. Several sulfonylurea herbicide-based mixtures can control both the R and S biotypes of C difformis, except sulfonylurea plus dimepiperate, molinate or pyriftalid, and pyrazolate plus butachlor. Although mixtures of sulfonylurea herbicides might be more effective, they should be avoided and used only in special cases. In terms of in vitro ALS activity, the R biotype was 1139-, 3583-, 1482-, 416-, 5- and 9-fold more resistant to bensulfuron-methyl, cyclosulfamuron, imazosulfuron, pyrazosulfuron-ethyl, bispyribac-sodium and imazapyr, respectively, than the S biotype. The in vivo ALS activity of the R biotype was also less affected by the sulfonylurea herbicides, imazosulfuron and pyrazosulfuron-ethyl, than the S biotype. Results of in vitro and in vivo ALS assays indicated that the resistance mechanism of C difformis to ALS inhibitor herbicides was primarily due to an alteration in the target enzyme, ALS. Greenhouse experiments showed delayed flowering and reduced seed production of the R biotype, which could possibly result in reduced fitness. This unusual observation needs to be confirmed in field situations. PMID:14727745

  20. Novel insights into the structural basis of pH-sensitivity in inward rectifier K+ channels Kir2.3.

    PubMed

    Ureche, Oana N; Baltaev, Ravshan; Ureche, Liviu; Strutz-Seebohm, Nathalie; Lang, Florian; Seebohm, Guiscard

    2008-01-01

    The Kir2 channels belong to a family of potassium selective channels with characteristic strong inward rectification. Heteromeric assemblies of Kir2.1, Kir2.2 and Kir2.3 channels underly membrane potential stabilizing currents in ventricular myocytes, neurons and skeletal muscle. Kir2 channels differ substantially in their sensitivity to extracellular pH. The extracellular histidine Kir2.3(H117) contributes to the pH dependence of K-channels containing Kir2.3. Here, we study the possibility of intramolecular interactions of the residue Kir2.3(H117) with conserved cysteines in close proximity to the selectivity filter. We engineered a cobalt coordination site and reduction/oxidation sensitivity in Kir2.3 by introduction of a cysteine into the putatively hydrogen bonding residue (Kir2.3(H117C)) confirming that this residue is in proximity to Kir2.3(C141). Using SCAM we determined the location of the Kir2.3(H117) in the outer pore mouth and incorporated these data into a 3D model. We conclude that formation of a hydrogen bond at low pH may stabilize the outer pore domain to favour the selectivity filter in a slightly distorted conformation thus reducing ion permeation. The data provide molecular insight into the unique pH regulation of inward rectifier channels. PMID:18453743

  1. Muscle KATP Channels: Recent Insights to Energy Sensing and Myoprotection

    PubMed Central

    Flagg, Thomas P.; Enkvetchakul, Decha; Koster, Joseph C.; Nichols, Colin G.

    2011-01-01

    ATP-sensitive (KATP) channels are present in the surface and internal membranes of cardiac, skeletal and smooth muscle cell, and provide a unique feedback between muscle cell metabolism and electrical activity. In so doing, they can play an important role in the control of contractility, particularly when cellular energetics are compromised, protecting the tissue against calcium overload and fiber damage, but the cost of this protection may be enhanced arrhythmic activity. Generated as complexes of Kir6.1 or Kir6.2 pore-forming subunits with regulatory sulfonylurea receptor subunits, SUR1 or SUR2, the differential assembly of KATP channels in different tissues gives rise to tissue-specific physiological and pharmacological regulation, and hence to the tissue-specific pharmacological control of contractility. The last ten years have provided insights to the regulation and role of muscle KATP channels, in large part driven by studies of mice in which the protein determinants of channel activity have been deleted or modified. As yet, few human diseases have been correlated with altered muscle KATP activity, but genetically modified animals give important insights to likely pathological roles of aberrant channel activity in different muscle types. PMID:20664073

  2. The cardioprotective effect of naringenin against ischemia-reperfusion injury through activation of ATP-sensitive potassium channel in rat.

    PubMed

    Meng, Li-Min; Ma, Hui-Jie; Guo, Hui; Kong, Qian-Qian; Zhang, Yi

    2016-09-01

    Naringenin (Nari) has antioxidative and anti-atherosclerosis effects, and activation of ATP-sensitive potassium channel (KATP) can offer cardiac protection. We hypothesized that Nari protects the heart against ischemia-reperfusion (I-R) injury through activation of KATP. Isolated hearts from adult male Sprague-Dawley rats experienced a 30-min global ischemia followed by 60-min reperfusion (120 min for the infarct size determination). The hearts were treated with Nari (NARI); Nari plus glibenclamide (GLI), a non-specific ATP-sensitive potassium channel blocker (NARI+GLI); and Nari plus 5-hydroxy decanoic acid (5-HD), a mitochondrial membrane ATP-sensitive potassium channel blocker (NARI+5-HD). The left ventricular pressure, lactate dehydrogenates (LDH) in coronary effluent, superoxide dismutase (SOD) and malondialdehyde (MDA) in myocardium, and myocardial infarct area were measured. Nari above 2.5 μmol/L improved the recovery of left ventricular function, decreased LDH in coronary effluent, and reduced myocardial infarct area. The SOD activity was increased and MDA was decreased in Nari-treated myocardium. The cardioprotective effect of Nari was canceled by GLI and 5-HD. In conclusion, Nari has a cardioprotective effect against I-R injury, which may be carried out through activating ATP-sensitive potassium channels in both cell and mitochondrial membrane, and enhancing myocardial antioxidant capacity. PMID:27408985

  3. Potent modulation of tetrodotoxin-sensitive and tetrodotoxin-resistant sodium channels by the type II pyrethroid deltamethrin.

    PubMed

    Tabarean, I V; Narahashi, T

    1998-03-01

    In both tetrodotoxin-resistant (TTX-R) and tetrodotoxin-sensitive (TTX-S) sodium channels, deltamethrin greatly prolonged the current during step depolarizing pulse and caused a large and prolonged slow tail current. The activation was shifted by 20 mV in the hyperpolarizing direction. These changes in channel kinetics account for the prolongation of action potential, membrane depolarization and spontaneous discharges in the deltamethrin-treated neurons. The slow tail current of TTX-S sodium channels rose and decayed slowly, showing a hook. By contrast, the slow tail current of TTX-R channels occurred quickly upon step repolarization. The slow tail current in deltamethrin-treated cells developed slowly during a depolarizing pulse, with a time constant in the order of several milliseconds. The percentages of sodium channels modified by deltamethrin were measured as a function of the deltamethrin concentration. The EC50 values were 0.53 microM and 0.37 microM for TTX-S and TTX-R sodium channels, respectively. However, when compared at the level of 5% modification, the potency of deltamethrin for TTX-R channels was 40 to 50 times higher than that for TTX-S channels. Deltamethrin-induced large and prolonged tail current was hardly reversed after prolonged washout with drug-free solution. However, after application of tetramethrin, it was converted into a much shorter tail current. Washout with solution devoid of tetramethrin and deltamethrin resulted in rapid reappearance of the deltamethrin-type tail current. These results suggest that the deltamethrin and tetramethrin share a binding site on the sodium channel and that the slow onset and offset of deltamethrin action are controlled by the rates at which deltamethrin moves and unbinds from the membrane lipid phase rather than by the rate of deltamethrin binding to the sodium channel site. PMID:9495855

  4. The Walter B. Cannon Physiology in Perspective Lecture, 2007. ATP-sensitive K+ channels and disease: from molecule to malady.

    PubMed

    Ashcroft, Frances M

    2007-10-01

    This essay is based on a lecture given to the American Physiological Society in honor of Walter B. Cannon, an advocate of homeostasis. It focuses on the role of the ATP-sensitive potassium K(+) (K(ATP)) channel in glucose homeostasis and, in particular, on its role in insulin secretion from pancreatic beta-cells. The beta-cell K(ATP) channel comprises pore-forming Kir6.2 and regulatory SUR1 subunits, and mutations in either type of subunit can result in too little or too much insulin release. Here, I review the latest information on the relationship between K(ATP) channel structure and function, and consider how mutations in the K(ATP) channel genes lead to neonatal diabetes or congenital hyperinsulinism. PMID:17652156

  5. The bile acid-sensitive ion channel (BASIC), the ignored cousin of ASICs and ENaC

    PubMed Central

    Wiemuth, Dominik; Assmann, Marc; Gründer, Stefan

    2014-01-01

    The DEG/ENaC gene family of ion channels is characterized by a high degree of structural similarity and an equally high degree of diversity concerning the physiological function. In humans and rodents, the DEG/ENaC family comprises 2 main subgroups: the subunits of the epithelial Na+ channel (ENaC) and the subunits of the acid sensing ion channels (ASICs). The bile acid-sensitive channel (BASIC), previously known as BLINaC or INaC, represents a third subgroup within the DEG/ENaC family. Although BASIC was identified more than a decade ago, very little is known about its physiological function. Recent progress in the characterization of this neglected member of the DEG/ENaC family, which is summarized in this focused review, includes the discovery of surprising species differences, its pharmacological characterization, and the identification of bile acids as putative natural activators. PMID:24365967

  6. Differential sensitivity of TREK–1, TREK–2 and TRAAK background potassium channels to the polycationic dye ruthenium red

    PubMed Central

    Braun, G; Lengyel, M; Enyedi, P; Czirják, G

    2015-01-01

    Background and Purpose Pharmacological separation of the background potassium currents of closely related K2P channels is a challenging problem. We previously demonstrated that ruthenium red (RR) inhibits TASK-3 (K2P9.1), but not TASK-1 (K2P3.1) channels. RR has been extensively used to distinguish between TASK currents in native cells. In the present study, we systematically investigate the RR sensitivity of a more comprehensive set of K2P channels. Experimental Approach K+ currents were measured by two-electrode voltage clamp in Xenopus oocytes and by whole-cell patch clamp in mouse dorsal root ganglion (DRG) neurons. Key Results RR differentiates between two closely related members of the TREK subfamily. TREK-2 (K2P10.1) proved to be highly sensitive to RR (IC50 = 0.2 μM), whereas TREK-1 (K2P2.1) was not affected by the compound. We identified aspartate 135 (D135) as the target of the inhibitor in mouse TREK-2c. D135 lines the wall of the extracellular ion pathway (EIP), a tunnel structure through the extracellular cap characteristic for K2P channels. TREK-1 contains isoleucine in the corresponding position. The mutation of this isoleucine (I110D) rendered TREK-1 sensitive to RR. The third member of the TREK subfamily, TRAAK (K2P4.1) was more potently inhibited by ruthenium violet, a contaminant in some RR preparations, than by RR. DRG neurons predominantly express TREK-2 and RR-resistant TREK-1 and TRESK (K2P18.1) background K+ channels. We detected the RR-sensitive leak K+ current component in DRG neurons. Conclusions and Implications We propose that RR may be useful for distinguishing TREK-2 (K2P10.1) from TREK-1 (K2P2.1) and other RR-resistant K2P channels in native cells. PMID:25409575

  7. Expression of temperature-sensitive ion channel TRPM8 in sperm cells correlates with vertebrate evolution

    PubMed Central

    Majhi, Rakesh Kumar; Saha, Somdatta; Kumar, Ashutosh; Ghosh, Arijit; Swain, Nirlipta; Goswami, Luna; Mohapatra, Pratyush; Maity, Apratim; Kumar Sahoo, Vivek

    2015-01-01

    Transient Receptor Potential cation channel, subfamily Melastatin, member 8 (TRPM8) is involved in detection of cold temperature, different noxious compounds and in execution of thermo- as well as chemo-sensitive responses at cellular levels. Here we explored the molecular evolution of TRPM8 by analyzing sequences from various species. We elucidate that several regions of TRPM8 had different levels of selection pressure but the 4th–5th transmembrane regions remain highly conserved. Analysis of synteny suggests that since vertebrate origin, TRPM8 gene is linked with SPP2, a bone morphogen. TRPM8, especially the N-terminal region of it, seems to be highly variable in human population. We found 16,656 TRPM8 variants in 1092 human genomes with top variations being SNPs, insertions and deletions. A total of 692 missense mutations are also mapped to human TRPM8 protein of which 509 seem to be delateroiours in nature as supported by Polyphen V2, SIFT and Grantham deviation score. Using a highly specific antibody, we demonstrate that TRPM8 is expressed endogenously in the testis of rat and sperm cells of different vertebrates ranging from fish to higher mammals. We hypothesize that TRPM8 had emerged during vertebrate evolution (ca 450 MYA). We propose that expression of TRPM8 in sperm cell and its role in regulating sperm function are important factors that have guided its molecular evolution, and that these understandings may have medical importance. PMID:26500819

  8. Surface-sensitive two-dimensional magneto-fingerprint in mesoscopic Bi2Se3 channels.

    PubMed

    Kandala, Abhinav; Richardella, Anthony; Zhang, Duming; Flanagan, Thomas C; Samarth, Nitin

    2013-06-12

    Periodic Aharonov–Bohm and Altshuler–Aronov–Spivak oscillations have traditionally been observed in lateral transport through patterned mesoscopic loops of diffusive conductors. However, our studies of perpendicular-to-plane magnetotransport in straight-channel, diffusive devices of epitaxial Bi2Se3 surprisingly reveal signatures of Aharonov–Bohm orbits, periodic conductance fluctuation magneto-fingerprints, even though the devices are not explicitly patterned into loops. We show that the length scale of these orbits corresponds to the typical perimeter of triangular terraces found on the surface of these thin film devices, strongly suggesting that the periodic magneto-fingerprint arises from coherent scattering of electron waves from the step-edges. Our interpretation is bolstered by control measurements in devices without such surface morphology that only show a conventional, aperiodic magneto-fingerprint. These results show that lithographically patterned Bi2Se3 devices provide a novel class of mesoscopic physical systems for systematic studies of coherent surface sensitive transport. PMID:23642037

  9. Evaluation of six channelized Hotelling observers in combination with a contrast sensitivity function to predict human observer performance

    NASA Astrophysics Data System (ADS)

    Goffi, Marco; Veldkamp, Wouter J. H.; van Engen, Ruben E.; Bouwman, Ramona W.

