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Sample records for channel sulfonylurea sensitivity

  1. Facilitation of ß-cell K(ATP) channel sulfonylurea sensitivity by a cAMP analog selective for the cAMP-regulated guanine nucleotide exchange factor Epac.

    PubMed

    Leech, Colin A; Dzhura, Igor; Chepurny, Oleg G; Schwede, Frank; Genieser, Hans-G; Holz, George G

    2010-01-01

    Clinical studies demonstrate that combined administration of sulfonylureas with exenatide can induce hypoglycemia in type 2 diabetic subjects. Whereas sulfonylureas inhibit ß-cell K(ATP) channels by binding to the sulfonylurea receptor-1 (SUR1), exenatide binds to the GLP-1 receptor, stimulates ß-cell cAMP production and activates both PKA and Epac. In this study, we hypothesized that the adverse in vivo interaction of sulfonylureas and exenatide to produce hypoglycemia might be explained by Epac-mediated facilitation of K(ATP) channel sulfonylurea sensitivity. We now report that the inhibitory action of a sulfonylurea (tolbutamide) at K(ATP) channels was facilitated by 2’-O-Me-cAMP, a selective activator of Epac. Thus, under conditions of excised patch recording, the dose-response relationship describing the inhibitory action of tolbutamide at human ß-cell or rat INS-1 cell K(ATP) channels was left-shifted in the presence of 2’-O-Me-cAMP, and this effect was abolished in INS-1 cells expressing a dominant-negative Epac2. Using an acetoxymethyl ester prodrug of an Epac-selective cAMP analog (8-pCP T-2’-O-Me-cAMP-AM), the synergistic interaction of an Epac activator and tolbutamide to depolarize INS-1 cells and to raise [Ca²(+)](i) was also measured. This effect of 8-pCP T-2’-O-Me-cAMP-AM correlated with its ability to stimulate phosphatidylinositol 4,5-bisphosphate hydrolysis that might contribute to the changes in K(ATP) channel sulfonylurea-sensitivity reported here. On the basis of such findings, we propose that the adverse interaction of sulfonylureas and exenatide to induce hypoglycemia involves at least in part, a functional interaction of these two compounds to close K(ATP) channels, to depolarize ß-cells and to promote insulin secretion.

  2. 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

  3. [Activation of ATP-sensitive K+ channels by ADP and K+ channel openers: homology model of sulfonylurea receptor carboxyl-termini].

    PubMed

    Fujita, A; Matsuoka, T; Matsushita, K; Kurachi, Y

    2001-09-01

    The ATP-sensitive K+ channels (KATP) are composed of Kir6.0 subunits and sulfonylurea receptors (SUR1, 2A and 2B). SUR2A and SUR2B are splice variants and differ only in the C-terminal 42 amino acid residue (C42). SURs are supposed to be the subunit that determines the different response of KATPs to intracellular nucleotides, K+ channel openers and inhibitors. In this study, we report that C42 of SURs plays critical roles in differential activation of various KATPs by ADP and K+ channel openers such as diazoxide and nicorandil. KATPs containing distinct SURs and Kir6.2 were reconstructed on HEK293T cells. Much higher concentrations of ADP were necessary to activate channels which SUR1 or SUR2B. In all KATPs containing different SUR, diazoxide increased the potency of ADP for channel activity without affecting its efficacy. From the electrophysiological data obtained from C-terminal chimeras and point mutants in the second nucleotide binding domain (NBDs), we developed the homology model of each SUR-NBD2 based on the crystallgraphically determined structure of HisP, a member of the ABC protein superfamily. In this model, C42 is located just beneath the Walker A motif of NBD2 and regulates the binding of nucleotide to NBD2 by affecting the 3-D construct of NBD2. This homology model well explains the different response of KATPs to ADP. Based on this model, it will be possible to develop new ligands for KATPs.

  4. The nucleotide-binding domains of sulfonylurea receptor 2A and 2B play different functional roles in nicorandil-induced activation of ATP-sensitive K+ channels.

    PubMed

    Yamada, Mitsuhiko; Kurachi, Yoshihisa

    2004-05-01

    Nicorandil activates ATP-sensitive K(+) channels composed of Kir6.2 and either sulfonylurea receptor (SUR) 2A or 2B. Although SUR2A and SUR2B differ only in their C-terminal 42 amino acids (C42) and possess identical drug receptors and nucleotide-binding domains (NBDs), nicorandil more potently activates SUR2B/Kir6.2 than SUR2A/Kir6.2 channels. Here, we analyzed the roles of NBDs in these channels' response to nicorandil with the inside-out configuration of the patch-clamp method. Binding and hydrolysis of nucleotides by NBDs were impaired by mutations in the Walker A motif of NBD1 (K708A) and NBD2 (K1349A) and in the Walker B motif of NBD2 (D1470N). Experiments were done with internal ATP (1 mM). In SUR2A/Kir6.2 channels, the K708A mutation abolished, and the K1349A but not D1470N mutation reduced the sensitivity to nicorandil. ADP (100 microM) significantly increased the wild-type channels' sensitivity to nicorandil, which was abolished by the K1349A or D1470N mutation. Thus, the SUR2A/Kir6.2 channels' response to nicorandil critically depends on ATP-NBD1 interaction and is facilitated by interactions of ATP or ADP with NBD2. In SUR2B/Kir6.2 channels, either the K708A or K1349A mutation partially suppressed the response to nicorandil, and double mutations abolished it. The D1470N mutation also significantly impaired the response. ADP did not sensitize the channels. Thus, NBD2 hydrolyzes ATP, and NBD1 and NBD2 equally contribute to the response by interacting with ATP and ADP, accounting for the higher nicorandil sensitivity of SUR2B/Kir6.2 than SUR2A/Kir6.2 channels in the presence of ATP alone. Thus, C42 modulates the interaction of both NBDs with intracellular nucleotides.

  5. Sulfonylureas.

    PubMed

    Kalra, Sanjay; Gupta, Yashdeep

    2015-01-01

    This review describes the basic and clinical pharmacology of sulfonylureas. It undertakes a balanced assessment of the advantages and limitations of sulfonylureas, and compares the use of various sulfonylureas in different clinical situations. The authors suggest pragmatic guidance to facilitate safe and effective use of this class of drugs, and thus help make maximal use of this economical therapeutic option in resource challenged settings such as developed nations.

  6. 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

  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.

  8. Epac2: a sulfonylurea receptor?

    PubMed

    Rehmann, Holger

    2012-02-01

    Sulfonylureas are widely used oral drugs in the treatment of diabetes mellitus. They function by the inhibition of ATP-sensitive K+ channels in pancreatic β-cells, which are thus considered the 'classical' sulfonylurea receptor. Next to the ATP-sensitive K+ channels, additional sulfonylurea-interacting proteins were identified, which might contribute to the physiological effects of this drug family. Most recently, Epac2 (exchange protein directly activated by cAMP 2) was added to the list of sulfonylurea receptors. However, this finding caused controversy in the literature. The critical discussion of the present paper comes to the conclusion that sulfonylureas are not able to activate Epac2 directly and are unlikely to bind to Epac2. Increased blood glucose levels after food intake result in the secretion of insulin from pancreatic β-cells. Glucose levels are detected 'indirectly' by β-cells: owing to increased glycolysis rates, the ratio of cellular ATP/ADP increases and causes the closure of ATP-sensitive K+ channels. In consequence, cells depolarize and voltage-dependent Ca2+ channels open to cause an increase in the cellular Ca2+ concentration. Finally, Ca2+ induces the fusion of insulin-containing granules with the plasma membrane. Sulfonylureas, such as tolbutamide, glibenclamide or acetohexamide, form a class of orally applicable drugs used in the treatment of non-insulin-dependent diabetes mellitus.

  9. Sulfonylurea receptors inhibit the epithelial sodium channel (ENaC) by reducing surface expression.

    PubMed

    Konstas, A A; Bielfeld-Ackermann, A; Korbmacher, C

    2001-08-01

    In the kidney the epithelial Na+ channel (ENaC) is co-expressed with the sulfonylurea receptor (SUR), an ABC protein that shares a high degree of homology with the cystic fibrosis transmembrane conductance regulator (CFTR) and reportedly modifies ENaC in various preparations. To investigate a possible regulatory relationship between SUR and ENaC, we performed co-expression studies on Xenopus laevis oocytes, which were assayed for amiloride-sensitive currents (DeltaIami). Moreover, a chemiluminescence assay was used to investigate the surface expression of extracellular hemagglutinin-tagged SUR1 (SUR1-HA) or HA-tagged ENaC (ENaC-HA). In oocytes co-injected with SUR1/ENaC (or SUR2B/ENaC) DeltaIami was reduced by congruent with 53% (or congruent with 45%) compared to DeltaIami measured in matched control oocytes injected with ENaC alone. The inhibitory effect of SUR on DeltaIami was preserved in oocytes expressing ENaC with C-terminally truncated subunits. Co-expression of SURs did not confer sensitivity of DeltaIami to diazoxide, pinacidil, tolbutamide, or glibenclamide. ENaC does not facilitate the surface expression of SUR1-HA, which is known to be retained in the endoplasmatic reticulum (ER) by an ER-retention/retrieval signal. SUR1-HAAAA, a mutant that lacks this signal, still inhibits ENaC currents. Chemiluminescence was reduced by congruent with 49% in oocytes co-expressing ENaC-HA/SUR1 compared to that in control oocytes expressing ENaC-HA alone. We conclude that SUR does not interact with ENaC at the level of the plasma membrane but that it inhibits DeltaIami by reducing surface expression of the channel.

  10. 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

  11. Optical control of insulin release using a photoswitchable sulfonylurea.

    PubMed

    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

    2014-10-14

    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.

  12. Pharmacological rescue of trafficking-impaired ATP-sensitive potassium channels

    PubMed Central

    Martin, Gregory M.; Chen, Pei-Chun; Devaraneni, Prasanna; Shyng, Show-Ling

    2013-01-01

    ATP-sensitive potassium (KATP) channels link cell metabolism to membrane excitability and are involved in a wide range of physiological processes including hormone secretion, control of vascular tone, and protection of cardiac and neuronal cells against ischemic injuries. In pancreatic β-cells, KATP channels play a key role in glucose-stimulated insulin secretion, and gain or loss of channel function results in neonatal diabetes or congenital hyperinsulinism, respectively. The β-cell KATP channel is formed by co-assembly of four Kir6.2 inwardly rectifying potassium channel subunits encoded by KCNJ11 and four sulfonylurea receptor 1 subunits encoded by ABCC8. Many mutations in ABCC8 or KCNJ11 cause loss of channel function, thus, congenital hyperinsulinism by hampering channel biogenesis and hence trafficking to the cell surface. The trafficking defects caused by a subset of these mutations can be corrected by sulfonylureas, KATP channel antagonists that have long been used to treat type 2 diabetes. More recently, carbamazepine, an anticonvulsant that is thought to target primarily voltage-gated sodium channels has been shown to correct KATP channel trafficking defects. This article reviews studies to date aimed at understanding the mechanisms by which mutations impair channel biogenesis and trafficking and the mechanisms by which pharmacological ligands overcome channel trafficking defects. Insight into channel structure-function relationships and therapeutic implications from these studies are discussed. PMID:24399968

  13. Congenital Hyperinsulinism–Associated ABCC8 Mutations That Cause Defective Trafficking of ATP-Sensitive K+ Channels

    PubMed Central

    Yan, Fei-Fei; Lin, Yu-Wen; MacMullen, Courtney; Ganguly, Arupa; Stanley, Charles A.; Shyng, Show-Ling

    2008-01-01

    Congenital hyperinsulinism (CHI) is a disease characterized by persistent insulin secretion despite severe hypoglycemia. Mutations in the pancreatic ATP-sensitive K+ (KATP) channel proteins sulfonylurea receptor 1 (SUR1) and Kir6.2, encoded by ABCC8 and KCNJ11, respectively, is the most common cause of the disease. Many mutations in SUR1 render the channel unable to traffic to the cell surface, thereby reducing channel function. Previous studies have shown that for some SUR1 trafficking mutants, the defects could be corrected by treating cells with sulfonylureas or diazoxide. The purpose of this study is to identify additional mutations that cause channel biogenesis/trafficking defects and those that are amenable to rescue by pharmacological chaperones. Fifteen previously uncharacterized CHI-associated missense SUR1 mutations were examined for their biogenesis/trafficking defects and responses to pharmacological chaperones, using a combination of immunological and functional assays. Twelve of the 15 mutations analyzed cause reduction in cell surface expression of KATP channels by >50%. Sulfonylureas rescued a subset of the trafficking mutants. By contrast, diazoxide failed to rescue any of the mutants. Strikingly, the mutations rescued by sulfonylureas are all located in the first transmembrane domain of SUR1, designated as TMD0. All TMD0 mutants rescued to the cell surface by the sulfonylurea tolbutamide could be subsequently activated by metabolic inhibition on tolbutamide removal. Our study identifies a group of CHI-causing SUR1 mutations for which the resulting KATP channel trafficking and expression defects may be corrected pharmacologically to restore channel function. PMID:17575084

  14. Permanent neonatal diabetes by a new mutation in KCNJ11: unsuccessful switch to sulfonylurea.

    PubMed

    Lau, Eva; Correia, Cintia; Freitas, Paula; Nogueira, Claúdia; Costa, Maria; Saavedra, Ana; Costa, Carla; Carvalho, Davide; Fontoura, Manuel

    2015-12-01

    Permanent neonatal diabetes (PNDM) can result from activating heterozygous mutations in KCNJ11 gene, encoding the Kir6.2 subunit of the pancreatic ATP-sensitive potassium channels (KATP). Sulfonylureas promote KATP closure and stimulate insulin secretion, being an alternative therapy in PNDM, instead of insulin. Male, 20 years old, diagnosed with diabetes at 3 months of age. The genetic study identified a novel heterozygous mutation in exon 1 of the KCNJ11 gene - KCNJ11:c1001G>7 (p.Gly334Val) - and confirmed the diagnosis of PNDM. Therefore it was attempted to switch from insulin therapy to sulfonylurea. During glibenclamide institution C-peptide levels increased, however the suboptimal glycemic control lead us to restart an intensive insulin scheme. This new variant of KCNJ11 mutation had a phenotypic lack of response to sulfonylurea therapy. Age, prior poor metabolic control and functional change of KATP channel induced by this specific mutation may explain the observed unsuccessful switch to sulfonylurea. Interestingly, C-peptide levels raise during glibenclamide administration support some degree of improvement in insulin secretory capacity induced by the treatment. Understanding the response to sulfonylurea is crucial as successful treatment may be life-changing in these patients.

  15. Sulfonylurea receptor 1 mutations that cause opposite insulin secretion defects with chemical chaperone exposure.

    PubMed

    Pratt, Emily B; Yan, Fei-Fei; Gay, Joel W; Stanley, Charles A; Shyng, Show-Ling

    2009-03-20

    The beta-cell ATP-sensitive potassium (K(ATP)) channel composed of sulfonylurea receptor SUR1 and potassium channel Kir6.2 serves a key role in insulin secretion regulation by linking glucose metabolism to cell excitability. Mutations in SUR1 or Kir6.2 that decrease channel function are typically associated with congenital hyperinsulinism, whereas those that increase channel function are associated with neonatal diabetes. Here we report that two hyperinsulinism-associated SUR1 missense mutations, R74W and E128K, surprisingly reduce channel inhibition by intracellular ATP, a gating defect expected to yield the opposite disease phenotype neonatal diabetes. Under normal conditions, both mutant channels showed poor surface expression due to retention in the endoplasmic reticulum, accounting for the loss of channel function phenotype in the congenital hyperinsulinism patients. This trafficking defect, however, could be corrected by treating cells with the oral hypoglycemic drugs sulfonylureas, which we have shown previously to act as small molecule chemical chaperones for K(ATP) channels. The R74W and E128K mutants thus rescued to the cell surface paradoxically exhibited ATP sensitivity 6- and 12-fold lower than wild-type channels, respectively. Further analyses revealed a nucleotide-independent decrease in mutant channel intrinsic open probability, suggesting the mutations may reduce ATP sensitivity by causing functional uncoupling between SUR1 and Kir6.2. In insulin-secreting cells, rescue of both mutant channels to the cell surface led to hyperpolarized membrane potentials and reduced insulin secretion upon glucose stimulation. Our results show that sulfonylureas, as chemical chaperones, can dictate manifestation of the two opposite insulin secretion defects by altering the expression levels of the disease mutants.

  16. Sulfonylurea Receptor 1 Mutations That Cause Opposite Insulin Secretion Defects with Chemical Chaperone Exposure*S⃞

    PubMed Central

    Pratt, Emily B.; Yan, Fei-Fei; Gay, Joel W.; Stanley, Charles A.; Shyng, Show-Ling

    2009-01-01

    The β-cell ATP-sensitive potassium (KATP) channel composed of sulfonylurea receptor SUR1 and potassium channel Kir6.2 serves a key role in insulin secretion regulation by linking glucose metabolism to cell excitability. Mutations in SUR1 or Kir6.2 that decrease channel function are typically associated with congenital hyperinsulinism, whereas those that increase channel function are associated with neonatal diabetes. Here we report that two hyperinsulinism-associated SUR1 missense mutations, R74W and E128K, surprisingly reduce channel inhibition by intracellular ATP, a gating defect expected to yield the opposite disease phenotype neonatal diabetes. Under normal conditions, both mutant channels showed poor surface expression due to retention in the endoplasmic reticulum, accounting for the loss of channel function phenotype in the congenital hyperinsulinism patients. This trafficking defect, however, could be corrected by treating cells with the oral hypoglycemic drugs sulfonylureas, which we have shown previously to act as small molecule chemical chaperones for KATP channels. The R74W and E128K mutants thus rescued to the cell surface paradoxically exhibited ATP sensitivity 6- and 12-fold lower than wild-type channels, respectively. Further analyses revealed a nucleotide-independent decrease in mutant channel intrinsic open probability, suggesting the mutations may reduce ATP sensitivity by causing functional uncoupling between SUR1 and Kir6.2. In insulin-secreting cells, rescue of both mutant channels to the cell surface led to hyperpolarized membrane potentials and reduced insulin secretion upon glucose stimulation. Our results show that sulfonylureas, as chemical chaperones, can dictate manifestation of the two opposite insulin secretion defects by altering the expression levels of the disease mutants. PMID:19151370

  17. [ATP-sensitive K(+)-channels in muscle cells: features and physiological role].

    PubMed

    Vadziuk, O B

    2014-01-01

    ATP-sensitive K(+)-channels of plasma membranes belong to the inward rectifier potassium channels type. They are involved in coupling of electrical activity of muscle cell with its metabolic state. These channels are heterooctameric and consist of two types of subunits: four poreforming (Kir 6.x) and four regulatory (SUR, sulfonylurea receptor). The Kir subunits contain highly selective K+ filter and provide for high-velocity K+ currents. The SUR subunits contain binding sites for activators and blockers and have metabolic sensor, which enables channel activation under conditions of metabolic stress. ATP blocks K+ currents through the ATP-sensitive K(+)-channels in the most types of muscle cells. However, functional activity of these channels does not depend on absolute concentration of ATP but on the ATP/ADP ratio and presence of Mg2+. Physiologically active substances, such as phosphatidylinositol bisphosphate and fatty acid esters can regulate the activity of these structures in muscle cells. Activation of these channels under ischemic conditions underlies their cytoprotective action, which results in prevention of Ca2+ overload in cytosol. In contrast to ATP-sensitive K(+)-channels of plasma membranes, the data regarding the structure and function of ATP-sensitive K(+)-channels of mitochondrial membrane are contradictory. Pore-forming subunits of this channel have not been firmly identified yet. ATP-sensitive K+ transport through the mitochondrial membrane is easily tested by different methods, which are briefly reviewed in this paper. Interaction of mitoK(ATP) with physiological and pharmacological ligands is discussed as well.

  18. Simple, cost-effective and sensitive liquid chromatography diode array detector method for simultaneous determination of eight sulfonylurea herbicides in soya milk samples.

    PubMed

    Rejczak, Tomasz; Tuzimski, Tomasz

    2016-11-18

    In this study authors propose a simple, cost-effective and sensitive liquid chromatography diode array detector (HPLC-DAD) method for the simultaneous determination of eight sulfonylurea herbicides (oxasulfuron, metsulfuron-methyl, triasulfuron, rimsulfuron, chlorsulfuron, mesosulfuron-methyl, amidosulfuron and bensulfuron-methyl) in soya milk samples. QuEChERS-based extraction procedure presents good performance for all of the analytes with recoveries in the range of 61-108% and relative standard deviations (RSD%) less than 15%. No significant matrix effect was observed thanks to application of effective zirconium-dioxide based sorbent (Z-Sep). Method LOQs for all investigated analytes were set at satisfactory low value of 10ngmL(-1) of food product. The procedure was evaluated in terms of natural samples analysis. Chlorsulfuron was determined at concentration of 14.2ngmL(-1) of soya milk product, which was qualitatively confirmed via LC coupled with tandem mass spectrometry (LC-MS/MS). The most important steps of procedure optimization are presented.

  19. Carbamazepine as a novel small molecule corrector of trafficking-impaired ATP-sensitive potassium channels identified in congenital hyperinsulinism.

    PubMed

    Chen, Pei-Chun; Olson, Erik M; Zhou, Qing; Kryukova, Yelena; Sampson, Heidi M; Thomas, David Y; Shyng, Show-Ling

    2013-07-19

    ATP-sensitive potassium (KATP) channels consisting of sulfonylurea receptor 1 (SUR1) and the potassium channel Kir6.2 play a key role in insulin secretion by coupling metabolic signals to β-cell membrane potential. Mutations in SUR1 and Kir6.2 that impair channel trafficking to the cell surface lead to loss of channel function and congenital hyperinsulinism. We report that carbamazepine, an anticonvulsant, corrects the trafficking defects of mutant KATP channels previously identified in congenital hyperinsulinism. Strikingly, of the 19 SUR1 mutations examined, only those located in the first transmembrane domain of SUR1 responded to the drug. We show that unlike that reported for several other protein misfolding diseases, carbamazepine did not correct KATP channel trafficking defects by activating autophagy; rather, it directly improved the biogenesis efficiency of mutant channels along the secretory pathway. In addition to its effect on channel trafficking, carbamazepine also inhibited KATP channel activity. Upon subsequent removal of carbamazepine, however, the function of rescued channels was recovered. Importantly, combination of the KATP channel opener diazoxide and carbamazepine led to enhanced mutant channel function without carbamazepine washout. The corrector effect of carbamazepine on mutant KATP channels was also demonstrated in rat and human β-cells with an accompanying increase in channel activity. Our findings identify carbamazepine as a novel small molecule corrector that may be used to restore KATP channel expression and function in a subset of congenital hyperinsulinism patients.

  20. Carbamazepine as a Novel Small Molecule Corrector of Trafficking-impaired ATP-sensitive Potassium Channels Identified in Congenital Hyperinsulinism*

    PubMed Central

    Chen, Pei-Chun; Olson, Erik M.; Zhou, Qing; Kryukova, Yelena; Sampson, Heidi M.; Thomas, David Y.; Shyng, Show-Ling

    2013-01-01

    ATP-sensitive potassium (KATP) channels consisting of sulfonylurea receptor 1 (SUR1) and the potassium channel Kir6.2 play a key role in insulin secretion by coupling metabolic signals to β-cell membrane potential. Mutations in SUR1 and Kir6.2 that impair channel trafficking to the cell surface lead to loss of channel function and congenital hyperinsulinism. We report that carbamazepine, an anticonvulsant, corrects the trafficking defects of mutant KATP channels previously identified in congenital hyperinsulinism. Strikingly, of the 19 SUR1 mutations examined, only those located in the first transmembrane domain of SUR1 responded to the drug. We show that unlike that reported for several other protein misfolding diseases, carbamazepine did not correct KATP channel trafficking defects by activating autophagy; rather, it directly improved the biogenesis efficiency of mutant channels along the secretory pathway. In addition to its effect on channel trafficking, carbamazepine also inhibited KATP channel activity. Upon subsequent removal of carbamazepine, however, the function of rescued channels was recovered. Importantly, combination of the KATP channel opener diazoxide and carbamazepine led to enhanced mutant channel function without carbamazepine washout. The corrector effect of carbamazepine on mutant KATP channels was also demonstrated in rat and human β-cells with an accompanying increase in channel activity. Our findings identify carbamazepine as a novel small molecule corrector that may be used to restore KATP channel expression and function in a subset of congenital hyperinsulinism patients. PMID:23744072

  1. Cryo-EM structure of the ATP-sensitive potassium channel illuminates mechanisms of assembly and gating

    PubMed Central

    Martin, Gregory M; Yoshioka, Craig; Rex, Emily A; Fay, Jonathan F; Xie, Qing; Whorton, Matthew R; Chen, James Z; Shyng, Show-Ling

    2017-01-01

    KATP channels are metabolic sensors that couple cell energetics to membrane excitability. In pancreatic β-cells, channels formed by SUR1 and Kir6.2 regulate insulin secretion and are the targets of antidiabetic sulfonylureas. Here, we used cryo-EM to elucidate structural basis of channel assembly and gating. The structure, determined in the presence of ATP and the sulfonylurea glibenclamide, at ~6 Å resolution reveals a closed Kir6.2 tetrameric core with four peripheral SUR1s each anchored to a Kir6.2 by its N-terminal transmembrane domain (TMD0). Intricate interactions between TMD0, the loop following TMD0, and Kir6.2 near the proposed PIP2 binding site, and where ATP density is observed, suggest SUR1 may contribute to ATP and PIP2 binding to enhance Kir6.2 sensitivity to both. The SUR1-ABC core is found in an unusual inward-facing conformation whereby the two nucleotide binding domains are misaligned along a two-fold symmetry axis, revealing a possible mechanism by which glibenclamide inhibits channel activity. DOI: http://dx.doi.org/10.7554/eLife.24149.001 PMID:28092267

  2. Role of Hsp90 in Biogenesis of the β-Cell ATP-sensitive Potassium Channel Complex

    PubMed Central

    Yan, Fei-Fei; Pratt, Emily B.; Chen, Pei-Chun; Wang, Fang; Skach, William R.; David, Larry L.

    2010-01-01

    The pancreatic β-cell ATP-sensitive potassium (KATP) channel is a multimeric protein complex composed of four inwardly rectifying potassium channel (Kir6.2) and four sulfonylurea receptor 1 (SUR1) subunits. KATP channels play a key role in glucose-stimulated insulin secretion by linking glucose metabolism to membrane excitability. Many SUR1 and Kir6.2 mutations reduce channel function by disrupting channel biogenesis and processing, resulting in insulin secretion disease. To better understand the mechanisms governing KATP channel biogenesis, a proteomics approach was used to identify chaperone proteins associated with KATP channels. We report that chaperone proteins heat-shock protein (Hsp)90, heat-shock cognate protein (Hsc)70, and Hsp40 are associated with β-cell KATP channels. Pharmacologic inhibition of Hsp90 function by geldanamycin reduces, whereas overexpression of Hsp90 increases surface expression of wild-type KATP channels. Coimmunoprecipitation data indicate that channel association with the Hsp90 complex is mediated through SUR1. Accordingly, manipulation of Hsp90 protein expression or function has significant effects on the biogenesis efficiency of SUR1, but not Kir6.2, expressed alone. Interestingly, overexpression of Hsp90 selectively improved surface expression of mutant channels harboring a subset of disease-causing SUR1 processing mutations. Our study demonstrates that Hsp90 regulates biogenesis efficiency of heteromeric KATP channels via SUR1, thereby affecting functional expression of the channel in β-cell membrane. PMID:20427569

  3. Antidiabetic sulfonylureas and cAMP cooperatively activate Epac2A.

    PubMed

    Takahashi, Toshimasa; Shibasaki, Tadao; Takahashi, Harumi; Sugawara, Kenji; Ono, Aika; Inoue, Naoko; Furuya, Toshio; Seino, Susumu

    2013-10-22

    Sulfonylureas are widely used drugs for treating insulin deficiency in patients with type 2 diabetes. Sulfonylureas bind to the regulatory subunit of the pancreatic β cell potassium channel that controls insulin secretion. Sulfonylureas also bind to and activate Epac2A, a member of the Epac family of cyclic adenosine monophosphate (cAMP)-binding proteins that promote insulin secretion through activation of the Ras-like guanosine triphosphatase Rap1. Using molecular docking simulation, we identified amino acid residues in one of two cyclic nucleotide-binding domains, cNBD-A, in Epac2A predicted to mediate the interaction with sulfonylureas. We confirmed the importance of the identified residues by site-directed mutagenesis and analysis of the response of the mutants to sulfonylureas using two assays: changes in fluorescence resonance energy transfer (FRET) of an Epac2A-FRET biosensor and direct sulfonylurea-binding experiments. These residues were also required for the sulfonylurea-dependent Rap1 activation by Epac2A. Binding of sulfonylureas to Epac2A depended on the concentration of cAMP and the structures of the drugs. Sulfonylureas and cAMP cooperatively activated Epac2A through binding to cNBD-A and cNBD-B, respectively. Our data suggest that sulfonylureas stabilize Epac2A in its open, active state and provide insight for the development of drugs that target Epac2A.

  4. Effects of sulfonylureas on tumor growth: a review of the literature.

    PubMed

    Pasello, Giulia; Urso, Loredana; Conte, Pierfranco; Favaretto, Adolfo

    2013-01-01

    Type 2 diabetes mellitus patients are at higher cancer risk, probably because of hyperinsulinemia and insulin growth factor 1 pathway activation. The effects of antidiabetic drugs on cancer risk have been described and discussed in several studies suggesting opposite effects of the biguanide metformin and sulfonylureas on cancer incidence and mortality. The anticancer mechanisms of metformin have been clarified, and some clinical studies, particularly in breast cancer patients, have been published or are currently ongoing; however, data about the effects of sulfonylureas on cancer growth are less consistent. The aims of this work are to review preclinical evidence of second-generation sulfonylureas effects on tumor growth, to clarify the potential mechanisms of action, and to identify possible metabolic targets for patient selection. Most evidence is on the adenosine triphosphate-sensitive potassium channels inhibitor glibenclamide, which interacts with reactive oxygen species production thus inducing cancer cell death. Among diarylsulfonylureas, next-generation DW2282 derivatives are particularly promising because of the proapoptotic activity in multidrug-resistant cells.

  5. Characterization of dihydropyridine-sensitive calcium channels

    SciTech Connect

    Horne, W.A.

    1989-01-01

    The structural and regulatory properties of the dihydropyridine-sensitive calcium channel were studied by isolating protein components of the channel complex from both cardiac and skeletal muscle. Hydrodynamic characterization of the (+)-({sup 3}H)PN200-110-labeled cardiac calcium channel revealed that the protein components of the complex had a total molecular mass of 370,000 daltons, a Stokes radius of 86 {angstrom}, and a frictional ratio of 1.3. A technique is described for the rapid incorporation of the CHAPS solubilized skeletal muscle calcium channel complex into phospholipid vesicles. {sup 45}Ca{sup 2+} uptake into phospholipid vesicles containing calcium channels was inhibited by phenylalkalamine calcium antagonists. Wheat germ lectin followed by DEAE chromatography of the CHAPS solubilized complex resulted in the dissociation of regulatory components of the complex from channel components. The DEAE preparation gave rise to {sup 45}Ca{sup 2+} uptake that was not inhibited by verapamil but was inhibited by GTPgS activated G{sub 0}. The inhibition of {sup 45}Ca{sup 2+} uptake by verapamil was restored by co-reconstitution of wash fractions from wheat germ lectin chromatography. Phosphorylation of polypeptides in this fraction by polypeptide-dependent protein kinase prevented the restoration of verapamil sensitivity. The partial purification of an endogenous skeletal muscle ADP-ribosyltransferase is also described. ADP-ribosylation of the {alpha}{sub 2} subunit of the calcium channel complex is enhanced by polylysine and inhibited by GTP{gamma}S, suggesting that regulation of this enzyme is under the control of GTP binding proteins. These results suggest a complex model, involving a number of different protein components, for calcium channel regulation in skeletal muscle.

  6. 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.

  7. Mechanism of Sulfonylurea Herbicide Resistance in the Broadleaf Weed, Kochia scoparia

    PubMed Central

    Saari, Leonard L.; Cotterman, Josephine C.; Primiani, Michael M.

    1990-01-01

    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 resistant 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 I50 to susceptible I50) 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 [14C]chlorsulfuron uptake, translocation, and metabolism between susceptible and resistant kochia biotypes. The Km 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. PMID:16667465

  8. 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.

  9. Endosulfine, an endogenous peptidic ligand for the sulfonylurea receptor: purification and partial characterization from ovine brain.

    PubMed Central

    Virsolvy-Vergine, A; Leray, H; Kuroki, S; Lupo, B; Dufour, M; Bataille, D

    1992-01-01

    Antidiabetic sulfonylureas act through receptors coupled to ATP-dependent potassium channels. Using the binding of [3H]glibenclamide, a highly potent sulfonylurea, to rat brain membranes to follow the purification procedure, we extracted from ovine brain, purified, and partially characterized two peptides that are endogenous ligands for the central nervous system sulfonylurea receptors. These peptides, referred to as alpha and beta endosulfine, differ by their isoelectric points, the beta form being more basic. Each form of endosulfine is recognized equally by the sulfonylurea receptors from the central nervous system and from insulin-secreting beta cells. In the same concentration range that is active on the receptors, beta endosulfine releases insulin from a beta-cell line. Endosulfine is a good candidate for being implicated in the physiology of beta cells and their disorders (e.g., type II diabetes) and in certain pathologies related to modifications of ion fluxes. Images PMID:1631165

  10. SulE, a sulfonylurea herbicide de-esterification esterase from Hansschlegelia zhihuaiae S113.

    PubMed

    Hang, Bao-Jian; Hong, Qing; Xie, Xiang-Ting; Huang, Xing; Wang, Cheng-Hong; He, Jian; Li, Shun-Peng

    2012-03-01

    De-esterification is an important degradation or detoxification mechanism of sulfonylurea herbicide in microbes and plants. However, the biochemical and molecular mechanisms of sulfonylurea herbicide de-esterification are still unknown. In this study, a novel esterase gene, sulE, responsible for sulfonylurea herbicide de-esterification, was cloned from Hansschlegelia zhihuaiae S113. The gene contained an open reading frame of 1,194 bp, and a putative signal peptide at the N terminal was identified with a predicted cleavage site between Ala37 and Glu38, resulting in a 361-residue mature protein. SulE minus the signal peptide was synthesized in Escherichia coli BL21 and purified to homogeneity. SulE catalyzed the de-esterification of a variety of sulfonylurea herbicides that gave rise to the corresponding herbicidally inactive parent acid and exhibited the highest catalytic efficiency toward thifensulfuron-methyl. SulE was a dimer without the requirement of a cofactor. The activity of the enzyme was completely inhibited by Ag(+), Cd(2+), Zn(2+), methamidophos, and sodium dodecyl sulfate. A sulE-disrupted mutant strain, ΔsulE, was constructed by insertion mutation. ΔsulE lost the de-esterification ability and was more sensitive to the herbicides than the wild type of strain S113, suggesting that sulE played a vital role in the sulfonylurea herbicide resistance of the strain. The transfer of sulE into Saccharomyces cerevisiae BY4741 conferred on it the ability to de-esterify sulfonylurea herbicides and increased its resistance to the herbicides. This study has provided an excellent candidate for the mechanistic study of sulfonylurea herbicide metabolism and detoxification through de-esterification, construction of sulfonylurea herbicide-resistant transgenic crops, and bioremediation of sulfonylurea herbicide-contaminated environments.