    2015-03-01

    Standard methods to quantify image quality (IQ) may not be adequate for clinical images since they depend on uniform backgrounds and linearity. Statistical model observers are not restricted to these limitations and might be suitable for IQ evaluation of clinical images. One of these statistical model observers is the channelized Hotelling observer (CHO), where the images are filtered by a set of channels. The aim of this study was to evaluate six different channel sets, with an additional filter to simulate the human contrast sensitivity function (CSF), in their ability to predict human observer performance. For this evaluation a two alternative forced choice experiment was performed with two types of background structures (white noise (WN) and clustered lumpy background (CLB)), 5 disk-shaped objects with different diameters and 3 different signal energies. The results show that the correlation between human and model observers have a diameter dependency for some channel sets in combination with CLBs. The addition of the CSF reduces this diameter dependency and in some cases improves the correlation coefficient between human- and model observer. For the CLB the Partial Least Squares channel set shows the highest correlation with the human observer (r2=0.71) and for WN backgrounds it was the Gabor-channel set with CSF (r2=0.72). This study showed that for some channels there is a high correlation between human and model observer, which suggests that the CHO has potential as a tool for IQ analysis of digital mammography systems.

  10. Influence of Thromboxane A2 on the Regulation of Adenosine Triphosphate-Sensitive Potassium Channels in Mouse Ventricular Myocytes

    PubMed Central

    Jeong, In Seok; Cho, Hwa Jin; Cho, Jeong Gwan; Kim, Sang Hyung; Na, Kook Joo

    2016-01-01

    Background and Objectives Adenosine triphosphate (ATP)-sensitive potassium (KATP) channels play an important role in myocardial protection. We examined the effects of thromboxane A2 on the regulation of KATP channel activity in single ventricular myocytes. Subjects and Methods Single ventricular myocytes were isolated from the hearts of adult Institute of Cancer Research (ICR) mice by enzymatic digestion. Single channel activity was recorded by excised inside-out and cell-attached patch clamp configurations at −60 mV holding potential during the perfusion of an ATP-free K-5 solution. Results In the excised inside-out patches, the thromboxane A2 analog, U46619, decreased the KATP channel activity in a dose-dependent manner; however, the thromboxane A2 receptor antagonist, SQ29548, did not significantly attenuate the inhibitory effect of U46619. In the cell-attached patches, U46619 inhibited dinitrophenol (DNP)-induced KATP channel activity in a dose-dependent manner, and SQ29548 attenuated the inhibitory effects of U46619 on DNP-induced KATP channel activity. Conclusion Thromboxane A2 may inhibit KATP channel activity, and may have a harmful effect on ischemic myocardium. PMID:27482267

  11. TMEM16F is a component of a Ca2+-activated Cl- channel but not a volume-sensitive outwardly rectifying Cl- channel.

    PubMed

    Shimizu, Takahiro; Iehara, Takahiro; Sato, Kaori; Fujii, Takuto; Sakai, Hideki; Okada, Yasunobu

    2013-04-15

    TMEM16 (transmembrane protein 16) proteins, which possess eight putative transmembrane domains with intracellular NH2- and COOH-terminal tails, are thought to comprise a Cl(-) channel family. The function of TMEM16F, a member of the TMEM16 family, has been greatly controversial. In the present study, we performed whole cell patch-clamp recordings to investigate the function of human TMEM16F. In TMEM16F-transfected HEK293T cells but not TMEM16K- and mock-transfected cells, activation of membrane currents with strong outward rectification was found to be induced by application of a Ca(2+) ionophore, ionomycin, or by an increase in the intracellular free Ca(2+) concentration. The free Ca(2+) concentration for half-maximal activation of TMEM16F currents was 9.6 μM, which is distinctly higher than that for TMEM16A/B currents. The outwardly rectifying current-voltage relationship for TMEM16F currents was not changed by an increase in the intracellular Ca(2+) level, in contrast to TMEM16A/B currents. The Ca(2+)-activated TMEM16F currents were anion selective, because replacing Cl(-) with aspartate(-) in the bathing solution without changing cation concentrations caused a positive shift of the reversal potential. The anion selectivity sequence of the TMEM16F channel was I(-) > Br(-) > Cl(-) > F(-) > aspartate(-). Niflumic acid, a Ca(2+)-activated Cl(-) channel blocker, inhibited the TMEM16F-dependent Cl(-) currents. Neither overexpression nor knockdown of TMEM16F affected volume-sensitive outwardly rectifying Cl(-) channel (VSOR) currents activated by osmotic swelling or apoptotic stimulation. These results demonstrate that human TMEM16F is an essential component of a Ca(2+)-activated Cl(-) channel with a Ca(2+) sensitivity that is distinct from that of TMEM16A/B and that it is not related to VSOR activity. PMID:23426967

  12. Three mutations identified in the voltage-sensitive sodium channel alpha-subunit gene of permethrin-resistant human head lice reduce the permethrin sensitivity of house fly Vssc1 sodium channels expressed in Xenopus oocytes.

    PubMed

    SupYoon, Kyong; Symington, Steven B; Hyeock Lee, Si; Soderlund, David M; Marshall Clark, J

    2008-03-01

    Point mutations in the para-orthologous sodium channel alpha-subunit of the head louse (M815I, T917I, and L920F) are associated with permethrin resistance and DDT resistance. These mutations were inserted in all combinations using site-directed mutagenesis at the corresponding amino acid sequence positions (M827I, T929I, and L932F) of the house fly para-orthologous voltage-sensitive sodium channel alpha-subunit (Vssc1(WT)) gene and heterologously co-expressed with the sodium channel auxiliary subunit of house fly (Vsscbeta) in Xenopus oocytes. The double mutant possessing M827I and T929I (Vssc1(MITI)/Vsscbeta) caused a approximately 4.0mV hyperpolarizing shift and the triple mutant, Vssc1(MITILF)/Vsscbeta, caused a approximately 3.2mV depolarizing shift in the voltage dependence of activation curves. Vssc1(MITI)/Vsscbeta, Vssc1(TILF)/Vsscbeta, and Vssc1(MITILF)/Vsscbeta caused depolarizing shifts ( approximately 6.6, approximately 7.6, and approximately 8.8mV, respectively) in the voltage dependence of steady-state inactivation curves. The M827I and L932F mutations reduced permethrin sensitivity when expressed alone but the T929I mutation, either alone or in combination, virtually abolished permethrin sensitivity. Thus, the T929I mutation is the principal cause of permethrin resistance in head lice. Comparison of the expression rates of channels containing single, double and triple mutations with that of Vssc1(WT)/Vsscbeta channels indicates that the M827I mutation may play a role in rescuing the decreased expression of channels containing T929I. PMID:18252244

  13. Regulation of Cardiac ATP-sensitive Potassium Channel Surface Expression by Calcium/Calmodulin-dependent Protein Kinase II*

    PubMed Central

    Sierra, Ana; Zhu, Zhiyong; Sapay, Nicolas; Sharotri, Vikas; Kline, Crystal F.; Luczak, Elizabeth D.; Subbotina, Ekaterina; Sivaprasadarao, Asipu; Snyder, Peter M.; Mohler, Peter J.; Anderson, Mark E.; Vivaudou, Michel; Zingman, Leonid V.; Hodgson-Zingman, Denice M.

    2013-01-01

    Cardiac ATP-sensitive potassium (KATP) channels are key sensors and effectors of the metabolic status of cardiomyocytes. Alteration in their expression impacts their effectiveness in maintaining cellular energy homeostasis and resistance to injury. We sought to determine how activation of calcium/calmodulin-dependent protein kinase II (CaMKII), a central regulator of calcium signaling, translates into reduced membrane expression and current capacity of cardiac KATP channels. We used real-time monitoring of KATP channel current density, immunohistochemistry, and biotinylation studies in isolated hearts and cardiomyocytes from wild-type and transgenic mice as well as HEK cells expressing wild-type and mutant KATP channel subunits to track the dynamics of KATP channel surface expression. Results showed that activation of CaMKII triggered dynamin-dependent internalization of KATP channels. This process required phosphorylation of threonine at 180 and 224 and an intact 330YSKF333 endocytosis motif of the KATP channel Kir6.2 pore-forming subunit. A molecular model of the μ2 subunit of the endocytosis adaptor protein, AP2, complexed with Kir6.2 predicted that μ2 docks by interaction with 330YSKF333 and Thr-180 on one and Thr-224 on the adjacent Kir6.2 subunit. Phosphorylation of Thr-180 and Thr-224 would favor interactions with the corresponding arginine- and lysine-rich loops on μ2. We concluded that calcium-dependent activation of CaMKII results in phosphorylation of Kir6.2, which promotes endocytosis of cardiac KATP channel subunits. This mechanism couples the surface expression of cardiac KATP channels with calcium signaling and reveals new targets to improve cardiac energy efficiency and stress resistance. PMID:23223335

  14. AKAP150, a switch to convert mechano-, pH- and arachidonic acid-sensitive TREK K+ channels into open leak channels

    PubMed Central

    Sandoz, Guillaume; Thümmler, Susanne; Duprat, Fabrice; Feliciangeli, Sylvain; Vinh, Joëlle; Escoubas, Pierre; Guy, Nicolas; Lazdunski, Michel; Lesage, Florian

    2006-01-01

    TREK channels are unique among two-pore-domain K+ channels. They are activated by polyunsaturated fatty acids (PUFAs) including arachidonic acid (AA), phospholipids, mechanical stretch and intracellular acidification. They are inhibited by neurotransmitters and hormones. TREK-1 knockout mice have impaired PUFA-mediated neuroprotection to ischemia, reduced sensitivity to volatile anesthetics and altered perception of pain. Here, we show that the A-kinase-anchoring protein AKAP150 is a constituent of native TREK-1 channels. Its binding to a key regulatory domain of TREK-1 transforms low-activity outwardly rectifying currents into robust leak conductances insensitive to AA, stretch and acidification. Inhibition of the TREK-1/AKAP150 complex by Gs-coupled receptors such as serotonin 5HT4sR and noradrenaline β2AR is as extensive as for TREK-1 alone, but is faster. Inhibition of TREK-1/AKAP150 by Gq-coupled receptors such as serotonin 5HT2bR and glutamate mGluR5 is much reduced when compared to TREK-1 alone. The association of AKAP150 with TREK channels integrates them into a postsynaptic scaffold where both G-protein-coupled membrane receptors (as demonstrated here for β2AR) and TREK-1 dock simultaneously. PMID:17110924

  15. Functional implications of the localization and activity of acid-sensitive channels in rat peripheral nervous system.

    PubMed

    Alvarez de la Rosa, Diego; Zhang, Ping; Shao, Deren; White, Fletcher; Canessa, Cecilia M

    2002-02-19

    Acid-sensitive ion channels (ASIC) are proton-gated ion channels expressed in neurons of the mammalian central and peripheral nervous systems. The functional role of these channels is still uncertain, but they have been proposed to constitute mechanoreceptors and/or nociceptors. We have raised specific antibodies for ASIC1, ASIC2, ASIC3, and ASIC4 to examine the distribution of these proteins in neurons from dorsal root ganglia (DRG) and to determine their subcellular localization. Western blot analysis demonstrates that all four ASIC proteins are expressed in DRG and sciatic nerve. Immunohistochemical experiments and functional measurements of unitary currents from the ASICs with the patch-clamp technique indicate that ASIC1 localizes to the plasma membrane of small-, medium-, and large-diameter cells, whereas ASIC2 and ASIC3 are preferentially in medium to large cells. Neurons coexpressing ASIC2 and ASIC3 form predominantly heteromeric ASIC2-3 channels. Two spliced forms, ASIC2a and ASIC2b, colocalize in the same population of DRG neurons. Within cells, the ASICs are present mainly on the plasma membrane of the soma and cellular processes. Functional studies indicate that the pH sensitivity for inactivation of ASIC1 is much higher than the one for activation; hence, increases in proton concentration will inactivate the channel. These functional properties and localization in DRG have profound implications for the putative functional roles of ASICs in the nervous system. PMID:11842212

  16. Functional implications of the localization and activity of acid-sensitive channels in rat peripheral nervous system

    PubMed Central

    Alvarez de la Rosa, Diego; Zhang, Ping; Shao, Deren; White, Fletcher; Canessa, Cecilia M.

    2002-01-01

    Acid-sensitive ion channels (ASIC) are proton-gated ion channels expressed in neurons of the mammalian central and peripheral nervous systems. The functional role of these channels is still uncertain, but they have been proposed to constitute mechanoreceptors and/or nociceptors. We have raised specific antibodies for ASIC1, ASIC2, ASIC3, and ASIC4 to examine the distribution of these proteins in neurons from dorsal root ganglia (DRG) and to determine their subcellular localization. Western blot analysis demonstrates that all four ASIC proteins are expressed in DRG and sciatic nerve. Immunohistochemical experiments and functional measurements of unitary currents from the ASICs with the patch–clamp technique indicate that ASIC1 localizes to the plasma membrane of small-, medium-, and large-diameter cells, whereas ASIC2 and ASIC3 are preferentially in medium to large cells. Neurons coexpressing ASIC2 and ASIC3 form predominantly heteromeric ASIC2–3 channels. Two spliced forms, ASIC2a and ASIC2b, colocalize in the same population of DRG neurons. Within cells, the ASICs are present mainly on the plasma membrane of the soma and cellular processes. Functional studies indicate that the pH sensitivity for inactivation of ASIC1 is much higher than the one for activation; hence, increases in proton concentration will inactivate the channel. These functional properties and localization in DRG have profound implications for the putative functional roles of ASICs in the nervous system. PMID:11842212

  17. Comparison of vildagliptin as an add-on therapy and sulfonylurea dose-increasing therapy in patients with inadequately controlled type 2 diabetes using metformin and sulfonylurea (VISUAL study): A randomized trial.

    PubMed

    Hong, A Ram; Lee, Jeun; Ku, Eu Jeong; Hwangbo, Yul; Kim, Kyoung Min; Moon, Jae Hoon; Choi, Sung Hee; Jang, Hak Chul; Lim, Soo

    2015-07-01

    The aim of present study is to compare the efficacy and safety of adding vildagliptin with sulfonylurea dose-increasing as an active comparator in patients who had inadequately controlled type 2 diabetes mellitus (T2DM) using metformin plus sulfonylurea in real clinical practice. Patients using metformin plus sulfonylurea were assigned to either vildagliptin add-on (50 mg twice a day, n=172) or sulfonylurea dose-increasing by 50% (n=172) treatment groups. The primary endpoint was a change in HbA(1c) after 24 weeks. The secondary endpoints were patients achieving HbA(1c)≤7.0% (53 mmol/mol) and changes in the fasting plasma glucose (FPG), 2-h postprandial glucose (2pp), lipid profiles, and urine albumin-to-creatinine ratio. Body weight and hypoglycemia were also investigated. The mean HbA(1c) at baseline was 8.6% (70 mmol/mol) in both groups. At week 24, the adjusted mean HbA(1c) levels decreased by -1.19% (-13.09 mmol/mol) with vildagliptin add-on and -0.46% (-5.06 mmol/mol) with sulfonylurea (P<0.001). Significantly more vildagliptin add-on patients achieved HbA(1c)≤7.0% (53 mmol/mol) than did sulfonylurea patients (40.1% vs. 7.9%; P<0.001). Greater reductions in FPG and 2pp were observed with vildagliptin add-on than with sulfonylurea (P<0.001). The vildagliptin add-on group exhibited no clinically relevant weight gain and had a lower incidence of hypoglycemia compared with the sulfonylurea group. Vildagliptin add-on therapy might be a suitable option for patients with T2DM that is controlled inadequately by metformin and sulfonylurea, based on its greater glucose control and better safety profile (ClinicalTrial.gov: NCT01099137). PMID:26003889

  18. Single Nisoldipine-Sensitive Calcium Channels in Smooth Muscle Cells Isolated from Rabbit Mesenteric Artery

    NASA Astrophysics Data System (ADS)

    Worley, Jennings F.; Deitmer, Joachim W.; Nelson, Mark T.

    1986-08-01

    Single smooth muscle cells were enzymatically isolated from the rabbit mesenteric artery. At physiological levels of external Ca, these cells were relaxed and contracted on exposure to norepinephrine, caffeine, or high levels of potassium. The patch-clamp technique was used to measure unitary currents through single channels in the isolated cells. Single channels were selective for divalent cations and exhibited two conductance levels, 8 pS and 15 pS. Both types of channels were voltage-dependent, and channel activity occurred at potentials positive to -40 mV. The activity of both channel types was almost completely inhibited by 50 nM nisoldipine. These channels appear to be the pathways for voltage-dependent Ca influx in vascular smooth muscle and may be the targets of the clinically used dihydropyridines.

  19. A Comparative Proteomic Analysis of the KATP channel Complex in Different Tissue Types

    PubMed Central

    Kefaloyianni, Eirini; Lyssand, John S.; Moreno, Cesar; Delaroche, Diane; Hong, Miyoun; Fenyö, David; Mobbs, Charles V.; Neubert, Thomas A.; Coetzee, William A.

    2013-01-01

    ATP-sensitive K+ (KATP) channels are expressed ubiquitously, but have diverse roles in various organs and cells. Their diversity can partly be explained by distinct tissue-specific compositions of four copies of the pore-forming inward rectifier potassium channel subunits (Kir6.1 and/or Kir6.2) and four regulatory sulfonylurea receptor subunits (SUR1 and/or SUR2). Channel function and/or subcellular localization also can be modified by the proteins with which they transiently or permanently interact to generate even more diversity. We performed a quantitative proteomic analysis of KATP channel complexes in the heart, endothelium, insulin-secreting min6 cells (pancreatic β-cell like) and the hypothalamus to identify proteins with which they interact in different tissues. Glycolysis is an over-represented pathway in identified proteins of the heart, min6 cells and the endothelium. Proteins with other energy metabolic functions were identified in the hypothalamic samples. These data suggest that the metabolo-electrical coupling conferred by KATP channels is conferred partly by proteins with which they interact. A large number of identified cytoskeletal and trafficking proteins suggests endocytic recycling may help control KATP channel surface density and/or subcellular localization. Overall, our data demonstrate that KATP channels in different tissues may assemble with proteins having common functions, but that tissue-specific complex organization also occurs. PMID:23197389

  20. Tetrodotoxin-sensitive calcium-conducting channels in the rat hippocampal CA1 region.

    PubMed Central

    Akaike, N; Takahashi, K

    1992-01-01

    1. Tetrodotoxin (TTX)-sensitive Ca2+ conducting channels which produce a transient inward current were investigated in pyramidal neurones freshly dissociated from the dorsal part of rat hippocampal CA1 region by the use of the suction-pipette technique, which allows for intracellular perfusion under a single-electrode voltage clamp. 2. In all cells superfused with Na(+)- and K(+)-free external solution containing 10 mM-Ca2+ and 10(-5) M-La3+, a transient inward Ca2+ current was evoked by a step depolarization to potentials more positive than about -50 mV from a holding potential (VH) of -100 mV. This current was inhibited by either removing the extracellular Ca2+ or adding TTX (termed as 'TTX-ICa'). 3. Activation and inactivation processes of the TTX-ICa were highly potential dependent at 20-22 degrees C, and the latter was fitted by a double exponential function. The time to peak of the current decreased from 5.0 to 2.3 ms at a test potential change from -50 to 0 mV. The time constants of the current decay decreased from 2.8 to 2.2 ms for fast component (tau if) and from 16.0 to 8.2 ms for slow component (tau is) at a potential change from -35 to -10 mV. 4. The TTX-ICa was activated at threshold potential of about -55 mV and reached full activation at -30 mV. The steady-state inactivation of TTX-ICa could be fitted by a Boltzmann equation with a slope factor of 6.0 mV and a half-inactivation voltage of -72.5 mV. 5. Biphasic recovery (reactivation) from the complete inactivation of TTX-ICa was observed. The time constant of the major component (78.8 to 91.6% of total) of the reactivation was 13.1 ms, and that of the minor one was 120 to 240 ms. Therefore, TTX-ICa remained fairly constant at a train of stimulation up to 3 Hz. However, the inhibition of current amplitude occurred as the repetitive stimulation increased more than 10 Hz, and considerable tonic inhibition occurred with increasing stimulation frequency. 6. When the peak amplitudes in the individual

  1. 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.

  2. Oxygen-Sensitive Calcium Channels in Vascular Smooth Muscle and Their Possible Role in Hypoxic Arterial Relaxation

    NASA Astrophysics Data System (ADS)

    Franco-Obregon, A.; Urena, J.; Lopez-Barneo, J.

    1995-05-01

    We have investigated the modifications of cytosolic [Ca2+] and the activity of Ca2+ channels in freshly dispersed arterial myocytes to test whether lowering O_2 tension (PO_2) directly influences Ca2+ homeostasis in these cells. Unclamped cells loaded with fura-2 AM exhibit oscillations of cytosolic Ca2+ whose frequency depends on extracellular Ca2+ influx. Switching from a PO_2 of 150 to 20 mmHg leads to a reversible attenuation of the Ca2+ oscillations. In voltage-clamped cells, hypoxia reversibly reduces the influx of Ca2+ through voltage-dependent channels, which can account for the inhibition of the Ca2+ oscillations. Low PO_2 selectively inhibits L-type Ca2+ channel activity, whereas the current mediated by T-type channels is unaltered by hypoxia. The effect of low PO_2 on the L-type channels is markedly voltage dependent, being more apparent with moderate depolarizations. These findings demonstrate the existence of O_2-sensitive, voltage-dependent, Ca2+ channels in vascular smooth muscle that may critically contribute to the local regulation of circulation.

  3. The ATP-sensitive K+-channel (KATP) controls early left-right patterning in Xenopus and chick embryos

    PubMed Central

    Aw, Sherry; Koster, Joseph; Pearson, Wade; Nichols, Colin; Shi, Nian-Qing; Carneiro, Katia; Levin, Michael

    2010-01-01

    Consistent left-right asymmetry requires specific ion currents. We characterize a novel laterality determinant in Xenopus laevis: the ATP-sensitive K+-channel (KATP). Expression of specific dominant-negative mutants of the Xenopus Kir6.1 pore subunit of the KATP channel induced randomization of asymmetric organ positioning. Spatio-temporally controlled loss-of-function experiments revealed that the KATP channel functions asymmetrically in LR patterning during very early cleavage stages, and also symmetrically during the early blastula stages, a period when heretofore largely unknown events transmit LR patterning cues. Blocking KATP channel activity randomizes the expression of the left-sided transcription of Nodal. Immunofluorescence analysis revealed that XKir6.1 is localized to basal membranes on the blastocoel roof and cell-cell junctions. A tight junction integrity assay showed that KATP channels are required for proper tight junction function in early Xenopus embryos. We also present evidence that this function may be conserved to the chick, as inhibition of KATP in the primitive streak of chick embryos randomizes the expression of the left-sided gene Sonic hedgehog. We propose a model by which KATP channels control LR patterning via regulation of tight junctions. PMID:20643119

  4. Highly Sensitive Multi-Channel IDC Sensor Array for Low Concentration Taste Detection

    PubMed Central

    Khan, Md. Rajibur Rahaman; Kang, Shin-Won

    2015-01-01

    In this study, we designed and developed an interdigitated capacitor (IDC)-based taste sensor array to detect different taste substances. The designed taste sensing array has four IDC sensing elements. The four IDC taste sensing elements of the array are fabricated by incorporating four different types of lipids into the polymer, dioctyl phenylphosphonate (DOPP) and tetrahydrofuran (THF) to make the respective dielectric materials that are individually placed onto an interdigitated electrode (IDE) via spin coating. When the dielectric material of an IDC sensing element comes into contact with a taste substance, its dielectric properties change with the capacitance of the IDC sensing element; this, in turn, changes the voltage across the IDC, as well as the output voltage of each channel of the system. In order to assess the effectiveness of the sensing system, four taste substances, namely sourness (HCl), saltiness (NaCl), sweetness (glucose) and bitterness (quinine-HCl), were tested. The IDC taste sensor array had rapid response and recovery times of about 12.9 s and 13.39 s, respectively, with highly stable response properties. The response property of the proposed IDC taste sensor array was linear, and its correlation coefficient R2 was about 0.9958 over the dynamic range of the taste sensor array as the taste substance concentration was varied from 1 μM to 1 M. The proposed IDC taste sensor array has several other advantages, such as real-time monitoring capabilities, high sensitivity 45.78 mV/decade, good reproducibility with a standard deviation of about 0.029 and compactness, and the circuitry is based on readily available and inexpensive electronic components. The proposed IDC taste sensor array was compared with the potentiometric taste sensor with respect to sensitivity, dynamic range width, linearity and response time. We found that the proposed IDC sensor array has better performance. Finally, principal component analysis (PCA) was applied to

  5. Evaluation of tribenuron-methyl on sulfonylurea herbicide tolerant lettuce germplasm

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The gene for sulfonylurea (SU) herbicide resistance discovered in a prickly lettuce population in Idaho was transferred to domestic lettuce by University of Idaho researchers. California researchers acquired the Idaho lettuce germplasm, “IDBR-1” and transferred the SU resistance gene to five common ...

  6. Crystal structures of two novel sulfonylurea herbicides in complex with Arabidopsis thaliana acetohydroxyacid synthase

    SciTech Connect

    Wang, Jian-Guo; Lee, Patrick K.-M.; Dong, Yu-Hui; Pang, Siew Siew; Duggleby, Ronald G.; Li, Zheng-Ming; Guddat, Luke W.