  11. Dysregulation of Insulin Secretion in Children With Congenital Hyperinsulinism due to Sulfonylurea Receptor Mutations

    PubMed Central

    Grimberg, A.; Ferry, R.J.; Kelly, A.; Koo-McCoy, S.; Polonsky, K.; Glaser, B.; Permutt, M.A.; Aguilar-Bryan, L.; Stafford, D.; Thornton, P.S.; Baker, L.; Stanley, Charles A.

    2012-01-01

    Mutations in the high-affinity sulfonylurea receptor (SUR)-1 cause one of the severe recessively inherited diffuse forms of congenital hyperinsulinism or, when associated with loss of heterozygosity, focal adenomatosis. We hypothesized that SUR1 mutations would render the β-cell insensitive to sulfonylureas and to glucose. Stimulated insulin responses were compared among eight patients with diffuse hyperinsulinism (two mutations), six carrier parents, and ten normal adults. In the patients with diffuse hyperinsulinism, the acute insulin response to intravenous tolbutamide was absent and did not overlap with the responses seen in either adult group. There was positive, albeit significantly blunted, acute insulin response to intravenous dextrose in the patients with diffuse hyperinsulinism. Graded infusions of glucose, to raise and then lower plasma glucose concentrations over 4 h, caused similar rises in blood glucose but lower peak insulin levels in the hyperinsulinemic patients. Loss of acute insulin response to tolbutamide can identify children with diffuse SUR1 defects. The greater response to glucose than to tolbutamide indicates that ATP-sensitive potassium (KATP) channel–independent pathways are involved in glucose-mediated insulin release in patients with diffuse SUR1 defects. The diminished glucose responsiveness suggests that SUR1 mutations and lack of KATP channel activity may contribute to the late development of diabetes in patients with hyperinsulinism independently of subtotal pancreatectomy. PMID:11272143

  12. Topical sulfonylurea as a novel therapy for hypertrichosis secondary to diazoxide, and potentially for other conditions with excess hair growth.

    PubMed

    Newfield, Ron S

    2015-12-01

    It is hypothesized that a topical application of a sulfonylurea drug, which can inhibit the ATP-sensitive potassium-gated channels (Kir6.X/SUR) present in human hair bulb tissues, will inhibit hair growth in a targeted manner. Diazoxide is used to treat severe hypoglycemia due to hyperinsulinism of infancy. However, this often results in hypertrichosis that can be severe enough to prevent its use. Diazoxide blocks insulin release from the pancreas by opening the SUR1/Kir6.2 channels in ß-cells. Diazoxide can also act on two potassium-gated channels in the skin that affect hair growth, namely SUR1/Kir6.2 and SUR2B/Kir6.1, thus causing hypertrichosis. It is proposed that a topical sulfonylurea will inhibit the excessive hair growth due to diazoxide, but will not impact the beneficial effects of diazoxide on beta cells. This approach can also be applied to rare cases of Cantú syndrome, caused by mutations in ABCC9 (coding for SUR2) or in KCNJ8 (coding for Kir6.1) that is characterized by congenital hypertrichosis. More importantly, this approach may also be effective in treating other forms of hypertrichosis or hirsutism, that are quite common, yet very distressing to patients worldwide.

  13. 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.

  14. Sulfonylureas: a new look at old therapy.

    PubMed

    Thulé, Peter M; Umpierrez, Guillermo

    2014-04-01

    Sulfonylurea compounds were the first available oral antidiabetic agents and they remain an important tool in our quest for optimal glucose control. The sulfonylureas stimulate the release of insulin from pancreatic β-cells and have a number of extrapancreatic effects, including decreasing hepatic insulin clearance and reducing glucagon secretion in patients with type 2 diabetes. Although these agents have been the mainstay of pharmacotherapy for patients with type 2 diabetes mellitus (T2DM), their safety and clinical utility has been a matter of active debate in recent years, as their use is associated with risks of hypoglycemia and weight gain. We review the discovery and mechanisms of action of sulfonylureas, and the results of clinical trials to provide practical information on the pros and cons of their use in clinical practice. This review addresses advances in our understanding of mechanisms of action of sulfonylurea agents, their efficacy in T2DM, side effects, and impact on cardiovascular disease outcomes.

  15. 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.

  16. [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.

  17. 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

  18. Stromal Interaction Molecule 1 (STIM1) Regulates ATP-sensitive Potassium (KATP) and Store-operated Ca(2+) Channels in MIN6 β-Cells.

    PubMed

    Leech, Colin A; Kopp, Richard F; Nelson, Heather A; Nandi, Jyotirmoy; Roe, Michael W

    2017-02-10

    Stromal interaction molecule 1 (STIM1) regulates store-operated Ca(2+) entry (SOCE) and other ion channels either as an endoplasmic reticulum Ca(2+)-sensing protein or when present in the plasma membrane. However, the role of STIM1 in insulin-secreting β-cells is unresolved. We report that lowering expression of STIM1, the gene that encodes STIM1, in insulin-secreting MIN6 β-cells with RNA interference inhibits SOCE and ATP-sensitive K(+) (KATP) channel activation. The effects of STIM1 knockdown were reversed by transduction of MIN6 cells with an adenovirus gene shuttle vector that expressed human STIM1 Immunoprecipitation studies revealed that STIM1 binds to nucleotide binding fold-1 (NBF1) of the sulfonylurea receptor 1 (SUR1) subunit of the KATP channel. Binding of STIM1 to SUR1 was enhanced by poly-lysine. Our data indicate that SOCE and KATP channel activity are regulated by STIM1. This suggests that STIM1 is a multifunctional signaling effector that participates in the control of membrane excitability and Ca(2+) signaling events in β-cells.

  19. N-terminal transmembrane domain of SUR1 controls gating of Kir6.2 by modulating channel sensitivity to PIP2.

    PubMed

    Pratt, Emily B; Tewson, Paul; Bruederle, Cathrin E; Skach, William R; Shyng, Show-Ling

    2011-03-01

    Functional integrity of pancreatic adenosine triphosphate (ATP)-sensitive potassium (K(ATP)) channels depends on the interactions between the pore-forming potassium channel subunit Kir6.2 and the regulatory subunit sulfonylurea receptor 1 (SUR1). Previous studies have shown that the N-terminal transmembrane domain of SUR1 (TMD0) interacts with Kir6.2 and is sufficient to confer high intrinsic open probability (P(o)) and bursting patterns of activity observed in full-length K(ATP) channels. However, the nature of TMD0-Kir6.2 interactions that underlie gating modulation is not well understood. Using two previously described disease-causing mutations in TMD0 (R74W and E128K), we performed amino acid substitutions to study the structural roles of these residues in K(ATP) channel function in the context of full-length SUR1 as well as TMD0. Our results revealed that although R74W and E128K in full-length SUR1 both decrease surface channel expression and reduce channel sensitivity to ATP inhibition, they arrive there via distinct mechanisms. Mutation of R74 uniformly reduced TMD0 protein levels, suggesting that R74 is necessary for stability of TMD0. In contrast, E128 mutations retained TMD0 protein levels but reduced functional coupling between TMD0 and Kir6.2 in mini-K(ATP) channels formed by TMD0 and Kir6.2. Importantly, E128K full-length channels, despite having a greatly reduced P(o), exhibit little response to phosphatidylinositol 4,5-bisphosphate (PIP(2)) stimulation. This is reminiscent of Kir6.2 channel behavior in the absence of SUR1 and suggests that TMD0 controls Kir6.2 gating by modulating Kir6.2 interactions with PIP(2). Further supporting this notion, the E128W mutation in full-length channels resulted in channel inactivation that was prevented or reversed by exogenous PIP(2). These results identify a critical determinant in TMD0 that controls Kir6.2 gating by controlling channel sensitivity to PIP(2). Moreover, they uncover a novel mechanism of K

  20. N-terminal transmembrane domain of SUR1 controls gating of Kir6.2 by modulating channel sensitivity to PIP2

    PubMed Central

    Pratt, Emily B.; Tewson, Paul; Bruederle, Cathrin E.; Skach, William R.

    2011-01-01

    Functional integrity of pancreatic adenosine triphosphate (ATP)-sensitive potassium (KATP) channels depends on the interactions between the pore-forming potassium channel subunit Kir6.2 and the regulatory subunit sulfonylurea receptor 1 (SUR1). Previous studies have shown that the N-terminal transmembrane domain of SUR1 (TMD0) interacts with Kir6.2 and is sufficient to confer high intrinsic open probability (Po) and bursting patterns of activity observed in full-length KATP channels. However, the nature of TMD0–Kir6.2 interactions that underlie gating modulation is not well understood. Using two previously described disease-causing mutations in TMD0 (R74W and E128K), we performed amino acid substitutions to study the structural roles of these residues in KATP channel function in the context of full-length SUR1 as well as TMD0. Our results revealed that although R74W and E128K in full-length SUR1 both decrease surface channel expression and reduce channel sensitivity to ATP inhibition, they arrive there via distinct mechanisms. Mutation of R74 uniformly reduced TMD0 protein levels, suggesting that R74 is necessary for stability of TMD0. In contrast, E128 mutations retained TMD0 protein levels but reduced functional coupling between TMD0 and Kir6.2 in mini-KATP channels formed by TMD0 and Kir6.2. Importantly, E128K full-length channels, despite having a greatly reduced Po, exhibit little response to phosphatidylinositol 4,5-bisphosphate (PIP2) stimulation. This is reminiscent of Kir6.2 channel behavior in the absence of SUR1 and suggests that TMD0 controls Kir6.2 gating by modulating Kir6.2 interactions with PIP2. Further supporting this notion, the E128W mutation in full-length channels resulted in channel inactivation that was prevented or reversed by exogenous PIP2. These results identify a critical determinant in TMD0 that controls Kir6.2 gating by controlling channel sensitivity to PIP2. Moreover, they uncover a novel mechanism of KATP channel inactivation

  1. 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

  2. Nocistatin sensitizes TRPA1 channels in peripheral sensory neurons.

    PubMed

    Avenali, Luca; Abate Fulas, Oli; Sondermann, Julia; Narayanan, Pratibha; Gomez-Varela, David; Schmidt, Manuela

    2017-01-02

    The ability of sensory neurons to detect potentially harmful stimuli relies on specialized molecular signal detectors such as transient receptor potential (TRP) A1 ion channels. TRPA1 is critically implicated in vertebrate nociception and different pain states. Furthermore, TRPA1 channels are subject to extensive modulation and regulation - processes which consequently affect nociceptive signaling. Here we show that the neuropeptide Nocistatin sensitizes TRPA1-dependent calcium influx upon application of the TRPA1 agonist mustard oil (MO) in cultured sensory neurons of dorsal root ganglia (DRG). Interestingly, TRPV1-mediated cellular calcium responses are unaffected by Nocistatin. Furthermore, Nocistatin-induced TRPA1-sensitization is likely independent of the Nocistatin binding partner 4-Nitrophenylphosphatase domain and non-neuronal SNAP25-like protein homolog 1 (NIPSNAP1) as assessed by siRNA-mediated knockdown in DRG cultures. In conclusion, we uncovered the sensitization of TRPA1 by Nocistatin, which may represent a novel mechanism how Nocistatin can modulate pain.

  3. Fast activation of dihydropyridine-sensitive calcium channels of skeletal muscle. Multiple pathways of channel gating

    PubMed Central

    1996-01-01

    Dihydropyridine (DHP) receptors of the transverse tubule membrane play two roles in excitation-contraction coupling in skeletal muscle: (a) they function as the voltage sensor which undergoes fast transition to control release of calcium from sarcoplasmic reticulum, and (b) they provide the conducting unit of a slowly activating L-type calcium channel. To understand this dual function of the DHP receptor, we studied the effect of depolarizing conditioning pulse on the activation kinetics of the skeletal muscle DHP-sensitive calcium channels reconstituted into lipid bilayer membranes. Activation of the incorporated calcium channel was imposed by depolarizing test pulses from a holding potential of -80 mV. The gating kinetics of the channel was studied with ensemble averages of repeated episodes. Based on a first latency analysis, two distinct classes of channel openings occurred after depolarization: most had delayed latencies, distributed with a mode of 70 ms (slow gating); a small number of openings had short first latencies, < 12 ms (fast gating). A depolarizing conditioning pulse to +20 mV placed 200 ms before the test pulse (-10 mV), led to a significant increase in the activation rate of the ensemble averaged-current; the time constant of activation went from tau m = 110 ms (reference) to tau m = 45 ms after conditioning. This enhanced activation by the conditioning pulse was due to the increase in frequency of fast open events, which was a steep function of the intermediate voltage and the interval between the conditioning pulse and the test pulse. Additional analysis demonstrated that fast gating is the property of the same individual channels that normally gate slowly and that the channels adopt this property after a sojourn in the open state. The rapid secondary activation seen after depolarizing prepulses is not compatible with a linear activation model for the calcium channel, but is highly consistent with a cyclical model. A six- state cyclical model is

  4. Leptin Suppression of Insulin Secretion by the Activation of ATP-Sensitive K+ Channels in Pancreatic β-Cells

    PubMed Central

    Kieffer, Timothy J.; Heller, R. Scott; Leech, Colin A.; Holz, George G.; Habener, Joel F.

    2010-01-01

    In the genetic mutant mouse models ob/ob or db/db, leptin deficiency or resistance, respectively, results in severe obesity and the development of a syndrome resembling NIDDM. One of the earliest manifestations in these mutant mice is hyperinsulinemia, suggesting that leptin may normally directly suppress the secretion of insulin. Here, we show that pancreatic islets express a long (signal-transducing) form of leptin-receptor mRNA and that β-cells bind a fluorescent derivative of leptin (Cy3-leptin). The expression of leptin receptors on insulin-secreting β-cells was also visualized utilizing antisera generated against an extracellular epitope of the receptor. A functional role for the β-cell leptin receptor is indicated by our observation that leptin (100 ng/ml) suppressed the secretion of insulin from islets isolated from ob/ob mice. Furthermore, leptin produced a marked lowering of [Ca2+]i in ob/ob β-cells, which was accompanied by cellular hyperpolarization and increased membrane conductance. Cell-attached patch measurements of ob/ob β-cells demonstrated that leptin activated ATP-sensitive potassium channels (KATP) by increasing the open channel probability, while exerting no effect on mean open time. These effects were reversed by the sulfonylurea tolbutamide, a specific inhibitor of KATP. Taken together, these observations indicate an important physiological role for leptin as an inhibitor of insulin secretion and lead us to propose that the failure of leptin to inhibit insulin secretion from the β-cells of ob/ob and db/db mice may explain, in part, the development of hyperinsulinemia, insulin resistance, and the progression to NIDDM. PMID:9166685

  5. 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

  6. ClC-3 chloride channel functions as a mechanically sensitive channel in osteoblasts.

    PubMed

    Wang, Huan; Wang, Rong; Wang, Zhe; Liu, Qian; Mao, Yong; Duan, Xiaohong

    2015-12-01

    Mechanical stimulation usually causes the volume changes of osteoblasts. Whether these volume changes could be sensed by the ClC-3 chloride channel, a volume-sensitive ion channel, and further promote the osteodifferentiation in osteoblasts has not been determined. In this study, we applied persistent static compression on MC3T3-E1 cells to detect the expression changes of ClC-3, osteogenic markers, as well as some molecules related with signaling transduction pathway. We tested the key role of ClC-3 in transferring the mechanical signal to osteoinduction by ClC-3 overexpressing and siRNA technique. We found that ClC-3 level was up-regulated by mechanical stimulation in MC3T3-E1 cells. Mechanical force also up-regulated the mRNA level of osteogenic markers such as alkaline phosphatase (Alp), bone sialoprotein (Bsp), and osteocalcin (Oc), which could be blocked or strengthened by Clcn3 siRNA or overexpressing, and Alp expression was more sensitive to the changes of ClC-3 level. We also found that runt-related transcription factor 2 (Runx2), transforming growth factor beta 1 (TGF-β1), and Wnt pathway might be involved in ClC-3 mediated mechanical transduction in osteoblasts. The data from the current study suggest that the ClC-3 chloride channel acts as a mechanically sensitive channel to regulate osteodifferentiation in osteoblasts.

  7. Treatment of sulfonylurea and insulin overdose.

    PubMed

    Klein-Schwartz, Wendy; Stassinos, Gina L; Isbister, Geoffrey K

    2016-03-01

    The most common toxicity associated with sulfonylureas and insulin is hypoglycaemia. The article reviews existing evidence to better guide hypoglycaemia management. Sulfonylureas and insulin have narrow therapeutic indices. Small doses can cause hypoglycaemia, which may be delayed and persistent. All children and adults with intentional overdoses need to be referred for medical assessment and treatment. Unintentional supratherapeutic ingestions can be initially managed at home but if symptomatic or if there is persistent hypoglycaemia require medical referral. Patients often require intensive care and prolonged observation periods. Blood glucose concentrations should be assessed frequently. Asymptomatic children with unintentional sulfonylurea ingestions should be observed for 12 h, except if this would lead to discharge at night when they should be kept until the morning. Prophylactic intravenous dextrose is not recommended. The goal of therapy is to restore and maintain euglycaemia for the duration of the drug's toxic effect. Enteral feeding is recommended in patients who are alert and able to tolerate oral intake. Once insulin or sulfonylurea-induced hypoglycaemia has developed, it should be initially treated with an intravenous dextrose bolus. Following this the mainstay of therapy for insulin-induced hypoglycaemia is intravenous dextrose infusion to maintain the blood glucose concentration between 5.5 and 11 mmol l(-1) . After sulfonylurea-induced hypoglycaemia is initially corrected with intravenous dextrose, the main treatment is octreotide which is administered to prevent insulin secretion and maintain euglycaemia. The observation period varies depending on drug, product formulation and dose. A general guideline is to observe for 12 h after discontinuation of intravenous dextrose and, if applicable, octreotide.

  8. Acid-sensitive outwardly rectifying anion channels in human erythrocytes.

    PubMed

    Kucherenko, Yuliya V; Mörsdorf, Daniel; Lang, Florian

    2009-07-01

    Acid-sensitive outwardly rectifying anion channels (ASOR) have been described in several mammalian cell types. The present whole-cell patch-clamp study elucidated whether those channels are expressed in erythrocytes. To this end whole-cell recordings were made in human erythrocytes from healthy donors treated with low pH and high osmotic pressure. When the pipette solution had a reduced Cl(-) concentration, treatment of the cells with Cl(-)-containing normal and hyperosmotic (addition of sucrose and polyethelene glycol 1000 [PEG-1000] to the Ringer) media with low pH significantly increased the conductance of the cells at positive voltages. Channel activity was highest in the PEG-1000 media (95 and 300 mM PEG-1000, pH 4.5 and 4.3, respectively) where the current-voltage curves demonstrated strong outward rectification and reversed at -40 mV. Substitution of the Cl(-)-containing medium with Cl(-)-free medium resulted in a decrease of the conductance at hyperpolarizing voltages, a shift in reversal potential (to 0 mV) and loss of outward rectification. The chloride currents were inhibited by chloride channels blockers DIDS and NPPB (IC(50) for both was approximately 1 mM) but not with niflumic acid and amiloride. The observations reveal expression of ASOR in erythrocytes.

  9. 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

  10. Successful sulfonylurea treatment of an insulin-naïve neonate with diabetes mellitus due to a KCNJ11 mutation.

    PubMed

    Wambach, Jennifer A; Marshall, Bess A; Koster, Joseph C; White, Neil H; Nichols, Colin G

    2010-06-01

    Activating mutations in the K(ATP)-channel cause neonatal diabetes mellitus (NDM), and patients have been safely transitioned from insulin to sulfonylureas. We report a male infant with permanent NDM (PNDM), born to a PNDM mother. Blood glucose began to rise on day of life (DOL) 2, and sulfonylurea (glyburide) therapy was initiated on DOL 5. Glucose was subsequently well controlled and normal at 3 months. A K(ATP) mutation (R201H; KCNJ11) was detected in the infant, the mother, and 6-yr-old sister with PNDM; both were also subsequently transitioned off insulin onto glyburide. To our knowledge, this is the youngest NDM patient to receive oral glyburide and, importantly, the only one deliberately initiated on sulfonylureas. Strikingly, the current dose (0.017 mg/kg/d) is below the reported therapeutic range and approximately 75-fold lower than doses required by the affected sister and mother. Pancreatic insulin disappears in an animal model of K(ATP)-induced NDM, unless glycemia is well controlled, thus, a dramatically lower glyburide requirement in the infant may reflect preserved insulin content because of early sulfonylurea intervention. Safe and effective initiation of glyburide in an insulin-naïve neonatal patient with K(ATP)-dependent PNDM argues for early detection and sulfonylurea intervention.

  11. Structure and function of the low conductance KATP channel, ROMK.

    PubMed

    Hebert, S C; Wang, W H

    1997-06-27

    The renal ATP-sensitive low-conductance K+ channel (KATP) plays an important role in K+ recycling in the thick ascending limb and in K+ secretion in the collecting duct. The low-conductance KATP is stimulated by cAMP-dependent protein kinase A and inhibited by protein kinase C, arachidonic acid, acidic pH and sulfonylurea agents. We reviewed the progress concerning the properties of the recently cloned inward-rectifying K+ channel (ROMK or KirI) and compared their regulatory mechanisms with the native low-conductance KATP. The results are important to gain insight into molecular mechanisms by which ROMK channels are regulated.

  12. 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

  13. Trypsin and alpha-chymotrypsin treatment abolishes glibenclamide sensitivity of KATP channels in rat ventricular myocytes.

    PubMed

    Nichols, C G; Lopatin, A N

    1993-03-01

    Cytoplasmic trypsin-treatment of voltage-sensitive potassium channels has been shown to cleave domains of the channel responsible for inactivation of the channel. Trypsin has also been reported to remove slow, irreversible inactivation, or run-down in ATP-sensitive potassium (KATP) channels. Cytoplasmic treatment of rat ventricular KATP channels with either crude, or pure trypsin (1-2 mg/ml) failed to prevent a slow run-down of channel activity. However, trypsin (porcine pancreatic type IX, or type II (Sigma Chem. Co.), or alpha-chymotrypsin (Sigma Chem. Co.) rapidly and irreversibly removed, or substantiallly decreased glibenclamide and tolbutamide-sensitivity of the channels without removing sensitivity to ATP. We conclude that glibenclamide must bind to either a separate protein, or to a separate domain on the channel in order to effect channel inhibition, and this domain is functionally disconnected from the channel by trypsin-, or alpha-chymotrypsin treatment.

  14. Sulfonylurea receptor -1 (SUR1): genetic and metabolic evidences for a role in the susceptibility to type 2 diabetes mellitus.

    PubMed

    Reis, A F; Velho, G

    2002-02-01

    The pancreatic B-cell ATP-sensitive potassium channel (K(ATP)) is composed of two distinct subunits, an inwardly rectifying ion channel forming the pore (Kir6.2), and a regulatory subunit, namely the sulfonylurea receptor-1 (SUR1), which binds this widely used class of insulin-secreting drugs. Mutations in the genes encoding Kir6.2 and SUR1 may result in familial persistent hyperinsulinemic hypoglycaemia of infancy, demonstrating their role in the regulation of insulin secretion. Studies in various populations with different ethnic background provided evidence that various alleles of single nucleotide polymorphisms (SNPs) in the SUR1 gene, and to a less extent in the Kir6.2 gene, confer a significantly increased risk for the development of type 2 diabetes mellitus (T2DM). Allelic variations of these SNPs were shown to modulate insulin secretion and insulin sensitivity in vivo, thus providing a pathophysiological background to explain their contribution to the genetic susceptibility to T2DM. The aim of this review is to summarise and discuss the significant results of recent literature on the implication of K(ATP), and particularly of SUR1, in the genetic and pathopysiological mechanisms of T2DM.

  15. Sulfonylureas and meglitinides: historical and contemporary issues.

    PubMed

    Lamos, E L; Stein, S A; Davis, S N

    2013-09-01

    Insulin secretagogue therapy is commonly used in clinical practice. These agents may be utilized as first, second-line or adjunct therapy behind metformin for treatment of type 2 diabetes mellitus. Sulfonylureas and meglitinides are effective treatments, but cumulative data over decades of research raise concerns regarding universal prescribing. The role of insulin secretagogue therapy in β-cell failure, blunting of ischemic pre-conditioning, the incidence of hypoglycemia - specifically in at-risk populations, modest weight gain and the unproven link to cancer are discussed. Ultimately, many of the concerns appear to be agent and not class-specific with glibenclamide fairing the worst amongst all of the agents discussed.

  16. Developmental regulation of voltage-sensitive sodium channels in rat skeletal muscle

    SciTech Connect

    Sherman, S.J.

    1985-01-01

    The developmental regulation of the voltage-sensitive Na/sup +/ channel in rat skeletal muscle was studied in vivo and in vitro. In triceps surae muscle developing in vivo the development of TTX-sensitive Na/sup +/ channel occurred primarily during the first three postnatal weeks as determined by the specific binding of (/sup 3/H)saxitoxin. This development proceeded in two separate phases. The first phase occurs independently of continuing motor neuron innervation and accounts for 60% of the adult density of TTX-sensitive Na/sup +/ channels. The second phase, which begins about day 11, requires innervation. Muscle cells in primary culture were found to have both TTX-sensitive and insensitive Na/sup +/ channels. The development of the TTX-sensitive channel, in vitro, paralleled the initial innervation-independent phase of development observed in vivo. The density of TTX-sensitive Na/sup +/ channels in cultured muscle cells was regulated by electrical activity and cytosolic Ca/sup + +/ levels. Pharmacological blockade of the spontaneous electrical activity present in these cells lead to a nearly 2-fold increase in the surface density of TTX-sensitive channels. The turnover time of the TTX-sensitive Na/sup +/ channel was measured by blocking the incorporation of newly synthesized channels with tunicamycin, an inhibitor of N-linked protein glycosylation. The regulation of channel density by electrical activity, cytosolic Ca/sup + +/levels, and agents affecting cyclic neucleotide levels had no effect on the turnover time of the TTX-sensitive Na/sup +/ channel, indicating that these regulatory agents instead affect the synthesis of the channel.

  17. Comparative analysis of cholesterol sensitivity of Kir channels: role of the CD loop

    PubMed Central

    Rosenhouse-Dantsker, Avia; Leal-Pinto, Edgar; Logothetis, Diomedes E.; Levitan, Irena

    2010-01-01

    Kir channels are important in setting the resting membrane potential and modulating membrane excitability. A common feature of Kir2 channels and several other ion channels that has emerged in recent years is that they are regulated by cholesterol, a major lipid component of the plasma membrane whose excess is associated with multiple pathological conditions. Yet, the mechanism by which cholesterol affects channel function is not clear. We have recently shown that the sensitivity of Kir2 channels to cholesterol depends on residues in the CD loop of the cytosolic domain of the channels with one of the mutations, L222I, abrogating cholesterol sensitivity of the channels completely. Here we show that in addition to Kir2 channels, members of other Kir subfamilies are also regulated by cholesterol. Interestingly, while similarly to Kir2 channels, several Kir channels, Kir1.1, Kir4.1 and Kir6.2Δ36 were suppressed by an increase in membrane cholesterol, the function of Kir3.4* and Kir7.1 was enhanced following cholesterol enrichment. Furthermore, we show that independent of the impact of cholesterol on channel function, mutating residues in the corresponding positions of the CD loop in Kir2.1 and Kir3.4*, inhibits cholesterol sensitivity of Kir channels, thus extending the critical role of the CD loop beyond Kir2 channels. PMID:19923917

  18. 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).

  19. Biophysical Properties of ATP-sensitive Potassium Channels in CA3 Hippocampal Neurons

    NASA Astrophysics Data System (ADS)

    Obregón-Herrera, Armando; Márquez-Gamiño, Sergio; Onetti, Carlos G.

    2004-09-01

    Single-channel activity of glucose-sensitive channels from CA3 neurons of the rat hippocampus, was studied in cell-attached membrane patches. Single-channel activity was totally abolished at 20 mM external glucose. Glucose-sensitive channels were selective to K+ ions; the unitary conductance was 170 pS in 140 mM K+, and the K+ permeability was 3.86×10-13 cmṡs-1. The open-state probability (PO) increased with membrane depolarization as a result of mean open time enhancement and shortening of the closure periods. The activation midpoint was -79 mV. Glucose-sensitive K+ channel of CA3 neurons could be considered as an ATP-sensitive potassium channel.

  20. ATP-sensitive Potassium Channels and L-type Calcium Channels are Involved in Morphine-induced Hyperalgesia after Nociceptive Sensitization in Mice

    PubMed Central

    Ahmadi, Shamseddin; Azarian, Shaho; Ebrahimi, Sayede Shohre; Rezayof, Ameneh

    2014-01-01

    Introduction We investigated the role of ATP-sensitive potassium channels and L-type calcium channels in morphine-induced hyperalgesia after nociceptive sensitization. Methods We used a hotplate apparatus to assess pain behavior in male NMRI mice. Nociceptive sensitization was induced by three days injection of morphine and five days of drug free. On day 9 of the schedule, pain behavior test was performed for evaluating the effects of morphine by itself and along with nimodipine, a blocker of L-type calcium channels and diazoxide, an opener of ATP-sensitive potassium channels. All drugs were injected through an intraperitoneal route. Results The results showed that morphine (7.5, 10 and 15 mg/kg) induced analgesia in normal mice, which was prevented by naloxone (1 mg/kg). After nociceptive sensitization, analgesic effect of morphine (10 and 15 mg/kg) was significantly decreased in sensitized mice. The results showed that nimodipine (2.5, 5, 10 and 20 mg/kg) had no significant effect on pain behavior test in either normal or sensitized mice. However, nimodipine (20 mg/ kg) along with morphine (10 and 15 mg/kg) caused more decrease in morphine analgesia in sensitized mice. Furthermore, diazoxide by itself (0.25, 1, 5 and 20 mg/kg) had also no significant effect on pain behavior in both normal and sensitized mice, but at dose of 20 mg/kg along with morphine (10 and 15 mg/kg) decreased analgesic effect of morphine in sensitized mice. Discussion It can be concluded that potassium and calcium channels have some roles in decrease of analgesic effect of morphine after nociceptive sensitization induced by pretreatment of morphine. PMID:25337379

  1. A functional role of the C-terminal 42 amino acids of SUR2A and SUR2B in the physiology and pharmacology of cardiovascular ATP-sensitive K(+) channels.

    PubMed

    Yamada, Mitsuhiko; Kurachi, Yoshihisa

    2005-07-01

    The ATP-sensitive K(+) (K(ATP)) channel is composed of four pore-forming Kir6.2 subunits and four sulfonylurea receptors (SUR). Intracellular ATP inhibits K(ATP) channels through Kir6.2. SUR is an ABC protein bearing transmembrane domains and two nucleotide-binding domains (NBD1 and NBD2). SUR increases the open probability of K(ATP) channels by interacting with ATP and ADP through NBDs and with K(+) channel openers such as nicorandil through its transmembrane domain. Because NBDs and the drug receptor allosterically interact with each other, nucleotides and drugs probably activate K(ATP) channels by causing the same conformational change of SUR. SUR2A and SUR2B have the identical drug receptor and NBDs and differ only in the C-terminal 42 amino acids (C42). Nonetheless, nicorandil ~100 times more potently activates SUR2B/Kir6.2 than SUR2A/Kir6.2 channels. Based on our allosteric model, we have analyzed the interaction between NBDs and the drug receptor in SUR2A and SUR2B and found that both nucleotide-bound NBD1 and NBD2 more strongly induce the conformational change in SUR2B than SUR2A. Therefore, C42 modulates the function of not only NBD2 which is close to C42 in a primary structure but NBD1 which is more than 630 amino acid N-terminal to C42. This raises the possibility that in the presence of nucleotides, NBD1 and NBD2 dimerize to induce the conformational change and that the dimerization enables C42 to gain access to both NBDs. Modulation of the nucleotide-NBD1 and -NBD2 interactions by C42 would determine the stability of the nucleotide-dependent dimer and thus, the physiological and pharmacological properties of K(ATP) channels.

  2. Actions of Ethanol on Voltage-Sensitive Sodium Channels: Effects on Neurotoxin Binding

    DTIC Science & Technology

    1987-01-01

    Exprnmantal Trherpeutics Ped in I.SA. Actions of Ethanol on Voltage-Sensitive Sodium Channels: Effects on Neurotoxin Binding1 MICHAEL J. MULLIN 2 and... sodium channels. This indirect allosteric mechanism for inhibition of [H]BTX-B binding. effect orethanol was concentration-dependent and was affected...ethanol increased the equilibrium binding constant without af- that ethanol can affect the voltage-sensitive sodium channels in fecting the apparent

  3. The effects of inorganic lead on voltage-sensitive calcium channels differ among cell types and among channel subtypes.