    2009-08-17

    Acetohydroxyacid synthase (AHAS; EC 2.2.1.6) is the first enzyme in the biosynthetic pathway of the branched-chain amino acids. It catalyzes the conversion of two molecules of pyruvate into 2-acetolactate or one molecule of pyruvate and one molecule of 2-ketobutyrate into 2-aceto-2-hydroxybutyrate. AHAS requires the cofactors thiamine diphosphate (ThDP), Mg{sup 2+} and FAD for activity. The herbicides that target this enzyme are effective in protecting a broad range of crops from weed species. However, resistance in the field is now a serious problem worldwide. To address this, two new sulfonylureas, monosulfuron and monosulfuron ester, have been developed as commercial herbicides in China. These molecules differ from the traditional sulfonylureas in that the heterocyclic ring attached to the nitrogen atom of the sulfonylurea bridge is monosubstituted rather than disubstituted. The structures of these compounds in complex with the catalytic subunit of Arabidopsis thaliana AHAS have been determined to 3.0 and 2.8 {angstrom}, respectively. In both complexes, these molecules are bound in the tunnel leading to the active site, such that the sole substituent of the heterocyclic ring is buried deepest and oriented towards the ThDP. Unlike the structures of Arabidopsis thaliana AHAS in complex with the classic disubstituted sulfonylureas, where ThDP is broken, this cofactor is intact and present most likely as the hydroxylethyl intermediate.

  7. Mass spectrometry of the photolysis of sulfonylurea herbicides in Prairie waters.

    PubMed

    Headley, John V; Du, Jing-Long; Peru, Kerry M; McMartin, Dena W

    2010-01-01

    This review of mass spectrometry of sulfonylurea herbicides includes a focus on studies relevant to Canadian Prairie waters. Emphasis is given to data gaps in the literature for the rates of photolysis of selected sulfonylurea herbicides in different water matrices. Specifically, results are evaluated for positive ion electrospray tandem mass spectrometry with liquid chromatography separation for the study of the photolysis of chlorsulfuron, tribenuron-methyl, thifensulfuron-methyl, metsulfuron-methyl, and ethametsulfuron-methyl. LC-MS/MS is shown to be the method of choice for the quantification of sulfonylurea herbicides with instrumental detection limits ranging from 1.3 to 7.2 pg (on-column). Tandem mass spectrometry coupled with the use of authentic standards likewise has proven to be well suited for the identification of transformation products. To date, however, the power of time-of-flight MS and ultrahigh resolution MS has not been exploited fully for the identification of unknown photolysis products. Dissipation of the herbicides under natural sunlight fit pseudo-first-order kinetics with half-life values ranging from 4.4 to 99 days. For simulated sunlight, radiation wavelengths shorter than 400 nm are required to induce significant photolytic reactions. The correlation between field dissipation studies and laboratory photolysis experiments suggests that photolysis is a major pathway for the dissipation of some sulfonylurea herbicides in natural Prairie waters. PMID:19890978

  8. Transformation kinetics and mechanism of the sulfonylurea herbicides pyrazosulfuron ethyl and halosulfuron methyl in aqueous solutions

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Pyrazosulfuron ethyl (PE) and halosulfuron methyl (HM) are two new highly active sulfonylurea herbicides which have been widely used for weed control in a variety of vegetables and other crops. These two herbicides have similar molecular structure, differing only in the substitutions on the pyrazole...

  9. Heteromeric TASK-1/TASK-3 is the major oxygen-sensitive background K+ channel in rat carotid body glomus cells.

    PubMed

    Kim, Donghee; Cavanaugh, Eric J; Kim, Insook; Carroll, John L

    2009-06-15

    Carotid body (CB) glomus cells from rat express a TASK-like background K+ channel that is believed to play a critical role in the regulation of excitability and hypoxia-induced increase in respiration. Here we studied the kinetic behaviour of single channel openings from rat CB cells to determine the molecular identity of the 'TASK-like' K+ channels. In outside-out patches, the TASK-like background K+ channel in CB cells was inhibited >90% by a reduction of pH(o) from 7.3 to 5.8. In cell-attached patches with 140 mM KCl and 1 mM Mg2+ in the bath and pipette solutions, two main open levels with conductance levels of approximately 14 pS and approximately 32 pS were recorded at a membrane potential of -60 mV. The K+ channels showed kinetic properties similar to TASK-1 (approximately 14 pS), TASK-3 (approximately 32 pS) and TASK-1/3 heteromer (approximately 32 pS). The presence of three TASK isoforms was tested by reducing [Mg2+](o) to approximately 0 mM, which had no effect on the conductance of TASK-1, but increased those of TASK-1/3 and TASK-3 to 42 pS and 74 pS, respectively. In CB cells, the reduction of [Mg2+](o) to approximately 0 mM also caused the appearance of approximately 42 pS (TASK-1/3-like) and approximately 74 pS (TASK-3-like) channels, in addition to the approximately 14 pS (TASK-1-like) channel. The 42 pS channel was the most abundant, contributing approximately 75% of the current produced by TASK-like channels. Ruthenium red (5 microM) had no effect on TASK-1 and TASK-1/3, but inhibited TASK-3 by 87%. In CB cells, ruthenium red caused approximately 12% inhibition of TASK-like activity. Methanandamide reduced the activity of all three TASKs by 80-90%, and that of TASK-like channels in CB cell also by approximately 80%. In CB cells, hypoxia caused inhibition of TASK-like channels, including TASK-1/3-like channels. These results show that TASK-1, TASK-1/3 and TASK-3 are all functionally expressed in isolated CB cells, and that the TASK-1/3 heteromer

  10. Reach-scale channel sensitivity to multiple human activities and natural events: Lower Santa Clara River, California, USA

    NASA Astrophysics Data System (ADS)

    Downs, Peter W.; Dusterhoff, Scott R.; Sears, William A.

    2013-05-01

    Understanding the cumulative impact of natural and human influences on the sensitivity of channel morphodynamics, a relative measure between the drivers for change and the magnitude of channel response, requires an approach that accommodates spatial and temporal variability in the suite of primary stressors. Multiple historical data sources were assembled to provide a reach-scale analysis of the lower Santa Clara River (LSCR) in Ventura County, California, USA. Sediment supply is naturally high due to tectonic activity, earthquake-generated landslides, wildfires, and high magnitude flow events during El Niño years. Somewhat typically for the region, the catchment has been subject to four reasonably distinct land use and resource management combinations since European-American settlement. When combined with analysis of channel morphological response (quantifiable since ca. 1930), reach-scale and temporal differences in channel sensitivity become apparent. Downstream reaches have incised on average 2.4 m and become narrower by almost 50% with changes focused in a period of highly sensitive response after about 1950 followed by forced insensitivity caused by structural flood embankments and a significant grade control structure. In contrast, the middle reaches have been responsive but are morphologically resilient, and the upstream reaches show a mildly sensitive aggradational trend. Superimposing the natural and human drivers for change reveals that large scale stressors (related to ranching and irrigation) have been replaced over time by a suite of stressors operating at multiple spatial scales. Lower reaches have been sensitive primarily to 'local' scale impacts (urban growth, flood control, and aggregate mining) whereas, upstream, catchment-scale influences still prevail (including flow regulation and climate-driven sediment supply factors). These factors illustrate the complexity inherent to cumulative impact assessment in fluvial systems, provide evidence for a

  11. Function and Mechanism of Axonal Targeting of Voltage-sensitive Potassium Channels

    PubMed Central

    Gu, Chen; Barry, Joshua

    2011-01-01

    Precise localization of various ion channels into proper subcellular compartments is crucial for neuronal excitability and synaptic transmission. Axonal K+ channels that are activated by depolarization of the membrane potential participate in the repolarizing phase of the action potential, and hence regulate action potential firing patterns, which encode output signals. Moreover, some of these channels can directly control neurotransmitter release at axonal terminals by constraining local membrane excitability and limiting Ca2+ influx. K+ channels differ not only in biophysical and pharmacological properties, but in expression and subcellular distribution as well. Importantly, proper targeting of channel proteins is a prerequisite for electrical and chemical functions of axons. In this review, we first highlight recent studies that demonstrate different roles of axonal K+ channels in the local regulation of axonal excitability. Next, we focus on research progress in identifying axonal targeting motifs and machinery of several different types of K+ channels present in axons. Regulation of K+ channel targeting and activity may underlie a novel form of neuronal plasticity. This research field can contribute to generating novel therapeutic strategies through manipulating neuronal excitability in treating neurological diseases, such as multiple sclerosis, neuropathic pain, and Alzheimer’s disease. PMID:21530607

  12. Differential Regulation of Proton-Sensitive Ion Channels by Phospholipids: A Comparative Study between ASICs and TRPV1

    PubMed Central

    Kweon, Hae-Jin; Yu, Soo-Young; Kim, Dong-Il; Suh, Byung-Chang

    2015-01-01

    Protons are released in pain-generating pathological conditions such as inflammation, ischemic stroke, infection, and cancer. During normal synaptic activities, protons are thought to play a role in neurotransmission processes. Acid-sensing ion channels (ASICs) are typical proton sensors in the central nervous system (CNS) and the peripheral nervous system (PNS). In addition to ASICs, capsaicin- and heat-activated transient receptor potential vanilloid 1 (TRPV1) channels can also mediate proton-mediated pain signaling. In spite of their importance in perception of pH fluctuations, the regulatory mechanisms of these proton-sensitive ion channels still need to be further investigated. Here, we compared regulation of ASICs and TRPV1 by membrane phosphoinositides, which are general cofactors of many receptors and ion channels. We observed that ASICs do not require membrane phosphatidylinositol 4-phosphate (PI(4)P) or phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) for their function. However, TRPV1 currents were inhibited by simultaneous breakdown of PI(4)P and PI(4,5)P2. By using a novel chimeric protein, CF-PTEN, that can specifically dephosphorylate at the D3 position of phosphatidylinositol 3,4,5-trisphosphate (PI(3,4,5)P3), we also observed that neither ASICs nor TRPV1 activities were altered by depletion of PI(3,4,5)P3 in intact cells. Finally, we compared the effects of arachidonic acid (AA) on two proton-sensitive ion channels. We observed that AA potentiates the currents of both ASICs and TRPV1, but that they have different recovery aspects. In conclusion, ASICs and TRPV1 have different sensitivities toward membrane phospholipids, such as PI(4)P, PI(4,5)P2, and AA, although they have common roles as proton sensors. Further investigation about the complementary roles and respective contributions of ASICs and TRPV1 in proton-mediated signaling is necessary. PMID:25781982

  13. Genetic analysis of a synaptic calcium channel in Drosophila: intragenic modifiers of a temperature-sensitive paralytic mutant of cacophony.

    PubMed Central

    Brooks, I M; Felling, R; Kawasaki, F; Ordway, R W

    2003-01-01

    Our previous genetic analysis of synaptic mechanisms in Drosophila identified a temperature-sensitive paralytic mutant of the voltage-gated calcium channel alpha1 subunit gene, cacophony (cac). Electrophysiological studies in this mutant, designated cac(TS2), indicated cac encodes a primary calcium channel alpha1 subunit functioning in neurotransmitter release. To further examine the functions and interactions of cac-encoded calcium channels, a genetic screen was performed to isolate new mutations that modify the cac(TS2) paralytic phenotype. The screen recovered 10 mutations that enhance or suppress cac(TS2), including second-site mutations in cac (intragenic modifiers) as well as mutations mapping to other genes (extragenic modifiers). Here we report molecular characterization of three intragenic modifiers and examine the consequences of these mutations for temperature-sensitive behavior, synaptic function, and processing of cac pre-mRNAs. These mutations may further define the structural basis of calcium channel alpha1 subunit function in neurotransmitter release. PMID:12750329

  14. Temperature-sensitive gating of TRPV1 channel as probed by atomistic simulations of its trans- and juxtamembrane domains.

    PubMed

    Chugunov, Anton O; Volynsky, Pavel E; Krylov, Nikolay A; Nolde, Dmitry E; Efremov, Roman G

    2016-01-01

    Heat-activated transient receptor potential channel TRPV1 is one of the most studied eukaryotic proteins involved in temperature sensation. Upon heating, it exhibits rapid reversible pore gating, which depolarizes neurons and generates action potentials. Underlying molecular details of such effects in the pore region of TRPV1 is of a crucial importance to control temperature responses of the organism. Despite the spatial structure of the channel in both open (O) and closed (C) states is known, microscopic nature of channel gating and mechanism of thermal sensitivity are still poorly understood. In this work, we used unrestrained atomistic molecular dynamics simulations of TRPV1 (without N- and C-terminal cytoplasmic domains) embedded into explicit lipid bilayer in its O- and C-states. We found that the pore domain with its neighboring loops undergoes large temperature-dependent conformational transitions in an asymmetric way, when fragments of only one monomer move with large amplitude, freeing the pore upon heating. Such an asymmetrical gating looks rather biologically relevant because it is faster and more reliable than traditionally proposed "iris-like" symmetric scheme of channel opening. Analysis of structural, dynamic, and hydrophobic organization of the pore domain revealed entropy growth upon TRPV1 gating, which is in line with current concepts of thermal sensitivity. PMID:27612191

  15. Temperature-sensitive gating of TRPV1 channel as probed by atomistic simulations of its trans- and juxtamembrane domains

    PubMed Central

    Chugunov, Anton O.; Volynsky, Pavel E.; Krylov, Nikolay A.; Nolde, Dmitry E.; Efremov, Roman G.