    PubMed

    Audesirk, G; Audesirk, T

    1993-01-01

    The whole-cell version of patch clamping was used to compare the effects of acute in vitro exposure to inorganic lead (Pb2+) on voltage-sensitive calcium channels in cultured N1E-115 mouse neuroblastoma cells and E18 rat hippocampal neurons. Free Pb2+ concentrations in salines with a high lead-buffering capacity were measured with a calibrated Pb(2+)-selective electrode. Previously, we found that N1E-115 neurons contain low voltage activated, rapidly inactivating (T) channels and high voltage activated, slowly inactivating (L) channels. Pb2+ inhibits both channel subtypes in N1E-115 cells, with some selectivity against L-type channels (IC50 approximately 700 nM free Pb2+ for L-type channels, 1300 nM free Pb2+ for T-type channels; Audesirk and Audesirk, 1991). In addition to T-type and L-type channels, cultured E18 rat hippocampal neurons have been reported to contain high voltage-activated, rapidly inactivating (N) channels. In our experiments with 5 to 20 day old cultures, almost all neurons showed substantial L-type current, approximately half showed significant N-type current, and fewer than 5% showed significant T-type current. We found that Pb2+ is somewhat selective against L-type channels (IC50 approximately 30 nM free Pb2+ in 10 mM Ba2+ as the charge carrier, 55 nM in 50 mM Ba2+) compared to N-channels (IC50 approximately 80 nM free Pb2+ in 10 mM Ba2+, 200 nM in 50 mM Ba2+). These results suggest that the effects of Pb2+ on calcium channels of vertebrate neurons vary both among cell types and among channel subtypes.

  4. Normal axonal ion channel function in large peripheral nerve fibers following chronic ciguatera sensitization.

    PubMed

    Vucic, Steve; Kiernan, Matthew C

    2008-03-01

    Although the acute clinical effects of ciguatera poisoning, due to ingestion of ciguatoxin, are mediated by activation of transient Na+ channels, the mechanisms underlying ciguatera sensitization remain undefined. Axonal excitability studies were performed by stimulating the median motor and sensory nerves in two patients with ciguatera sensitization. Excitability parameters were all within normal limits, thereby arguing against dysfunction of axonal membrane ion channels in large-diameter fibers in ciguatera sensitization.

  5. Electrophysiology of lead intoxication: effects on voltage-sensitive ion channels.

    PubMed

    Audesirk, G

    1993-01-01

    Neuronal function depends on the activity of a variety of voltage-sensitive, ion-specific membrane channels, including channels permeable chiefly to sodium, potassium, and calcium. The plasma membranes of many neurons contain several types of each class of channel. In general, heavy metal ions exert little effect on voltage-sensitive sodium or potassium channels, but inhibit ion flow through voltage-sensitive calcium channels (VSCC). The literature abounds with descriptions of different types of calcium channels in vertebrate neurons. These descriptions suggest that there are many physiologically and pharmacologically distinct calcium channels, some of them possibly cell-type specific. Among the heavy metals, Pb2+ is one of the most potent inhibitors of VSCC in both vertebrate and invertebrate neurons. Some heavy metals, including Ni2+ and Cd2+, are fairly selective against certain types of calcium channels. Limited evidence suggests that Pb2+ inhibits all calcium channel types within a given cell, with only minor differences in potency. However, there appear to be substantial differences among cell types in the concentration dependence of calcium channel inhibition by Pb2+. Therefore, to appreciate the range of effects of Pb2+ on calcium channels throughout the nervous system, it will be important to examine a large number of cell types. Pb2+ is highly permeable through at least some types of VSCC. Entry of Pb2+ into the neuronal cytoplasm through VSCC, followed by disturbance of intracellular functions, may be a major mechanism of Pb2+ neurotoxicity.

  6. 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.

  7. Successful sulfonylurea treatment of a neonate with neonatal diabetes mellitus due to a novel missense mutation, p.P1199L, in the ABCC8 gene.

    PubMed

    Oztekin, O; Durmaz, E; Kalay, S; Flanagan, S E; Ellard, S; Bircan, I

    2012-08-01

    Neonatal/infancy-onset diabetes mellitus is a monogenic form of diabetes with onset within 6 months of age. Two distinct types of neonatal diabetes mellitus have been recognized: permanent and transient. Mutations within the K(+)ATP channel and insulin genes are found in most patients with permanent diabetes mellitus. There have been several reports of the successful transition from insulin to sulfonylurea agents in patients with permanent diabetes mellitus caused by mutations in the KCNJ11 gene. We report on a term female neonate with a novel missense mutation, p.P1199L, in the ABCC8 gene that encodes the sulfonylurea receptor 1 whose treatment was successfully converted from insulin to sulfonylurea.

  8. Heteromeric heat-sensitive transient receptor potential channels exhibit distinct temperature and chemical response.

    PubMed

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

    2012-03-02

    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.

  9. Heteromeric Slick/Slack K+ channels show graded sensitivity to cell volume changes

    PubMed Central

    Hashem, Nadia; Calloe, Kirstine; Klaerke, Dan A.

    2017-01-01

    Slick and Slack high-conductance K+ channels are found in the CNS, kidneys, pancreas, among other organs, where they play an important role in cell excitability as well as in ion transport processes. They are both activated by Na+ and Cl- but show a differential regulation by cell volume changes. Slick has been shown to be regulated by cell volume changes, whereas Slack is insensitive. α-subunits of these channels form homomeric as well as heteromeric channels. It is the aim of this work to explore whether the subunit composition of the Slick/Slack heteromeric channel affects the response to osmotic challenges. In order to provide with the adequate water permeability to the cell membrane of Xenopus laevis oocytes, mRNA of aquaporin 1 was co-expressed with homomeric or heteromeric Slick and Slack α-subunits. Oocytes were superfused with hypotonic or hypertonic buffers and changes in currents were measured by two-electrode voltage clamp. This work presents the first heteromeric K+ channel with a characteristic graded sensitivity to small and fast changes in cell volume. Our results show that the cell volume sensitivity of Slick/Slack heteromeric channels is dependent on the number of volume sensitive Slick α-subunits in the tetrameric channels, giving rise to graded cell volume sensitivity. Regulation of the subunit composition of a channel may constitute a novel mechanism to determine volume sensitivity of cells. PMID:28222129

  10. A heat-sensitive TRP channel expressed in keratinocytes.

    PubMed

    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

    2002-06-14

    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.

  11. 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…

  12. Phosphorylation-dependent Changes in Nucleotide Binding, Conformation, and Dynamics of the First Nucleotide Binding Domain (NBD1) of the Sulfonylurea Receptor 2B (SUR2B)*

    PubMed Central

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

    2015-01-01

    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

  13. 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.

  14. Barium, TEA and sodium sensitive potassium channels are present in the human placental syncytiotrophoblast apical membrane.

    PubMed

    Díaz, P; Vallejos, C; Guerrero, I; Riquelme, G

    2008-10-01

    The human placental syncytiotrophoblast (hSTB) is a polarized epithelial structure, without paracellular routes, forming the main barrier for materno-fetal exchange. There is ample evidence suggesting the presence of potassium (K(+)) channels in the placental apical membrane; which could contribute to membrane potential and volume regulation. We have therefore examined the K(+) currents of isolated apical membranes from human term placenta using electrophysiological methods: reconstitution of ion channels from apical membranes into giant liposomes (single channel recordings, patch clamp method) or their functional transplantation into Xenopus laevis oocytes (total currents recording, voltage clamp method). Single channel recording experiments show the presence of K(+) channels in the hSTB microvillous membrane sensitive to Tetraethylammonium (TEA) and Barium (Ba(+2)). Patch current activity was diminished 50% and 70% by 20 mmol/L TEA and 5 mmol/L Ba(+2) respectively. The more frequent conductance was approximately 73pS, however several levels of current were detected suggesting the presence of more than one type of K(+) channel. In addition, sodium (Na(+)) sensitivity was detected in the patch current thus, over 10 mmol/L Na(+) reduced the seal current to 38%. These results were corroborated by the total current experiments where the K(+) current elicited in injected oocytes with apical purified membrane was blocked by Ba(+2) and TEA. The total current was also affected by Na(+), becoming larger when a Na(+)-free solution was used. Our results show the existence of at least two types of Ba(+2)-sensitive K(+) channels including a TEA sensitive sub-population, and some of them Na(+) sensitive K(+) channels. These channels could be the conductive pathways proposed previously for this cation in placental hSTB. Our novel contribution has been to successfully obtain K(+) channel recordings in systems suitable for electrophysiological studies of isolated apical membranes.

  15. Acid modulation of tetrodotoxin-sensitive Na(+) channels in large-sized trigeminal ganglion neurons.

    PubMed

    Nakamura, Michiko; Kim, Do-Yeon; Jang, Il-Sung

    2016-11-15

    Voltage-gated Na(+) channels in primary afferent neurons can be divided into tetrodotoxin-sensitive (TTX-S) and tetrodotoxin-resistant (TTX-R) Na(+) channels. Although previous studies have shown the acid modulation of TTX-R Na(+) channels, the effect of acidic pH on tetrodotoxin-sensitive (TTX-S) Na(+) channels is still unknown. Here we report the effect of acidic pH on TTX-S Na(+) channels expressed in large-sized trigeminal ganglion (TG) neurons using a whole-cell patch clamp technique. The application of acidic extracellular solution decreased the peak amplitude of TTX-S currents (INa) in a pH-dependent manner, but weak acid (≥pH 6.0) had no inhibitory effect on TTX-S INa. Acidic pH (pH 6.0) shifted both the activation and steady-state fast inactivation relationships of TTX-S Na(+) channels toward depolarized potentials. However, acidic pH (pH 6.0) had no effect on use-dependent inhibition in response to high-frequency stimuli, development of inactivation, and accelerated the recovery from inactivation of TTX-S Na(+) channels, suggesting that TTX-S Na(+) channels in large-sized TG neurons are less sensitive to acidic pH. Given that voltage-gated Na(+) channels play a pivotal role in the generation and conduction of action potentials in neural tissues, the insensitivity of TTX-S Na(+) channels expressed in large-sized TG neurons to acidic pH would ensure transmission of innocuous tactile sensation from orofacial regions at acidic pH conditions.

  16. 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

  17. Receptor-mediated glutamate release from volume sensitive channels in astrocytes

    NASA Astrophysics Data System (ADS)

    Takano, Takahiro; Kang, Jian; Jaiswal, Jyoti K.; Simon, Sanford M.; Lin, Jane H.-C.; Yu, Yufei; Li, Yuxing; Yang, Jay; Dienel, Gerald; Zielke, H. Ronald; Nedergaard, Maiken

    2005-11-01

    Several lines of work have shown that astrocytes release glutamate in response to receptor activation, which results in a modulation of local synaptic activity. Astrocytic glutamate release is Ca2+-dependent and occurs in conjunction with exocytosis of glutamate containing vesicles. However, astrocytes contain a millimolar concentration of cytosolic glutamate and express channels permeable to small anions, such as glutamate. Here, we tested the idea that astrocytes respond to receptor stimulation by dynamic changes in cell volume, resulting in volume-sensitive channel activation, and efflux of cytosolic glutamate. Confocal imaging and whole-cell recordings demonstrated that astrocytes exhibited a transient Ca2+-dependent cell volume increase, which activated glutamate permeable channels. HPLC analysis revealed that glutamate was released in conjunction with other amino acid osmolytes. Our observations indicate that volume-sensitive channel may constitute a previously uncharacterized target for modulation of astrocyte-neuronal interactions. electrophysiology | exocytosis | neurotransmitters | osmolarity | synapses

  18. The purified mechanosensitive channel TREK-1 is directly sensitive to membrane tension.

    PubMed

    Berrier, Catherine; Pozza, Alexandre; de Lacroix de Lavalette, Agnes; Chardonnet, Solenne; Mesneau, Agnes; Jaxel, Christine; le Maire, Marc; Ghazi, Alexandre

    2013-09-20

    Mechanosensitive channels are detected in all cells and are speculated to play a key role in many functions including osmoregulation, growth, hearing, balance, and touch. In prokaryotic cells, a direct gating of mechanosensitive channels by membrane tension was clearly demonstrated because the purified channels could be functionally reconstituted in a lipid bilayer. No such evidence has been presented yet in the case of mechanosensitive channels from animal cells. TREK-1, a two-pore domain K(+) channel, was the first animal mechanosensitive channel identified at the molecular level. It is the target of a large variety of agents such as volatile anesthetics, neuroprotective agents, and antidepressants. We have produced the mouse TREK-1 in yeast, purified it, and reconstituted the protein in giant liposomes amenable to patch clamp recording. The protein exhibited the expected electrophysiological properties in terms of kinetics, selectivity, and pharmacology. Negative pressure (suction) applied through the pipette had no effect on the channel, but positive pressure could completely and reversibly close the channel. Our interpretation of these data is that the intrinsic tension in the lipid bilayer is sufficient to maximally activate the channel, which can be closed upon modification of the tension. These results indicate that TREK-1 is directly sensitive to membrane tension.

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

    PubMed Central

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

    2015-01-01

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

  20. Subunit-specific mechanisms and proton sensitivity of NMDA receptor channel block.

    PubMed

    Dravid, Shashank M; Erreger, Kevin; Yuan, Hongjie; Nicholson, Katherine; Le, Phuong; Lyuboslavsky, Polina; Almonte, Antoine; Murray, Ernest; Mosely, Cara; Barber, Jeremy; French, Adam; Balster, Robert; Murray, Thomas F; Traynelis, Stephen F

    2007-05-15

    We have compared the potencies of structurally distinct channel blockers at recombinant NR1/NR2A, NR1/NR2B, NR1/NR2C and NR1/NR2D receptors. The IC50 values varied with stereochemistry and subunit composition, suggesting that it may be possible to design subunit-selective channel blockers. For dizocilpine (MK-801), the differential potency of MK-801 stereoisomers determined at recombinant NMDA receptors was confirmed at native receptors in vitro and in vivo. Since the proton sensor is tightly linked both structurally and functionally to channel gating, we examined whether blocking molecules that interact in the channel pore with the gating machinery can differentially sense protonation of the receptor. Blockers capable of remaining trapped in the pore during agonist unbinding showed the strongest dependence on extracellular pH, appearing more potent at acidic pH values that promote channel closure. Determination of pK(a) values for channel blockers suggests that the ionization of ketamine but not of other blockers can influence its pH-dependent potency. Kinetic modelling and single channel studies suggest that the pH-dependent block of NR1/NR2A by (-)MK-801 but not (+)MK-801 reflects an increase in the MK-801 association rate even though protons reduce channel open probability and thus MK-801 access to its binding site. Allosteric modulators that alter pH sensitivity alter the potency of MK-801, supporting the interpretation that the pH sensitivity of MK-801 binding reflects the changes at the proton sensor rather than a secondary effect of pH. These data suggest a tight coupling between the proton sensor and the ion channel gate as well as unique subunit-specific mechanisms of channel block.

  1. Highly Sensitive and Patchable Pressure Sensors Mimicking Ion-Channel-Engaged Sensory Organs.

    PubMed

    Chun, Kyoung-Yong; Son, Young Jun; Han, Chang-Soo

    2016-04-26

    Biological ion channels have led to much inspiration because of their unique and exquisite operational functions in living cells. Specifically, their extreme and dynamic sensing abilities can be realized by the combination of receptors and nanopores coupled together to construct an ion channel system. In the current study, we demonstrated that artificial ion channel pressure sensors inspired by nature for detecting pressure are highly sensitive and patchable. Our ion channel pressure sensors basically consisted of receptors and nanopore membranes, enabling dynamic current responses to external forces for multiple applications. The ion channel pressure sensors had a sensitivity of ∼5.6 kPa(-1) and a response time of ∼12 ms at a frequency of 1 Hz. The power consumption was recorded as less than a few μW. Moreover, a reliability test showed stability over 10 000 loading-unloading cycles. Additionally, linear regression was performed in terms of temperature, which showed no significant variations, and there were no significant current variations with humidity. The patchable ion channel pressure sensors were then used to detect blood pressure/pulse in humans, and different signals were clearly observed for each person. Additionally, modified ion channel pressure sensors detected complex motions including pressing and folding in a high-pressure range (10-20 kPa).

  2. 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.

  3. ATP binding cassette modulators control abscisic acid-regulated slow anion channels in guard cells

    PubMed Central

    Leonhardt, N; Vavasseur, A; Forestier, C

    1999-01-01

    In animal cells, ATP binding cassette (ABC) proteins are a large family of transporters that includes the sulfonylurea receptor and the cystic fibrosis transmembrane conductance regulator (CFTR). These two ABC proteins possess an ion channel activity and bind specific sulfonylureas, such as glibenclamide, but homologs have not been identified in plant cells. We recently have shown that there is an ABC protein in guard cells that is involved in the control of stomatal movements and guard cell outward K+ current. Because the CFTR, a chloride channel, is sensitive to glibenclamide and able to interact with K+ channels, we investigated its presence in guard cells. Potent CFTR inhibitors, such as glibenclamide and diphenylamine-2-carboxylic acid, triggered stomatal opening in darkness. The guard cell protoplast slow anion current that was recorded using the whole-cell patch-clamp technique was inhibited rapidly by glibenclamide in a dose-dependent manner; the concentration producing half-maximum inhibition was at 3 &mgr;M. Potassium channel openers, which bind to and act through the sulfonylurea receptor in animal cells, completely suppressed the stomatal opening induced by glibenclamide and recovered the glibenclamide-inhibited slow anion current. Abscisic acid is known to regulate slow anion channels and in our study was able to relieve glibenclamide inhibition of slow anion current. Moreover, in epidermal strip bioassays, the stomatal closure triggered by Ca2+ or abscisic acid was reversed by glibenclamide. These results suggest that the slow anion channel is an ABC protein or is tightly controlled by such a protein that interacts with the abscisic acid signal transduction pathway in guard cells. PMID:10368184

  4. On the sensitivity of Tropical Rainfall Measuring Mission (TRMM) Microwave Imager channels to overland rainfall

    NASA Astrophysics Data System (ADS)

    You, Yalei; Liu, Guosheng; Wang, Yu; Cao, Jie

    2011-06-01

    , and the V37 or V21 channel becomes the top responder to surface rain as the amount of hydrometeors in the atmospheric column reaches very high values. Additionally, it is found that land surface type and 2 m air temperature have significant skills in characterizing rain cloud types, so that the V19-V37 channel is more sensitive to surface rainfall for more vegetated warm surface, while the V85 channel is more sensitive to cold bare land. This finding implies that the above two parameters may be used to prioritize satellite observations at different channels, so that the channel that has the best rainfall sensitivity under a given condition receives the highest weight in retrieval algorithms.

  5. 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...

  6. Dual Regulation of Voltage-Sensitive Ion Channels by PIP(2).

    PubMed

    Rodríguez-Menchaca, Aldo A; Adney, Scott K; Zhou, Lei; Logothetis, Diomedes E

    2012-01-01

    Over the past 16 years, there has been an impressive number of ion channels shown to be sensitive to the major phosphoinositide in the plasma membrane, phosphatidylinositol 4,5-bisphosphate (PIP(2)). Among them are voltage-gated channels, which are crucial for both neuronal and cardiac excitability. Voltage-gated calcium (Cav) channels were shown to be regulated bidirectionally by PIP(2). On one hand, PIP(2) stabilized their activity by reducing current rundown but on the other hand it produced a voltage-dependent inhibition by shifting the activation curve to more positive voltages. For voltage-gated potassium (Kv) channels PIP(2) was first shown to prevent N-type inactivation regardless of whether the fast inactivation gate was part of the pore-forming α subunit or of an accessory β subunit. Careful examination of the effects of PIP(2) on the activation mechanism of Kv1.2 has shown a similar bidirectional regulation as in the Cav channels. The two effects could be distinguished kinetically, in terms of their sensitivities to PIP(2) and by distinct molecular determinants. The rightward shift of the Kv1.2 voltage dependence implicated basic residues in the S4-S5 linker and was consistent with stabilization of the inactive state of the voltage sensor. A third type of a voltage-gated ion channel modulated by PIP(2) is the hyperpolarization-activated cyclic nucleotide-gated (HCN) channel. PIP(2) has been shown to enhance the opening of HCN channels by shifting their voltage-dependent activation toward depolarized potentials. The sea urchin HCN channel, SpIH, showed again a PIP(2)-mediated bidirectional effect but in reverse order than the depolarization-activated Cav and Kv channels: a voltage-dependent potentiation, like the mammalian HCN channels, but also an inhibition of the cGMP-induced current activation. Just like the Kv1.2 channels, distinct molecular determinants underlied the PIP(2) dual effects on SpIH, with the proximal C-terminus implicated in the

  7. 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

  8. TRPV4, TRPC1, and TRPP2 assemble to form a flow-sensitive heteromeric channel

    PubMed Central

    Du, Juan; Ma, Xin; Shen, Bing; Huang, Yu; Birnbaumer, Lutz; Yao, Xiaoqiang

    2014-01-01

    Transient receptor potential (TRP) channels, a superfamily of ion channels, can be divided into 7 subfamilies, including TRPV, TRPC, TRPP, and 4 others. Functional TRP channels are tetrameric complexes consisting of 4 pore-forming subunits. The purpose of this study was to explore the heteromerization of TRP subunits crossing different TRP subfamilies. Two-step coimmunoprecipitation (co-IP) and fluorescence resonance energy transfer (FRET) were used to determine the interaction of the different TRP subunits. Patch-clamp and cytosolic Ca2+ measurements were used to determine the functional role of the ion channels in flow conditions. The analysis demonstrated the formation of a heteromeric TRPV4-C1-P2 complex in primary cultured rat mesenteric artery endothelial cells (MAECs) and HEK293 cells that were cotransfected with TRPV4, TRPC1, and TRPP2. In functional experiments, pore-dead mutants for each of these 3 TRP isoforms nearly abolished the flow-induced cation currents and Ca2+ increase, suggesting that all 3 TRPs contribute to the ion permeation pore of the channels. We identified the first heteromeric TRP channels composed of subunits from 3 different TRP subfamilies. Functionally, this heteromeric TRPV4-C1-P2 channel mediates the flow-induced Ca2+ increase in native vascular endothelial cells.—Du, J., Ma, X., Shen, B., Huang, Y., Birnbaumer, L., Yao, X. TRPV4, TRPC1, and TRPP2 assemble to form a flow-sensitive heteromeric channel. PMID:25114176

  9. The role of L-type calcium channels in the development and expression of behavioral sensitization to ethanol.

    PubMed

    Broadbent, Julie

    2013-10-11

    Behavioral sensitization is thought to play a significant role in drug addiction. L-type calcium channels have been implicated in sensitization to stimulant and opiate drugs but it is unclear if these channels also contribute to sensitization to ethanol. The effects of three L-type calcium channel blockers, nifedipine (1-7.5 mg/kg), diltiazem (12.5-50 mg/kg), and verapamil (12.5 and 25 mg/kg), on sensitization to ethanol (2 g/kg) were examined in DBA/2J mice. All three blockers reduced but did not prevent expression of sensitization. Only nifedipine blocked acquisition of sensitization. Nifedipine and verapamil decreased blood ethanol levels. The current findings suggest L-type calcium channels do not play a substantial role in sensitization to ethanol and that the neural mechanisms underlying sensitization to ethanol are distinct from those mediating sensitization to stimulants and opiates.

  10. The Role of L-Type Calcium Channels in the Development and Expression of Behavioral Sensitization to Ethanol

    PubMed Central

    Broadbent, Julie

    2013-01-01

    Behavioral sensitization is thought to play a significant role in drug addiction. L-type calcium channels have been implicated in sensitization to stimulant and opiate drugs but it is unclear if these channels also contribute to sensitization to ethanol. The effects of three L-type calcium channel blockers, nifedipine (1 – 7.5 mg/kg), diltiazem (12.5 – 50 mg/kg), and verapamil (12.5 and 25 mg/kg), on sensitization to ethanol (2 g/kg) were examined in DBA/2J mice. All three blockers reduced but did not prevent expression of sensitization. Only nifedipine blocked acquisition of sensitization. Nifedipine and verapamil decreased blood ethanol levels. The current findings suggest L-type calcium channels do not play a substantial role in sensitization to ethanol and that the neural mechanisms underlying sensitization to ethanol are distinct from those mediating sensitization to stimulants and opiates. PMID:23994059

  11. Immunocytochemical localization of amiloride-sensitive sodium channels in the lower intestine of the hen.

    PubMed

    Smith, P R; Bradford, A L; Dantzer, V; Benos, D J; Skadhauge, E

    1993-04-01

    We have used polyclonal antibodies generated against purified bovine renal amiloride-sensitive Na+ channels to localize amiloride-sensitive Na+ channels within the lower intestine (colon and coprodeum) of the hen. These antibodies cross-reacted with two polypeptides exhibiting M(r)'s of 235 and 150 kDa on immunoblots of detergent-solubilized apical membrane fractions from both the colon and coprodeum. The apparent molecular masses of theses polypeptides are in agreement with the M(r)'s of 2 of the subunits of the renal high amiloride-affinity Na+ channel, namely the alpha and the beta (= amiloride binding) subunits. The cellular distribution of Na+ channels was determined by immunoperoxidase and indirect immunofluorescence cytochemical techniques. The apical (luminal) membrane and cytoplasm of villar principal cells in both colon and coprodeum exhibited immunoreactivity, whereas goblet cells were negative. Both principal and goblet cells of the crypts were also negative. We conclude that the amiloride-sensitive Na+ channels are localized to the principal cells of the intestinal villi and that these cells are responsible for intestinal Na+ absorption.

  12. Spontaneously active NaV1.5 sodium channels may underlie odor sensitivity.

    PubMed

    Dionne, Vincent E

    2016-08-01

    The olfactory system is remarkably sensitive to airborne odor molecules, but precisely how very low odor concentrations bordering on just a few molecules per olfactory sensory neuron can trigger graded changes in firing is not clear. This report reexamines signaling in olfactory sensory neurons in light of the recent account of NaV1.5 sodium channel-mediated spontaneous firing. Using a model of spontaneous channel activity, the study shows how even submillivolt changes in membrane potential elicited by odor are expected to cause meaningful changes in NaV1.5-dependent firing. The results suggest that the random window currents of NaV1.5 channels may underpin not only spontaneous firing in olfactory sensory neurons but the cellular response to odor as well, thereby ensuring the robustness and sensitivity of signaling that is especially important for low odor concentrations.

  13. Activation of the ATP-sensitive K+ channel by decavanadate in guinea-pig ventricular myocytes.

    PubMed

    Nakashima, H; Kakei, M; Tanaka, H

    1993-03-23

    To evaluate the effects of decavanadate on the ATP-sensitive K+ (KATP) channel, we applied the inside-out membrane patch-clamp technique to ventricular myocytes isolated from guinea-pig hearts. Decavanadate increased the probability of the KATP channel being open in a dose-dependent manner over the range of 0.1 to 5 mM in the presence of 0.3 mM ATP. Half-maximal activation occurred at 540 microM decavanadate and a Hill coefficient of 1.3 was obtained when the Hill equation was used to fit the dose-dependent activation for the channel by decavanadate. The half-maximum inhibition for the channel by ATP (K1/2) in the presence of 2 mM Mg2+ was 19 and 74 microM in its absence. In the presence of decavanadate, both curves shifted toward the higher concentration of ATP without a change in steepness of the slope (Hill coefficient = 2). The effect of decavanadate could be expressed by a model in which its binding prevents ATP binding from closing the channel. The estimated dissociation constant of decavanadate was 1.5 microM in the presence and 22.8 microM in the absence of Mg2+. Decavanadate reactivated the rundown channel in the absence of Mg2+ and ATP. Neither the single channel slope conductance nor the mean open and closed lifetime within the bursts of channel openings were affected by decavanadate. We conclude that internal Mg2+ is not required for the modulation produced by decavanadate, but this ion influences the channel and changes the dissociation constant of both ATP and decavanadate to the channel.

  14. Alternative Splicing Governs Cone Cyclic Nucleotide-gated (CNG) Channel Sensitivity to Regulation by Phosphoinositides*

    PubMed Central

    Dai, Gucan; Sherpa, Tshering; Varnum, Michael D.

    2014-01-01

    Precursor mRNA encoding CNGA3 subunits of cone photoreceptor cyclic nucleotide-gated (CNG) channels undergoes alternative splicing, generating isoforms differing in the N-terminal cytoplasmic region of the protein. In humans, four variants arise from alternative splicing, but the functional significance of these changes has been a persistent mystery. Heterologous expression of the four possible CNGA3 isoforms alone or with CNGB3 subunits did not reveal significant differences in basic channel properties. However, inclusion of optional exon 3, with or without optional exon 5, produced heteromeric CNGA3 + CNGB3 channels exhibiting an ∼2-fold greater shift in K1/2,cGMP after phosphatidylinositol 4,5-biphosphate or phosphatidylinositol 3,4,5-trisphosphate application compared with channels lacking the sequence encoded by exon 3. We have previously identified two structural features within CNGA3 that support phosphoinositides (PIPn) regulation of cone CNG channels: N- and C-terminal regulatory modules. Specific mutations within these regions eliminated PIPn sensitivity of CNGA3 + CNGB3 channels. The exon 3 variant enhanced the component of PIPn regulation that depends on the C-terminal region rather than the nearby N-terminal region, consistent with an allosteric effect on PIPn sensitivity because of altered N-C coupling. Alternative splicing of CNGA3 occurs in multiple species, although the exact variants are not conserved across CNGA3 orthologs. Optional exon 3 appears to be unique to humans, even compared with other primates. In parallel, we found that a specific splice variant of canine CNGA3 removes a region of the protein that is necessary for high sensitivity to PIPn. CNGA3 alternative splicing may have evolved, in part, to tune the interactions between cone CNG channels and membrane-bound phosphoinositides. PMID:24675082

  15. 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.

  16. Acid-sensitive channel inhibition prevents fetal alcohol spectrum disorders cerebellar Purkinje cell loss.

    PubMed

    Ramadoss, Jayanth; Lunde, Emilie R; Ouyang, Nengtai; Chen, Wei-Jung A; Cudd, Timothy A

    2008-08-01

    Ethanol is now considered the most common human teratogen. Educational campaigns have not reduced the incidence of ethanol-mediated teratogenesis, leading to a growing interest in the development of therapeutic prevention or mitigation strategies. On the basis of the observation that maternal ethanol consumption reduces maternal and fetal pH, we hypothesized that a pH-sensitive pathway involving the TWIK-related acid-sensitive potassium channels (TASKs) is implicated in ethanol-induced injury to the fetal cerebellum, one of the most sensitive targets of prenatal ethanol exposure. Pregnant ewes were intravenously infused with ethanol (258+/-10 mg/dl peak blood ethanol concentration) or saline in a "3 days/wk binge" pattern throughout the third trimester. Quantitative stereological analysis demonstrated that ethanol resulted in a 45% reduction in the total number of fetal cerebellar Purkinje cells, the cell type most sensitive to developmental ethanol exposure. Extracellular pH manipulation to create the same degree and pattern of pH fall caused by ethanol (manipulations large enough to inhibit TASK 1 channels), resulted in a 24% decrease in Purkinje cell number. We determined immunohistochemically that TASK 1 channels are expressed in Purkinje cells and that the TASK 3 isoform is expressed in granule cells of the ovine fetal cerebellum. Pharmacological blockade of both TASK 1 and TASK 3 channels simultaneous with ethanol effectively prevented any reduction in fetal cerebellar Purkinje cell number. These results demonstrate for the first time functional significance of fetal cerebellar two-pore domain pH-sensitive channels and establishes them as a potential therapeutic target for prevention of ethanol teratogenesis.

  17. The role of pH-sensitive TASK channels in central respiratory chemoreception

    PubMed Central

    Bayliss, Douglas A.; Barhanin, Jacques; Gestreau, Christian; Guyenet, Patrice G.

    2014-01-01

    A number of the subunits within the family of K2P background K+ channels are sensitive to changes in extracellular pH in the physiological range, making them likely candidates to mediate various pH-dependent processes. Based on expression patterns within several brainstem neuronal cell groups that are believed to function in CO2/H+ regulation of breathing, three TASK subunits – TASK-1, TASK-2 and TASK-3 – were specifically hypothesized to contribute to this central respiratory chemoreflex. For the acid-sensitive TASK-1 and TASK-3 channels, despite widespread expression at multiple levels within the brainstem respiratory control system (including presumptive chemoreceptor populations), experiments in knockout mice provided no evidence for their involvement in CO2 regulation of breathing. By contrast, the alkaline-activated TASK-2 channel has a more restricted brainstem distribution and was localized to the Phox2b-expressing chemoreceptor neurons of the retrotrapezoid nucleus (RTN). Remarkably, in a Phox2b27Ala/+ mouse genetic model of congenital central hypoventilation syndrome (CCHS) that is characterized by reduced central respiratory chemosensitivity, selective ablation of Phox2b-expressing RTN neurons was accompanied by a corresponding loss of TASK-2 expression. Furthermore, genetic deletion of TASK-2 blunted RTN neuronal pH sensitivity in vitro, reduced alkaline-induced respiratory network inhibition in situ and diminished the ventilatory response to CO2/H+ in vivo. Notably, a subpopulation of RTN neurons from TASK-2−/− mice retained their pH sensitivity, at least in part due to a residual pH-sensitive background K+ current, suggesting that other mechanisms (and perhaps other K2P channels) for RTN neuronal pH sensitivity are yet to be identified. PMID:25346157

  18. The bile acid-sensitive ion channel (BASIC) is activated by alterations of its membrane environment.

    PubMed

    Schmidt, Axel; Lenzig, Pia; Oslender-Bujotzek, Adrienne; Kusch, Jana; Lucas, Susana Dias; Gründer, Stefan; Wiemuth, Dominik

    2014-01-01

    The bile acid-sensitive ion channel (BASIC) is a member of the DEG/ENaC family of ion channels. Channels of this family are characterized by a common structure, their physiological functions and modes of activation, however, are diverse. Rat BASIC is expressed in brain, liver and intestinal tract and activated by bile acids. The physiological function of BASIC and its mechanism of bile acid activation remain a puzzle. Here we addressed the question whether amphiphilic bile acids activate BASIC by directly binding to the channel or indirectly by altering the properties of the surrounding membrane. We show that membrane-active substances other than bile acids also affect the activity of BASIC and that activation by bile acids and other membrane-active substances is non-additive, suggesting that BASIC is sensitive for changes in its membrane environment. Furthermore based on results from chimeras between BASIC and ASIC1a, we show that the extracellular and the transmembrane domains are important for membrane sensitivity.