    2016-01-01

    Heat-activated transient receptor potential channel TRPV1 is one of the most studied eukaryotic proteins involved in temperature sensation. Upon heating, it exhibits rapid reversible pore gating, which depolarizes neurons and generates action potentials. Underlying molecular details of such effects in the pore region of TRPV1 is of a crucial importance to control temperature responses of the organism. Despite the spatial structure of the channel in both open (O) and closed (C) states is known, microscopic nature of channel gating and mechanism of thermal sensitivity are still poorly understood. In this work, we used unrestrained atomistic molecular dynamics simulations of TRPV1 (without N- and C-terminal cytoplasmic domains) embedded into explicit lipid bilayer in its O- and C-states. We found that the pore domain with its neighboring loops undergoes large temperature-dependent conformational transitions in an asymmetric way, when fragments of only one monomer move with large amplitude, freeing the pore upon heating. Such an asymmetrical gating looks rather biologically relevant because it is faster and more reliable than traditionally proposed “iris-like” symmetric scheme of channel opening. Analysis of structural, dynamic, and hydrophobic organization of the pore domain revealed entropy growth upon TRPV1 gating, which is in line with current concepts of thermal sensitivity. PMID:27612191

  16. A residue in the transmembrane segment 6 of domain I in insect and mammalian sodium channels regulate differential sensitivities to pyrethroid insecticides.

    PubMed

    Oliveira, Eugênio E; Du, Yuzhe; Nomura, Yoshiko; Dong, Ke

    2013-09-01

    Voltage-gated sodium channels are critical for electrical signaling in the nervous system. Pyrethroid insecticides exert their toxic action by modifying the gating of sodium channels. A valine to methionine mutation in the transmembrane segment 6 of domain I (IS6) of sodium channels from tobacco budworms (Heliothis virescens) has been shown to alter channel gating and reduce insect sodium channel sensitivity to pyrethroids. A valine to leucine substitution was subsequently reported in pyrethroid-resistant bedbug populations. Intriguingly, pyrethroid-resistant mammalian sodium channels possess an isoleucine at the corresponding position. To determine whether different substitutions at this position alter channel gating and confer pyrethroid resistance, we made valine to methionine, isoleucine or leucine substitutions at the corresponding position, V409, in a cockroach sodium channel and examined the gating properties and pyrethroid sensitivity of the three mutants in Xenopus oocytes. All three mutations reduced the channel sensitivity to three pyrethroids (permethrin, cismethrin and deltamethrin). V409M, but not V409I or V409L, caused 6-7mV depolarizing shifts in the voltage dependences of both activation and inactivation. V409M and V409L slowed channel activation kinetics and accelerated open-state deactivation kinetics, but V409I did not. Furthermore, the substitution of isoleucine with valine, but not with methionine nor leucine, at the corresponding position in a rat skeletal muscle sodium channel, rNav1.4, enhanced channel sensitivity to deltamethrin. Collectively, our study highlights an important role of residues at 409 in regulating not only sodium channel gating, but also the differential sensitivities of insect and mammalian sodium channels to pyrethroids. PMID:23764339

  17. A residue in the transmembrane segment 6 of domain I in insect and mammalian sodium channels regulate differential sensitivities to pyrethroid insecticides

    PubMed Central

    Oliveira, Eugênio E.; Du, Yuzhe; Nomura, Yoshiko; Dong, Ke

    2013-01-01

    Voltage-gated sodium channels are critical for electrical signaling in the nervous system. Pyrethroid insecticides exert their toxic action by modifying the gating of sodium channels. A valine to methionine mutation in the transmembrane segment 6 of domain I (IS6) of sodium channels from tobacco budworms (Heliothis virescens) has been shown to alter channel gating and reduce insect sodium channel sensitivity to pyrethroids. A valine to leucine substitution was subsequently reported in pyrethroid-resistant bedbug populations. Intriguingly, pyrethroid-resistant mammalian sodium channels possess an isoleucine at the corresponding position. To determine whether different substitutions at this position alter channel gating and confer pyrethroid resistance, we made valine to methionine, isoleucine or leucine substitutions at the corresponding position, V409, in a cockroach sodium channel and examined the gating properties and pyrethroid sensitivity of the three mutants in Xenopus oocytes. All three mutations reduced the channel sensitivity to three pyrethroids (permethrin, cismethrin and deltamethrin). V409M, but not V409I or V409L, caused 6-7 mV depolarizing shifts in the voltage dependences of both activation and inactivation. V409M and V409L slowed channel activation kinetics and accelerated open-state deactivation kinetics, but V409I did not. Furthermore, the substitution of isoleucine with valine, but not with methionine nor leucine, at the corresponding position in a rat skeletal muscle sodium channel, rNav1.4, enhanced channel sensitivity to deltamethrin. Collectively, our study highlights an important role of residues at 409 in regulating not only sodium channel gating, but also the differential sensitivities of insect and mammalian sodium channels to pyrethroids. PMID:23764339

  18. Phosphorylation of BK channels modulates the sensitivity to hydrogen sulfide (H2S)

    PubMed Central

    Sitdikova, Guzel F.; Fuchs, Roman; Kainz, Verena; Weiger, Thomas M.; Hermann, Anton

    2014-01-01

    Introduction: Gases, such as nitric oxide (NO), carbon monoxide (CO), or hydrogen sulfide (H2S), termed gasotransmitters, play an increasingly important role in understanding of how electrical signaling of cells is modulated. H2S is well-known to act on various ion channels and receptors. In a previous study we reported that H2S increased calcium-activated potassium (BK) channel activity. Aims: The goal of the present study is to investigate the modulatory effect of BK channel phosphorylation on the action of H2S on the channel as well as to recalculate and determine the H2S concentrations in aqueous sodium hydrogen sulfide (NaHS) solutions. Methods: Single channel recordings of GH3, GH4, and GH4 STREX cells were used to analyze channel open probability, amplitude, and open dwell times. H2S was measured with an anion selective electrode. Results: The concentration of H2S produced from NaHS was recalculated taking pH, temperature salinity of the perfusate, and evaporation of H2S into account. The results indicate that from a concentration of 300 μM NaHS, only 11–13%, i.e., 34–41 μM is effective as H2S in solution. GH3, GH4, and GH4 STREX cells respond differently to phosphorylation. BK channel open probability (Po) of all cells lines used was increased by H2S in ATP-containing solutions. PKA prevented the action of H2S on channel Po in GH4 and GH4 STREX, but not in GH3 cells. H2S, high significantly increased Po of all PKG pretreated cells. In the presence of PKC, which lowers channel activity, H2S increased channel Po of GH4 and GH4 STREX, but not those of GH3 cells. H2S increased open dwell times of GH3 cells in the absence of ATP significantly. A significant increase of dwell times with H2S was also observed in the presence of okadaic acid. Conclusions: Our results suggest that phosphorylation by PKG primes the channels for H2S activation and indicate that channel phosphorylation plays an important role in the response to H2S. PMID:25429270

  19. Studies of the voltage-sensitive calcium channels in smooth muscle, neuronal, and cardiac tissues using 1,4-dihydropyridine calcium channel antagonists and activators

    SciTech Connect

    Wei, X.

    1988-01-01

    This study describes the investigation of the voltage-sensitive Ca{sup +} channels in vascular and intestinal smooth muscle, chick neural retina cells and neonatal rat cardiac myocytes using 1,4-dihydropyridine Ca{sup 2+} channel antagonists and activators. In rat aorta, the tumor promoting phorbol ester, 12-O-tetradecanoylphorbol-13-acetate (TPA) produced Ca{sup 2+}-dependent contractile responses. The responses to TPA were blocked by the Ca{sup 2+} channel antagonists. The effects of the enantiomers of Bay K 8644 and 202-791 were characterized in both rat tail artery and guinea pig ileal longitudinal smooth muscle preparations using pharmacologic and radioligand binding assays. The (S)-enantiomers induced contraction and potentiated the responses to K{sup +} depolarization. The (R)-enantiomers inhibited the tension responses to K{sup +}. All the enantiomers inhibited specific ({sup 3}H)nitrendipine binding. The pharmacologic activities of both activator and antagonist ligands correlated on a 1:1 basis with the binding affinities. In chick neural retina cells the (S)-enantiomers of Bay K 8644 and 202-791 enhanced Ca{sup 2+} influx. In contrast, the (R)-enantiomers inhibited Ca{sup 2+} influx. The enantiomers of Bay K 8644 and 202-791 inhibited specific ({sup 3}H)PN 200-110 binding competitively. Binding of 1,4-dihydropyridines was characterized in neonatal rat heart cells.

  20. Reduced Nav1.6 Sodium Channel Activity in Mice Increases In Vivo Sensitivity to Volatile Anesthetics

    PubMed Central

    Pal, Dinesh; Jones, Julie M.; Wisidagamage, Stella

    2015-01-01

    Nav1.6 is a major voltage-gated sodium channel in the central and peripheral nervous systems. Within neurons, the channel protein is concentrated at the axon initial segment and nodes of Ranvier, where it functions in initiation and propagation of action potentials. We examined the role of Nav1.6 in general anesthesia using two mouse mutants with reduced activity of Nav1.6, Scn8amedJ/medJ and Scn8a9J/9J. The mice were exposed to the general anesthetics isoflurane and sevoflurane in step-wise increments; the concentration required to produce loss of righting reflex, a surrogate for anesthetic-induced unconsciousness in rodents, was determined. Mice homozygous for these mutations exhibited increased sensitivity to both isoflurane and sevoflurane. The increased sensitivity was observed during induction of unconsciousness but not during the recovery phase, suggesting that the effect is not attributable to compromised systemic physiology. Electroencephalographic theta power during baseline waking was lower in mutants, suggesting decreased arousal and reduced neuronal excitability. This is the first report linking reduced activity of a specific voltage-gated sodium channel to increased sensitivity to general anesthetics in vivo. PMID:26252017

  1. Okadaic acid-sensitive activation of Maxi Cl− channels by triphenylethylene antioestrogens in C1300 mouse neuroblastoma cells

    PubMed Central

    Diaz, Mario; Bahamonde, Maria I; Lock, Hagar; Muñoz, Francisco J; Hardy, Simon P; Posas, Francesc; Valverde, Miguel A

    2001-01-01

    The regulation of Maxi Cl− channels by 17β-oestradiol and non-steroidal triphenylethylene antioestrogens represents a rapid, non-classical effect of these compounds. In the present study we have investigated the signalling pathways used for the regulation of Maxi Cl− channel activity by oestrogens and antioestrogens in C1300 neuroblastoma cells.Whole-cell Maxi Cl− currents were readily and reversibly activated by tamoxifen, toremifene and the membrane-impermeant ethyl-bromide tamoxifen, only when applied to the extracellular medium.Pre-treatment of C1300 cells with oestrogen or cAMP prevented the antioestrogen-induced activation of Maxi Cl− channels. The inhibitory effect of 17β-oestradiol and cAMP was abolished by the kinase inhibitor staurosporine.Current activation was unaffected by the removal of intracellular Ca2+ and Mg2+, but was completely abolished in the presence of okadaic acid. These results are consistent with the participation of an okadaic acid-sensitive serine/threonine protein phosphatase in the activation of Maxi Cl− channels. However, neither oestrogen or antioestrogen treatment modified the total activity of the two major serine/threonine phosphatases, PP1 and PP2A, in C1300 cells.Although the role of these Maxi Cl− channels remains unknown, our findings suggest strongly that their modulation by oestrogens and antioestrogens is linked to intracellular signalling pathways. PMID:11579158

  2. Characteristics and roles of the volume-sensitive outwardly rectifying (VSOR) anion channel in the central nervous system.

    PubMed

    Akita, T; Okada, Y

    2014-09-01

    Cell volume regulation (CVR) is essential for all types of cells in the central nervous system (CNS) to counteract cell volume changes that may be associated with neuronal activities or diseases and with osmosensing in the hypothalamus, to facilitate morphological changes during cell proliferation, differentiation and migration, and to execute apoptosis of cells. The regulation is attained by regulating the net influx or efflux of solutes and water across the plasma membrane. The volume-sensitive outwardly rectifying (VSOR) anion channel plays a major role in providing a pathway for anion flux during the regulation. The VSOR anion channel is permeable not only to Cl(-) ions but also to amino acids like glutamate and taurine. This property confers a means of intercellular communications through the opening of the channel in the CNS. Thus exploring the roles of VSOR anion channels is crucial to understand the basic principles of cellular functions in the CNS. Here we review biophysical and pharmacological characteristics of the VSOR anion channel in the CNS, discuss its activation mechanisms and roles in the CNS reported so far, and give some perspectives on the next issues to be examined in the near future. PMID:24937753

  3. Opening of ATP-sensitive K(+) (KATP) channels enhance hydroxyl radical generation induced by MPP(+) in rat striatum.

    PubMed

    Obata, Toshio; Nakashima, Michiko

    2016-07-15

    The present study examined whether opening of adenosine triphosphate (ATP) sensitive K(+) (KATP) channels can enhance 1-methyl-4-phenylpyridinium (MPP(+))-induced hydroxyl radical (OH) generation in rat striatum. Rats were anesthetized, and sodium salicylate in Ringer's solution (0.5nmol/ml per min) was infused through a microdialysis probe to detect the generation of OH as reflected by the non-enzymatic formation of 2.3-dihydroxybenzoic acid (DHBA) in the striatum. MPP(+) (5mM) enhanced generation of OH with concomitant increased efflux of dopamine (DA). Cromakalim (100μM), a KATP channel opener, through the microdialysis probe significantly increased both DA efflux and OH formation induced by MPP(+). Another KATP channel opener, nicorandil (1mM), also increased the level DA or DHBA, but these changes were not significant. However, in the presence of glibenclamide (10μM), a KATP channel antagonist, and the increase of MPP(+)-induced DA or DHBA were not observed. Cromakalim (10, 50 and 100μM) increased MPP(+)-induced DHBA formation in a concentration-dependent manner. However, the effects of cromakalim in the presence of glibenclamide were abolished. These results suggest that opening of KATP channels may cause OH generation by MPP(+). PMID:27288802

  4. Kinetics of modulation of tetrodotoxin-sensitive and tetrodotoxin-resistant sodium channels by tetramethrin and deltamethrin.

    PubMed

    Tabarean, I V; Narahashi, T

    2001-12-01

    Pyrethroid insecticides may be classified into two groups: type I pyrethroids lack a cyano group in the alpha-position, whereas type II pyrethroids have a cyano group. Both types prolong the sodium channel current thereby causing hyperexcitability, yet details of modulation of current kinetics remain largely to be seen. The mechanism of pyrethroid modulation of sodium currents was studied by the whole-cell patch-clamp technique with rat dorsal root ganglion neurons. Both deltamethrin (type II) and tetramethrin (type I) acted on both tetrodotoxin-sensitive and tetrodotoxin-resistant channels in a qualitatively similar manner and some quantitative differences were derived from different kinetics. During repetitive stimulation in the presence of deltamethrin, leak current increased due to accumulation of prolonged tail currents, explaining the apparent use-dependent modification. For tetramethrin-modified channels, such accumulation was much less because of faster kinetics. Slowing of the kinetics of sodium channel activation by deltamethrin was revealed even after the fast inactivation had been removed by papain. The kinetics of deltamethrin-modified sodium channels was fitted better by the equation that contained two activation components than that with one component. Deltamethrin caused a large shift of the conductance-voltage curve in the direction of hyperpolarization. Cell-attached patch-clamp experiments revealed that deltamethrin had much smaller mobility in the cell membrane than tetramethrin. It was concluded that the apparent use dependence of deltamethrin modification of sodium channels was due primarily to the accumulation of prolonged tail currents during repetitive stimulation and that the sodium channel activation mechanism is the major target of pyrethroids. PMID:11714887

  5. ATP-Sensitive K+ Channels Regulate the Concentrative Adenosine Transporter CNT2 following Activation by A1 Adenosine Receptors

    PubMed Central

    Duflot, Sylvie; Riera, Bárbara; Fernández-Veledo, Sonia; Casadó, Vicent; Norman, Robert I.; Casado, F. Javier; Lluís, Carme; Franco, Rafael; Pastor-Anglada, Marçal

    2004-01-01

    This study describes a novel mechanism of regulation of the high-affinity Na+-dependent adenosine transporter (CNT2) via the activation of A1 adenosine receptors (A1R). This regulation is mediated by the activation of ATP-sensitive K+ (KATP) channels. The high-affinity Na+-dependent adenosine transporter CNT2 and A1R are coexpressed in the basolateral domain of the rat hepatocyte plasma membrane and are colocalized in the rat hepatoma cell line FAO. The transient increase in CNT2-mediated transport activity triggered by (−)-N6-(2-phenylisopropyl)adenosine is fully inhibited by KATP channel blockers and mimicked by a KATP channel opener. A1R agonist activation of CNT2 occurs in both hepatocytes and FAO cells, which express Kir6.1, Kir6.2, SUR1, SUR2A, and SUR2B mRNA channel subunits. With the available antibodies against Kir6.X, SUR2A, and SUR2B, it is shown that all of these proteins colocalize with CNT2 and A1R in defined plasma membrane domains of FAO cells. The extent of the purinergic modulation of CNT2 is affected by the glucose concentration, a finding which indicates that glycemia and glucose metabolism may affect this cross-regulation among A1R, CNT2, and KATP channels. These results also suggest that the activation of KATP channels under metabolic stress can be mediated by the activation of A1R. Cell protection under these circumstances may be achieved by potentiation of the uptake of adenosine and its further metabolization to ATP. Mediation of purinergic responses and a connection between the intracellular energy status and the need for an exogenous adenosine supply are novel roles for KATP channels. PMID:15024061

  6. Numerical sensitivity analysis of passive EHF and SMMW channels to tropospheric water vapor, clouds, and precipitation

    NASA Technical Reports Server (NTRS)

    Gasiewski, A. J.

    1992-01-01

    Potential uses of specific extremely High Frequency (EHF) and Sub-Millimeter-Wave (SMMW) channels at 90, 166, 183, 220, 325, 340, and 410 GHz for passive spaceborne remote sensing of the troposphere and lower stratosphere are investigated using an iterative numerical radiative transfer model. Collectively, these channels offer potential for high spatial resolution imaging using diffraction-limited apertures of practical size, along with the ability to profile water vapor, map precipitation beneath optically opaque cloud cover, and to measure nonprecipitating cloud (e.g., cirrus) parameters. A widely-spaced set of EHF and SMMW channels can yield observable degrees of freedom related to clouds and precipitation not available by exclusively using the more thoroughly studied microwave channels below 183 GHz. A new passive airborne imaging instrument for tropospheric meteorological sensing is described.

  7. Functional evidence for oxygen-sensitive voltage-gated potassium channels in human placental vasculature.

    PubMed

    Kiernan, M F; Barrie, A; Szkolar, J; Mills, T A; Wareing, M

    2010-06-01

    Hypoxic fetoplacental vasoconstriction (HFPV), involving voltage-gated potassium (K(V)) channels, has been suggested in human placenta; the identity of these channels remains unclear. Using wire myography, chorionic plate blood vessels were exposed to isoform-specific K(V) channel blockers. Dose-response curves (thromboxane mimetic U46619; 0.1-2000 nM) pre- and post-addition of K(V) channel modulator were analysed. Arterial U46619-induced contraction increased with margatoxin and stromatoxin-1, whilst only correolide increased U46619-induced contraction in veins (P < 0.05 two-way ANOVA). Basal tone was unaffected in arteries or veins. These data implicate K(V)1.2 and/or K(V)2.1 and K(V)1.5 in the control of agonist-induced contraction of human placental arteries and veins respectively. PMID:20451247

  8. PERTURBATION OF VOLTAGE-SENSITIVE Ca2+ CHANNEL FUNCTION BY VOLATILE ORGANIC SOLVENTS.

    EPA Science Inventory

    The mechanisms underlying the acute neurophysiological and behavioral effects of volatile organic compounds (VOCs) remain to be elucidated. However, the function of neuronal ion channels is perturbed by VOCs. The present study examined effects of toluene (TOL), trichloroethylene ...

  9. Dynamic subunit stoichiometry confers a progressive continuum of pharmacological sensitivity by KCNQ potassium channels

    PubMed Central

    Yu, Haibo; Lin, Zhihong; Mattmann, Margrith E.; Zou, Beiyan; Terrenoire, Cecile; Zhang, Hongkang; Wu, Meng; McManus, Owen B.; Kass, Robert S.; Lindsley, Craig W.; Hopkins, Corey R.; Li, Min

    2013-01-01

    Voltage-gated KCNQ1 (Kv7.1) potassium channels are expressed abundantly in heart but they are also found in multiple other tissues. Differential coassembly with single transmembrane KCNE beta subunits in different cell types gives rise to a variety of biophysical properties, hence endowing distinct physiological roles for KCNQ1–KCNEx complexes. Mutations in either KCNQ1 or KCNE1 genes result in diseases in brain, heart, and the respiratory system. In addition to complexities arising from existence of five KCNE subunits, KCNE1 to KCNE5, recent studies in heterologous systems suggest unorthodox stoichiometric dynamics in subunit assembly is dependent on KCNE expression levels. The resultant KCNQ1–KCNE channel complexes may have a range of zero to two or even up to four KCNE subunits coassembling per KCNQ1 tetramer. These findings underscore the need to assess the selectivity of small-molecule KCNQ1 modulators on these different assemblies. Here we report a unique small-molecule gating modulator, ML277, that potentiates both homomultimeric KCNQ1 channels and unsaturated heteromultimeric (KCNQ1)4(KCNE1)n (n < 4) channels. Progressive increase of KCNE1 or KCNE3 expression reduces efficacy of ML277 and eventually abolishes ML277-mediated augmentation. In cardiomyocytes, the slowly activating delayed rectifier potassium current, or IKs, is believed to be a heteromultimeric combination of KCNQ1 and KCNE1, but it is not entirely clear whether IKs is mediated by KCNE-saturated KCNQ1 channels or by channels with intermediate stoichiometries. We found ML277 effectively augments IKs current of cultured human cardiomyocytes and shortens action potential duration. These data indicate that unsaturated heteromultimeric (KCNQ1)4(KCNE1)n channels are present as components of IKs and are pharmacologically distinct from KCNE-saturated KCNQ1–KCNE1 channels. PMID:23650380

  10. Numerical sensitivity analysis of 3- and 2- dimensional rib-roughened channels

    NASA Astrophysics Data System (ADS)

    Keshmiri, Amir

    2012-07-01

    Rough surfaces have been used as a tool to enhance heat transfer by increasing the level of turbulence mixing in the flow. In numerically simulating such flows, it is common to simulate a 3D rib-roughened channel with a 2D domain in order to reduce the computational time and power. The main purpose of the present work is to investigate the accuracy of the above approximation. In order to do so, initially a 3D channel is simulated using Reynolds-Averaged Navier-Stokes technique and comparison is made against 2D simulations as well as experimental data. In addition, the effects of rib thermal boundary condition and near-wall treatments are also investigated. All computations are undertaken using the commercial CFD code `STAR-CD'. The Reynolds number, based on the channel bulk velocity and hydraulic diameter, is 30,000. Two low-Reynolds-number linear Eddy-Viscosity Models, namely the Lien-Chen-Leschziner k - ɛ model and a variant of Durbin's v 2 - f formulation are used. In the CFD simulations reported here, the focus is on the experimental data of Rau et al. (ASME J Turbomach 120:368-375, 1998). It was found that the present results for a 3D channel are in relatively good agreement with the data. It was also shown that a 2D channel can be used to represent the flow in the centre-line of a 3D channel with relatively good accuracy.

  11. Intracellular signalling mechanism responsible for modulation of sarcolemmal ATP-sensitive potassium channels by nitric oxide in ventricular cardiomyocytes.

    PubMed

    Zhang, Dai-Min; Chai, Yongping; Erickson, Jeffrey R; Brown, Joan Heller; Bers, Donald M; Lin, Yu-Fung

    2014-03-01

    The ATP-sensitive potassium (KATP) channels are crucial for stress adaptation in the heart. It has previously been suggested that the function of KATP channels is modulated by nitric oxide (NO), a gaseous messenger known to be cytoprotective; however, the underlying mechanism remains poorly understood. Here we sought to delineate the intracellular signalling mechanism responsible for NO modulation of sarcolemmal KATP (sarcKATP) channels in ventricular cardiomyocytes. Cell-attached patch recordings were performed in transfected human embryonic kidney (HEK) 293 cells and ventricular cardiomyocytes freshly isolated from adult rabbits or genetically modified mice, in combination with pharmacological and biochemical approaches. Bath application of the NO donor NOC-18 increased the single-channel activity of Kir6.2/SUR2A (i.e., the principal ventricular-type KATP) channels in HEK293 cells, whereas the increase was abated by KT5823 [a selective cGMP-dependent protein kinase (PKG) inhibitor], mercaptopropionyl glycine [MPG; a reactive oxygen species (ROS) scavenger], catalase (an H2O2-degrading enzyme), myristoylated autocamtide-2 related inhibitory peptide (mAIP) selective for Ca2+ / calmodulin-dependent protein kinase II (CaMKII) and U0126 [an extracellular signal-regulated protein kinase 1/2 (ERK1/2) inhibitor], respectively. The NO donors NOC-18 and N-(2-deoxy-α,β-d-glucopyranose-2-)-N2-acetyl-S-nitroso-d,l-penicillaminamide (glycol-SNAP-2) were also capable of stimulating native sarcKATP channels preactivated by the channel opener pinacidil in rabbit ventricular myocytes, through reducing the occurrence and the dwelling time of the long closed states whilst increasing the frequency of channel opening; in contrast, all these changes were reversed in the presence of inhibitors selective for soluble guanylyl cyclase (sGC), PKG, calmodulin, CaMKII or ERK1/2. Mimicking the action of NO donors, exogenous H2O2 potentiated pinacidil-preactivated sarcKATP channel activity in

  12. Intracellular signalling mechanism responsible for modulation of sarcolemmal ATP-sensitive potassium channels by nitric oxide in ventricular cardiomyocytes

    PubMed Central

    Zhang, Dai-Min; Chai, Yongping; Erickson, Jeffrey R; Brown, Joan Heller; Bers, Donald M; Lin, Yu-Fung