  19. 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

  20. Activation of ATP-sensitive K+ channels by epoxyeicosatrienoic acids in rat cardiac ventricular myocytes

    PubMed Central

    Lu, Tong; Hoshi, Toshinori; Weintraub, Neal L; Spector, Arthur A; Lee, Hon-Chi

    2001-01-01

    We examined the effects of epoxyeicosatrienoic acids (EETs), which are cytochrome P450 metabolites of arachidonic acid (AA), on the activities of the ATP-sensitive K+ (KATP) channels of rat cardiac myocytes, using the inside-out patch-clamp technique. In the presence of 100 μm cytoplasmic ATP, the KATP channel open probability (Po) was increased by 240 ± 60% with 0.1 μm 11,12-EET and by 400 ± 54% with 5 μm 11,12-EET (n = 5 –10, P < 0.05 vs. control), whereas neither 5 μm AA nor 5 μm 11,12-dihydroxyeicosatrienoic acid (DHET), which is the epoxide hydrolysis product of 11,12-EET, had any effect on Po. The half-maximal activating concentration (EC50) was 18.9 ± 2.6 nm for 11,12-EET (n = 5) and 19.1 ± 4.8 nm for 8,9-EET (n = 5), P = n.s. vs. 11,12-EET). Furthermore, 11,12-EET failed to alter the inhibition of KATP channels by glyburide. Application of 11,12-EET markedly decreased the channel sensitivity to cytoplasmic ATP. The half-maximal inhibitory concentration of ATP (IC50) was increased from 21.2 ± 2.0 μm at baseline to 240 ± 60 μm with 0.1 μm 11,12-EET (n = 5, P < 0.05 vs. control) and to 780 ± 30 μm with 5 μm 11,12-EET (n = 11, P < 0.05vs. control). Increasing the ATP concentration increased the number of kinetically distinguishable closed states, promoting prolonged closure durations. 11,12-EET antagonized the effects of ATP on the kinetics of the KATP channels in a dose and voltage-dependent manner. 11,12-EET (1 μm) reduced the apparent association rate constant of ATP to the channel by 135-fold. Application of 5 μm 11,12-EET resulted in hyperpolarization of the resting membrane potential in isolated cardiac myocytes, which could be blocked by glyburide. These results suggest that EETs are potent activators of the cardiac KATP channels, modulating channel behaviour by reducing the channel sensitivity to ATP. Thus, EETs could be important endogenous regulators of cardiac electrical excitability. PMID:11744757

  1. Novel nucleotide-binding sites in ATP-sensitive potassium channels formed at gating interfaces.

    PubMed

    Dong, Ke; Tang, Lie-Qi; MacGregor, Gordon G; Leng, Qiang; Hebert, Steven C

    2005-04-06

    The coupling of cell metabolism to membrane electrical activity is a vital process that regulates insulin secretion, cardiac and neuronal excitability and the responses of cells to ischemia. ATP-sensitive potassium channels (K(ATP); Kir6.x) are a major part of this metabolic-electrical coupling system and translate metabolic signals such as the ATP:ADP ratio to changes in the open or closed state (gate) of the channel. The localization of the nucleotide-binding site (NBS) on Kir6.x channels and how nucleotide binding gates these K(ATP) channels remain unclear. Here, we use fluorescent nucleotide binding to purified Kir6.x proteins to define the peptide segments forming the NBS on Kir6.x channels and show that unique N- and C-terminal interactions from adjacent subunits are required for high-affinity nucleotide binding. The short N- and C-terminal segments comprising the novel intermolecular NBS are next to helices that likely move with channel opening/closing, suggesting a lock-and-key model for ligand gating.

  2. Reduced voltage sensitivity in a K+-channel voltage sensor by electric field remodeling.

    PubMed

    González-Pérez, Vivian; Stack, Katherine; Boric, Katica; Naranjo, David

    2010-03-16

    Propagation of the nerve impulse relies on the extreme voltage sensitivity of Na(+) and K(+) channels. The transmembrane movement of four arginine residues, located at the fourth transmembrane segment (S4), in each of their four voltage-sensing domains is mostly responsible for the translocation of 12 to 13 e(o) across the transmembrane electric field. Inserting additional positively charged residues between the voltage-sensing arginines in S4 would, in principle, increase voltage sensitivity. Here we show that either positively or negatively charged residues added between the two most external sensing arginines of S4 decreased voltage sensitivity of a Shaker voltage-gated K(+)-channel by up to approximately 50%. The replacement of Val363 with a charged residue displaced inwardly the external boundaries of the electric field by at least 6 A, leaving the most external arginine of S4 constitutively exposed to the extracellular space and permanently excluded from the electric field. Both the physical trajectory of S4 and its electromechanical coupling to open the pore gate seemed unchanged. We propose that the separation between the first two sensing charges at resting is comparable to the thickness of the low dielectric transmembrane barrier they must cross. Thus, at most a single sensing arginine side chain could be found within the field. The conserved hydrophobic nature of the residues located between the voltage-sensing arginines in S4 may shape the electric field geometry for optimal voltage sensitivity in voltage-gated ion channels.

  3. TASK-2 Channels Contribute to pH Sensitivity of Retrotrapezoid Nucleus Chemoreceptor Neurons

    PubMed Central

    Wang, Sheng; Benamer, Najate; Zanella, Sébastien; Kumar, Natasha N.; Shi, Yingtang; Bévengut, Michelle; Penton, David; Guyenet, Patrice G.; Lesage, Florian

    2013-01-01

    Phox2b-expressing glutamatergic neurons of the retrotrapezoid nucleus (RTN) display properties expected of central respiratory chemoreceptors; they are directly activated by CO2/H+ via an unidentified pH-sensitive background K+ channel and, in turn, facilitate brainstem networks that control breathing. Here, we used a knock-out mouse model to examine whether TASK-2 (K2P5), an alkaline-activated background K+ channel, contributes to RTN neuronal pH sensitivity. We made patch-clamp recordings in brainstem slices from RTN neurons that were identified by expression of GFP (directed by the Phox2b promoter) or β-galactosidase (from the gene trap used for TASK-2 knock-out). Whereas nearly all RTN cells from control mice were pH sensitive (95%, n = 58 of 61), only 56% of GFP-expressing RTN neurons from TASK-2−/− mice (n = 49 of 88) could be classified as pH sensitive (>30% reduction in firing rate from pH 7.0 to pH 7.8); the remaining cells were pH insensitive (44%). Moreover, none of the recorded RTN neurons from TASK-2−/− mice selected based on β-galactosidase activity (a subpopulation of GFP-expressing neurons) were pH sensitive. The alkaline-activated background K+ currents were reduced in amplitude in RTN neurons from TASK-2−/− mice that retained some pH sensitivity but were absent from pH-insensitive cells. Finally, using a working heart–brainstem preparation, we found diminished inhibition of phrenic burst amplitude by alkalization in TASK-2−/− mice, with apneic threshold shifted to higher pH levels. In conclusion, alkaline-activated TASK-2 channels contribute to pH sensitivity in RTN neurons, with effects on respiration in situ that are particularly prominent near apneic threshold. PMID:24107938

  4. Actions of Ethanol on Voltage-Sensitive Sodium Channels. Effects on Neurotoxin-Stimulated Sodium Uptake in Synaptosomes

    DTIC Science & Technology

    1985-01-01

    concentration in the nonaqueuus (membrane) phase (Lyon et aL, 1981). Concentration- effect summarized in table 1 . When sodium channels were activated curves were...Voltage-Sensitive Sodium Channels : Effects on Neurotoxin-Stimulated Sodium Uptake in DT (7 Synaptosomes E L C MICHAEL J. MULLIN’ and WALTER A. HUNT...1984). At the present time, the 8 1 structural and functional properties of the voltage-sensitive sodium channels are understood most completely

  5. 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

  6. Genetic variations in TCF7L2 influence therapeutic response to sulfonylureas in Indian diabetics.

    PubMed

    Dhawan, Dipali; Padh, Harish

    2016-11-01

    Sulfonylureas are widely used to treat type 2 diabetes, with considerable inter-individual variation in the hypoglycaemic response to sulfonylureas. Genetic variants in the gene encoding for transcription factor-7-like 2 (TCF7L2) have been associated with type 2 diabetes. This study aimed to study the effect of variations in TCF7L2 on therapeutic response to sulfonylureas in Type 2 diabetes mellitus patients. The effect of TCF7L2 rs12255372, rs7903146 and rs4506565 genotypes on glycaemic response was observed in 250 diabetic patients treated with sulfonylureas and sulfonylureas along with metformin. The genotyping tests were done by allele-specific multiplex PCR. Glycated haemoglobin (HbA1c) levels were used as phenotypic marker. 60% of sulfonylurea users did not achieve a target HbA1c levels of ⩽6.5% (48mmol/mol) (which denotes good control in diabetics). Genotype influenced response to sulfonylureas, with more treatment failure in the TT homozygotes in case of rs12255372 and rs4506565. The GG genotype at rs12255372 favourably influences treatment success with sulfonylurea therapy in patients with type 2 diabetes (p⩽0.05). At rs12255372, 70.5% GT or TT genotype failed to achieve therapeutic target, an absolute difference of 19% compared to GG homozygotes. Our preliminary data show that genetic variation at rs12255372 has a direct correlation with therapeutic success with sulfonylureas in type 2 diabetes, hence paving the way for better treatment outcomes in diabetics.

  7. Sulfonylurea treatment of type 2 diabetes mellitus: focus on glimepiride.

    PubMed

    Korytkowski, Mary T

    2004-05-01

    Sulfonylureas, which have evolved through two generations since their introduction nearly 50 years ago, remain the most frequently prescribed oral agents for treatment of patients with type 2 diabetes mellitus. Glyburide, glipizide, and glimepiride, the newest sulfonylureas, are as effective at lowering plasma glucose concentrations as first-generation agents but are more potent, better tolerated, and associated with a lower risk of adverse effects. Differences in their binding affinity to the beta-cell sulfonylurea receptor have been described, with preservation of cardioprotective responses to ischemia with glimepiride. Clinical studies have shown glimepiride to be safe and effective in reducing fasting and postprandial glucose levels, as well as glycosylated hemoglobin concentrations, with dosages of 1-8 mg/day. In comparative trials, glimepiride was as effective in lowering glucose levels as glyburide and glipizide, but glimepiride was associated with a reduced likelihood of hypoglycemia and a smaller increase in fasting insulin and C-peptide levels than glyburide, and a more rapid lowering of fasting plasma glucose levels than glipizide. Glimepiride also improves first-phase insulin secretion, which plays an important role in reducing postprandial hyperglycemia. Insulin secretagogues, specifically glimepiride, merit consideration as first-line therapy for patients with type 2 diabetes.

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

    PubMed

    Winkler, Gábor

    2014-04-06

    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.

  9. 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

  10. 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.

  11. 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.

  12. 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.

  13. Complex voltage-dependent behavior of single unliganded calcium-sensitive potassium channels.

    PubMed Central

    Talukder, G; Aldrich, R W

    2000-01-01

    study and characterization of unliganded openings is of central significance for the elucidation of gating mechanisms for allosteric ligand-gated ion channels. Unliganded openings have been reported for many channel types, but their low open probability can make it difficult to study their kinetics in detail. Because the large conductance calcium-activated potassium channel mSlo is sensitive to both intracellular calcium and to membrane potential, we have been able to obtain stable unliganded single-channel recordings of mSlo with relatively high opening probability. We have found that the single-channel gating behavior of mSlo is complex, with multiple open and closed states, even when no ligand is present. Our results rule out a Monod-Wyman-Changeux allosteric mechanism with a central voltage-dependent concerted step, and they support the existence of quaternary states with less than the full number of voltage sensors activated, as has been suggested by previous work involving measurements of gating currents. PMID:10653789

  14. Micromolar-affinity benzodiazepine receptors regulate voltage-sensitive calcium channels in nerve terminal preparations.

    PubMed Central

    Taft, W C; DeLorenzo, R J

    1984-01-01

    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. PMID:6328498

  15. 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.

  16. Intracellular ATP can regulate afferent arteriolar tone via ATP-sensitive K+ channels in the rabbit.

    PubMed Central

    Lorenz, J N; Schnermann, J; Brosius, F C; Briggs, J P; Furspan, P B

    1992-01-01

    Studies were performed to assess whether ATP-sensitive K+ (KATP) channels on rabbit preglomerular vessels can influence afferent arteriolar (AA) tone. K+ channels with a slope conductance of 258 +/- 13 (n = 7) pS and pronounced voltage dependence were demonstrated in excised patches from vascular smooth muscle cells of microdissected preglomerular segments. Channel activity was markedly reduced by 1 mM ATP and in a dose-dependent fashion by glibenclamide (10(-9) M to 10(-6) M), a specific antagonist of KATP channels. 10(-5) M diazoxide, a K+ channel opener, activated these channels in the presence of ATP, and this effect was also blocked by glibenclamide. To determine the role of these KATP channels in the control of vascular tone, diazoxide was tested on isolated perfused AA. After preconstriction from a control diameter of 13.1 +/- 1.1 to 3.5 +/- 2.1 microns with phenylephrine (PE), addition of 10(-5) M diazoxide dilated vessels to 11.2 +/- 0.7 microns, which was not different from control. Further addition of 10(-5) M glibenclamide reconstricted the vessels to 5.8 +/- 1.5 microns (n = 5; P less than 0.03). In support of its specificity for KATP channels, glibenclamide did not reverse verapamil induced dilation in a separate series of experiments. To determine whether intracellular ATP levels can effect AA tone, studies were conducted to test the effect of the glycolytic inhibitor 2-deoxy-D-glucose. After preconstriction from 13.4 +/- 3.2 to 7.7 +/- 1.3 microns with PE, bath glucose was replaced with 6 mM 2-deoxy-D-glucose. Within 10 min, the arteriole dilated to a mean value of 11.8 +/- 1.4 microns (n = 6; NS compared to control). Subsequent addition of 10(-5) M glibenclamide significantly reconstricted the vessels to a diameter of 8.6 +/- 0.5 micron (P less than 0.04). These data demonstrate that KATP channels are present on the preglomerular vasculature and that changes in intracellular ATP can directly influence afferent arteriolar tone via these channels

  17. The development of sulfonylurea herbicide-resistant birdsfoot trefoil (Lotus corniculatus) plants from in vitro selection.

    PubMed

    Pofelis, S; Le, H; Grant, W F

    1992-02-01

    Herbicide-resistant lines of birdsfoot trefoil (Lotus corniculatus L. cv 'Leo') were isolated after sequential selection at the callus, shoot, and whole plant levels to the sulfonylurea (SU) herbicide Harmony {DPX-M6316; 3-[[[(4-methoxy-6methyl-1,3,5, triazine-2-yl) amino] carbonyl] amino] sulfonyl-2-thiophenecarboxylate}. In field and growth chamber tests the Harmony regenerant lines displayed an increased tolerance as compared to control plants from tissue culture and controls grown from seed. Results of evaluation of callus cultures of regenerated mutant lines signify stability of the resistance. Outcrossed seeds collected from field trials, and tested in vitro for herbicide resistance, indicate that the trait is heritable and that resistance may be due to reduced sensitivity of acetolactate synthase to SU inhibition. Genetically stable herbicide-resistant lines of birdsfoot trefoil were successfully isolated using in vitro selection.

  18. 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...

  19. Cell volume-sensitive sodium channels upregulated by glucocorticoids in U937 macrophages.

    PubMed

    Gamper, N; Huber, S M; Badawi, K; Lang, F

    2000-12-01

    Glucocorticoids exert their anti-inflammatory action in part by influencing macrophages. As regulation of macrophage function involves ion channels, the present study was performed to elucidate the influence of glucocorticoids on macrophage ion channel activity. To this end, the effects of corticosteroids on the sodium conductance in human monocytic cells (U937) was studied using whole-cell and outside-out patch-clamp techniques. Increasing extracellular osmolarity from 310 to 420 mosmol/kg led to cell shrinkage followed by marked activation of inward whole-cell current from -36+/-2 to -72+/-9 pA (n=13; recorded at -150 mV voltage with CsCl intracellular solution, NaCl extracellular solution) while outward current remained unchanged. The increase of inward current was accompanied by a positive shift of reversal potential and was sensitive to amiloride (100 microM). The activation of inward current by shrinkage was not observed when external sodium was replaced by potassium, indicating that the shrinkage-stimulated conductance is sodium selective. Outside-out single-channel measurements revealed a unitary conductance of 6+/-1 pS (n=5) for the sodium-selective amiloride-sensitive current. Pretreating the cells with deoxycorticosterone (100 nM/6 h) markedly upregulated the shrinkage-activated Na+ current. In conclusion, human macrophage-like U937 cells express a sodium-selective shrinkage-activated channel which is upregulated by corticosteroids. Activation of the channel may increase cell volume, an effect of glucocorticoids in other cells.

  20. Benzamil sensitive ion channels contribute to volume regulation in canine chondrocytes

    PubMed Central

    Lewis, R; Feetham, CH; Gentles, L; Penny, J; Tregilgas, L; Tohami, W; Mobasheri, A; Barrett-Jolley, R

    2013-01-01

    Background and Purpose Chondrocytes exist within cartilage and serve to maintain the extracellular matrix. It has been postulated that osteoarthritic (OA) chondrocytes lose the ability to regulate their volume, affecting extracellular matrix production. In previous studies, we identified expression of epithelial sodium channels (ENaC) in human chondrocytes, but their function remained unknown. Although ENaC typically has Na+ transport roles, it is also involved in the cell volume regulation of rat hepatocytes. ENaC is a member of the degenerin (Deg) family, and ENaC/Deg-like channels have a low conductance and high sensitivity to benzamil. In this study, we investigated whether canine chondrocytes express functional ENaC/Deg-like ion channels and, if so, what their function may be. Experimental Approach Canine chondrocytes were harvested from dogs killed for unassociated welfare reasons. We used immunohistochemistry and patch-clamp electrophysiology to investigate ENaC expression and video microscopy to analyse the effects of pharmacological inhibition of ENaC/Deg on cell volume regulation. Key Results Immunofluorescence showed that canine chondrocytes expressed ENaC protein. Single-channel recordings demonstrated expression of a benzamil-sensitive Na+ conductance (9 pS), and whole-cell experiments show this to be approximately 1.5 nS per cell with high selectivity for Na+. Benzamil hyperpolarized chondrocytes by approximately 8 mV with a pD2 8.4. Chondrocyte regulatory volume decrease (RVI) was inhibited by benzamil (pD2 7.5) but persisted when extracellular Na+ ions were replaced by Li+. Conclusion and Implications Our data suggest that benzamil inhibits RVI by reducing the influx of Na+ ions through ENaC/Deg-like ion channels and present ENaC/Deg as a possible target for pharmacological modulation of chondrocyte volume. PMID:22928819

  1. TASK channel deletion reduces sensitivity to local anesthetic-induced seizures

    PubMed Central

    Du, Guizhi; Chen, Xiangdong; Todorovic, Marko S.; Shu, Shaofang; Kapur, Jaideep; Bayliss, Douglas A.

    2011-01-01

    Background Local anesthetics (LAs) are typically used for regional anesthesia but can be given systemically to mitigate postoperative pain, supplement general anesthesia or prevent cardiac arrhythmias. However, systemic application or inadvertent intravenous injection can be associated with substantial toxicity, including seizure induction. The molecular basis for this toxic action remains unclear. Methods We characterized effects of different LAs on homomeric and heteromeric K+ channels containing TASK-1 (K2P3.1, KCNK3) and TASK-3 (K2P9.1, KCNK9) subunits in a mammalian expression system. In addition, we used TASK-1/TASK-3 knockout mice to test the possibility that TASK channels contribute to LA-evoked seizures. Results LAs inhibited homomeric and heteromeric TASK channels in a range relevant for seizure induction; channels containing TASK-1 subunits were most sensitive and IC50 values indicated a rank order potency of bupivacaine > ropivacaine ⟫ lidocaine. LAs induced tonic-clonic seizures in mice with the same rank order potency, but higher LA doses were required to evoke seizures in TASK knockout mice. For bupivacaine, which produced the longest seizure times, seizure duration was significantly shorter in TASK knockout mice; bupivacaine-induced seizures were associated with an increase in electroencephalogram power at frequencies <5 Hz in both wild type and TASK knockout mice. Conclusions These data suggest that increased neuronal excitability associated with TASK channel inhibition by LAs contributes to seizure induction. Since all LAs were capable of evoking seizures in TASK channel deleted mice, albeit at higher doses, the results imply that other molecular targets must also be involved in this toxic action. PMID:21946151

  2. Cell-type specific expression of ATP-sensitive potassium channels in the rat hippocampus

    PubMed Central

    Zawar, C; Plant, T D; Schirra, C; Konnerth, A; Neumcke, B

    1999-01-01

    The distribution of ATP-sensitive K+ channels (KATP channels) was investigated in four cell types in hippocampal slices prepared from 10- to 13-day-old rats: CA1 pyramidal cells, interneurones of stratum radiatum in CA1, complex glial cells of the same area and granule cells of the dentate gyrus. The neuronal cell types were identified visually and characterized by the shapes and patterns of their action potentials and by neurobiotin labelling.The patch-clamp technique was used to study the sensitivity of whole-cell currents to diazoxide (0·3 mm), a KATP channel opener, and to tolbutamide (0·5 mm) or glibenclamide (20 μm), two KATP channel inhibitors. The fraction of cells in which whole-cell currents were activated by diazoxide and inhibited by tolbutamide was 26% of pyramidal cells, 89% of interneurones, 100% of glial cells and 89% of granule cells. The reversal potential of the diazoxide-induced current was at the K+ equilibrium potential and a similar current activated spontaneously when cells were dialysed with an ATP-free pipette solution.Using the single-cell RT-PCR method, the presence of mRNA encoding KATP channel subunits (Kir6.1, Kir6.2, SUR1 and SUR2) was examined in CA1 pyramidal cells and interneurones. Subunit mRNA combinations that can result in functional KATP channels (Kir6.1 together with SUR1, Kir6.2 together with SUR1 or SUR2) were detected in only 17% of the pyramidal cells. On the other hand, KATP channelsmay be formed in 75%of the interneurones, mainly by the combination of Kir6.2 with SUR1 (58% of all interneurones).The results of these combined analyses indicate that functional KATP channels are present in principal neurones, interneurones and glial cells of the rat hippocampus, but at highly different densities in the four cell types studied. PMID:9852317

  3. A red-shifted photochromic sulfonylurea for the remote control of pancreatic beta cell function.

    PubMed

    Broichhagen, J; Frank, J A; Johnston, N R; Mitchell, R K; Šmid, K; Marchetti, P; Bugliani, M; Rutter, G A; Trauner, D; Hodson, D J

    2015-04-07

    Azobenzene photoresponsive elements can be installed on sulfonylureas, yielding optical control over pancreatic beta cell function and insulin release. An obstacle to such photopharmacological approaches remains the use of ultraviolet-blue illumination. Herein, we synthesize and test a novel yellow light-activated sulfonylurea based on a heterocyclic azobenzene bearing a push-pull system.

  4. Calcium-dependent inactivation of the dihydropyridine-sensitive calcium channels in GH3 cells

    PubMed Central

    1988-01-01

    The inactivation of calcium channels in mammalian pituitary tumor cells (GH3) was studied with patch electrodes under voltage clamp in cell- free membrane patches and in dialyzed cells. The calcium current elicited by depolarization from a holding potential of -40 mV passed predominantly through one class of channels previously shown to be modulated by dihydropyridines and cAMP-dependent phosphorylation (Armstrong and Eckert, 1987). When exogenous calcium buffers were omitted from the pipette solution, the macroscopic calcium current through those channels inactivated with a half time of approximately 10 ms to a steady state level 40-75% smaller than the peak. Inactivation was also measured as the reduction in peak current during a test pulse that closely followed a prepulse. Inactivation was largely reduced or eliminated by (a) buffering free calcium in the pipette solution to less than 10(-8) M; (b) replacing extracellular calcium with barium; (c) increasing the prepulse voltage from +10 to +60 mV; or (d) increasing the intracellular concentration of cAMP, either 'directly' with dibutyryl-cAMP or indirectly by activating adenylate cyclase with forskolin or vasoactive intestinal peptide. Thus, inactivation of the dihydropyridine-sensitive calcium channels in GH3 cells only occurs when membrane depolarization leads to calcium ion entry and intracellular accumulation. PMID:2849631

  5. Sulfonylurea receptor 1 contributes to the astrocyte swelling and brain edema in acute liver failure.

    PubMed

    Jayakumar, A R; Valdes, V; Tong, X Y; Shamaladevi, N; Gonzalez, W; Norenberg, M D

    2014-02-01

    Astrocyte swelling (cytotoxic brain edema) is the major neurological complication of acute liver failure (ALF), a condition in which ammonia has been strongly implicated in its etiology. Ion channels and transporters are known to be involved in cell volume regulation, and a disturbance in these systems may result in cell swelling. One ion channel known to contribute to astrocyte swelling/brain edema in other neurological disorders is the ATP-dependent, nonselective cation (NCCa-ATP) channel. We therefore examined its potential role in the astrocyte swelling/brain edema associated with ALF. Cultured astrocytes treated with 5 mM ammonia showed a threefold increase in the sulfonylurea receptor type 1 (SUR1) protein expression, a marker of NCCa-ATP channel activity. Blocking SUR1 with glibenclamide significantly reduced the ammonia-induced cell swelling in cultured astrocytes. Additionally, overexpression of SUR1 in ammonia-treated cultured astrocytes was significantly reduced by cotreatment of cells with BAY 11-7082, an inhibitor of NF-κB, indicating the involvement of an NF-κB-mediated SUR1 upregulation in the mechanism of ammonia-induced astrocyte swelling. Brain SUR1 mRNA level was also found to be increased in the thioacetamide (TAA) rat model of ALF. Additionally, we found a significant increase in SUR1 protein expression in rat brain cortical astrocytes in TAA-treated rats. Treatment with glibenclamide significantly reduced the brain edema in this model of ALF. These findings strongly suggest the involvement of NCCa-ATP channel in the astrocyte swelling/brain edema in ALF and that targeting this channel may represent a useful approach for the treatment of the brain edema associated with ALF.

  6. 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.

  7. 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.

  8. Role of ATP-sensitive potassium channels in the piracetam induced blockade of opioid effects.

    PubMed

    Rehni, Ashish K; Singh, Nirmal; Jindal, Seema

    2007-12-01

    The present study has been designed to investigate the effect of piracetam on morphine/ buprenorphine-induced antinociception in rats and effect of piracetam on morphine or minoxidil induced relaxation in KCl-precontracted isolated rat aortic ring preparation. Nociceptive threshold was measured by the tail flick test in rats. The cumulative dose responses of morphine or minoxidil were recorded in KCl-precontracted isolated rat aortic ring preparation. Piracetam attenuated buprenorphine-induced antinociception in rats. Piracetam significantly reduced the morphine and minoxidil induced relaxation in KCl precontracted isolated rat aortic ring preparation suggesting that piracetam interferes with opioid receptor and ATP-sensitive potassium channel (KATP) opener mediated responses in vitro. Thus, it may be suggested that piracetam attenuates opioid effects by an opioid receptor-KATP channel linked mechanism.

  9. Bidirectional effects of hydrogen sulfide via ATP-sensitive K(+) channels and transient receptor potential A1 channels in RIN14B cells.

    PubMed

    Ujike, Ayako; Otsuguro, Ken-ichi; Miyamoto, Ryo; Yamaguchi, Soichiro; Ito, Shigeo

    2015-10-05

    Hydrogen sulfide (H2S) reportedly acts as a gasotransmitter because it mediates various cellular responses through several ion channels including ATP-sensitive K(+) (KATP) channels and transient receptor potential (TRP) A1 channels. H2S can activate both KATP and TRPA1 channels at a similar concentration range. In a single cell expressing both channels, however, it remains unknown what happens when both channels are simultaneously activated by H2S. In this study, we examined the effects of H2S on RIN14B cells that express both KATP and TRPA1 channels. RIN14B cells showed several intracellular Ca(2+) concentration ([Ca(2+)]i) responses to NaHS (300 µM), an H2S donor, i.e., inhibition of spontaneous Ca(2+) oscillations (37%), inhibition followed by [Ca(2+)]i increase (24%), and a rapid increase in [Ca(2+)]i (25%). KATP channel blockers, glibenclamide or tolbutamide, abolished any inhibitory effects of NaHS and enhanced NaHS-mediated [Ca(2+)]i increases, which were inhibited by extracellular Ca(2+) removal, HC030031 (a TRPA1 antagonist), and disulfide bond-reducing agents. NaHS induced 5-hydroxytryptamine (5-HT) release from RIN14B cells, which was also inhibited by TRPA1 antagonists. These results indicate that H2S has both inhibitory and excitatory effects by opening KATP and TRPA1 channels, respectively, in RIN14B cells, suggesting potential bidirectional modulation of secretory functions.

  10. Intramembrane aromatic interactions influence the lipid sensitivities of pentameric ligand-gated ion channels.

    PubMed

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

    2015-01-23

    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.

  11. Actions of Ethanol on Voltage-Sensitive Sodium Channels: Effects of Acute and Chronic Ethanol Treatment

    DTIC Science & Technology

    1987-01-01

    No. 2 C4"ght 0 1967 by "he Amusca Soiety for Pharmacolog and Kxporuont Therpadu" Prud m U.S.A. Actions of Ethanol on Voltage-Sensitive Sodium ...inhibitory effect of ethanol in vitro on sodium benzoate binding to neuronal sodium channels were studi-d in uptake for up to 20 days after withdrawal...adapt rapidly to some sodium uptake in the absence of ethanol in vitro, however, a effects of ethanol and that chronic ethanol administration can

  12. Cyclic AMP-dependent phosphorylation of voltage-sensitive sodium channels in primary cultures of rat brain cells

    SciTech Connect

    Rossie, S.; Catterall, W.A.

    1986-03-05

    The ..cap alpha.. subunit of the voltage-sensitive Na channel from rat brain is phosphorylated by cAMP-dependent protein kinase in purified preparations and in synaptosomes. The authors have begun to study cAMP-dependent phosphorylation of Na channels in intact cells. Rat brain cells collected at embryonic day 15 and maintained in culture for approximately 21 days were subjected to treatments designed to increase intracellular cAMP. Cells were solubilized and Na channels were isolated by immunoprecipitation, then rephosphorylated with the catalytic subunit of cAMP-dependent protein kinase and /sup 32/P-ATP, to allow incorporation of /sup 32/P into available cAMP-dependent phosphorylation sites of Na channels. The amount of /sup 32/P incorporated into channel is inversely proportional to the extent of endogenous phosphorylation. Treatment of cells with forskolin inhibited rephosphorylation of Na channels, indicating that enhanced endogenous phosphorylation of channels had occurred. The effect of forskolin on cell surface Na channels occurred rapidly, was sustained over 30 min., and was half-maximal at 6..mu..M. 8-Br-cAMP, (EC/sub 50/-5mM) and isobutylmethylxanthine (EC/sub 50/-60..mu..M) also caused inhibition of /sup 32/P incorporation into Na channels. These results indicate that the extent of cAMP-dependent phosphorylation of voltage-sensitive Na channels in intact brain neurons is modified by changes in intracellular levels of cAMP.

  13. Effects of N-Glycosylation of the human cation channel TRPA1 on agonist-sensitivity.

    PubMed

    Egan, Timothy James; Acuña, Mario A; Zenobi-Wong, Marcy; Zeilhofer, Hanns Ulrich; Urech, David

    2016-08-31

    Determining the functional significance of post-translational modifications advances our understanding of many broadly-expressed proteins, and particularly ion channels. The enzymes that catalyze these modifications are often expressed in a cell-type specific manner, resulting in considerable structural diversity among post-translationally modified proteins that are expressed across a variety of cell types. TRP channels exhibit notably variable behavior between cell types in vitro and in vivo , and they are frequently modified with N-glycans that contribute to protein function. TRPA1 possesses two putative N-linked glycosylation sites at N747 and N753 that have not yet been studied in detail. Here, we show that both of these sites can be modified with an N-glycan and that the glycan at position N747 modulates agonist-sensitivity of TRPA1 in vitro Additionally, we found that N-glycosylation also modulates cooperative effects of temperature and the agonist cinnamaldehyde on TRPA1 channel activation. Collectively, these findings suggest a dynamic role played by the N-glycosylation of human TRPA1. They also provide further evidence of the versatility of N-glycans and will assist in efforts to fully understand the complex regulation of TRPA1 activity.