    2014-01-01

    The ATP-sensitive potassium (KATP) channels are crucial for stress adaptation in the heart. It has previously been suggested that the function of KATP channels is modulated by nitric oxide (NO), a gaseous messenger known to be cytoprotective; however, the underlying mechanism remains poorly understood. Here we sought to delineate the intracellular signalling mechanism responsible for NO modulation of sarcolemmal KATP (sarcKATP) channels in ventricular cardiomyocytes. Cell-attached patch recordings were performed in transfected human embryonic kidney (HEK) 293 cells and ventricular cardiomyocytes freshly isolated from adult rabbits or genetically modified mice, in combination with pharmacological and biochemical approaches. Bath application of the NO donor NOC-18 increased the single-channel activity of Kir6.2/SUR2A (i.e. the principal ventricular-type KATP) channels in HEK293 cells, whereas the increase was abated by KT5823 [a selective cGMP-dependent protein kinase (PKG) inhibitor], mercaptopropionyl glycine [MPG; a reactive oxygen species (ROS) scavenger], catalase (an H2O2-degrading enzyme), myristoylated autocamtide-2 related inhibitory peptide (mAIP) selective for Ca2+/calmodulin-dependent protein kinase II (CaMKII) and U0126 [an extracellular signal-regulated protein kinase 1/2 (ERK1/2) inhibitor], respectively. The NO donors NOC-18 and N-(2-deoxy-α,β-d-glucopyranose-2-)-N2-acetyl-S-nitroso-d,l-penicillaminamide (glycol-SNAP-2) were also capable of stimulating native sarcKATP channels preactivated by the channel opener pinacidil in rabbit ventricular myocytes, through reducing the occurrence and the dwelling time of the long closed states whilst increasing the frequency of channel opening; in contrast, all these changes were reversed in the presence of inhibitors selective for soluble guanylyl cyclase (sGC), PKG, calmodulin, CaMKII or ERK1/2. Mimicking the action of NO donors, exogenous H2O2 potentiated pinacidil-preactivated sarcKATP channel activity in

  13. ATP-sensitive K(+) channels (Kir6.1/SUR1) regulate gap junctional coupling in cochlear-supporting cells.

    PubMed

    Blödow, Alexander; Begandt, Daniela; Bader, Almke; Becker, Annegret; Burghard, Alice; Kühne, Daniela; Kral, Andrej; Ngezahayo, Anaclet

    2016-07-01

    Using the double whole-cell patch-clamp technique, we found that the absence of intracellular ATP led to gap junction uncoupling in cochlear-supporting Hensen cells. The uncoupling was observed as a progressive reduction of the gap junctional electrical conductance from a starting value of approximately 40 nS to less than 0.04 nS within 10-20 min. The conductance rundown was partly avoided by at least 3 mM ATP and completely suppressed by 5 mM ATP or 5'-adenylyl-imidodiphosphate (AMP-PNP), the non-hydrolysable ATP analog, in the pipette filling solution, suggesting that ATP was needed as ligand and not as a hydrolysable energy supplier or substrate for enzymatic reactions. The effect of intracellular ATP was mimicked by the external application of barium, a nonselective blocker of inwardly rectifying K(+) (Kir) channels, and glibenclamide, an inhibitor of the ATP-sensitive Kir channels (KATP). Moreover a Ba(2+)-sensitive whole-cell inward current was observed in absence of internal ATP. We propose that the internal ATP kept the KATP channels in a closed state, thereby maintaining the gap junction coupling of Hensen cells. The immunostaining of guinea pig cochlear tissue revealed for the first time the expression of the KATP channel subunits Kir6.1 and SUR1 in Hensen cells and supported the proposed hypothesis. The results suggest that KATP channels, as regulator of the gap junction coupling in Hensen cells, could be the physiological link between the metabolic state of the supporting cells and K(+) recycling in the organ of Corti. PMID:27030354

  14. 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

  15. Evidence for a Ca(2+)-gated ryanodine-sensitive Ca2+ release channel in visceral smooth muscle.

    PubMed Central

    Xu, L; Lai, F A; Cohn, A; Etter, E; Guerrero, A; Fay, F S; Meissner, G

    1994-01-01

    Although a role for the ryanodine receptor (RyR) in Ca2+ signaling in smooth muscle has been inferred, direct information on the biochemical and functional properties of the receptor has been largely lacking. Studies were thus carried out to purify and characterize the RyR in stomach smooth muscle cells from the toad Bufo marinus. Intracellular Ca2+ measurements with the Ca(2+)-sensitive fluorescent indicator fura-2 under voltage clamp indicated the presence of a caffeine- and ryanodine-sensitive internal store for Ca2+ in these cells. The (CHAPS)-solubilized, [3H]ryanodine-labeled RyR of toad smooth muscle was partially purified from microsomal membranes by rate density centrifugation as a 30-S protein complex. SDS/PAGE indicated the comigration of a high molecular weight polypeptide with the peak attributed to 30-S RyR, which had a mobility similar to the cardiac RyR and on immunoblots cross-reacted with a monoclonal antibody to the canine cardiac RyR. Following planar lipid bilayer reconstitution of 30-S stomach muscle RyR fractions, single-channel currents (830 pS with 250 mM K+ as the permeant ion) were observed that were activated by Ca2+ and modified by ryanodine. In vesicle-45Ca2+ efflux measurements, the toad channel was activated to a greater extent at 100-1000 microM than 1-10 microM Ca2+. These results suggest that toad stomach muscle contains a ryanodine-sensitive Ca2+ release channel with properties similar but not identical to those of the mammalian skeletal and cardiac Ca(2+)-release channels. Images PMID:8159742

  16. An amiloride-sensitive H+-gated Na+ channel in Caenorhabditis elegans body wall muscle cells

    PubMed Central

    Jospin, Maëlle; Allard, Bruno

    2004-01-01

    About 30 genes are predicted to encode degenerin/epithelial sodium channels (DEG/ENaCs) in Caenorhabditis elegans but the gating mode of these channels has not been determined. Using the whole-cell configuration of the patch-clamp technique in acutely dissected C. elegans, we investigated the effects of H+ as a potential activating factor of DEG/ENaCs on electrical properties of body wall muscle cells. Under current-clamp conditions, decreasing external pH from 7.2 to 6.1 led to a reversible depolarization of muscle cells associated with a decrease in input resistance which was partially inhibited by amiloride. Under voltage-clamp conditions, extracellular acidification activated an inward desensitizing current at −60 mV. In the absence of external Ca2+, H+-gated channels were found to be slightly more permeable to Na+ than to K+ and were blocked by amiloride with a K0.5 of 31 μm at −60 mV. An inward current could be also activated by protons in a GABA receptor null mutant in the presence of d-tubocurare and in an unc-105 null mutant. These results demonstrate that ion channels sharing common properties with mammalian acid-sensing ion channels (ASICs) are functional in C. elegans muscle which should prove useful for understanding proton sensing in animals. PMID:15254157

  17. Single molecule FRET reveals pore size and opening mechanism of a mechano-sensitive ion channel

    PubMed Central

    Wang, Yong; Liu, Yanxin; DeBerg, Hannah A; Nomura, Takeshi; Hoffman, Melinda Tonks; Rohde, Paul R; Schulten, Klaus; Martinac, Boris; Selvin, Paul R

    2014-01-01

    The mechanosensitive channel of large conductance, which serves as a model system for mechanosensitive channels, has previously been crystallized in the closed form, but not in the open form. Ensemble measurements and electrophysiological sieving experiments show that the open-diameter of the channel pore is >25 Å, but the exact size and whether the conformational change follows a helix-tilt or barrel-stave model are unclear. Here we report measurements of the distance changes on liposome-reconstituted MscL transmembrane α-helices, using a ‘virtual sorting’ single-molecule fluorescence energy transfer. We observed directly that the channel opens via the helix-tilt model and the open pore reaches 2.8 nm in diameter. In addition, based on the measurements, we developed a molecular dynamics model of the channel structure in the open state which confirms our direct observations. DOI: http://dx.doi.org/10.7554/eLife.01834.001 PMID:24550255

  18. Quinidine-sensitive K+ channels in the basolateral membrane of embryonic coprodeum epithelium: regulation by aldosterone and thyroxine.

    PubMed

    Illek, B; Fischer, H; Clauss, W

    1993-01-01

    Basolateral K+ channels and their regulation during aldosterone- and thyroxine-stimulated Na+ transport were studied in the lower intestinal epithelium (coprodeum) of embryonic chicken in vitro. Isolated tissues of the coprodeum were mounted in Ussing chambers and investigated under voltage-clamped conditions. Simultaneous stimulation with aldosterone (1 mumol.l-1) and thyroxine (1 mumol.l-1) raised short-circuit current after a 1- to 2-h latent period. Maximal values were reached after 6-7 h of hormonal treatment, at which time transepithelial Na+ absorption was more than tripled (77 +/- 11 microA.cm-2) compared to control (24 +/- 8 microA.cm-2). K+ currents across the basolateral membrane were investigated after permeabilizing the apical membrane with the pore-forming antibiotic amphotericin B and application of a mucosal-to-serosal K+ gradient. This K+ current could be dose dependently depressed by the K+ channel blocker quinidine. Fluctuation analysis of the short-circuit current revealed a spontaneous and a blocker-induced Lorentzian noise component in the power density spectra. The Lorentzian corner frequencies increased linearly with the applied blocker concentration. This enabled the calculation of single K+ channel current and K+ channel density. Single K+ channel current was not affected by stimulation, whereas the number of quinidine-sensitive K+ channels in the basolateral membrane increased from 11 to 26.10(6).cm-2 in parallel to the hormonal stimulation transepithelial Na+ transport. This suggests that the basolateral membrane is a physiological target during synergistic aldosterone and thyroxine regulation of transepithelial Na+ transport for maintaining intracellular K+ homeostasis. PMID:8151014

  19. Ca2+ -calmodulin feedback mediates sensory adaptation and inhibits pheromone-sensitive ion channels in the vomeronasal organ.

    PubMed

    Spehr, Jennifer; Hagendorf, Silke; Weiss, Jan; Spehr, Marc; Leinders-Zufall, Trese; Zufall, Frank

    2009-02-18

    The mammalian vomeronasal organ (VNO) mediates the regulation of social behaviors by complex chemical signals. These cues trigger transient elevations of intracellular Ca(2+) in vomeronasal sensory neurons (VSNs), but the functional role of such Ca(2+) elevations is unknown. We show that stimulus-induced Ca(2+) entry plays an essential role as a negative feedback regulator of VSN sensitivity. Electrophysiological VSN responses undergo effective sensory adaptation that requires the influx of Ca(2+) and is mediated by calmodulin (CaM). Removal of the Ca(2+)-CaM feedback eliminates this form of adaptation. A key target of this feedback module is the pheromone-sensitive TRPC2-dependent cation channel of VSNs, as its activation is strongly inhibited by Ca(2+)-CaM. Our results reveal a previously unrecognized CaM-signaling pathway that endows the VSNs with a mechanism for adjusting gain and sensitivity of chemosensory signaling in the VNO. PMID:19228965

  20. [Sulfonylureas in today's blood glucose lowering therapy. New data on advantages and potential barriers of an "old" antidiabetic group].

    PubMed

    Winkler, Gábor

    2015-03-29

    Sulfonylurea compounds have been basic elements of antidiabetic treatment in type 2 diabetes for a long time. However, with the introduction of incretin type insulin secretagogues it is often arises, whether is still there a place for sulfonylureas in the today's therapy. To answer this question the author overviews general pharmaceutical characteristics of the sulfonylurea compounds as well as individual particularities of the second generation derivatives used at present in Hungary. The author details also the most important differences between incretin type drugs - first of all dipeptidyl peptidase-4 inhibitors - and sulfonylureas. On the basis of available data it can be concluded in accordance with the latest international guidelines, that sulfonylureas have still role in the blood glucose lowering therapy of type 2 diabetes, though they became somewhat pushed back among insulin secretagogue type drugs. If a sulfonylurea compound is the drug of choice, it is important to select the appropriate molecule (in case of normal renal function gliclazide or glimepiride). It is also important to re-educate the patient, as well as to apply the minimal dose providing the desired glycaemic effect. PMID:25796278

  1. Role of Epac2A/Rap1 signaling in interplay between incretin and sulfonylurea in insulin secretion.

    PubMed

    Takahashi, Harumi; Shibasaki, Tadao; Park, Jae-Hyung; Hidaka, Shihomi; Takahashi, Toshimasa; Ono, Aika; Song, Dae-Kyu; Seino, Susumu

    2015-04-01

    Incretin-related drugs and sulfonylureas are currently used worldwide for the treatment of type 2 diabetes. We recently found that Epac2A, a cAMP binding protein having guanine nucleotide exchange activity toward Rap, is a target of both incretin and sulfonylurea. This suggests the possibility of interplay between incretin and sulfonylurea through Epac2A/Rap1 signaling in insulin secretion. In this study, we examined the combinatorial effects of incretin and various sulfonylureas on insulin secretion and activation of Epac2A/Rap1 signaling. A strong augmentation of insulin secretion by combination of GLP-1 and glibenclamide or glimepiride, which was found in Epac2A(+/+) mice, was markedly reduced in Epac2A(-/-) mice. In contrast, the combinatorial effect of GLP-1 and gliclazide was rather mild, and the effect was not altered by Epac2A ablation. Activation of Rap1 was enhanced by the combination of an Epac-selective cAMP analog with glibenclamide or glimepiride but not gliclazide. In diet-induced obese mice, ablation of Epac2A reduced the insulin secretory response to coadministration of the GLP-1 receptor agonist liraglutide and glimepiride. These findings clarify the critical role of Epac2A/Rap1 signaling in the augmenting effect of incretin and sulfonylurea on insulin secretion and provide the basis for the effects of combination therapies of incretin-related drugs and sulfonylureas. PMID:25315008

  2. In Liddle Syndrome, Epithelial Sodium Channel Is Hyperactive Mainly in the Early Part of the Aldosterone-Sensitive Distal Nephron.