  14. Effects of N-glycosylation of the human cation channel TRPA1 on agonist-sensitivity

    PubMed Central

    Egan, Timothy J.; Acuña, Mario A.; Zenobi-Wong, Marcy; Zeilhofer, Hanns Ulrich; Urech, David

    2016-01-01

    Determining the functional significance of post-translational modifications advances our understanding of many broadly-expressed proteins, and particularly ion channels. The enzymes that catalyse these modifications are often expressed in a cell-type specific manner, resulting in considerable structural diversity among post-translationally modified proteins that are expressed across a variety of cell types. TRP channels exhibit notably variable behaviour between cell types in vitro and in vivo, and they are frequently modified with N-glycans that contribute to protein function. TRPA1 possesses two putative N-linked glycosylation sites at Asn747 and Asn753 that have not yet been studied in detail. In the present study, we show that both of these sites can be modified with an N-glycan and that the glycan at position Asn747 modulates agonist-sensitivity of TRPA1 in vitro. Additionally, we found that N-glycosylation also modulates cooperative effects of temperature and the agonist cinnamaldehyde (CA) on TRPA1 channel activation. Collectively, these findings suggest a dynamic role played by the N-glycosylation of human TRPA1. They also provide further evidence of the versatility of N-glycans and will assist in efforts to fully understand the complex regulation of TRPA1 activity. PMID:27582506

  15. TRPV3 is a calcium-permeable temperature-sensitive cation channel.

    PubMed

    Xu, Haoxing; Ramsey, I Scott; Kotecha, Suhas A; Moran, Magdalene M; Chong, Jayhong A; Lawson, Deborah; Ge, Pei; Lilly, Jeremiah; Silos-Santiago, Inmaculada; Xie, Yu; DiStefano, Peter S; Curtis, Rory; Clapham, David E

    2002-07-11

    Transient receptor potential (TRP) proteins are cation-selective channels that function in processes as diverse as sensation and vasoregulation. Mammalian TRP channels that are gated by heat and capsaicin (>43 degrees C; TRPV1 (ref. 1)), noxious heat (>52 degrees C; TRPV2 (ref. 2)), and cooling (< 22 degrees C; TRPM8 (refs 3, 4)) have been cloned; however, little is known about the molecular determinants of temperature sensing in the range between approximately 22 degrees C and 40 degrees C. Here we have identified a member of the vanilloid channel family, human TRPV3 (hTRPV3) that is expressed in skin, tongue, dorsal root ganglion, trigeminal ganglion, spinal cord and brain. Increasing temperature from 22 degrees C to 40 degrees C in mammalian cells transfected with hTRPV3 elevated intracellular calcium by activating a nonselective cationic conductance. As in published recordings from sensory neurons, the current was steeply dependent on temperature, sensitized with repeated heating, and displayed a marked hysteresis on heating and cooling. On the basis of these properties, we propose that hTRPV3 is thermosensitive in the physiological range of temperatures between TRPM8 and TRPV1.

  16. 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.

  17. 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.

  18. A proinflammatory chemokine, CCL3, sensitizes the heat- and capsaicin-gated ion channel TRPV1.

    PubMed

    Zhang, Ning; Inan, Saadet; Inan, Sadeet; Cowan, Alan; Sun, Ronghua; Wang, Ji Ming; Rogers, Thomas J; Caterina, Michael; Oppenheim, Joost J

    2005-03-22

    Pain, a critical component of host defense, is one hallmark of the inflammatory response. We therefore hypothesized that pain might be exacerbated by proinflammatory chemokines. To test this hypothesis, CCR1 was cotransfected into human embryonic kidney (HEK)293 cells together with transient receptor potential vanilloid 1 (TRPV1), a cation channel required for certain types of thermal hyperalgesia. In these cells, capsaicin and anandamide induced Ca(2+) influx mediated by TRPV1. When CCR1:TRPV1/HEK293 cells were pretreated with CCL3, the sensitivity of TRPV1, as indicated by the Ca(2+) influx, was increased approximately 3-fold. RT-PCR analysis showed that a spectrum of chemokine and cytokine receptors is expressed in rat dorsal root ganglia (DRG). Immunohistochemical staining of DRG showed that CCR1 is coexpressed with TRPV1 in >85% of small-diameter neurons. CCR1 on DRG neurons was functional, as demonstrated by CCL3-induced Ca(2+) ion influx and PKC activation. Pretreatment with CCL3 enhanced the response of DRG neurons to capsaicin or anandamide. This sensitization was inhibited by pertussis toxin, U73122, or chelerythrine chloride, inhibitors of Gi-protein, phospholipase C, and protein kinase C, respectively. Intraplantar injection of mice with CCL3 decreased their hot-plate response latency. That a proinflammatory chemokine, by interacting with its receptor on small-diameter neurons, sensitizes TRPV1 reveals a previously undescribed mechanism of receptor cross-sensitization that may contribute to hyperalgesia during inflammation.

  19. Drosophila mushroom body Kenyon cells generate spontaneous calcium transients mediated by PLTX-sensitive calcium channels.

    PubMed

    Jiang, Shaojuan Amy; Campusano, Jorge M; Su, Hailing; O'Dowd, Diane K

    2005-07-01

    Spontaneous calcium oscillations in mushroom bodies of late stage pupal and adult Drosophila brains have been implicated in memory consolidation during olfactory associative learning. This study explores the cellular mechanisms regulating calcium dynamics in Kenyon cells, principal neurons in mushroom bodies. Fura-2 imaging shows that Kenyon cells cultured from late stage Drosophila pupae generate spontaneous calcium transients in a cell autonomous fashion, at a frequency similar to calcium oscillations in vivo (10-20/h). The expression of calcium transients is up regulated during pupal development. Although the ability to generate transients is a property intrinsic to Kenyon cells, transients can be modulated by bath application of nicotine and GABA. Calcium transients are blocked, and baseline calcium levels reduced, by removal of external calcium, addition of cobalt, or addition of Plectreurys toxin (PLTX), an insect-specific calcium channel antagonist. Transients do not require calcium release from intracellular stores. Whole cell recordings reveal that the majority of voltage-gated calcium channels in Kenyon cells are PLTX-sensitive. Together these data show that influx of calcium through PLTX-sensitive voltage-gated calcium channels mediates spontaneous calcium transients and regulates basal calcium levels in cultured Kenyon cells. The data also suggest that these calcium transients represent cellular events underlying calcium oscillations in the intact mushroom bodies. However, spontaneous calcium transients are not unique to Kenyon cells as they are present in approximately 60% of all cultured central brain neurons. This suggests the calcium transients play a more general role in maturation or function of adult brain neurons.

  20. Lactate modulates the intracellular pH sensitivity of human TREK1 channels.

    PubMed

    Ghatak, Swagata; Sikdar, Sujit Kumar

    2016-05-01

    Tissue acidosis and high lactate concentrations are associated with cerebral ischaemia. The degree of acidosis is dependent on circulating glucose concentration, hyperglycaemia being associated with increased acidosis. Among other agents, lactate and protons have been shown to activate the leak potassium channel; TREK1 (TWIK related potassium channel 1) from the intracellular side and its increased activity is implicated in tolerance towards ischaemic cell damage. In the present study, we show that ischaemic concentrations of lactate (30 mM) at pH 7.0 and 6.5, commonly observed during ischemia, cause robust potentiation of human TREK1 (hTREK1) activity at single-channel level in cell-free inside-out membrane patches, while 30 mM lactate at pH 6.0 to 5.5, commonly observed during hyperglycaemic ischemia, reduces hTREK1 channel activity significantly. The biphasic effect of 30 mM lactate (ischaemic concentrations) on modulation of hTREK1 by varying pH conditions is specific since basal concentrations of lactate (3 mM) and 30 mM pyruvate at pH 7.0 and 5.5 failed to show similar effect as lactate. Experiments with deletion and point mutants of hTREK1 channel suggest that lactate changes the pH modulation of hTREK1 by interacting differently with the histidine residue at 328th position (H328) above and below its pKa (∼6.0) in the intracellular carboxyl-terminal domain of TREK1. This lactate-induced pH modulation of hTREK1 is absent in C-terminal deletion mutant, CTDΔ100, and is similar in E321A-hTREK1 mutant as in wild-type hTREK1 suggesting that it is independent of pH-sensitive glutamate residue at 321st position. Such a differential pH-dependent effect of lactate on an ion channel function has not been reported earlier and has important implications in different stages of ischaemia.

  1. Flow-activated chloride channels in vascular endothelium. Shear stress sensitivity, desensitization dynamics, and physiological implications.

    PubMed

    Gautam, Mamta; Shen, Yue; Thirkill, Twanda L; Douglas, Gordon C; Barakat, Abdul I

    2006-12-01

    Although activation of outward rectifying Cl(-) channels is one of the fastest responses of endothelial cells (ECs) to shear stress, little is known about these channels. In this study, we used whole-cell patch clamp recordings to characterize the flow-activated Cl(-) current in bovine aortic ECs (BAECs). Application of shear stress induced rapid development of a Cl(-) current that was effectively blocked by the Cl(-) channel antagonist 5-nitro-2-(3-phenopropylamino)benzoic acid (100 microM). The current initiated at a shear stress as low as 0.3 dyne/cm(2), attained its peak within minutes of flow onset, and saturated above 3.5 dynes/cm(2) approximately 2.5-3.5-fold increase over pre-flow levels). The Cl(-) current desensitized slowly in response to sustained flow, and step increases in shear stress elicited increased current only if the shear stress levels were below the 3.5 dynes/cm(2) saturation level. Oscillatory flow with a physiological oscillation frequency of 1 Hz, as occurs in disturbed flow zones prone to atherosclerosis, failed to elicit the Cl(-) current, whereas lower oscillation frequencies led to partial recovery of the current. Nonreversing pulsatile flow, generally considered protective of atherosclerosis, was as effective in eliciting the current as steady flow. Measurements using fluids of different viscosities indicated that the Cl(-) current is responsive to shear stress rather than shear rate. Blocking the flow-activated Cl(-) current abolished flow-induced Akt phosphorylation in BAECs, whereas blocking flow-sensitive K(+) currents had no effect, suggesting that flow-activated Cl(-) channels play an important role in regulating EC flow signaling.

  2. 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.

  3. Predictors of Insulin Initiation in Metformin and Sulfonylurea Users in Primary Care Practices

    PubMed Central

    Dippel, Franz-Werner; Rathmann, Wolfgang

    2014-01-01

    The aims were to investigate predictors of insulin initiation in new users of metformin or sulfonylureas in primary care practices, in particular, its association with decreased renal function. Data from 9103 new metformin and 1120 sulfonylurea users with normal baseline glomerular filtration rate (eGFR) >90 ml/min/1.73 m2 from 1072 practices were retrospectively analyzed (Disease Analyzer Germany: 01/2003-06/2012). Cox regression models and propensity score matching was used to adjust for confounders (age, sex, practice characteristics, comorbidity). Insulin treatment was started in 394 (4.3%) metformin and in 162 (14.5%) sulfonylurea users within 6 years (P < .001). Kaplan-Meier curves (propensity score matched patients) showed that the metformin group was at a lower risk of insulin initiation compared to sulfonylurea users throughout the study period. A substantial eGFR decline (category: 15-<30 ml/min/1.73 m2) was significantly associated with a higher likelihood to have insulin initiated (adjusted hazard ratio [HR]: 2.39; 95% CI: 1.09-5.23) in metformin but not in sulfonylurea (HR: 0.45; 95% CI: 0.16-1.30) users. New users of sulfonylurea monotherapy in primary care practices in Germany were about 3-fold more likely to start insulin therapy than those with metformin. Kidney function decline was associated with earlier insulin initiation in metformin but not in sulfonylurea users. PMID:24876433

  4. Mitochondrial ATP-sensitive potassium channel activity and hypoxic preconditioning are independent of an inwardly rectifying potassium channel subunit in C. elegans

    PubMed Central

    Wojtovich, Andrew P.; DiStefano, Peter; Sherman, Teresa; Brookes, Paul S.; Nehrke, Keith

    2012-01-01

    Hypoxic preconditioning (HP) is an evolutionarily-conserved mechanism that protects an organism against stress. The mitochondrial ATP-sensitive K+ channel (mKATP) plays an essential role in the protective signaling, but remains molecularly undefined. Several lines of evidence suggest that mKATP may arise from an inward rectifying K+ channel (Kir). The genetic model organism C. elegans exhibits HP and displays mKATP activity. Here, we investigate the tissue expression profile of the three C. elegans Kir genes and demonstrate that mutant strains where the irk genes have been deleted either individually or in combination can be protected by HP and exhibit robust mKATP channel activity in purified mitochondria. These data suggest that the mKATP in C. elegans does not arise from a Kir derived channel. PMID:22281198

  5. Mitochondrial ATP-sensitive potassium channel activity and hypoxic preconditioning are independent of an inwardly rectifying potassium channel subunit in Caenorhabditis elegans.

    PubMed

    Wojtovich, Andrew P; DiStefano, Peter; Sherman, Teresa; Brookes, Paul S; Nehrke, Keith

    2012-02-17

    Hypoxic preconditioning (HP) is an evolutionarily-conserved mechanism that protects an organism against stress. The mitochondrial ATP-sensitive K(+) channel (mK(ATP)) plays an essential role in the protective signaling, but remains molecularly undefined. Several lines of evidence suggest that mK(ATP) may arise from an inward rectifying K(+) channel (Kir). The genetic model organism Caenorhabditis elegans exhibits HP and displays mK(ATP) activity. Here, we investigate the tissue expression profile of the three C. elegans Kir genes and demonstrate that mutant strains where the irk genes have been deleted either individually or in combination can be protected by HP and exhibit robust mK(ATP) channel activity in purified mitochondria. These data suggest that the mK(ATP) in C. elegans does not arise from a Kir derived channel.

  6. Naphthalene/quinoline amides and sulfonylureas as potent and selective antagonists of the EP4 receptor.

    PubMed

    Burch, Jason D; Farand, Julie; Colucci, John; Sturino, Claudio; Ducharme, Yves; Friesen, Richard W; Lévesque, Jean-François; Gagné, Sébastien; Wrona, Mark; Therien, Alex G; Mathieu, Marie-Claude; Denis, Danielle; Vigneault, Erika; Xu, Daigen; Clark, Patsy; Rowland, Steve; Han, Yongxin

    2011-02-01

    Two new series of EP(4) antagonists based on naphthalene/quinoline scaffolds have been identified as part of our on-going efforts to develop treatments for inflammatory pain. One series contains an acidic sulfonylurea pharmacophore, whereas the other is a neutral amide. Both series show subnanomolar intrinsic binding potency towards the EP(4) receptor, and excellent selectivity towards other prostanoid receptors. While the amide series generally displays poor pharmacokinetic parameters, the sulfonylureas exhibit greatly improved profile. MF-592, the optimal compound from the sulfonylurea series, has a desirable overall preclinical profile that suggests it is suitable for further development.

  7. 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.

  8. Mapping of dihydropyridine binding residues in a less sensitive invertebrate L-type calcium channel (LCa v 1).

    PubMed

    Senatore, Adriano; Boone, Adrienne; Lam, Stanley; Dawson, Taylor F; Zhorov, Boris; Spafford, J David

    2011-01-01

    Invertebrate L-type calcium channel, LCa(v) 1, isolated from the pond snail Lymnaea stagnalis is nearly indistinguishable from mammalian Ca(v) 1.2 (α1C) calcium channel in biophysical characteristics observed in vitro. These L-type channels are likely constrained within a narrow range of biophysical parameters to perform similar functions in the snail and mammalian cardiovascular systems. What distinguishes snail and mammalian L-type channels is a difference in dihydropyridine sensitivity: 100 nM isradipine exhibits a significant block of mammalian Ca(v) 1.2 currents without effect on snail LCa(v)1 currents. The native snail channel serves as a valuable surrogate for validating key residue differences identified from previous experimental and molecular modeling work. As predicted, three residue changes in LCa(v)1 (N_3o18, F_3i10, and I_4i12) replaced with DHP-sensing residues in respective positions of Ca(v) 1.2, (Q_3o18, Y_3i10, and M_4i12) raises the potency of isradipine block of LCa(v)1 channels to that of mammalian Ca(v) 1.2. Interestingly, the single N_3o18_Q mutation in LCa(v) 1 channels lowers DHP sensitivity even further and the triple mutation bearing enhanced isradipine sensitivity, still retains a reduced potency of agonist, (S)-Bay K8644.

  9. Adenine nucleotides and intracellular Ca2+ regulate a voltage-dependent and glucose-sensitive potassium channel in neurosecretory cells.

    PubMed

    Onetti, C G; Lara, J; García, E

    1996-05-01

    Effects of membrane potential, intracellular Ca2+ and adenine nucleotides on glucose-sensitive channels from X organ (XO) neurons of the crayfish were studied in excised inside-out patches. Glucose- sensitive channels were selective to K+ ions; the unitary conductance was 112 pS in symmetrical K+, and the K+ permeability (PK) was 1.3 x 10(-13) cm x s(-1). An inward rectification was observed when intracellular K+ was reduced. Using a quasi-physiological K+ gradient, a non-linear K+ current/voltage relationship was found showing an outward rectification and a slope conductance of 51 pS. The open-state probability (Po) increased with membrane depolarization as a result of an enhancement of the mean open time and a shortening of the longer period of closures. In quasi-physio- logical K+ concentrations, the channel was activated from a threshold of about -60 mV, and the activation midpoint was -2 mV. Po decreased noticeably at 50 microM internal adenosine 5'-triphosphate (ATP), and single-channel activity was totally abolished at 1 mM ATP. Hill analysis shows that this inhibition was the result of simultaneous binding of two ATP molecules to the channel, and the half-blocking concentration of ATP was 174 microM. Internal application of 5'-adenylylimidodiphosphate (AMP-PNP) as well as glibenclamide also decreased Po. By contrast, the application of internal ADP (0.1 to 2 mM) activated this channel. An optimal range of internal free Ca2+ ions (0.1 to 10 microM) was required for the activation of this channel. The glucose--sensitive K+ channel of XO neurons could be considered as a subtype of ATP-sensitive K+ channel, contributing substantially to macroscopic outward current.

  10. CNGA3 achromatopsia-associated mutation potentiates the phosphoinositide sensitivity of cone photoreceptor CNG channels by altering intersubunit interactions

    PubMed Central

    Dai, Gucan

    2013-01-01

    Cyclic nucleotide-gated (CNG) channels are critical for sensory transduction in retinal photoreceptors and olfactory receptor cells; their activity is modulated by phosphoinositides (PIPn) such as phosphatidylinositol 4,5-bisphosphate (PIP2) and phosphatidylinositol 3,4,5-trisphosphate (PIP3). An achromatopsia-associated mutation in cone photoreceptor CNGA3, L633P, is located in a carboxyl (COOH)-terminal leucine zipper domain shown previously to be important for channel assembly and PIPn regulation. We determined the functional consequences of this mutation using electrophysiological recordings of patches excised from cells expressing wild-type and mutant CNG channel subunits. CNGA3-L633P subunits formed functional channels with or without CNGB3, producing an increase in apparent cGMP affinity. Surprisingly, L633P dramatically potentiated PIPn inhibition of apparent cGMP affinity for these channels. The impact of L633P on PIPn sensitivity depended on an intact amino (NH2) terminal PIPn regulation module. These observations led us to hypothesize that L633P enhances PIPn inhibition by altering the coupling between NH2- and COOH-terminal regions of CNGA3. A recombinant COOH-terminal fragment partially restored normal PIPn sensitivity to channels with COOH-terminal truncation, but L633P prevented this effect. Furthermore, coimmunoprecipitation of channel fragments, and thermodynamic linkage analysis, also provided evidence for NH2-COOH interactions. Finally, tandem dimers of CNGA3 subunits that specify the arrangement of subunits containing L633P and other mutations indicated that the putative interdomain interaction occurs between channel subunits (intersubunit) rather than exclusively within the same subunit (intrasubunit). Collectively, these studies support a model in which intersubunit interactions control the sensitivity of cone CNG channels to regulation by phosphoinositides. Aberrant channel regulation may contribute to disease progression in patients with the

  11. Effects of inorganic lead on voltage-sensitive calcium channels in N1E-115 neuroblastoma cells.

    PubMed

    Audesirk, G; Audesirk, T

    1991-01-01

    N1E-115 mouse neuroblastoma cells have been reported to possess two types of voltage-sensitive calcium channels: Low voltage activated, rapidly inactivating T-type (type I) and high voltage activated, slowly inactivating L-type (type II). We studied the effects of acute in vitro exposure to inorganic lead on these calcium channels, using the whole-cell variant of patch clamping. Using salines with a high lead-buffering capacity, we found that both T-type and L-type channels are reversibly inhibited in a dose-dependent manner at free Pb2+ concentrations ranging from 20 nM to 14 microM. L-type channels are somewhat more sensitive to Pb2+ than T-type channels are (L-type: IC50 approx. 0.7 microM; T-type: IC50 approx. 1.3 microM). Both channels show small but significant inhibition (approx. 10%) at 20 nM free Pb2+. Pb2+ affects neither activation nor inactivation of T-type channels, but enhances inactivation of L-type channels at holding potentials around -60 to -40 mV. A peculiar phenomenon was observed in cells exposed to 2.3 microM free Pb2+. T-type channels were inhibited in all 20 cells studied. In 15 cells, L-type channels were also inhibited, but in the remaining 5 cells, current flow through L-type channels was enhanced by Pb2+ exposure.

  12. Suppression of the Eag1 potassium channel sensitizes glioblastoma cells to injury caused by temozolomide

    PubMed Central

    Sales, Thais Torquato; Resende, Fernando Francisco Borges; Chaves, Natália Lemos; Titze-De-Almeida, Simoneide Souza; Báo, Sônia Nair; Brettas, Marcella Lemos; Titze-De-Almeida, Ricardo

    2016-01-01

    Glioblastoma multiforme (GBM) is the most aggressive type of human primary brain tumor. The standard treatment protocol includes radiotherapy in combination with temozolomide (TMZ). Despite advances in GBM treatment, the survival time of patients diagnosed with glioma is 14.5 months. Regarding tumor biology, various types of cancer cell overexpress the ether à go-go 1 (Eag1) potassium channel. Therefore, the present study examined the role of Eag1 in the cell damage caused by TMZ on the U87MG glioblastoma cell line. Eag1 was inhibited using a channel blocker (astemizole) or silenced by a short-hairpin RNA expression vector (pKv10.1-3). pKv10.1-3 (0.2 µg) improved the Eag1 silencing caused by 250 µM TMZ, as determined by reverse transcription-quantitative polymerase chain reaction and immunocytochemistry. Additionally, inhibiting Eag1 with the vector or astemizole (5 µM) reduced glioblastoma cell viability and sensitized cells to TMZ. Cell viability decreased by 63% for pKv10.1-3 + TMZ compared with 34% for TMZ alone, and by 77% for astemizole + TMZ compared with 46% for TMZ alone, as determined by MTT assay. In addition, both the vector and astemizole increased the apoptosis rate of glioblastoma cells triggered by TMZ, as determined by an Annexin V apoptosis assay. Collectively, the current data reveal that Eag1 has a role in the damage caused to glioblastoma by TMZ. Furthermore, suppression of this channel can improve the action of TMZ on U87MG glioblastoma cells. Thus, silencing Eag1 is a promising strategy to improve GBM treatment and merits additional studies in animal models of glioma. PMID:27698831

  13. Allosteric modulation of neurotoxin binding to voltage-sensitive sodium channels by Ptychodiscus brevis toxin 2.

    PubMed

    Sharkey, R G; Jover, E; Couraud, F; Baden, D G; Catterall, W A

    1987-03-01

    The effects of Ptychodiscus brevis toxin 2 (PbTx-2) on the binding of neurotoxins at four different neurotoxin receptor sites on voltage-sensitive sodium channels in rat brain synaptosomes were examined. Binding of saxitoxin at neurotoxin receptor site 1 and Leiurus quinquestriatus alpha-scorpion toxin (LqTx) at neurotoxin receptor site 3 was unaffected. PbTx-2 enhanced binding of batrachotoxinin A 20-alpha-benzoate (BTX-B) to neurotoxin receptor site 2 and Centruroides suffusus suffusus beta-scorpion toxin (CsTx II) to site 4 on sodium channels. These results support the proposal that PbTx-2 and related toxins act at a new receptor site (site 5) that has not been previously analyzed in binding experiments. Half-maximal effects of PbTx-2 were observed in the range of 20-50 nM PbTx-2. The enhancement of BTX-B binding was reduced by depolarization. Saturating concentrations of PbTx-2 reduced KD values for binding of BTX-B and CsTx-II 2.9-fold and 2.6-fold, respectively. The effects of PbTx-2 and LqTx in enhancing BTX-B binding were synergistic. A model involving both preferential binding of BTX-B, PbTx-2, LqTx, and CsTx II to active states of sodium channels and allosteric interactions among the four receptor sites at which these toxins act accommodates these and previous results.

  14. Taurine-induced modulation of voltage-sensitive Na+ channels in rat dorsal root ganglion neurons.

    PubMed

    Yu, Shan-Shan; Yu, Kuai; Gu, Yan; Ruan, Di-Yun

    2005-08-15

    The physiological role of taurine, an abundant free amino acid in the neural system, is still poorly understood. The aim of this study was to investigate its effect on TTX-sensitive (TTX-S) and TTX-resistant (TTX-R) Na+ currents in enzymatically dissociated neurons from rat dorsal root ganglion (DRG) with conventional whole-cell recording manner under voltage-clamp conditions. A TTX-S Na+ current was recorded preferentially from large DRG neurons and a TTX-R Na+ current preferentially from small ones. For TTX-S Na+ channel, taurine of the concentration > or = 10 mM shifted the activation curve in the depolarizing direction and the inactivation curve in the hyperpolarizing direction. There was no change in the activation curve for TTX-R Na+ channel and the inactivation curve was shifted in the hyperpolarizing direction slightly in the presence of taurine > or = 20 mM. When the recovery kinetics was examined, the presence of taurine resulted in a slower recovery from inactivation of TTX-S currents and no change of TTX-R ones. All the effects of taurine were weakly concentration-dependent and partly recovered quite slowly after washout. Our data indicate that taurine alters the properties of Na+ currents in intact DRG neurons. These may contribute to the understanding of taurine as a natural neuroprotectant and the potential of taurine as a useful medicine for the treatment of sensory neuropathies.

  15. Channels, carriers, and pumps in the pathogenesis of sodium-sensitive hypertension.

    PubMed

    Capasso, Giovambattista; Cantone, Alessandra; Evangelista, Ciriana; Zacchia, Miriam; Trepiccione, Francesco; Acone, Daria; Rizzo, Maria

    2005-11-01

    Sodium-sensitive hypertension is thought to be dependent on primary alterations in renal tubular sodium reabsorption. The major apical plasma membrane Na(+) transporters include the proximal tubular Na(+)-H(+) exchanger, the thick ascending limb Na(+)-K(+)-2Cl(-) cotransport system, the distal tubular Na(+)-Cl(-) cotransporter, and the collecting duct epithelial sodium channel (ENaC). This article explores the role of each transporter in the pathogenesis of hypertension. Although the contribution of the proximal tubule Na(+)-H(+) exchanger is not yet defined completely, more convincing data have been generated about the importance of the Na(+)-K(+)-2Cl(-). Indeed at least 2 forms of hypertension appear to be related to the up-regulation of the transporter: the so-called programmed hypertension induced by low-protein diet during pregnancy and the early phase of hypertension in the Milan strain of rats. With respect to the Na(+)-Cl(-) cotransporter this may be overactive caused by inactivating mutation of WNK4 as in the Gordon syndrome, although it is the main actor for the maintenance phase of the hypertension found in the Milan strain of rats. Finally, the contribution of the ENaC has been established clearly; indeed, in the Liddle syndrome the mutation of the ENaC gene leads to a longer retention of the channel on the cell surface of collecting duct principal cells, thus inducing stronger sodium reabsorption along this segment. All these examples clearly indicate that renal sodium transporters may be responsible for various types of sodium-sensitive hypertension.

  16. Environment-Sensitive Fluorescent Probe for the Human Ether-a-go-go-Related Gene Potassium Channel

    PubMed Central

    2016-01-01

    A novel environment-sensitive probe S2 with turn-on switch for Human Ether-a-go-go-Related Gene (hERG) potassium channel was developed herein. After careful evaluation, this fluorescent probe showed high binding affinity with hERG potassium channel with an IC50 value of 41.65 nM and can be well applied to hERG channel imaging or cellular distribution study for hERG channel blockers. Compared with other imaging techniques, such as immunofluorescence and fluorescent protein-based approaches, this method is convenient and affordable, especially since a washing procedure is not needed. Meanwhile, this environment-sensitive turn-on design strategy may provide a good example for the probe development for these targets that have no reactive or catalytic activity. PMID:26730746

  17. Environment-Sensitive Fluorescent Probe for the Human Ether-a-go-go-Related Gene Potassium Channel.

    PubMed

    Liu, Zhenzhen; Jiang, Tianyu; Wang, Beilei; Ke, Bowen; Zhou, Yubin; Du, Lupei; Li, Minyong

    2016-02-02

    A novel environment-sensitive probe S2 with turn-on switch for Human Ether-a-go-go-Related Gene (hERG) potassium channel was developed herein. After careful evaluation, this fluorescent probe showed high binding affinity with hERG potassium channel with an IC50 value of 41.65 nM and can be well applied to hERG channel imaging or cellular distribution study for hERG channel blockers. Compared with other imaging techniques, such as immunofluorescence and fluorescent protein-based approaches, this method is convenient and affordable, especially since a washing procedure is not needed. Meanwhile, this environment-sensitive turn-on design strategy may provide a good example for the probe development for these targets that have no reactive or catalytic activity.

  18. A Cytosolic Amphiphilic α-Helix Controls the Activity of the Bile Acid-sensitive Ion Channel (BASIC).

    PubMed

    Schmidt, Axel; Löhrer, Daniel; Alsop, Richard J; Lenzig, Pia; Oslender-Bujotzek, Adrienne; Wirtz, Monika; Rheinstädter, Maikel C; Gründer, Stefan; Wiemuth, Dominik

    2016-11-18

    The bile acid-sensitive ion channel (BASIC) is a member of the degenerin/epithelial Na(+) channel (Deg/ENaC) family of ion channels. It is mainly found in bile duct epithelial cells, the intestinal tract, and the cerebellum and is activated by alterations of its membrane environment. Bile acids, one class of putative physiological activators, exert their effect by changing membrane properties, leading to an opening of the channel. The physiological function of BASIC, however, is unknown. Deg/ENaC channels are characterized by a trimeric subunit composition. Each subunit is composed of two transmembrane segments, which are linked by a large extracellular domain. The termini of the channels protrude into the cytosol. Many Deg/ENaC channels contain regulatory domains and sequence motifs within their cytosolic domains. In this study, we show that BASIC contains an amphiphilic α-helical structure within its N-terminal domain. This α-helix binds to the cytosolic face of the plasma membrane and stabilizes a closed state. Truncation of this domain renders the channel hyperactive. Collectively, we identify a cytoplasmic domain, unique to BASIC, that controls channel activity via membrane interaction.

  19. Distribution of diuretics and hypoglycemic sulfonylureas in rabbit erythrocytes.

    PubMed

    Yoshitomi, H; Kiko, S; Ikeda, K; Goto, S

    1983-03-01

    The distribution of three sulfonylureas and six diuretics in rabbit erythrocytes was studied in vitro at 37 degrees C. The drugs were taken up by the erythrocyte compartment, and distribution equilibrium was reached within 60 min of incubation. A distribution percentage in erythrocyte compartment was maintained at roughly constant value over the whole concentration range of drugs. Therefore, a linear relationship was established between total concentrations of drug in whole blood or erythrocyte suspension and in the erythrocyte compartment. Bovine serum albumin combined with the erythrocyte suspension appeared to reduce drug distribution in the erythrocyte compartment. Whole blood obtained from renal failure rabbits showed greater distribution of drug in the erythrocyte compartment compared with the whole blood of a normal rabbit. This might be due to a change in plasma protein binding ability related to the progress of renal failure.

  20. 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.

  1. The effect of ATP-sensitive potassium channel modulation on heart rate in isolated muskrat and guinea pig hearts.

    PubMed

    Streeby, D R; McKean, T A

    1994-12-01

    Muskrats (Ondontra zibethicus) are common freshwater diving mammals exhibiting a bradycardia with both forced and voluntary diving. This bradycardia is mediated by vagal innervation; however, if hypoxia is present there may be local factors that also decrease heart rate. Some of these local factors may include ATP-sensitive potassium channel activation and extracellular accumulation of potassium ions, hydrogen ions and lactate. The purpose of this study was to investigate the role of these factors in the isolated perfused hearts of muskrats and of a non-diving mammal, the guinea pig. Although lactate and proton administration reduced heart rate in isolated muskrat and guinea pig hearts, there was no difference in the response to lactate and proton infusion between the two species. Muskrat hearts were more sensitive to the heart-rate-lowering effects of exogenously applied potassium than were guinea pig hearts. Early increases in extracellular potassium concentration during hypoxia are thought to be mediated by the ATP-sensitive potassium channel. Activation of these channels under normoxic conditions had a mildly negative chronotropic effect in both species; however, activation of these channels with Lemakalim under hypoxic conditions caused the guinea pig heart to respond with an augmented bradycardia similar to that seen in the hypoxic muskrat heart in the absence of drugs. Inhibition of these channels by glibenclamide during hypoxia was partially successful in blocking the bradycardia in guinea pig hearts, but inhibition of the same channels in hypoxic muskrat hearts had a damaging effect as two of five hearts went into contracture during the hypoxia. Thus, although ATP-sensitive potassium channels appear to have a major role in the bradycardia of hypoxia in guinea pigs, the failure to prevent the bradycardia by inhibition of these channels in muskrat hearts suggests that multiple factors are involved in the hypoxia-induced bradycardia in this species.

  2. Novel sulfonylurea derivatives as H3 receptor antagonists. Preliminary SAR studies.

    PubMed

    Ceras, Javier; Cirauqui, Nuria; Pérez-Silanes, Silvia; Aldana, Ignacio; Monge, Antonio; Galiano, Silvia

    2012-06-01

    The combination of antagonism at histamine H(3) receptor and the stimulation of insulin secretion have been proposed as an approach to new dual therapeutic agents for the treatment of type 2 diabetes mellitus associated with obesity. We have designed and synthesized a new series of non-imidazole derivatives, based on a basic amine ring connected through an alkyl spacer of variable length to a phenoxysulfonylurea moiety. These compounds were initially evaluated for histamine H(3) receptor binding affinities, suggesting that a propoxy chain linker between the amine and the core ring could be essential for optimal binding affinity. Compound 56, 1-(naphthalen-1-yl)-3-[(p-(3-pyrrolidin-1-ylpropoxy)benzene)]sulfonylurea exhibited the best H(3) antagonism affinity. However, since all these derivatives failed to block K(ATP) channels, the link of these two related moieties should not be considered a good pharmacophore for obtaining new dual H(3) antagonists with insulinotropic activity, suggesting the necessity to propose a new chemical hybrid prototype.

  3. 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.

  4. TRPA1 Channels in Drosophila and Honey Bee Ectoparasitic Mites Share Heat Sensitivity and Temperature-Related Physiological Functions

    PubMed Central

    Peng, Guangda; Kashio, Makiko; Li, Tianbang; Dong, Xiaofeng; Tominaga, Makoto; Kadowaki, Tatsuhiko

    2016-01-01

    The transient receptor potential cation channel, subfamily A, member 1 (TRPA1) is conserved between many arthropods, and in some has been shown to function as a chemosensor for noxious compounds. Activation of arthropod TRPA1 channels by temperature fluctuations has been tested in only a few insect species, and all of them were shown to be activated by heat. The recent identification of chemosensitive TRPA1 channels from two honey bee ectoparasitic mite species (VdTRPA1 and TmTRPA1) have provided an opportunity to study the temperature-dependent activation and the temperature-associated physiological functions of TRPA1 channels in non-insect arthropods. We found that both mite TRPA1 channels are heat sensitive and capable of rescuing the temperature-related behavioral defects of a Drosophila melanogaster trpA1 mutant. These results suggest that heat-sensitivity of TRPA1 could be conserved between many arthropods despite its amino acid sequence diversity. Nevertheless, the ankyrin repeats (ARs) 6 and 7 are well-conserved between six heat-sensitive arthropod TRPA1 channels and have critical roles for the heat activation of VdTRPA1. PMID:27761115

  5. TRPA1 Channels in Drosophila and Honey Bee Ectoparasitic Mites Share Heat Sensitivity and Temperature-Related Physiological Functions.

    PubMed

    Peng, Guangda; Kashio, Makiko; Li, Tianbang; Dong, Xiaofeng; Tominaga, Makoto; Kadowaki, Tatsuhiko

    2016-01-01

    The transient receptor potential cation channel, subfamily A, member 1 (TRPA1) is conserved between many arthropods, and in some has been shown to function as a chemosensor for noxious compounds. Activation of arthropod TRPA1 channels by temperature fluctuations has been tested in only a few insect species, and all of them were shown to be activated by heat. The recent identification of chemosensitive TRPA1 channels from two honey bee ectoparasitic mite species (VdTRPA1 and TmTRPA1) have provided an opportunity to study the temperature-dependent activation and the temperature-associated physiological functions of TRPA1 channels in non-insect arthropods. We found that both mite TRPA1 channels are heat sensitive and capable of rescuing the temperature-related behavioral defects of a Drosophila melanogaster trpA1 mutant. These results suggest that heat-sensitivity of TRPA1 could be conserved between many arthropods despite its amino acid sequence diversity. Nevertheless, the ankyrin repeats (ARs) 6 and 7 are well-conserved between six heat-sensitive arthropod TRPA1 channels and have critical roles for the heat activation of VdTRPA1.

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

    PubMed

    Cui, Yuanyuan; Yang, Fan; Cao, Xu; Yarov-Yarovoy, Vladimir; Wang, KeWei; Zheng, Jie

    2012-04-01

    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.

  7. Antagonistic regulation of native Ca2+- and ATP-sensitive cation channels in brain capillaries by nucleotides and decavanadate.

    PubMed

    Csanády, László; Adam-Vizi, Vera

    2004-06-01

    Regulation by cytosolic nucleotides of Ca2+- and ATP-sensitive nonselective cation channels (CA-NSCs) in rat brain capillary endothelial cells was studied in excised inside-out patches. Open probability (Po) was suppressed by cytosolic nucleotides with apparent KI values of 17, 9, and 2 microM for ATP, ADP, and AMP, as a consequence of high-affinity inhibition of channel opening rate and low-affinity stimulation of closing rate. Cytosolic [Ca2+] and voltage affected inhibition of Po, but not of opening rate, by ATP, suggesting that the conformation of the nucleotide binding site is influenced only by the state of the channel gate, not by that of the Ca2+ and voltage sensors. ATP inhibition was unaltered by channel rundown. Nucleotide structure affected inhibitory potency that was little sensitive to base substitutions, but was greatly diminished by 3'-5' cyclization, removal of all phosphates, or complete omission of the base. In contrast, decavanadate potently (K1/2 = 90 nM) and robustly stimulated Po, and functionally competed with inhibitory nucleotides. From kinetic analyses we conclude that (a) ATP, ADP, and AMP bind to a common site; (b) inhibition by nucleotides occurs through simple reversible binding, as a consequence of tighter binding to the closed-channel relative to the open-channel conformation; (c) the conformation of the nucleotide binding site is not directly modulated by Ca2+ and voltage; (d) the differences in inhibitory potency of ATP, ADP, and AMP reflect their different affinities for the closed channel; and (e) though decavanadate is the only example found to date of a compound that stimulates Po with high affinity even in the presence of millimolar nucleotides, apparently by competing for the nucleotide binding site, a comparable mechanism might allow CA-NSC channels to open in living cells despite physiological levels of nucleotides. Decavanadate now provides a valuable tool for studying native CA-NSC channels and for screening cloned

  8. ATP-sensitive potassium channels mediate contraction-induced attenuation of sympathetic vasoconstriction in rat skeletal muscle.

    PubMed

    Thomas, G D; Hansen, J; Victor, R G

    1997-06-01

    Sympathetic vasoconstriction is sensitive to inhibition by metabolic events in contracting rat and human skeletal muscle, but the underlying cellular mechanisms are unknown. In rats, this inhibition involves mainly alpha2-adrenergic vasoconstriction, which relies heavily on Ca2+ influx through voltage-dependent Ca2+ channels. We therefore hypothesized that contraction-induced inhibition of sympathetic vasoconstriction is mediated by ATP-sensitive potassium (KATP) channels, a hyperpolarizing vasodilator mechanism that could be activated by some metabolic product(s) of skeletal muscle contraction. We tested this hypothesis in anesthetized rats by measuring femoral artery blood flow responses to lumbar sympathetic nerve stimulation or intraarterial hindlimb infusion of the specific alpha2-adrenergic agonist UK 14,304 during KATP channel activation with diazoxide in resting hindlimb and during KATP channel block with glibenclamide in contracting hindlimb. The major new findings are twofold. First, like muscle contraction, pharmacologic activation of KATP channels with diazoxide in resting hindlimb dose dependently attenuated the vasoconstrictor responses to either sympathetic nerve stimulation or intraarterial UK 14,304. Second, the large contraction-induced attenuation in sympathetic vasoconstriction elicited by nerve stimulation or UK 14,304 was partially reversed when the physiologic activation of KATP channels produced by muscle contraction was prevented with glibenclamide. We conclude that contraction-induced activation of KATP channels is a major mechanism underlying metabolic inhibition of sympathetic vasoconstriction in exercising skeletal muscle.

  9. 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.

  10. Molecular and functional characterization of a calcium-sensitive chloride channel from mouse lung.

    PubMed

    Gandhi, R; Elble, R C; Gruber, A D; Schreur, K D; Ji, H L; Fuller, C M; Pauli, B U

    1998-11-27

    A protein (mCLCA1) has been cloned from a mouse lung cDNA library that bears strong sequence homology with the recently described bovine tracheal, Ca2+-sensitive chloride channel protein (bCLCA1), bovine lung endothelial cell adhesion molecule-1 (Lu-ECAM-1), and the human intestinal Ca2+-sensitive chloride channel protein (hCLCA1). In vitro, its 3.1-kilobase message translates into a 100-kDa protein that can be glycosylated to an approximately 125-kDa product. SDS-polyacrylamide gel electrophoresis from lysates of mCLCA1 cDNA-transfected transformed human embryonic kidney cells (HEK293) reveals proteins of 130, 125, and 90 kDa as well as a protein triplet in the 32-38 kDa size range. Western analyses with antisera raised against Lu-ECAM-1 peptides show that the N-terminal region of the predicted open reading frame is present only in the larger size proteins (i.e. 130, 125, and 90 kDa), whereas the C-terminal region of the open reading frame is observed in the 32-38 kDa size proteins, suggesting a posttranslational, proteolytic processing of a precursor protein (125/130 kDa) into 90 kDa and 32-38 kDa components similar to that reported for Lu-ECAM-1. Hydrophobicity analyses predict four transmembrane domains for the 90-kDa protein. The mCLCA1 mRNA is readily detected by Northern analysis and by in situ hybridization in the respiratory epithelia of trachea and bronchi. Transient expression of mCLCA1 in HEK293 cells was associated with an increase in whole cell Cl- current that could be activated by Ca2+ and ionomycin and inhibited by 4, 4'-diisothiocyanatostilbene-2,2'-disulfonic acid, dithiothreitol, and niflumic acid. The discovery of mCLCA1 opens the door for further investigating the possible contribution of a Ca2+-sensitive chloride conductance to the pathogenesis of cystic fibrosis.

  11. [Polymorphism of the sulfonylurea receptor gene in type 2 diabetes mellitus].

    PubMed

    Owecki, Maciej; Horst-Sikorska, Wanda; Kaczmarek, Marta; Słomski, Ryszard; Sowiński, Jerzy

    2003-02-01

    Sulfonylureas are used in treatment of diabetes. Resistance to these derivatives is a therapeutical problem. Sulfonylureas act through sulfonylurea receptor 1 (SUR1) in the beta cell. SUR1 also enhances a physiological secretion of insulin induced by an increase of glucose concentration. It may be expected that polymorphism of SUR1 gene can lead to beta cell dysfunction and resistance to sulfonylureas. The aim of this study was to examine the frequency of polymorphism in exon 22 of SUR1 gene and its correlation with type 2 diabetes mellitus and sulfonylurea treatment failure. The group consisted of 42 patients with type 2 diabetes. The controls were 46 persons with proper glucose tolerance. Polymorphism was found in 5 patients and in 1 control person. Neither statistically significant difference of polymorphism frequency nor correlation between polymorphism and sulfonylurea failure was found due to a low number of cases. Polymorphism of exon 22 of SUR1 gene appeared more frequent in diabetic than in non-diabetic subjects but this was statistically not significant.

  12. Effect of Sulfonylureas Administered Centrally on the Blood Glucose Level in Immobilization Stress Model

    PubMed Central

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

    2015-01-01

    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

  13. IP3 receptor binds to and sensitizes TRPV4 channel to osmotic stimuli via a calmodulin-binding site.

    PubMed

    Garcia-Elias, Anna; Lorenzo, Ivan M; Vicente, Rubén; Valverde, Miguel A

    2008-11-14

    Activation of the non-selective cation channel TRPV4 by mechanical and osmotic stimuli requires the involvement of phospholipase A2 and the subsequent production of the arachidonic acid metabolites, epoxieicosatrienoic acids (EET). Previous studies have shown that inositol trisphosphate (IP3) sensitizes TRPV4 to mechanical, osmotic, and direct EET stimulation. We now search for the IP3 receptor-binding site on TRPV4 and its relevance to IP3-mediated sensitization. Three putative sites involved in protein-protein interactions were evaluated: a proline-rich domain (PRD), a calmodulin (CaM)-binding site, and the last four amino acids (DAPL) that show a PDZ-binding motif-like. TRPV4-DeltaCaM-(Delta812-831) channels preserved activation by hypotonicity, 4alpha-phorbol 12,13-didecanoate, and EET but lost their physical interaction with IP3 receptor 3 and IP3-mediated sensitization. Deletion of a PDZ-binding motif-like (TRPV4-DeltaDAPL) did not affect channel activity or IP3-mediated sensitization, whereas TRPV4-DeltaPRD-(Delta132-144) resulted in loss of channel function despite correct trafficking. We conclude that IP3-mediated sensitization requires IP3 receptor binding to a TRPV4 C-terminal domain that overlaps with a previously described calmodulin-binding site.

  14. External copper inhibits the activity of the large-conductance calcium- and voltage-sensitive potassium channel from skeletal muscle.

    PubMed

    Morera, F J; Wolff, D; Vergara, C

    2003-03-01

    We have characterized the effect of external copper on the gating properties of the large-conductance calcium- and voltage-sensitive potassium channel from skeletal muscle, incorporated into artificial bilayers. The effect of Cu2+ was evaluated as changes in the gating kinetic properties of the channel after the addition of this ion. We found that, from concentrations of 20 microM and up, copper induced a concentration- and time-dependent decrease in channel open probability. The inhibition of channel activity by Cu2+ could not be reversed by washing or by addition of the copper chelator, bathocuproinedisulfonic acid. However, channel activity was appreciably restored by the sulfhydryl reducing agent dithiothreitol. The effect of copper was specific since other transition metal divalent cations such as Ni2+, Zn2+ or Cd2+ did not affect BK(Ca) channel activity in the same concentration range. These results suggest that external Cu2+-induced inhibition of channel activity was due to direct or indirect oxidation of key amino-acid sulfhydryl groups that might have a role in channel gating.

  15. Primary structure of an apical protein from Xenopus laevis that participates in amiloride-sensitive sodium channel activity

    PubMed Central

    1992-01-01

    High resistance epithelia express on their apical side an amiloride- sensitive sodium channel that controls sodium reabsorption. A cDNA was found to encode a 1,420-amino acid long polypeptide with no signal sequence, a putative transmembrane segment, and three predicted amphipathic alpha helices. A corresponding 5.2-kb mRNA was detected in Xenopus laevis kidney, intestine, and oocytes, with weak expression in stomach and eyes. An antibody directed against a fusion protein containing a COOH-terminus segment of the protein and an antiidiotypic antibody known to recognize the amiloride binding site of the epithelial sodium channel (Kleyman, T. R., J.-P. Kraehenbuhl, and S. A. Ernst. 1991. J. Biol. Chem. 266:3907-3915) immunoprecipitated a similar protein complex from [35S]methionine-labeled and from apically radioiodinated Xenopus laevis kidney-derived A6 cells. A single integral of 130-kD protein was recovered from samples reduced with DTT. The antibody also cross-reacted by ELISA with the putative amiloride- sensitive sodium channel isolated from A6 cells (Benos, D. J., G. Saccomani, and S. Sariban-Sohraby. 1987. J. Biol. Chem. 262:10613- 10618). Although the protein is translated, cRNA injected into oocytes did not reconstitute amiloride-sensitive sodium transport, while antisense RNA or antisense oligodeoxynucleotides specific for two distinct sequences of the cloned cDNA inhibited amiloride-sensitive sodium current induced by injection of A6 cell mRNA. We propose that the cDNA encodes an apical plasma membrane protein that plays a role in the functional expression of the amiloride-sensitive epithelial sodium channel. It may represent a subunit of the Xenopus laevis sodium channel or a regulatory protein essential for sodium channel function. PMID:1334959

  16. Insulin modulation of ATP-sensitive K+ channel of rat skeletal muscle is impaired in the hypokalaemic state.

    PubMed

    Tricarico, D; Capriulo, R; Conte Camerino, D

    1999-01-01

    In the present work, we examined the effects of in vivo administration of insulin to rats made hypokalaemic by feeding a K+-free diet. The i.p. injection of insulin in the hypokalaemic rats provoked muscle paralysis within 3-5 h. Consistent with this observation, the skeletal muscle fibres of the paralysed rats were depolarized. In contrast, in the normokalaemic animals, insulin neither provoked paralysis nor produced significant fibre hyperpolarization. In the hypokalaemic rats, insulin almost completely abolished the sarcolemma adenosine triphosphate (ATP)-sensitive K+ currents without altering the sensitivity of the channels to ATP or glibenclamide. In contrast, in the normokalaemic rats, insulin enhanced ATP-sensitive K+ currents that became also resistant to ATP and glibenclamide. Our experiments indicate that the modulation of the sarcolemma ATP-sensitive K+ channels by insulin is impaired in the hypokalaemic state. This phenomenon appears to be related to the fibre depolarization and paralysis observed in the same animals.

  17. 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

  18. Amiloride-sensitive channels are a major contributor to mechanotransduction in mammalian muscle spindles

    PubMed Central

    Simon, Anna; Shenton, Fiona; Hunter, Irene; Banks, Robert W; Bewick, Guy S

    2010-01-01

    We investigated whether channels of the epithelial sodium/amiloride-sensitive degenerin (ENaC/DEG) family are a major contributor to mechanosensory transduction in primary mechanosensory afferents, using adult rat muscle spindles as a model system. Stretch-evoked afferent discharge was reduced in a dose-dependent manner by amiloride and three analogues – benzamil, 5-(N-ethyl-N-isopropyl) amiloride (EIPA) and hexamethyleneamiloride (HMA), reaching ≥85% inhibition at 1 mm. Moreover, firing was slightly but significantly increased by ENaC δ subunit agonists (icilin and capsazepine). HMA's profile of effects was distinct from that of the other drugs. Amiloride, benzamil and EIPA significantly decreased firing (P < 0.01 each) at 1 μm, while 10 μm HMA was required for highly significant inhibition (P < 0.0001). Conversely, amiloride, benzamil and EIPA rarely blocked firing entirely at 1 mm, whereas 1 mm HMA blocked 12 of 16 preparations. This pharmacology suggests low-affinity ENaCs are the important spindle mechanotransducer. In agreement with this, immunoreactivity to ENaC α, β and γ subunits was detected both by Western blot and immunocytochemistry. Immunofluorescence intensity ratios for ENaC α, β or γ relative to the vesicle marker synaptophysin in the same spindle all significantly exceeded controls (P < 0.001). Ratios for the related brain sodium channel ASIC2 (BNaC1α) were also highly significantly greater (P < 0.005). Analysis of confocal images showed strong colocalisation within the terminal of ENaC/ASIC2 subunits and synaptophysin. This study implicates ENaC and ASIC2 in mammalian mechanotransduction. Moreover, within the terminals they colocalise with synaptophysin, a marker for the synaptic-like vesicles which regulate afferent excitability in these mechanosensitive endings. PMID:19917568

  19. Reconstitution of highly purified saxitoxin-sensitive Na+-channels into planar lipid bilayers.

    PubMed Central

    Hanke, W; Boheim, G; Barhanin, J; Pauron, D; Lazdunski, M

    1984-01-01

    Highly purified Na+-channels isolated from rat brain have been reconstituted into virtually solvent-free planar lipid bilayer membranes. Two different types of electrically excitable channels were detected in the absence of any neurotoxins. The activity of both channels was blocked by saxitoxin. The first channel type is highly selective for Na+ over K+ (approximately 10:1), it shows a bursting behavior, a conductance of 25 pS in Na+-Ringer and undergoes continuous opening and closing events for periods of minutes within a defined range of negative membranes voltages. The second channel type has a conductance of 150 pS and a lower selectivity for Na+ and K+ (2.2:1); only a few opening and closing events are observed with this channel after one voltage jump. The latter type of channel is also found with highly purified Na+-channel from Electrophorus electricus electroplax. A qualitative analysis of the physicochemical and pharmacological properties of the high conductance channel has been carried out. Channel properties are affected not only by saxitoxin but also by a scorpion (Centruroides suffusus suffusus) toxin and a sea anemone (Anemonia sulcata) toxin both known to be selective for the Na+-channel. The spontaneous transformation of the large conductance channel type into the small one has been considered; the two channel types may represent the expression of activity of different conformational states of the same protein. Images Fig. 1. PMID:6325173

  20. Reconstitution of highly purified saxitoxin-sensitive Na+-channels into planar lipid bilayers.

    PubMed

    Hanke, W; Boheim, G; Barhanin, J; Pauron, D; Lazdunski, M

    1984-03-01

    Highly purified Na+-channels isolated from rat brain have been reconstituted into virtually solvent-free planar lipid bilayer membranes. Two different types of electrically excitable channels were detected in the absence of any neurotoxins. The activity of both channels was blocked by saxitoxin. The first channel type is highly selective for Na+ over K+ (approximately 10:1), it shows a bursting behavior, a conductance of 25 pS in Na+-Ringer and undergoes continuous opening and closing events for periods of minutes within a defined range of negative membranes voltages. The second channel type has a conductance of 150 pS and a lower selectivity for Na+ and K+ (2.2:1); only a few opening and closing events are observed with this channel after one voltage jump. The latter type of channel is also found with highly purified Na+-channel from Electrophorus electricus electroplax. A qualitative analysis of the physicochemical and pharmacological properties of the high conductance channel has been carried out. Channel properties are affected not only by saxitoxin but also by a scorpion (Centruroides suffusus suffusus) toxin and a sea anemone (Anemonia sulcata) toxin both known to be selective for the Na+-channel. The spontaneous transformation of the large conductance channel type into the small one has been considered; the two channel types may represent the expression of activity of different conformational states of the same protein.

  1. Differential modulation of TWIK-related K(+) channel (TREK) and TWIK-related acid-sensitive K(+) channel 2 (TASK2) activity by pyrazole compounds.

    PubMed

    Kim, Hyun Jong; Woo, Joohan; Nam, Yuran; Nam, Joo Hyun; Kim, Woo Kyung

    2016-11-15

    Pyrazole derivatives were originally suggested as selective blockers of the transient receptor potential cation 3 (TRPC3) and channel. In particular, pyr3 and 10 selectively inhibit TRPC3, whereas pyr2 (BTP2) and 6 inhibit ORAI1. However, their effects on background K(+) channel activity have not been elucidated. In this study, the effects of BTP2, pyr3, pyr6, and pyr10 were studied on cloned human TWIK-related K(+) channels (TREKs) and TWIK-related acid-sensitive K(+) channel 2 (TASK-2) channels, which modulate Ca(2+) signaling by controlling membrane potential, in HEK293T-overexpressing cells by using a whole-cell patch clamp technique. Pyr3 potently inhibited TREK-1 (ITREK1), TREK-2 (ITREK2), and TASK2 current (ITASK-2) with half-maximal inhibitory concentrations (IC50) of 0.89±0.27, 1.95±1.44, and 2.42±0.39µM, respectively. BTP2 slightly inhibited ITASK-2 (80.3±2.5% at 100μM). In contrast, pyr6 at 100µM potentiated ITREK1 and ITREK2 by approximately 2.6- and 3.6-fold compared to the control and inhibited ITASK2 (38.7±9.2%). Pyr10 showed a subtype-specific inhibition of ITREK1 but not ITREK2. It also inhibited ITASK2 (70.9±3.1% at 100μM). To the best of our knowledge, this study is the first to describe the differential modulation of TREKs and TASK2 channels by pyrazole derivatives, previously used as inhibitors of TRPC3 and ORAI1. Therefore, studies using these drugs should consider their modulation of other channels such as TREK and TASK-2.

  2. Label-free cell phenotypic profiling decodes the composition and signaling of an endogenous ATP-sensitive potassium channel

    PubMed Central

    Sun, Haiyan; Wei, Ying; Deng, Huayun; Xiong, Qiaojie; Li, Min; Lahiri, Joydeep; Fang, Ye

    2014-01-01

    Current technologies for studying ion channels are fundamentally limited because of their inability to functionally link ion channel activity to cellular pathways. Herein, we report the use of label-free cell phenotypic profiling to decode the composition and signaling of an endogenous ATP-sensitive potassium ion channel (KATP) in HepG2C3A, a hepatocellular carcinoma cell line. Label-free cell phenotypic agonist profiling showed that pinacidil triggered characteristically similar dynamic mass redistribution (DMR) signals in A431, A549, HT29 and HepG2C3A, but not in HepG2 cells. Reverse transcriptase PCR, RNAi knockdown, and KATP blocker profiling showed that the pinacidil DMR is due to the activation of SUR2/Kir6.2 KATP channels in HepG2C3A cells. Kinase inhibition and RNAi knockdown showed that the pinacidil activated KATP channels trigger signaling through Rho kinase and Janus kinase-3, and cause actin remodeling. The results are the first demonstration of a label-free methodology to characterize the composition and signaling of an endogenous ATP-sensitive potassium ion channel. PMID:24816792

  3. Two critical residues in p-loop regions of puffer fish Na+ channels on TTX sensitivity.

    PubMed

    Maruta, Satoshi; Yamaoka, Kaoru; Yotsu-Yamashita, Mari

    2008-03-01

    We previously showed that Asn-383 and Thr-1569 residues of p-loop regions in domains I and IV, respectively, of the puffer fish, Fugu pardialis, skeletal muscle Na(v) (fNa(v)1.4a), are anomalous to those of other species of TTX-sensitive Na(+) channels, where the aromatic residues of Phe or Tyr, and Gly are the counterparts [Yotsu-Yamashita, M., Nishimori, K., Nitanai, Y., Isemura, M., Sugimoto, A., Yasumoto, T., 2000. Binding properties of (3)H-PbTx-3 and (3)H-saxitoxin to brain membranes and to skeletal muscle membranes of puffer fish Fugu pardalis and the primary structure of a voltage-gated Na(+) channel alpha-subunit (fMNa1) from skeletal muscle of F. pardalis. Biochem. Biophys. Res. Commun. 267, 403-412]. The former was suggested to confer TTX resistance by using Y401N mutant of rNa(v)1.4 [Venkatesh, V., Lu, S.Q., Dandona, N., See, S.L., Benner, S., Soong, T.W., 2005. Genetic basis of tetrodotoxin resistance in pufferfishes. Curr. Biol. 15, 2069-2072]. The latter function remained to be elucidated. Thus, we further explored the function of these two residues, electrophysiologically, by evaluating the K(d) (dissociation constants) values of TTX for F385N, F385A, F385Q, G1718T, and F385N/G1718T mutants of rNa(v)1.2a, transiently expressed in HEK-293 cells. F385N caused 3000-fold increase of the K(d), while G1718T and F385N/G1718T caused 2- and 3-fold increases compared with those of WT and F385N, respectively, suggesting that G1718T further enhanced TTX resistivity caused by F385N. The K(d) for F385A and F385Q were 2- and 11-fold larger than that of F385N, respectively, suggesting that the longer side chain in the non-aromatic amino acid residue causes the larger decrease of TTX sensitivity. Despite drastic changes in the K(d), the mutations at F385 caused only small changes in the k(off) from that of WT, suggesting that the K(d) for TTX receptors are mainly determined by the k(on).

  4. Sulfonylurea Receptor 1 in Humans with Post-Traumatic Brain Contusions

    PubMed Central

    Martínez-Valverde, Tamara; Vidal-Jorge, Marian; Martínez-Saez, Elena; Castro, Lidia; Arikan, Fuat; Cordero, Esteban; Rădoi, Andreea; Poca, Maria-Antonia; Simard, J. Marc

    2015-01-01

    Abstract Post-traumatic brain contusions (PTBCs) are traditionally considered primary injuries and can increase in size, generate perilesional edema, cause mass effect, induce neurological deterioration, and cause death. Most patients experience a progressive increase in pericontusional edema, and nearly half, an increase in the hemorrhagic component itself. The underlying molecular pathophysiology of contusion-induced brain edema and hemorrhagic progression remains poorly understood. The aim of this study was to investigate sulfonylurea 1/transient receptor potential melastatin 4 (SUR1-TRPM4) ion channel SUR1 expression in various cell types (neurons, astrocytes, endothelial cells, microglia, macrophages, and neutrophils) of human brain contusions and whether SUR1 up-regulation was related to time postinjury. Double immunolabeling of SUR1 and cell-type– specific proteins was performed in 26 specimens from traumatic brain injury patients whose lesions were surgically evacuated. Three samples from limited brain resections performed for accessing extra-axial skull-base tumors or intraventricular lesions were controls. We found SUR1 was significantly overexpresed in all cell types and was especially prominent in neurons and endothelial cells (ECs). The temporal pattern depended on cell type: 1) In neurons, SUR1 increased within 48 h of injury and stabilized thereafter; 2) in ECs, there was no trend; 3) in glial cells and microglia/macrophages, a moderate increase was observed over time; and 4) in neutrophils, it decreased with time. Our results suggest that up-regulation of SUR1 in humans point to this channel as one of the important molecular players in the pathophysiology of PTBCs. Our findings reveal opportunities to act therapeutically on the mechanisms of growth of traumatic contusions and therefore reduce the number of patients with neurological deterioration and poor neurological outcomes. PMID:26398596

  5. 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

  6. 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

  7. Functional control of cold- and menthol-sensitive TRPM8 ion channels by phosphatidylinositol 4,5-bisphosphate.

    PubMed

    Liu, Beiying; Qin, Feng

    2005-02-16

    Cold is detected by a small subpopulation of peripheral thermoreceptors. TRPM8, a cloned menthol- and cold-sensitive ion channel, has been suggested to mediate cold transduction in the innocuous range. The channel shows a robust response in whole-cell recordings but exhibits markedly reduced activity in excised membrane patches. Here we report that phosphatidylinositol 4,5-bisphosphate (PIP2) is an essential regulator of the channel function. The rundown of the channel is prevented by lipid phosphatase inhibitors. Application of exogenous PIP2 both activates the channel directly and restores rundown activity. Whole-cell experiments involving intracellular dialysis with polyvalent cations, inhibition of PIP2 synthesis kinases, and receptor-mediated hydrolysis of PIP2 show that PIP2 also modulates the channel activity in the intact cells. The crucial role of PIP2 on the function of TRPM8 suggests that the membrane PIP2 level may be an important regulator of cold transduction in vivo. The opposite effects of PIP2 on the vanilloid receptor TRPV1 and TRPM8 also implies that the membrane lipid may have dual actions as a bimodal switch to selectively control the heat- and cold-induced responses in nociceptors expressing both channels.

  8. 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.

  9. Voltage-gated K+ channels sensitive to stromatoxin-1 regulate myogenic and neurogenic contractions of rat urinary bladder smooth muscle

    PubMed Central

    Chen, Muyan; Kellett, Whitney F.

    2010-01-01

    Members of the voltage-gated K+ (KV) channel family are suggested to control the resting membrane potential and the repolarization phase of the action potential in urinary bladder smooth muscle (UBSM). Recent studies report that stromatoxin-1, a peptide isolated from tarantulas, selectively inhibits KV2.1, KV2.2, KV4.2, and KV2.1/9.3 channels. The objective of this study was to investigate whether KV channels sensitive to stromatoxin-1 participate in the regulation of rat UBSM contractility and to identify their molecular fingerprints. Stromatoxin-1 (100 nM) increased the spontaneous phasic contraction amplitude, muscle force, and tone in isolated UBSM strips. However, stromatoxin-1 (100 nM) had no effect on the UBSM contractions induced by depolarizing agents such as KCl (20 mM) or carbachol (1 μM). This indicates that, under conditions of sustained membrane depolarization, the KV channels sensitive to stromatoxin-1 have no further contribution to the membrane excitability and contractility. Stromatoxin-1 (100 nM) increased the amplitude of the electrical field stimulation-induced contractions, suggesting also a role for these channels in neurogenic contractions. RT-PCR experiments on freshly isolated UBSM cells showed mRNA expression of KV2.1, KV2.2, and KV9.3, but not KV4.2 channel subunits. Protein expression of KV2.1 and KV2.2 channels was detected using Western blot and was further confirmed by immunocytochemical detection in freshly isolated UBSM cells. These novel findings indicate that KV2.1 and KV2.2, but not KV4.2, channel subunits are expressed in rat UBSM and play a key role in opposing both myogenic and neurogenic UBSM contractions. PMID:20393158

  10. Electromagnetic fields (UHF) increase voltage sensitivity of membrane ion channels; possible indication of cell phone effect on living cells.

    PubMed

    Ketabi, N; Mobasheri, H; Faraji-Dana, R

    2015-03-01

    The effects of ultra high frequency (UHF) nonionizing electromagnetic fields (EMF) on the channel activities of nanopore forming protein, OmpF porin, were investigated. The voltage clamp technique was used to study the single channel activity of the pore in an artificial bilayer in the presence and absence of the electromagnetic fields at 910 to 990 MHz in real time. Channel activity patterns were used to address the effect of EMF on the dynamic, arrangement and dielectric properties of water molecules, as well as on the hydration state and arrangements of side chains lining the channel barrel. Based on the varied voltage sensitivity of the channel at different temperatures in the presence and absence of EMF, the amount of energy transferred to nano-environments of accessible groups was estimated to address the possible thermal effects of EMF. Our results show that the effects of EMF on channel activities are frequency dependent, with a maximum effect at 930 MHz. The frequency of channel gating and the voltage sensitivity is increased when the channel is exposed to EMF, while its conductance remains unchanged at all frequencies applied. We have not identified any changes in the capacitance and permeability of membrane in the presence of EMF. The effect of the EMF irradiated by cell phones is measured by Specific Absorption Rate (SAR) in artificial model of human head, Phantom. Thus, current approach applied to biological molecules and electrolytes might be considered as complement to evaluate safety of irradiating sources on biological matter at molecular level.

  11. 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

  12. ATP-sensitive potassium channel modulators and cardiac arrhythmias: an update.

    PubMed

    Muntean, Danina M; Kiss, Loránd; Jost, Norbert; Baczko, István

    2015-01-01

    Ischemia and heart failure-related cardiac arrhythmias, both atrial (e.g., atrial fibrillation) and ventricular (e.g., malignant tachyarrhythmias) represent a leading cause of morbidity and mortality worldwide. Despite the progress made in the last decade in understanding their pathophysiological mechanisms there is still an unmet need for safer and more efficacious pharmacological treatment, especially when considering the drawbacks and complications of implantable devices. Cardiac ATP-sensitive potassium channels located in the sarcolemmal membrane (sarcKATP) and the inner mitochondrial membrane (mitoKATP) have emerged as crucial controllers of several key cellular functions. In the past three decades a tremendous amount of research led to their structural and functional characterization unveiling both a protective role in cardiac adaptive responses to metabolic stress and a seemingly paradoxical role in promoting as well as protecting against atrial and ventricular arrhythmias. On the other hand, several KATP inhibitors have emerged as potential ischemia selective antiarrhythmic drugs. In this respect, cardioselective, chamber specific and combined sarcKATP and mitoKATP modulators currently represent a promising field for drug development.

  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. Sulfonylureas and on-clopidogrel platelet reactivity in type 2 diabetes mellitus patients.