    PubMed

    Nesterov, Viatcheslav; Krueger, Bettina; Bertog, Marko; Dahlmann, Anke; Palmisano, Ralf; Korbmacher, Christoph

    2016-06-01

    The epithelial sodium channel (ENaC) is rate limiting for Na(+) absorption in the aldosterone-sensitive distal nephron comprising the late distal convoluted tubule (DCT2), the connecting tubule (CNT), and the entire collecting duct. Liddle syndrome (pseudohyperaldosteronism), a severe form of salt-sensitive hypertension, is caused by gain-of-function mutations of ENaC, but the precise tubular site of increased ENaC function is unknown. In the cortical collecting duct (CCD), ENaC is known to be regulated by aldosterone. In contrast, we recently reported aldosterone-independent ENaC regulation in the early part of the aldosterone-sensitive distal nephron. Here, we investigated ENaC function in the transition zone of DCT2/CNT or CNT/CCD microdissected from mice homozygous for Liddle syndrome mutation or from wild-type control mice. Whole-cell patch-clamp recordings were used to measure amiloride-sensitive ENaC currents in nephron fragments from mice maintained on different sodium diets to vary plasma aldosterone levels. Our data indicate that in mice with Liddle syndrome, the primary site of increased Na(+) reabsorption is the DCT2/CNT. In addition, increased aldosterone responsiveness of ENaC in CNT/CCD may contribute to salt-sensitive hypertension in Liddle syndrome. Single channel properties of ENaC were similar in Liddle syndrome mutation and wild-type mice, but ENaC expression at the apical membrane was increased in Liddle syndrome mutation when compared with wild-type mice, in particular, in animals maintained on a high salt diet. Our findings highlight the importance of ENaC function and regulation in the early part of the aldosterone-sensitive distal nephron for the maintenance of sodium balance and blood pressure control. PMID:27170740

  3. Proton channels and renal hypertensive injury: a key piece of the Dahl salt-sensitive rat puzzle?

    PubMed

    O'Connor, Paul M; Guha, Avirup; Stilphen, Carly A; Sun, Jingping; Jin, Chunhua

    2016-04-15

    Hv1 is a voltage-gated proton channel highly expressed in phagocytic cells, where it participates in the NADPH oxidase-dependent respiratory burst. We have recently identified Hv1 as a novel renal channel, expressed in the renal medullary thick ascending limb that appears to importantly contribute to the pathogenesis of renal hypertensive injury in the Dahl salt-sensitive rat model. The purpose of this review is to describe the experimental approaches that we have undertaken to identify the source of excess reactive oxygen species production in the renal outer medulla of Dahl salt-sensitive rats and the resulting evidence that the voltage-gated proton channel Hv1 mediates augmented superoxide production and contributes to renal medullary oxidative stress and renal injury. In addition, we will attempt to point out areas of current controversy, as well as propose areas in which further experimental studies are likely to move the field forward. The content of the following review was presented as part of the Water and Electrolyte Homeostasis Section New Investigator Award talk at Experimental Biology 2014. PMID:26843580

  4. Mitochondrial ATP-sensitive potassium channel opening inhibits isoproterenol-induced cardiac hypertrophy by preventing oxidative damage.

    PubMed

    Lemos Caldas, Francisco Rodrigo; Rocha Leite, Iago Mateus; Tavarez Filgueiras, Ana Beatriz; de Figueiredo Júnior, Isaias Lima; Gomes Marques de Sousa, Tereza Amália; Martins, Pamela Reis; Kowaltowski, Alicia Juliana; Fernandes Facundo, Heberty di Tarso

    2015-04-01

    Cardiac hypertrophy is a chronic complex disease that occurs in response to hemodynamic load and is accompanied by oxidative stress and mitochondrial dysfunction. Mitochondrial ATP-sensitive K channels (mitoKATPs) have previously been shown to prevent oxidative cardiac damage under conditions of ischemia/reperfusion. However, the effect of these channels on cardiac hypertrophy has not been tested to date. In this study, we show that treatment of Swiss mice with isoproterenol (30 mg·kg·d) induces cardiac hypertrophy while significantly decreasing the levels of reduced protein thiols, glutathione, catalase, and superoxide dismutase activity, indicative of a condition of oxidative imbalance. Treatment with diazoxide (a mitoKATP opener, 5 mg·kg·d) normalized the levels of protein thiols and reduced glutathione, rescued superoxide dismutase activity, and significantly prevented cardiac hypertrophy. The protective effects of diazoxide were mitigated by the mitoKATP blockers 5-hydroxydecanoate (5 mg·kg·d) and glibenclamide (3 mg·kg·d), demonstrating that they were related to activation of the channel. Taken together, our results establish that mitoKATP activation promotes very robust prevention of cardiac hypertrophy and associated oxidative imbalance and suggest that these channels can be important drug targets for the pharmacological control of cardiac hypertrophy. PMID:25850726

  5. Involvement of ATP-sensitive potassium channels and the opioid system in the anticonvulsive effect of zolpidem in mice.

    PubMed

    Sheikhi, Mehdi; Shirzadian, Armin; Dehdashtian, Amir; Amiri, Shayan; Ostadhadi, Sattar; Ghasemi, Mehdi; Dehpour, Ahmad Reza

    2016-09-01

    Zolpidem is a hypnotic medication that mainly exerts its function through activating γ-aminobutyric acid (GABA)A receptors. There is some evidence that zolpidem may have anticonvulsive effects. However, the mechanisms underlying this effect have not been elucidated yet. In the present study, we used the pentylentetrazole (PTZ)-induced generalized seizure model in mice to investigate whether zolpidem can affect seizure threshold. We also further evaluated the roles of ATP-sensitive potassium (KATP) channels as well as μ-opioid receptors in the effects of zolpidem on seizure threshold. Our data showed that zolpidem in a dose-dependent manner increased the PTZ-induced seizure threshold. The noneffective (i.e., did not significantly alter the PTZ-induced seizure threshold by itself) doses of KATP channel blocker (glibenclamide) and nonselective opioid receptor antagonist (naloxone) were able to inhibit the anticonvulsive effect of zolpidem. Additionally, noneffective doses of either KATP channel opener (cromakalim) or nonselective μ-opioid receptor agonist (morphine) in combination with a noneffective dose of zolpidem exerted a significant anticonvulsive effect on PTZ-induced seizures in mice. A combination of noneffective doses of naloxone and glibenclamide, which separately did not affect zolpidem effect on seizure threshold, inhibited the anticonvulsive effects of zolpidem. These results suggest a role for KATP channels and the opioid system, alone or in combination, in the anticonvulsive effects of zolpidem. PMID:27521722

  6. Rational design and validation of a vanilloid-sensitive TRPV2 ion channel.

    PubMed

    Yang, Fan; Vu, Simon; Yarov-Yarovoy, Vladimir; Zheng, Jie

    2016-06-28

    Vanilloids activation of TRPV1 represents an excellent model system of ligand-gated ion channels. Recent studies using cryo-electron microcopy (cryo-EM), computational analysis, and functional quantification revealed the location of capsaicin-binding site and critical residues mediating ligand-binding and channel activation. Based on these new findings, here we have successfully introduced high-affinity binding of capsaicin and resiniferatoxin to the vanilloid-insensitive TRPV2 channel, using a rationally designed minimal set of four point mutations (F467S-S498F-L505T-Q525E, termed TRPV2_Quad). We found that binding of resiniferatoxin activates TRPV2_Quad but the ligand-induced open state is relatively unstable, whereas binding of capsaicin to TRPV2_Quad antagonizes resiniferatoxin-induced activation likely through competition for the same binding sites. Using Rosetta-based molecular docking, we observed a common structural mechanism underlying vanilloids activation of TRPV1 and TRPV2_Quad, where the ligand serves as molecular "glue" that bridges the S4-S5 linker to the S1-S4 domain to open these channels. Our analysis revealed that capsaicin failed to activate TRPV2_Quad likely due to structural constraints preventing such bridge formation. These results not only validate our current working model for capsaicin activation of TRPV1 but also should help guide the design of drug candidate compounds for this important pain sensor. PMID:27298359

  7. Ca2+ Changes the Force Sensitivity of the Hair-Cell Transduction Channel

    PubMed Central

    Cheung, Eunice L. M.; Corey, David P.

    2006-01-01

    The mechanically gated transduction channels of vertebrate hair cells tend to close in ∼1 ms after their activation by hair bundle deflection. This fast adaptation is correlated with a quick negative movement of the bundle (a “twitch”), which can exert force and may mediate an active mechanical amplification of sound stimuli in hearing organs. We used an optical trap to deflect bullfrog hair bundles and to measure bundle movement while controlling Ca2+ entry with a voltage clamp. The twitch elicited by repolarization of the cell varied with force applied to the bundle, going to zero where channels were all open or closed. The force dependence is quantitatively consistent with a model in which a Ca2+-bound channel requires ∼3 pN more force to open, and rules out other models for the site of Ca2+ action. In addition, we characterized a faster, voltage-dependent “flick”, which requires intact tip links but not current through transduction channels. PMID:16214875

  8. Influence of dihydrotestosterone on sex determination in channel catfish and blue catfish: period of developmental sensitivity.

    PubMed

    Davis, K B; Goudie, C A; Simco, B A; Tiersch, T R; Carmichael, G J

    1992-04-01

    Treatment of channel catfish with 0.2, 20, or 200 mg/liter of dihydrotestosterone (DHT) in the water during the egg stage or during egg and sac-fry stages did not alter the expected 1:1 sex ratio of the progeny. Feeding DHT at 200 mg/kg of feed for the first 21 days after yolk sac absorption resulted in 80% females; this proportion was increased by combining feeding with treatment of 200 mg DHT/liter in the sac-fry stage (90%) or in the egg and sac-fry stage (97%). In contrast, treatment of blue catfish sac-fry with 200 mg DHT/liter, with or without the combination of feeding DHT at 200 mg/kg food, resulted in 100% female populations. Neither clomiphene citrate, an estrogen-receptor blocking agent, nor clofibrate, an inhibitor of hepatic synthesis of cholesterol, affected the sex ratio of channel catfish, and neither of these compounds altered the feminizing effect of 200 mg DHT/kg when fed in combination with DHT. The nonaromatizable androgen DHT is not as effective as many other androgens in producing paradoxical female populations of channel catfish. However, feminization of blue catfish by treatment of sac-fry indicates that this species is more susceptible to hormonal manipulation and that the period of sex determination may occur earlier in development than in channel catfish. PMID:1505724

  9. Unfolding of a Temperature-Sensitive Domain Controls Voltage-Gated Channel Activation.

    PubMed

    Arrigoni, Cristina; Rohaim, Ahmed; Shaya, David; Findeisen, Felix; Stein, Richard A; Nurva, Shailika Reddy; Mishra, Smriti; Mchaourab, Hassane S; Minor, Daniel L

    2016-02-25

    Voltage-gated ion channels (VGICs) are outfitted with diverse cytoplasmic domains that impact function. To examine how such elements may affect VGIC behavior, we addressed how the bacterial voltage-gated sodium channel (BacNa(V)) C-terminal cytoplasmic domain (CTD) affects function. Our studies show that the BacNa(V) CTD exerts a profound influence on gating through a temperature-dependent unfolding transition in a discrete cytoplasmic domain, the neck domain, proximal to the pore. Structural and functional studies establish that the BacNa(V) CTD comprises a bi-partite four-helix bundle that bears an unusual hydrophilic core whose integrity is central to the unfolding mechanism and that couples directly to the channel activation gate. Together, our findings define a general principle for how the widespread four-helix bundle cytoplasmic domain architecture can control VGIC responses, uncover a mechanism underlying the diverse BacNa(V) voltage dependencies, and demonstrate that a discrete domain can encode the temperature-dependent response of a channel. PMID:26919429

  10. Streaming potentials reveal a short ryanodine-sensitive selectivity filter in cardiac Ca2+ release channel.

    PubMed Central

    Tu, Q; Vélez, P; Brodwick, M; Fill, M

    1994-01-01

    Single cardiac sarcoplasmic reticulum Ca2+ release channels were reconstituted into planar bilayer membranes. Streaming potentials were measured in osmotically asymmetric solutions as a shift in the reversal potential. Potential changes induced by water movement through the bilayer (concentration polarization) and reduced ion activity in the concentrated non-electrolyte solutions were determined using valinomycin. In 400 mM symmetrical CsCH3SO3, the average streaming potential was 2.74 +/- 0.2 mV (n = 5, mean +/- SE; 2 osmol/kg) and independent of the osmoticant used (sucrose or diglycine). Identical streaming potential magnitudes were obtained regardless of which side of the membrane the nonelectrolyte was placed. This suggests that the narrow part of the pore where single file diffusion occurs is relatively short (i.e., accommodates a minimum of 3 H2O molecules). This value is comparable to similar measurements in a variety of surface membrane channels. Ryanodine-modified channels had no measurable streaming potential, an increased Tris+ permeability relative to Cs+, and decreased divalent selectivity (PCs/PTris 5.1 +/- 1.1 to 1.7 +/- 0.3, n = 3; PBa/PCs 8.2 +/- 0.7 to 1.8 +/- 0.5, n = 4). Cation/anion selectivity was essentially unaltered in ryanodine-modified channels. These results suggests that the narrow region of the permeation pathway (i.e., the selectivity filter) is relatively short and widens after ryanodine modification. PMID:7696468