    PubMed

    Harmsze, Ankie M; Van Werkum, Jochem W; Moral, Fulya; Ten Berg, Jurri N M; Hackeng, Christian M; Klungel, Olaf H; De Boer, Anthonius; Deneer, Vera H M

    2011-01-01

    Clopidogrel is a prodrug that needs to be converted in?vivo by several cytochrome (CYP) P450 iso-enzymes to become active. Both clopidogrel and the oral hypoglycemic drug class sulfonylureas are metabolized by the iso-enzyme CYP2C9. The objective of the study was to evaluate the relationship of sulfonylureas and on-clopidogrel platelet reactivity in type 2 diabetes mellitus patients undergoing elective coronary stent implantation. In this prospective, observational study, on-clopidogrel platelet reactivity was quantified using adenosine diphosphate (ADP)-induced light transmittance aggregometry in 139 type 2 diabetes mellitus patients undergoing elective coronary stent implantation treated with clopidogrel and aspirin. High on-clopidogrel platelet reactivity was defined as >70.7% platelet reactivity to 20 μmol/L ADP. A total of 53 patients (38.1%) were on concomitant treatment with sulfonylureas. The remaining 86 patients were on other hypoglycemic drugs. On-clopidogrel platelet reactivity was significantly higher in patients with concomitant sulfonylurea treatment as compared to patients without concomitant sulfonylurea treatment (for 5 μmol/L ADP: 46.0% ± 11.8 vs. 40.6% ± 16.0; p=0.035, adjusted p=0.032 and for 20 μmol/L ADP: 64.6% ± 10.8 vs. 58.7% ± 15.5; p=0.019, adjusted p=0.017). The concomitant use of sulfonylureas was associated with a 2.2-fold increased risk of high on-clopidogrel platelet reactivity (OR 2.2, 95% CI 1.1-4.7, p=0.039 and after adjustment for confounders: OR(adj) 2.0, 95% CI 1.0-5.7, p=0.048). Concomitant treatment with sulfonylureas might be associated with decreased platelet inhibition by clopidogrel in type 2 diabetes mellitus patients on dual antiplatelet therapy undergoing elective coronary stent implantation.

  15. A wide range and high sensitivity four-channel compact electrochemical biosensor for neurotransmitter detection on a microfluidic platform.

    PubMed

    Ghodsevali, Elnaz; Landari, Hamza; Boukadoum, Mounir; Gosselin, Benoit; Miled, Amine

    2016-08-01

    We present a four-channel, high-sensitivity and linearity electrochemical biosensor for neurotransmitter (NT) detection and measurement. Using a multi-channel microfluidic platform makes this biosensor capable of detecting NT-related currents going from nanoamperes to milliamperes, with a sensitivity of the order of picoamperes. Moreover, by using a fully differential potentiostat architecture, the biosensor offers a high common-mode rejection ratio (90 dB), making it appropriate for low-noise and high-sensitive applications. The system was implemented on a 15 mm × 15 mm PCB with direct interface to the microfluidic chambers. It was calibrated with a 5 mM ferrocyanide solution and successfully tested with dopamine at three concentrations. The system shows a minimum sensistivity of 100 pA and consumes 60 mW.

  16. 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.

  17. Taurine blocks ATP-sensitive potassium channels of rat skeletal muscle fibres interfering with the sulphonylurea receptor.

    PubMed

    Tricarico, D; Barbieri, M; Camerino, D C

    2000-06-01

    Taurine is a sulphonic aminoacid present in high amounts in various tissues including cardiac and skeletal muscles showing different properties such as antioxidative, antimyotonic and anti-schaemic effects. The cellular mechanism of action of taurine is under investigation and appears to involve the interaction of the sulphonic aminoacid with several ion channels. Using the patch-clamp technique we studied the effects of taurine in rat skeletal muscle fibres on ATP-sensitive K(+) channel (K(ATP)) immediately after excision and on channels that underwent rundown. The cytoplasmic application of 20 mM of taurine reduced the K(ATP) current; this effect was reverted by washout of the drug solution. In this experimental condition the IC(50) was 20.1 mM. After rundown, taurine inhibited the K(ATP) current with similar efficacy. Competition experiments showed that taurine shifted the dose-response inhibition curve of glybenclamide to the left on the log-dose axis without significantly affecting those of ATP or Ca(2+) ion. Single channel recording revealed that taurine affects the close state of the channel prolonging it and reducing the bursts duration. Our data indicate that taurine inhibits the muscular K(ATP) channel interfering with the glybenclamide site on the sulphonylurea receptor of the channel or on the site allosterically coupled to it. During ischaemia and hypoxia, the skeletal and heart muscles undergo several changes; for example, the activation of K(ATP) channels and loss of the intracellular taurine content. The depletion of taurine during ischaemia would contribute to the early activation of K(ATP) channels and salvage the intracellular ATP content.

  18. ATP-sensitive K+ channels in rat pancreatic beta-cells: modulation by ATP and Mg2+ ions.

    PubMed Central

    Ashcroft, F M; Kakei, M

    1989-01-01

    1. The inside-out configuration of the patch-clamp method was used to study the effects of MgATP, free ATP and Mg2+ on single ATP-sensitive K+ channel currents in rat pancreatic beta-cells. 2. Magnesium ions caused a marked reduction of channel activity: 5 mM-free Mg2+ produced a 50% reduction in the activity of inward currents recorded at -60 mV in symmetrical K+ concentrations. 3. Inhibition of channel activity by MgATP does not involve phosphorylation as both free ATP (i.e. ATP in the absence of divalent cations) and non-hydrolysable ATP analogues were effective inhibitors. 4. Magnesium ions produced a striking reduction in the ability of ATP (total) to inhibit channel activity. When channel activity was plotted as a function of the total ATP concentration, the Ki for channel inhibition was 4 microM in Mg2(+)-free solution, compared to a Ki of 26 microM in the presence of 2 mM-Mg2+. The shape of the relationship between channel activity and the total ATP concentration was not changed by Mg2+. When channel activity was plotted as a function of the free ATP concentration, however, Mg2+ had little effect on Ki. This suggests that free ATP is the more potent inhibitor of channel activity and that MgATP has little inhibitory effect. 5. ATP analogues that dissociate only as far as the tribasic form were also able to inhibit channel activity. This suggests that both ATP4- and ATPH3- can block the channel. 6. Like ATP, ADP was more effective at inhibiting channel activity in the absence of Mg2+, that is as the free base. The non-hydrolysable ATP analogues AMP-PNP and AMP-PCP, however, were more effective in the presence of Mg2+. 7. It is suggested that (1) the potency of inhibition is related to the amount of negative charge carried by the ion and (2) the intracellular concentration of free ATP will be an important modulator of channel activity in the intact beta-cell. PMID:2691645

  19. The selectivity filter of the tandem pore potassium channel TASK-1 and its pH-sensitivity and ionic selectivity.

    PubMed

    Yuill, K; Ashmole, I; Stanfield, P R

    2004-04-01

    We have studied pH sensitivity and ionic selectivity of the tandem pore K(+) channel TASK-1 heterologously expressed in Xenopus oocytes. We fit pH sensitivity assuming that only one of the two residues H98 need be protonated for channels to be shut. The effect of protons was weakly voltage dependent with a p K(a) of 6.02 at +40 mV. Replacement of His (H98D, H98N) reduced pH sensitivity but did not abolish it. Use of a concatameric channel permitted replacement of one His residue only; this concatamer was fully pH-sensitive. Increasing the number of His residues to 4 (mutant D204H) abolished pH sensitivity over the physiological range. The implication that D204 plays a role in pH-sensitivity was confirmed by the finding that pH sensitivity over the physiological range was also abolished in the mutant D204N. Ionic selectivity was also altered in D204H, D204N and H98D mutants. P(Rb)/ P(K) was increased from 0.80+/-0.04 (n=19) in wild type to 1.06+/-0.04 (n=19) in D204H. H98D, D204H and D204N were permeable to Na(+) with P(Na)/ P(K)=0.39+/-0.03 (n=14) in H98D, 0.64+/-0.04 (n=18) in D204H and 0.33+/-0.07 (n=3) in D204N. Thus, the arrangement of ring of residues HDHD appears to optimise both pH sensitivity and ionic selectivity.

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

    PubMed

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

    2006-12-13

    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 beta2AR 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 beta2AR) and TREK-1 dock simultaneously.

  1. 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.

  2. 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.

  3. Hemicalcin, a new toxin from the Iranian scorpion Hemiscorpius lepturus which is active on ryanodine-sensitive Ca2+ channels

    PubMed Central

    Shahbazzadeh, Delavar; Srairi-Abid, Najet; Feng, Wei; Ram, Narendra; Borchani, Lamia; Ronjat, Michel; Akbari, Abolfazl; Pessah, Isaac N.; De Waard, Michel; El Ayeb, Mohamed

    2007-01-01

    In the present work, we purified and characterized a novel toxin named hemicalcin from the venom of the Iranian chactoid scorpion Hemiscorpius lepturus where it represents 0.6% of the total protein content. It is a 33-mer basic peptide reticulated by three disulfide bridges, and that shares between 85 and 91% sequence identity with four other toxins, all known or supposed to be active on ryanodine-sensitive calcium channels. Hemicalcin differs from these other toxins by seven amino acids at positions 9 (leucine/arginine), 12 (alanine/glutamic acid), 13 (aspartic acid/asparagine), 14 (lysine/asparagine), 18 (serine/glycine), 26 (threonine/alanine) and 28 (proline/isoleucine/alanine). In spite of these differences, hemicalcin remains active on ryanodine-sensitive Ca2+ channels, since it increases [3H]ryanodine binding on RyR1 (ryanodine receptor type 1) and triggers Ca2+ release from sarcoplasmic vesicles. Bilayer lipid membrane experiments, in which the RyR1 channel is reconstituted and its gating properties are analysed, indicate that hemicalcin promotes an increase in the opening probability at intermediate concentration and induces a long-lasting subconductance level of 38% of the original amplitude at higher concentrations. Mice intracerebroventricular inoculation of 300 ng of hemicalcin induces neurotoxic symptoms in vivo, followed by death. Overall, these data identify a new biologically active toxin that belongs to a family of peptides active on the ryanodine-sensitive channel. PMID:17291197

  4. Interactive domains between pore loops of the yeast K+ channel TOK1 associate with extracellular K+ sensitivity

    PubMed Central

    Johansson, Ingela; Blatt, Michael R.

    2005-01-01

    Gating of the outward-rectifying K+ channel TOK1 of Saccharomyces cerevisiae is controlled by membrane voltage and extracellular K+ concentration. Previous studies identified two kinetically distinct effects of K+, and site-mutagenic analysis associated these K+-dependencies with domains of the extracellular turrets of the channel protein. We have mapped the TOK1 pore domains to extant K+ channel crystal structures to target additional residues contributing to TOK1 gating. Leu270, located in the first pore domain of TOK1, was found to be critical for gating and its K+ sensitivity. Analysis of amino acid substitutions indicated that spatial position of the polypeptide backbone is a primary factor determining gating sensitivity to K+. The strongest effects, with L270Y, L270F and L270W, led to more than a 30-fold decrease in apparent K+ affinity and an inversion in the apparent K+-dependence of voltage-dependent gating compared with the wild-type current. A partial rescue of wild-type gating was obtained on substitution in the second pore domain with the double mutant L270D/A428Y. These, and additional results, demarcate extracellular domains that are associated with the K+-sensitivity of TOK1 and they offer primary evidence for a synergy in gating between the two pore domains of TOK1, demonstrating an unexpected degree of long-distance interaction across the mouth of the K+ channel. PMID:16287426

  5. 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.

  6. Coupling of activation and inactivation gate in a K+-channel: potassium and ligand sensitivity.

    PubMed

    Ader, Christian; Schneider, Robert; Hornig, Sönke; Velisetty, Phanindra; Vardanyan, Vitya; Giller, Karin; Ohmert, Iris; Becker, Stefan; Pongs, Olaf; Baldus, Marc

    2009-09-16

    Potassium (K(+))-channel gating is choreographed by a complex interplay between external stimuli, K(+) concentration and lipidic environment. We combined solid-state NMR and electrophysiological experiments on a chimeric KcsA-Kv1.3 channel to delineate K(+), pH and blocker effects on channel structure and function in a membrane setting. Our data show that pH-induced activation is correlated with protonation of glutamate residues at or near the activation gate. Moreover, K(+) and channel blockers distinctly affect the open probability of both the inactivation gate comprising the selectivity filter of the channel and the activation gate. The results indicate that the two gates are coupled and that effects of the permeant K(+) ion on the inactivation gate modulate activation-gate opening. Our data suggest a mechanism for controlling coordinated and sequential opening and closing of activation and inactivation gates in the K(+)-channel pore.

  7. Bacterial mechanosensitive channels--MscS: evolution's solution to creating sensitivity in function.

    PubMed

    Naismith, James H; Booth, Ian R

    2012-01-01

    The discovery of mechanosensing channels has changed our understanding of bacterial physiology. The mechanosensitive channel of small conductance (MscS) is perhaps the most intensively studied of these channels. MscS has at least two states: closed, which does not allow solutes to exit the cytoplasm, and open, which allows rapid efflux of solvent and solutes. The ability to appropriately open or close the channel (gating) is critical to bacterial survival. We briefly review the science that led to the isolation and identification of MscS. We concentrate on the structure-function relationship of the channel, in particular the structural and biochemical approaches to understanding channel gating. We highlight the troubling discrepancies between the various models developed to understand MscS gating.

  8. Phosphatidic acid stimulates cardiac KATP channels like phosphatidylinositols, but with novel gating kinetics.

    PubMed

    Fan, Zheng; Gao, Lizhi; Wang, Wenxia

    2003-01-01

    Membrane-bound anionic phospholipids such as phosphatidylinositols have the capacity to modulate ATP-sensitive potassium (K(ATP)) channels through a mechanism involving long-range electrostatic interaction between the lipid headgroup and channel. However, it has not yet been determined whether the multiple effects of phosphatidylinositols reported in the literature all result from this general electrostatic interaction or require a specific headgroup structure. The present study investigated whether phosphatidic acid (PA), an anionic phospholipid substantially different in structure from phosphatidylinositols, evokes effects similar to phosphatidylinositols on native K(ATP) channels of rat heart and heterogeneous Kir6.2/SUR2A channels. Channels treated with PA (0.2-1 mg/ml applied to the cytoplasmic side of the membrane) exhibited higher activity, lower sensitivity to ATP inhibition, less Mg(2+)-dependent nucleotide stimulation, and poor sulfonylurea inhibition. These effects match the spectrum of phosphatidylinositols' effects, but, in addition, PA also induced a novel pattern in gating kinetics, represented by a decreased mean open time (from 12.2 +/- 2.0 to 3.3 +/- 0.7 ms). This impact on gating kinetics clearly distinguishes PA's effects from those of phosphatidylinositols. Results indicate that multiple effects of anionic phospholipids on K(ATP) channels are related phenomena and can likely be attributed to a common mechanism, but additional specific effects due to other mechanisms may also coincide.

  9. The role of ATP-sensitive potassium channels in cellular function and protection in the cardiovascular system.

    PubMed

    Tinker, Andrew; Aziz, Qadeer; Thomas, Alison

    2014-01-01

    ATP-sensitive potassium channels (K(ATP)) are widely distributed and present in a number of tissues including muscle, pancreatic beta cells and the brain. Their activity is regulated by adenine nucleotides, characteristically being activated by falling ATP and rising ADP levels. Thus, they link cellular metabolism with membrane excitability. Recent studies using genetically modified mice and genomic studies in patients have implicated K(ATP) channels in a number of physiological and pathological processes. In this review, we focus on their role in cellular function and protection particularly in the cardiovascular system.

  10. Structure-activity relationships for a new family of sulfonylurea herbicides

    NASA Astrophysics Data System (ADS)

    Wang, Jian-Guo; Li, Zheng-Ming; Ma, Ning; Wang, Bao-Lei; Jiang, Lin; Pang, Siew Siew; Lee, Yu-Ting; Guddat, Luke W.; Duggleby, Ronald G.

    2005-11-01

    Acetohydroxyacid synthase (AHAS; EC 2.2.1.6) catalyzes the first common step in branched-chain amino acid biosynthesis. The enzyme is inhibited by several chemical classes of compounds and this inhibition is the basis of action of the sulfonylurea and imidazolinone herbicides. The commercial sulfonylureas contain a pyrimidine or a triazine ring that is substituted at both meta positions, thus obeying the initial rules proposed by Levitt. Here we assess the activity of 69 monosubstituted sulfonylurea analogs and related compounds as inhibitors of pure recombinant Arabidopsis thaliana AHAS and show that disubstitution is not absolutely essential as exemplified by our novel herbicide, monosulfuron (2-nitro- N-(4'-methyl-pyrimidin-2'-yl) phenyl-sulfonylurea), which has a pyrimidine ring with a single meta substituent. A subset of these compounds was tested for herbicidal activity and it was shown that their effect in vivo correlates well with their potency in vitro as AHAS inhibitors. Three-dimensional quantitative structure-activity relationships were developed using comparative molecular field analysis and comparative molecular similarity indices analysis. For the latter, the best result was obtained when steric, electrostatic, hydrophobic and H-bond acceptor factors were taken into consideration. The resulting fields were mapped on to the published crystal structure of the yeast enzyme and it was shown that the steric and hydrophobic fields are in good agreement with sulfonylurea-AHAS interaction geometry.

  11. Activation of steroid-sensitive TRPM3 channels potentiates glutamatergic transmission at cerebellar Purkinje neurons from developing rats.

    PubMed

    Zamudio-Bulcock, Paula A; Everett, Julie; Harteneck, Christian; Valenzuela, C Fernando

    2011-11-01

    The functional implications of transient receptor potential melastatin 3 (TRPM3) activation, the most recently described member of the melastatin subfamily of cation permeable TRP channels, have begun to be elucidated in recent years. The discovery of TRPM3 activation by the steroid pregnenolone sulfate (PregS) has shed new light on the physiological role of this channel. For example, TRPM3 activation enhances insulin secretion from β pancreatic cells, induces contraction of vascular smooth muscle, and is also involved in the detection of noxious heat. Although TRPM3 expression has been detected in several regions of the developing and mature brain, little is known about the roles of TRPM3 in brain physiology. In this study, we demonstrate the abundant expression of TRPM3 steroid-sensitive channels in the developing cerebellar cortex. We also show that TRPM3-like channels are expressed at glutamatergic synapses in neonatal Purkinje cells. We recently showed that PregS potentiates spontaneous glutamate release onto neonatal Purkinje cells during a period of active glutamatergic synapse formation; we now show that this effect of PregS is mediated by TRPM3-like channels. Mefenamic acid, a recently discovered TRPM3 antagonist, blocked the effect of PregS on glutamate release. The PregS effect on glutamate release was mimicked by other TRPM3 agonists (nifedipine and epipregnanolone sulfate) but not by a TRMP3-inactive steroid (progesterone). Our findings identify TRPM3 channels as novel modulators of glutamatergic transmission in the developing brain.

  12. Sodium-level-sensitive sodium channel Na(x) is expressed in glial laminate processes in the sensory circumventricular organs.

    PubMed

    Watanabe, Eiji; Hiyama, Takeshi Y; Shimizu, Hidetada; Kodama, Ryuji; Hayashi, Noriko; Miyata, Seiji; Yanagawa, Yuchio; Obata, Kunihiko; Noda, Masaharu

    2006-03-01

    Na(x) is an atypical sodium channel that is assumed to be a descendant of the voltage-gated sodium channel family. Our recent studies on the Na(x)-gene-targeting mouse revealed that Na(x) channel is localized to the circumventricular organs (CVOs), the central loci for the salt and water homeostasis in mammals, where the Na(x) channel serves as a sodium-level sensor of the body fluid. To understand the cellular mechanism by which the information sensed by Na(x) channels is transferred to the activity of the organs, we dissected the subcellular localization of Na(x) in the present study. Double-immunostaining and immunoelectron microscopic analyses revealed that Na(x) is exclusively localized to perineuronal lamellate processes extended from ependymal cells and astrocytes in the organs. In addition, glial cells isolated from the subfornical organ, one of the CVOs, were sensitive to an increase in the extracellular sodium level, as analyzed by an ion-imaging method. These results suggest that glial cells bearing the Na(x) channel are the first to sense a physiological increase in the level of sodium in the body fluid, and they regulate the neural activity of the CVOs by enveloping neurons. Close communication between inexcitable glial cells and excitable neural cells thus appears to be the basis of the central control of the salt homeostasis.

  13. 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.

  14. Role of ATP-sensitive potassium channels in modulating nociception in rat model of bone cancer pain.

    PubMed

    Xia, Hui; Zhang, Dengwen; Yang, Shijie; Wang, Yu; Xu, Lin; Wu, Jinjing; Ren, Jing; Yao, Wenlong; Fan, Longchang; Zhang, Chuanhan; Tian, Yuke; Pan, Hui-Lin; Wang, Xueren

    2014-03-20

    Bone cancer pain is a major clinical problem and remains difficult to treat. ATP-sensitive potassium (KATP) channels may be involved in regulating nociceptive transmission at the spinal cord level. We determined the role of spinal KATP channels in the control of mechanical hypersensitivity in a rat model of bone cancer pain. The rat model of bone cancer pain was induced by implanting rat mammary gland carcinoma cells (Walker256) into the tibias. KATP modulators (pinacidil and glibenclamide) or the specific Kir6.2-siRNA were injected via an intrathecal catheter. The mechanical withdrawal threshold of rats was tested using von Frey filaments. The Kir6.2 mRNA and protein levels were measured by quantitative PCR and western blots, respectively. Intrathecal injection of pinacidil, a KATP channel opener, significantly increased the tactile withdrawal threshold of cancer cell-injected rats in a dose-dependent manner. In contrast, intrathecal delivery of glibenclamide, a KATP channel blocker, or the specific Kir6.2-siRNA significantly reduced the tactile withdrawal threshold of cancer cell-injected rats. The mRNA and protein levels of Kir6.2 in the spinal cord of cancer cell-injected rats were significantly lower than those in control rats. Our findings suggest that the KATP channel expression level in the spinal cord is reduced in bone cancer pain. Activation of KATP channels at the spinal level reduces pain hypersensitivity associated with bone cancer pain.

  15. Thiazolidinedione addition reduces the serum retinol-binding protein 4 in type 2 diabetic patients treated with metformin and sulfonylurea.

    PubMed

    Lin, Kun-Der; Chang, Yu-Hung; Wang, Chiao-Ling; Yang, Yi-Hsin; Hsiao, Pi-Jung; Li, Tzu-Hui; Shin, Shyi-Jang

    2008-06-01

    Retinol-binding protein 4 (RBP4) has been found to induce insulin resistance and to be increased in type 2 diabetes. Thiazolidinediones (TZDs) can improve insulin sensitivity through the activation of peroxisome proliferators-activated receptor-gamma (PPAR-gamma) and have been suggested as an adjunct to metformin (MF) and sulfonylurea (SU) in type 2 diabetes in a consensus statement from the ADA and EASD. Therefore, we investigated whether TZD could affect serum RBP4 level in type 2 diabetes already treated with MF and/or SU. Eighty-one type 2 diabetic patients were divided into 2 groups: (1) TZD group (n = 55): Pioglitazone 30 mg/day was given as an add-on medication; (2) SU group (n = 26): Gliclazide MR 30-120 mg or glimepiride 2-8 mg/day was prescribed. The average period of study was 97.1 days. Serum RBP4 and adiponectin were measured by enzyme-linked immunosorbent assay and radioimmunoassay, respectively. The addition of pioglitazone (TZD group) markedly decreased homeostasis model assessment of insulin resistance (HOMA-IR) (P = 0.021) compared with the SU group (P = 0.688). The change of RBP4 in the TZD group (-3.87 +/- 11.27 microg/mL) significantly differed from that in the SU group (2.52 +/- 8.24 microg/mL, P < 0.012). The increase of adiponectin in the TZD group (11.49 +/- 7.85 microg/mL) was apparently higher than that in the SU group (1.54 +/- 5.62 microg/mL, P < 0.001). Despite the change of glycosylated hemoglobin (HbA1c) did not differ (-0.77 +/- 1.3 vs -0.50 +/- 1.7, P = 0.446), the addition of pioglitazone could significantly lower serum RBP4 and HOMA-IR values, whereas an increased dosage of sulfonylurea agents did not alter HOMA-IR, RBP4, or adiponectin in type 2 diabetic patients who had been treated with metformin and/or sulfonylurea.

  16. 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

  17. [Amiloride sensitive sodium channels (ENaC) and their regulation by proteases].

    PubMed

    Galizia, Luciano; Ojea, Alejandro; Kotsias, Basilio A

    2011-01-01

    ENaC is a channel that mediates entry of Na+ from the luminal fluid into the cells in many reabsorbing epithelia and it is also expressed in human placenta. ENaC is crucial in the control of electrolyte and extracellular volume homeostasis. ENaC is regulated by several hormones, including aldosterone and blocked by amiloride and its analogs. ENaC channels are composed by three homologous subunits, α, β and γ that form the pore where Na ions are transported. Two factors regulate the activity of ENaC channels: 1) the number of channels inserted in the membrane and 2) the open probability of the channels or time that the channel is open. The number of channels is the result of a balance between the synthesis and degradation of ENaC channels. The open probability depends on the proteolysis of specific segments in the α and γ subunits of ENaC by multiple proteases inside of the cell or in the extracellular space. Among the most studied proteases are furin, prostasin, elastase, plasmin and trypsin. There are endogenous substances that block the activity of these proteases such as aprotinin, bikunin and nexin-1 and the expression of both, proteases and their inhibitors are controlled by the rate of Na+ movement, aldosterone and TFG-β levels. In this work we present some examples of this regulation and the potential role that this process may play under normal and pathological conditions such as cystic fibrosis, kidney diseases and hypertension.

  18. 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

  19. On the Sensitivity of TRMM Microwave Imager Channels to Over-Land Rainfall

    NASA Astrophysics Data System (ADS)

    Liu, G.; You, Y.

    2010-12-01

    In this study we investigate the response to over land precipitation from brightness temperatures at different microwave frequencies by using the Tropical Rainfall Measuring Mission (TRMM) Precipitation Radar (PR) and Microwave Imager (TMI) data. The spearman rank correlation coefficients between observations of TMI channels or channel-combinations and PR-measured near surface rain are first computed using three years of TRMM data, which shows that the brightness temperature combinations from 19 and 37 GHz, i.e., V19-V37 (where the letter V denotes vertical polarization, and the numbers denote frequency in GHz) or V21-V37 can explain for 10% more variation of near surface rain than the V85 brightness temperature. Also, the global distribution of the above correlation reveals that over almost all of the tropical area covered by TRMM the V19-V37 channel has stronger responses to the over-land rainfall than the V85 channel. This result is somewhat counter-intuitive, because it has been long believed that the dominant signature of over-land rainfall is the brightness temperature depression caused by ice scattering at high microwave frequencies (e.g., 85 GHz). To understand the underlying physics of this better low-frequency response, data analysis and radiative transfer modeling have been conducted to assess the influence to brightness temperatures from clouds with different ice and liquid water partitions. The results showed that V85 channel can respond to the low rainfall intensities, although the signature is weaker; while at moderate rainfall intensities, the signal from V19-V37 channel responded better to rainfall rate than the one from V85 channel because in addition to some ice scattering signature, the V19-V37 channel contains liquid water information as well, which is more directly related to surface rain than ice water aloft; Also, the V37 channel rises to be the top responder to surface rain as the amount of hydrometeors in the atmospheric column reaches

  20. Activation of inositol trisphosphate-sensitive Ca2+ channels of sarcoplasmic reticulum from frog skeletal muscle.

    PubMed Central

    Suárez-Isla, B A; Alcayaga, C; Marengo, J J; Bull, R

    1991-01-01

    1. The modulation by Ca2+ of the activation by inositol 1,4,5-trisphosphate (IP3) of Ca2+ channels present in native sarcoplasmic reticulum membranes from frog skeletal muscle was studied after channel incorporation into planar phospholipid bilayers in the presence of Ca2+ or Ba2+ as current carrier species. 2. Channel activity expressed as fractional open time (Po) was low (less than or equal to 0.15) in the presence of varying free Ca2+ concentrations bathing the myoplasmic face of the channel (cis side), and did not increase significantly between 0.01 and 30 microM-Ca2+. 3. Channel activation mediated by IP3 could be elicited from free Ca2+ levels similar to those of resting skeletal muscle (about 0.1 microM) and was found to be strongly regulated by the free Ca2+ concentration present at the myoplasmic moiety of the channel. 4. Channel activation by 10 microM-IP3 depended on the Ca2+ concentration on the cis side. Po reached a maximum between pCa 7.0 and 6.0, but decreased at higher concentrations of free Ca2+. Thus, Ca2+ exerted a modulatory influence on IP3-mediated activation in a concentration range where the channel was insensitive to Ca2+. 5. The results indicate that Ca2+ ions act as modulators of IP3 efficacy to open the channel. This could arise from an interaction of Ca2+ with the channel gating mechanism or with the agonist binding site. PMID:1667801

  1. Intron retention in mRNA encoding ancillary subunit of insect voltage-gated sodium channel modulates channel expression, gating regulation and drug sensitivity.

    PubMed

    Bourdin, Céline M; Moignot, Bénédicte; Wang, Lingxin; Murillo, Laurence; Juchaux, Marjorie; Quinchard, Sophie; Lapied, Bruno; Guérineau, Nathalie C; Dong, Ke; Legros, Christian

    2013-01-01

    Insect voltage-gated sodium (Nav) channels are formed by a well-known pore-forming α-subunit encoded by para-like gene and ancillary subunits related to TipE from the mutation "temperature-induced-paralysis locus E." The role of these ancillary subunits in the modulation of biophysical and pharmacological properties of Na(+) currents are not enough documented. The unique neuronal ancillary subunit TipE-homologous protein 1 of Drosophila melanogaster (DmTEH1) strongly enhances the expression of insect Nav channels when heterologously expressed in Xenopus oocytes. Here we report the cloning and functional expression of two neuronal DmTEH1-homologs of the cockroach, Periplaneta americana, PaTEH1A and PaTEH1B, encoded by a single bicistronic gene. In PaTEH1B, the second exon encoding the last 11-amino-acid residues of PaTEH1A is shifted to 3'UTR by the retention of a 96-bp intron-containing coding-message, thus generating a new C-terminal end. We investigated the gating and pharmacological properties of the Drosophila Nav channel variant (DmNav1-1) co-expressed with DmTEH1, PaTEH1A, PaTEH1B or a truncated mutant PaTEH1Δ(270-280) in Xenopus oocytes. PaTEH1B caused a 2.2-fold current density decrease, concomitant with an equivalent α-subunit incorporation decrease in the plasma membrane, compared to PaTEH1A and PaTEH1Δ(270-280). PaTEH1B positively shifted the voltage-dependences of activation and slow inactivation of DmNav1-1 channels to more positive potentials compared to PaTEH1A, suggesting that the C-terminal end of both proteins may influence the function of the voltage-sensor and the pore of Nav channel. Interestingly, our findings showed that the sensitivity of DmNav1-1 channels to lidocaine and to the pyrazoline-type insecticide metabolite DCJW depends on associated TEH1-like subunits. In conclusion, our work demonstrates for the first time that density, gating and pharmacological properties of Nav channels expressed in Xenopus oocytes can be modulated by an

  2. Intron Retention in mRNA Encoding Ancillary Subunit of Insect Voltage-Gated Sodium Channel Modulates Channel Expression, Gating Regulation and Drug Sensitivity

    PubMed Central

    Bourdin, Céline M.; Moignot, Bénédicte; Wang, Lingxin; Murillo, Laurence; Juchaux, Marjorie; Quinchard, Sophie; Lapied, Bruno; Guérineau, Nathalie C.; Dong, Ke; Legros, Christian

    2013-01-01

    Insect voltage-gated sodium (Nav) channels are formed by a well-known pore-forming α-subunit encoded by para-like gene and ancillary subunits related to TipE from the mutation “temperature-induced-paralysis locus E.” The role of these ancillary subunits in the modulation of biophysical and pharmacological properties of Na+ currents are not enough documented. The unique neuronal ancillary subunit TipE-homologous protein 1 of Drosophila melanogaster (DmTEH1) strongly enhances the expression of insect Nav channels when heterologously expressed in Xenopus oocytes. Here we report the cloning and functional expression of two neuronal DmTEH1-homologs of the cockroach, Periplaneta americana, PaTEH1A and PaTEH1B, encoded by a single bicistronic gene. In PaTEH1B, the second exon encoding the last 11-amino-acid residues of PaTEH1A is shifted to 3′UTR by the retention of a 96-bp intron-containing coding-message, thus generating a new C-terminal end. We investigated the gating and pharmacological properties of the Drosophila Nav channel variant (DmNav1-1) co-expressed with DmTEH1, PaTEH1A, PaTEH1B or a truncated mutant PaTEH1Δ(270-280) in Xenopus oocytes. PaTEH1B caused a 2.2-fold current density decrease, concomitant with an equivalent α-subunit incorporation decrease in the plasma membrane, compared to PaTEH1A and PaTEH1Δ(270-280). PaTEH1B positively shifted the voltage-dependences of activation and slow inactivation of DmNav1-1 channels to more positive potentials compared to PaTEH1A, suggesting that the C-terminal end of both proteins may influence the function of the voltage-sensor and the pore of Nav channel. Interestingly, our findings showed that the sensitivity of DmNav1-1 channels to lidocaine and to the pyrazoline-type insecticide metabolite DCJW depends on associated TEH1-like subunits. In conclusion, our work demonstrates for the first time that density, gating and pharmacological properties of Nav channels expressed in Xenopus oocytes can be modulated by

  3. Glucose modulates the amount, but not the kinetics, of insulin released by sulfonylureas.

    PubMed

    Gregorio, F; Ambrosi, F; Filipponi, P; Cristallini, S; Santeusanio, F

    1994-01-01

    This study compares the insulin-secretory profiles induced by therapeutical concentrations of four different sulfonylureas--tolbutamide, gliquidone, gliclazide, and glibenclamide--and the amount of hormone released by each under different ambient glucose concentrations, using the isolated perfused rat pancreas model. All four sulfonylureas stimulated B-cell function, but the kinetics varied. Tolbutamide, gliquidone, and gliclazide produced a quick, biphasic release, whereas glibenclamide stimulated a delayed monophasic insulin secretion. Dramatic falls in insulin release were observed when ambient glucose concentrations were lowered. Glucagon release was not influenced by any of the sulfonylureas whatever the metabolic condition, neither directly nor indirectly, via an insulin-mediated paracrine inhibition of A-cell activity.

  4. 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.

  5. Oxygen-sensitive reduction in Ca²⁺-activated K⁺ channel open probability in turtle cerebrocortex.

    PubMed

    Rodgers-Garlick, C I; Hogg, D W; Buck, L T

    2013-05-01

    In response to low ambient oxygen levels the western painted turtle brain undergoes a large depression in metabolic rate which includes a decrease in neuronal action potential frequency. This involves the arrest of N-methyl-D-aspartate receptor (NMDAR) and α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid receptor (AMPAR) currents and paradoxically an increase in γ-aminobutyric acid receptor (GABAR) currents in turtle cortical neurons. In a search for other oxygen-sensitive channels we discovered a Ca(2+)-activated K(+) channel (K(Ca)) that exhibited a decrease in open time in response to anoxia. Single-channel recordings of K(Ca) activity were obtained in cell-attached and excised inside-out patch configurations from neurons in cortical brain sheets bathed in either normoxic or anoxic artificial cerebrospinal fluid (aCSF). The channel has a slope conductance of 223pS, is activated in response to membrane depolarization, and is controlled in a reversible manner by free [Ca(2+)] at the intracellular membrane surface. In the excised patch configuration anoxia had no effect on K(Ca) channel open probability (P(open)); however, in cell-attached mode, there was a reversible fivefold reduction in P(open) (from 0.5 ± 0.05 to 0.1 ± 0.03) in response to 30-min anoxia. The inclusion of the potent protein kinase C (PKC) inhibitor chelerythrine prevented the anoxia-mediated decrease in P(open) while drip application of a phorbol ester PKC activator decreased P(open) during normoxia (from normoxic 0.4 ± 0.05 to phorbol-12-myristate-13-acetate (PMA) 0.1 ± 0.02). Anoxia results in a slight depolarization of turtle pyramidal neurons (∼8 mV) and an increase in cytosolic [Ca(2+)]; therefore, K(Ca) arrest is likely important to prevent Ca(2+) activation during anoxia and to reduce the energetic cost of maintaining ion gradients. We conclude that turtle pyramidal cell Ca(2+)-activated K(+) channels are oxygen-sensitive channels regulated by cytosolic factors and are likely

  6. Mutation in pore domain uncovers cation- and voltage-sensitive recovery from inactivation in KAT1 channel.

    PubMed Central

    Moroni, A; Gazzarrini, S; Cerana, R; Colombo, R; Sutter, J U; DiFrancesco, D; Gradmann, D; Thiel, G

    2000-01-01

    Effects of threonine substitution by glutamine at position 256 in the pore of the KAT1 channel have been investigated by voltage-clamp, using heterologous gene expression in Xenopus oocytes. The major discrepancy in T256Q from the wild-type channel (wt) was cation specific. While K(+) currents were reduced in a largely scalar fashion, the NH(4)(+) current exhibited slow, voltage-dependent inhibition during hyperpolarization. The same effects could be induced in wt, or intensified in T256Q, by addition of the impermeant cation methylammonium (MA(+)) to the bath. This stresses that both the mutation and MA(+) affect a mechanism already present in the wt. Assuming that current inhibition could be described as entry of the channel into an inactive state, we modeled in both wt and in T256Q the relaxation kinetics of the clamp currents by a C-O-I gating scheme, where C (closed) and I (inactivated) are nonconductive states, and O is an open state allowing K(+) and NH(4)(+) passage. The key reaction is the transition I-O. This cation-sensitive transition step ensures release of the channel from the inactive state and is approximately 30 times smaller in T256Q compared to wt. It can be inhibited by external MA(+) and is stimulated strongly by K(+) and weakly by NH(4)(+). This sensitivity of gating to external cations may prevent K(+) leakage from cation-starved cells. PMID:10733966

  7. 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

  8. 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

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

    NASA Astrophysics Data System (ADS)

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

    2016-09-01

    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.

  10. Measuring and Evaluating the Role of ATP-Sensitive K+ Channels in Cardiac Muscle

    PubMed Central

    Kefaloyianni, Eirini; Bao, Li; Rindler, Michael J.; Hong, Miyoun; Patel, Tejaskumar; Taskin, Eylem; Coetzee, William A.

    2012-01-01

    Since ion channels move electrical charge during their activity, they have traditionally been studied using electrophysiological approaches. This was sometimes combined with mathematical models, for example with the description of the ionic mechanisms underlying the initiation and propagation of action potentials in the squid giant axon by Hodgkin and Huxley. The methods for studying ion channels also have strong roots in protein chemistry (limited proteolysis, the use of antibodies, etc). The advent of the molecular cloning and the identification of genes coding for specific ion channel subunits in the late 1980’s introduced a multitude of new techniques with which to study ion channels and the field has been rapidly expanding ever since (e.g. antibody development against specific peptide sequences, mutagenesis, the use of gene targeting in animal models, determination of their protein structures) and new methods are still in development. This review focuses on techniques commonly employed to examine ion channel function in a electrophysiological laboratory. The focus is on the KATP channel, but many of the techniques described are also used to study other ion channels. PMID:22245446

  11. Increased pressure-induced tone in rat parenchymal arterioles vs. middle cerebral arteries: role of ion channels and calcium sensitivity.

    PubMed

    Cipolla, Marilyn J; Sweet, Julie; Chan, Siu-Lung; Tavares, Matthew J; Gokina, Natalia; Brayden, Joseph E

    2014-07-01

    Brain parenchymal arterioles (PAs) are high-resistance vessels that branch off pial arteries and perfuse the brain parenchyma. PAs are the target of cerebral small vessel disease and have been shown to have greater pressure-induced tone at lower pressures than pial arteries. We investigated mechanisms by which brain PAs have increased myogenic tone compared with middle cerebral arteries (MCAs), focusing on differences in vascular smooth muscle (VSM) calcium and ion channel function. The amount of myogenic tone and VSM calcium was measured using Fura 2 in isolated and pressurized PAs and MCAs. Increases in intraluminal pressure caused larger increases in tone and cytosolic calcium in PAs compared with MCAs. At 50 mmHg, myogenic tone was 37 ± 5% for PAs vs. 6.5 ± 4% for MCAs (P < 0.01), and VSM calcium was 200 ± 20 nmol/l in PAs vs. 104 ± 15 nmol/l in MCAs (P < 0.01). In vessels permeabilized with Staphylococcus aureus α-toxin, PAs were not more sensitive to calcium, suggesting calcium sensitization was not at the level of the contractile apparatus. PAs were 30-fold more sensitive to the voltage-dependent calcium channel (VDCC) inhibitor nifedipine than MCAs (EC50 for PAs was 3.5 ± 0.4 vs. 82.1 ± 2.1 nmol/l for MCAs;P < 0.01); however, electrophysiological properties of the VDCC were not different in VSM. PAs had little to no response to the calcium-activated potassium channel inhibitor iberiotoxin, whereas MCAs constricted ∼15%. Thus increased myogenic tone in PAs appears related to differences in ion channel activity that promotes VSM membrane depolarization but not to a direct sensitization of the contractile apparatus to calcium.

  12. Kinin Receptors Sensitize TRPV4 Channel and Induce Mechanical Hyperalgesia: Relevance to Paclitaxel-Induced Peripheral Neuropathy in Mice.

    PubMed

    Costa, Robson; Bicca, Maíra A; Manjavachi, Marianne N; Segat, Gabriela C; Dias, Fabiana Chaves; Fernandes, Elizabeth S; Calixto, João B

    2017-03-10

    Kinin B1 (B1R) and B2 receptors (B2R) and the transient receptor potential vanilloid 4 (TRPV4) channel are known to play a critical role in the peripheral neuropathy induced by paclitaxel (PTX) in rodents. However, the downstream pathways activated by kinin receptors as well as the sensitizers of the TRPV4 channel involved in this process remain unknown. Herein, we investigated whether kinins sensitize TRPV4 channels in order to maintain PTX-induced peripheral neuropathy in mice. The mechanical hyperalgesia induced by bradykinin (BK, a B2R agonist) or des-Arg(9)-BK (DABK, a B1R agonist) was inhibited by the selective TRPV4 antagonist HC-067047. Additionally, BK was able to sensitize TRPV4, thus contributing to mechanical hyperalgesia. This response was dependent on phospholipase C/protein kinase C (PKC) activation. The selective kinin B1R (des-Arg(9)-[Leu(8)]-bradykinin) and B2R (HOE 140) antagonists reduced the mechanical hyperalgesia induced by PTX, with efficacies and time response profiles similar to those observed for the TRPV4 antagonist (HC-067047). Additionally, both kinin receptor antagonists inhibited the overt nociception induced by hypotonic solution in PTX-injected animals. The same animals presented lower PKCε levels in skin and dorsal root ganglion samples. The selective PKCε inhibitor (εV1-2) reduced the hypotonicity-induced overt nociception in PTX-treated mice with the same magnitude observed for the kinin receptor antagonists. These findings suggest that B1R or B2R agonists sensitize TRPV4 channels to induce mechanical hyperalgesia in mice. This mechanism of interaction may contribute to PTX-induced peripheral neuropathy through the activation of PKCε. We suggest these targets represent new opportunities for the development of effective analgesics to treat chronic pain.

  13. 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

  14. 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.

  15. Effects of ATP-sensitive potassium channel opener on potassium transport and alveolar fluid clearance in the resected human lung.

    PubMed

    Sakuma, T; Takahashi, K; Ohya, N; Nakada, T; Matthay, M A

    1998-07-01

    Since the effect of an ATP-sensitive potassium channel (KATP channel) opener on the function of alveolar epithelial cells is unknown, the effect of YM934, a newly synthesized KATP channel opener, on potassium influx into the alveolar spaces and alveolar fluid clearance was determined in the resected human lung. An isosmolar albumin solution with a low potassium concentration was instilled into the distal airspaces of resected human lungs. Alveolar fluid clearance was measured by the progressive increase in alveolar protein concentration. Net potassium transport was measured by the change in potassium concentration and alveolar fluid volume. YM934 (10(-4) M) increased net influx of potassium by 140% into the alveolar spaces and also increased alveolar fluid clearance by 60% in the experiments with a potassium concentration of 0.3 mEq/1. Glibenclamide (10(-4) M), a KATP channel blocker, inhibited the YM934-increased influx of potassium transport and the increase in alveolar fluid clearance. Also amiloride (10(-5) M), an inhibitors of apical sodium uptake, blocked the YM934 stimulated increase in net alveolar fluid clearance. These results indicate that a KATP channel opener can effect potassium transport and net vectorial fluid movement across the human alveolar epithelium.

  16. 1,4-Benzothiazine ATP-sensitive potassium channel openers: modifications at the C-2 and C-6 positions.

    PubMed

    Martelli, Alma; Manfroni, Giuseppe; Sabbatini, Paola; Barreca, Maria Letizia; Testai, Lara; Novelli, Michela; Sabatini, Stefano; Massari, Serena; Tabarrini, Oriana; Masiello, Pellegrino; Calderone, Vincenzo; Cecchetti, Violetta

    2013-06-13

    ATP-sensitive potassium (KATP) channels play a prominent role in controlling cardiovascular function. In this paper, a novel series of 4-(1-oxo-2-cyclopentenyl)-1,4-benzothiazine derivatives modified at the C-2, and C-6 positions were synthesized as openers of vascular KATP channels. Most of the tested compounds evoked vasorelaxing effects on rat aortic rings and membrane hyperpolarization in human vascular smooth muscle cells, with potency similar or superior to that of the reference levcromakalim (LCRK). The selective KATP blocker glibenclamide antagonized the above vascular effects, confirming that KATP channels are closely involved in the mechanism of action. The experimental results confirmed the 1,4-benzothiazine nucleus as an optimal scaffold for activators of vascular KATP channels; moreover, the high level of potency exhibited by the 6-acetyl substituted benzothiazine 8, along with the lack of any significant interference with insulin secretion from pancreatic β-cells, paves the way to further develop a new series of potent activators of vascular KATP channels.

  17. Disruption of ATP-sensitive potassium channel function in skeletal muscles promotes production and secretion of musclin

    PubMed Central

    Sierra, Ana; Subbotina, Ekaterina; Zhu, Zhiyong; Gao, Zhan; Koganti, Siva Rama Krishna; Coetzee, William; Goldhamer, David; Hodgson-Zingman, Denice M.; Zingman, Leonid V.

    2016-01-01

    Sarcolemmal ATP-sensitive potassium (KATP) channels control skeletal muscle energy use through their ability to adjust membrane excitability and related cell functions in accordance with cellular metabolic status. Mice with disrupted skeletal muscle KATP channels exhibit reduced adipocyte size and increased fatty acid release into the circulation. As yet, the molecular mechanisms underlying this link between skeletal muscle KATP channel function and adipose mobilization have not been established. Here, we demonstrate that skeletal muscle-specific disruption of KATP channel function in transgenic (TG) mice promotes production and secretion of musclin. Musclin is a myokine with high homology to atrial natriuretic peptide (ANP) that enhances ANP signaling by competing for elimination. Augmented musclin production in TG mice is driven by a molecular cascade resulting in enhanced acetylation and nuclear exclusion of the transcription factor forkhead box O1 (FOXO1) – an inhibitor of transcription of the musclin encoding gene. Musclin production/secretion in TG is paired with increased mobilization of fatty acids and a clear trend toward increased circulating ANP, an activator of lipolysis. These data establish KATP channel-dependent musclin production as a potential mechanistic link coupling “local” skeletal muscle energy consumption with mobilization of bodily resources from fat. Understanding such mechanisms is an important step toward designing interventions to manage metabolic disorders including those related to excess body fat and associated co-morbidities. PMID:26828268

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

    PubMed

    Akita, T; Okada, Y

    2014-09-05

    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.

  19. The endogenous mitochondrial complex II inhibitor malonate regulates mitochondrial ATP-sensitive potassium channels: implications for ischemic preconditioning.

    PubMed

    Wojtovich, Andrew P; Brookes, Paul S

    2008-01-01

    Ischemic preconditioning (IPC) affords cardioprotection against ischemia-reperfusion (IR) injury, and while the molecular mechanisms of IPC are debated, the mitochondrial ATP-sensitive K(+) channel (mK(ATP)) has emerged as a candidate effector for several IPC signaling pathways. The molecular identity of this channel is unknown, but significant pharmacologic overlap exists between mK(ATP) and mitochondrial respiratory complex II (succinate dehydrogenase). In this investigation, we utilized isolated cardiac mitochondria, Langendorff perfused hearts, and a variety of biochemical methods, to make the following observations: (i) The competitive complex II inhibitor malonate is formed in mitochondria under conditions resembling IPC. (ii) IPC leads to a reversible inhibition of complex II that has likely been missed in previous investigations due to the use of saturating concentrations of succinate. (iii) Malonate opens mK(ATP) channels even when mitochondria are respiring on complex I-linked substrates, suggesting an effect of this inhibitor on the mK(ATP) channel independent of complex II inhibition. Together, these observations suggest that complex II inhibition by endogenously formed malonate may represent an important activation pathway for mK(ATP) channels during IPC.

  20. Design, Synthesis and in Vivo Evaluation of Novel Glycosylated Sulfonylureas as Antihyperglycemic Agents.

    PubMed

    Suaifan, Ghadeer A R Y; Shehadeh, Mayadah B; Darwish, Rula M; Al-Ijel, Hebah; Abbate, Vincenzo

    2015-11-06

    Sulphonylurea compounds have versatile activities such as antidiabetic, diuretic, herbicide, oncolytic, antimalarial, antifungal and anticancer. The present study describes the design, synthesis and in vivo testing of novel glycosylated aryl sulfonylurea compounds as antihyperglycaemic agents in streptozocine-induced diabetic mice. The rational for the introduction of the glucosamine moiety is to enhance selective drug uptake by pancreatic β-cells in order to decrease the cardiotoxic side effect commonly associated with sulfonylurea agents. 2-Deoxy-2-(4-chlorophenylsulfonylurea)-D-glucopyranose was found to be the most potent antihyperglycaemic agents among the synthesized compounds in diabetic mice. This investigation indicates the importance of this novel class as potential antihyperglycaemic agents.

  1. Effects of intracellular pH on ATP-sensitive K+ channels in mouse pancreatic beta-cells.

    PubMed Central

    Proks, P; Takano, M; Ashcroft, F M

    1994-01-01

    1. The effects of intracellular pH (pHi) on the ATP-sensitive K+ channel (K+ATP channel) from mouse pancreatic beta-cells were examined in inside-out patches exposed to symmetrical 140 mM K+ solutions. 2. The relationship between channel activity and pHi was described by the Hill equation with half-maximal inhibition (Ki) at pHi 6.25 and a Hill coefficient of 3.7. 3. Following exposure to pHi < 6.8, channel activity did not recover to its original level. Subsequent application of trypsin to the intracellular membrane surface restored channel activity to its initial level or above. 4. At -60 mV the relationship between pHi and the single-channel current amplitude was described by a modified Hill equation with a Hill coefficient of 2.1, half-maximal inhibition at pHi 6.48 and a maximum inhibition of 18.5%. 5. A decrease in pHi reduced the extent of channel inhibition by ATP: Ki was 18 microM at pH 7.2 and 33 microM at pH 6.4. The Hill coefficient was also reduced, being 1.65 at pH 7.2 and 1.17 at pH 6.4. 6. When channel activity was plotted as a function of ATP4- (rather than total ATP) there was no effect of pHi on the relationship. This suggests that ATP4- is the inhibitory ion species and that the effects of reducing pHi are due to the lowered concentration of ATP4-. 7. Changes in external pH had little effect on either single-channel or whole-cell K+ATP currents. 8. The effects of pHi do not support a role for H+ in linking glucose metabolism to K+ATP channel inhibition in pancreatic beta-cells. PMID:8189391

  2. Redox-sensitive extracellular gates formed by auxiliary beta subunits of calcium-activated potassium channels.

    PubMed

    Zeng, Xu-Hui; Xia, Xiao-Ming; Lingle, Christopher J

    2003-06-01

    An important step to understanding ion channels is identifying the structural components that act as the gates to ion movement. Here we describe a new channel gating mechanism, produced by the beta3 auxiliary subunits of Ca2+-activated, large-conductance BK-type K+ channels when expressed with their pore-forming alpha subunits. BK beta subunits have a cysteine-rich extracellular segment connecting two transmembrane segments, with small cytosolic N and C termini. The extracellular segments of the beta3 subunits form gates to block ion permeation, providing a mechanism by which current can be rapidly diminished upon cellular repolarization. Furthermore, this gating mechanism is abolished by reduction of extracellular disulfide linkages, suggesting that endogenous mechanisms may regulate this gating behavior. The results indicate that auxiliary beta subunits of BK channels reside sufficiently close to the ion permeation pathway defined by the alpha subunits to influence or block access of small molecules to the permeation pathway.

  3. 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 ...

  4. Molecular Basis of Paralytic Neurotoxin Action on Voltage-Sensitive Sodium Channels

    DTIC Science & Technology

    1988-10-20

    channels in intact membranes (Messner et al, 1986; Tejedor & Catterall, 1988). The a subunits of the sodium channels in these reconstimtpd vesicles...1986; Tejedor & Catterall, 1988). In preliminary experiments, proteases were screened to find experimental conditions under which the a subunit could...1688, Tejctor, FJ. and Catterall, W.A. (1988) Biochemistry, in press, Tejedor , F.J. and Catterall, W.A. (1988) Proc. Nail, Acad. Sci. USA, in press

  5. Niflumic acid differentially modulates two types of skeletal ryanodine-sensitive Ca(2+)-release channels.

    PubMed

    Oba, T

    1997-11-01

    The effects of niflumic acid on ryanodine receptors (RyRs) of frog skeletal muscle were studied by incorporating sarcoplasmic reticulum (SR) vesicles into planar lipid bilayers. Frog muscle had two distinct types of RyRs in the SR: one showed a bell-shaped channel activation curve against cytoplasmic Ca2+ or niflumic acid, and its mean open probability (Po) was increased by perchlorate at 20-30 mM (termed "alpha-like" RyR); the other showed a sigmoidal activation curve against Ca2+ or niflumic acid, with no effect on perchlorate (termed "beta-like" RyR). The unitary conductance and reversal potential of both channel types were unaffected after exposure to niflumic acid when clamped at 0 mV. When clamped at more positive potentials, the beta-like RyR channel rectified this, increasing the unitary current. Treatment with niflumic acid did not inhibit the response of both channels to Ca2+ release channel modulators such as caffeine, ryanodine, and ruthenium red. The different effects of niflumic acid on Po and the unitary current amplitude in both types of channels may be attributable to the lack or the presence of inactivation sites and/or distinct responses to agonists.

  6. Selective phosphorylation modulates the PIP2 sensitivity of the CaM-SK channel complex

    PubMed Central

    Zhang, Miao; Meng, Xuan-Yu; Cui, Meng; Pascal, John M.; Logothetis, Diomedes E.; Zhang, Ji-Fang

    2015-01-01

    Phosphatidylinositol bisphosphate (PIP2) regulates the activities of many membrane proteins including ion channels through direct interactions. However, the affinity of PIP2 is so high for some channel proteins that its physiological role as a modulator has been questioned. Here we show that PIP2 is an important cofactor for activation of small conductance Ca2+-activated potassium channels (SK) by Ca2+-bound calmodulin (CaM). Removal of the endogenous PIP2 inhibits SK channels. The PIP2-binding site resides at the interface of CaM and the SK C-terminus. We further demonstrate that the affinity of PIP2 for its target proteins can be regulated by cellular signaling. Phosphorylation of CaM T79, located adjacent to the PIP2-binding site, by Casein Kinase 2 reduces the affinity of PIP2 for the CaM-SK channel complex by altering the dynamic interactions among amino acid residues surrounding the PIP2-binding site. This effect of CaM phosphorylation promotes greater channel inhibition by G-protein-mediated hydrolysis of PIP2. PMID:25108821

  7. 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.

  8. Dihydropyridine-sensitive skeletal muscle Ca channels in polarized planar bilayers. 3. Effects of phosphorylation by protein kinase C.

    PubMed Central

    Ma, J.; Gutiérrez, L. M.; Hosey, M. M.; Ríos, E.

    1992-01-01

    The effects of protein kinase C (PKC) were studied on dihydropyridine (DHP)-sensitive Ca channels from rabbit skeletal muscle T tubule membranes. To determine which channel subunits become phosphorylated under the conditions used for electrophysiological studies, we first performed biochemical studies of phosphorylation. T tubular membranes were fused with vesicles of the lipid mixture used in the planar bilayers, and phosphorylation was assessed using the same concentrations of PKC, adenosine 5'-triphosphate, and buffers as were used in the electrophysiological experiments. The alpha 1 subunit of the DHP receptors was phosphorylated by PKC to an extent of 1 mol phosphate/mol protein. The beta subunit was also phosphorylated but to a significantly lesser extent. The DHP-sensitive Ca channel activity was studied after fusing T tubule membranes with planar bilayers (Ma, J., C. Mundiña-Weilenmann, M. M. Hosey, and E. Ríos. 1991. Biophys. J. 60:890-901). The bilayers were held at -80 mV and activated by depolarizing voltage clamp pulses. The observed Ca channels exhibited two open states (tau o1 = 5 ms and tau o2 = 25 ms). On addition of purified PKC to the intracellular side, the proportion of the longer open state increased threefold. The average open probability during a 2-s, maximally activating pulse (Pmax) increased from 10 to 15%. The voltage dependence of activation was not changed by PKC; the Boltzmann parameters were V1 = -20.5 mV and K = 10.5 mV, which were not significantly different from the reference channels. The deactivation (closing) time constant was increased from 7 to 12 ms after PKC. The inactivation time constant during the pulse was slightly increased(from 1.2 to 1.6 s), and the channel availability at the holding potential was decreased from 76 to 71%. Taken together, the results revealed that PKC increased Pmax largely through a shift in the voltage independent open-close equilibrium of the fully activated channels.This is in contrast with

  9. Molecular packing and magnetic properties of lithium naphthalocyanine crystals: hollow channels enabling permeability and paramagnetic sensitivity to molecular oxygen

    PubMed Central

    Pandian, Ramasamy P.; Dolgos, Michelle; Marginean, Camelia; Woodward, Patrick M.; Hammel, P. Chris; Manoharan, Periakaruppan T.; Kuppusamy, Periannan

    2009-01-01

    The synthesis, structural framework, magnetic and oxygen-sensing properties of a lithium naphthalocyanine (LiNc) radical probe are presented. LiNc was synthesized in the form of a microcrystalline powder using a chemical method and characterized by electron paramagnetic resonance (EPR) spectroscopy, magnetic susceptibility, powder X-ray diffraction analysis, and mass spectrometry. X-Ray powder diffraction studies revealed a structural framework that possesses long, hollow channels running parallel to the packing direction. The channels measured approximately 5.0 × 5.4 Å2 in the two-dimensional plane perpendicular to the length of the channel, enabling diffusion of oxygen molecules (2.9 × 3.9 Å2) through the channel. The powdered LiNc exhibited a single, sharp EPR line under anoxic conditions, with a peak-to-peak linewidth of 630 mG at room temperature. The linewidth was sensitive to surrounding molecular oxygen, showing a linear increase in pO2 with an oxygen sensitivity of 31.2 mG per mmHg. The LiNc microcrystals can be further prepared as nano-sized crystals without the loss of its high oxygen-sensing properties. The thermal variation of the magnetic properties of LiNc, such as the EPR linewidth, EPR intensity and magnetic susceptibility revealed the existence of two different temperature regimes of magnetic coupling and hence differing columnar packing, both being one-dimensional antiferromagnetic chains but with differing magnitudes of exchange coupling constants. At a temperature of ∼50 K, LiNc crystals undergo a reversible phase transition. The high degree of oxygen-sensitivity of micro- and nano-sized crystals of LiNc, combined with excellent stability, should enable precise and accurate measurements of oxygen concentration in biological systems using EPR spectroscopy. PMID:19809598

  10. Voltage-dependent magnesium block of adenosine-triphosphate-sensitive potassium channel in guinea-pig ventricular cells.

    PubMed Central

    Horie, M; Irisawa, H; Noma, A

    1987-01-01

    1. The adenosine-5'-triphosphate (ATP)-sensitive K+ channel of guinea-pig ventricular cells was examined in the presence and absence of internal Mg2+ or Na+ using an open cell-attached configuration of the patch-clamp technique. 2. Millimolar concentrations of internal Mg2+ ([Mg2+]i) produced marked fluctuations in the outward current, and the amplitude of the open-channel current was reduced with increasing [Mg2+]i. Millimolar Na+ applied internally also decreased the mean amplitude of the outward current, but the increase in current noise was not obvious. These effects became larger when the membrane potential was shifted to be more positive from the K+ equilibrium potential (EK). At potentials negative to EK the inward current was affected by neither internal Mg2+ nor Na+. 3. The external application of Na+, Mg2+ or Ca2+, however, failed to affect the single-channel current. 4. After removal of both internal Mg2+ and Na+, the mean open-channel current-voltage relationship became virtually linear. Referring to these unblocked values, relative amplitudes were determined at different levels of [Mg2+]i or [Na+]i. The dose-response relations gave a Hill coefficient of approximately 1 for Mg2+ block and approximately 2 for Na+ block. The half-maximum concentrations (Kh) for both Mg2+ and Na+ block were shifted to lower values with increasing positive potentials. 5. The power-density spectrum of the open-channel current noise induced by internal Mg2+ showed a Lorentzian function with a corner frequency above 1 kHz, suggesting that the current noise is due to rapid fluctuations of open-channel current between blocked and unblocked states. The corner frequencies gave Mg2+ block and unblock rate constants which were of the order of 10(7) M-1 s-1 and 10(4) s-1, respectively. 6. With increasing external K+ concentration ([K+]o) from 0 to 140 mM the current fluctuations became less prominent, and Kh for Mg2+ block was shifted to higher values. Raising [K+]o enhanced the

  11. Long-range interactions, voltage sensitivity, and ion conduction in S4 segments of excitable channels.

    PubMed Central

    Leuchtag, H R

    1994-01-01

    Forces acting on the S4 segments of the channel, the voltage-sensing structures, are analyzed. The conformational change in the Na channel is modeled as a helix-coil transition in the four S4 segments, coupled to the membrane voltage by electrical forces. In the model, repulsions between like charges make the S4 segment unstable, but field-dependent forces hold it in an alpha-helix configuration at resting potential. At threshold depolarization, the S4 helices cooperatively expand into random coils, breaking the hydrogen bonds connecting adjacent loops of the alpha helices. Exposed electron pairs left on the carbonyl oxygens constitute sites at which cations can bind selectively. The first hydrogen bond to break is at the channel exterior, then the second breaks, and so on in a zipper-like motion along the entire segment. The Na+ ions hop from one site to the next until all H bonds are broken and all sites are filled with ions. This completes the pathway over which the permeant ions move through the channel, driven by the electrochemical potential difference across the membrane. This microscopic mechanism is consistent with the thermodynamic explanation of ion-channel gating previously formulated as the ferroelectric-superionic transition hypothesis. PMID:7510528

  12. 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

  13. 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).

  14. Activation of volume-sensitive Cl− channel mediates autophagy-related cell death in myocardial ischaemia/reperfusion injury

    PubMed Central

    Li, Xing; Huo, Cong; Jia, Xin; Wang, Lin; Xu, Rong; Wang, Ning; Zhang, Mingming; Li, Hong; Wang, Xiaoming

    2016-01-01

    Excessive reactive oxygen species (ROS) plays an important role in myocardial ischemia/reperfusion (I/R) injury, which triggers not only myocardial cellular apoptosis but also autophagy-related cell death, in which volume-sensitive outwardly rectifying (VSOR) Cl− channel-activated by ROS contributes to cell apoptotic volume decrease, playing an incipient incident of cellular apoptosis. However, whether VSOR Cl− channel concurrently participates in autophagy-related cell death regulation remains unclear. To illuminate the issue, studies underwent in myocardial vitro and vivo I/R model. Rats were performed to ischemia 30 minutes and subsequent reperfusion 24-96 hours, ROS scavenger (NAC), VSOR Cl− channel blocker (DCPIB) and autophagy inhibitor (3MA) were administered respectively. Results showed that oxidative stress, LC3-II stain and inflammation in myocardial tissue were markedly increased, lysosome associated membrane protein-2 (LAMP2) were significantly reduced with I/R group as compared with sham group, reperfusion significantly led to damage in myocardial tissue and heart function, whereas the disorder could be rescued through these agents. Moreover, primary neonatal rat cardiomyocytes hypoxia/reoxygenation model were administered, results showed that VSOR Cl− channel-activated by reoxygenation could cause both cell volume decrease and intracellular acidification, which further increased LC3 and depleted of LAMP2, resulting in autophagy-related cell death. Interestingly, VSOR Cl− channel-blocked by DCPIB could stably maintain the cell volume, intracellular pH, abundant LAMP2 and autophagic intensity regardless of ROS intension derived from reoxygenation injury or adding H2O2. These results first demonstrate that VSOR Cl− channel-activated is a pivotal event to trigger autophagy-related death, which reveals a novel therapeutic target to decrease myocardial I/R injury. PMID:27322431

  15. Wanderlust kinetics and variable Ca(2+)-sensitivity of Drosophila, a large conductance Ca(2+)-activated K+ channel, expressed in oocytes.

    PubMed Central

    Silberberg, S D; Lagrutta, A; Adelman, J P; Magleby, K L

    1996-01-01

    Cloned large conductance Ca(2+)-activated K+ channels (BK or maxi-K+ channels) from Drosophila (dSlo) were expressed in Xenopus oocytes and studied in excised membrane patches with the patch-clamp technique. Both a natural variant and a mutant that eliminated a putative cyclic AMP-dependent protein kinase phosphorylation site exhibited large, slow fluctuations in open probability with time. These fluctuations, termed "wanderlust kinetics," occurred with a time course of tens of seconds to minutes and had kinetic properties inconsistent with simple gating models. Wanderlust kinetics was still observed in the presence of 5 mM caffeine or 50 nM thapsigargin, or when the Ca2+ buffering capacity of the solution was increased by the addition of 5 mM HEDTA, suggesting that the wanderlust kinetics did not arise from Ca2+ release from caffeine and thapsigargin sensitive internal stores in the excised patch. The slow changes in kinetics associated with wanderlust kinetics could be generated with a discrete-state Markov model with transitions among three or more kinetic modes with different levels of open probability. To average out the wanderlust kinetics, large amounts of data were analyzed and demonstrated up to a threefold difference in the [Ca2+]i required for an open probability of 0.5 among channels expressed from the same injected mRNA. These findings indicate that cloned dSlo channels in excised patches from Xenopus oocytes can exhibit large variability in gating properties, both within a single channel and among channels. PMID:8744301

  16. A three ion channel genes-based signature predicts prognosis of primary glioblastoma patients and reveals a chemotherapy sensitive subtype

    PubMed Central

    Liu, Xiu; Yan, Xiao-Yan; Wang, Wen; Wu, Fan; Liang, Ting-Yu; Yang, Fan; Hu, Hui-Min; Mao, Heng-Xu; Liu, Yan-Wei; Zhang, Shi-Zhong

    2016-01-01

    Increasing evidence suggests that ion channels not only regulate electric signaling in excitable cells but also play important roles in the development of brain tumor. However, the roles of ion channels in glioma remain controversial. In the present study, we systematically analyzed the expression patterns of ion channel genes in a cohort of Chinese patients with glioma using RNAseq expression profiling. First, a molecular signature comprising three ion channel genes (KCNN4, KCNB1 and KCNJ10) was identified using Univariate Cox regression and two-tailed student's t test conducted in overall survival (OS) and gene expression. We assigned a risk score based on three ion channel genes to each primary Glioblastoma multiforme (pGBM) patient. We demonstrated that pGBM patients who had a high risk of unfavorable outcome were sensitive to chemotherapy. Next, we screened the three ion genes-based signature in different molecular glioma subtypes. The signature showed a Mesenchymal subtype and wild-type IDH1 preference. Gene ontology (GO) analysis for the functional annotation of the signature showed that patients with high-risk scores tended to exhibit the increased expression of proteins associated with apoptosis, immune response, cell adhesion and motion and vasculature development. Gene Set Enrichment Analysis (GSEA) results showed that pathways associated with negative regulation of programmed cell death, cell proliferation and locomotory behavior were highly expressed in the high-risk group. These results suggest that ion channel gene expression could improve the subtype classification in gliomas at the molecular level. The findings in the present study have been validated in two independent cohorts. PMID:27713134

  17. Baclofen, an agonist at peripheral GABAB receptors, induces antinociception via activation of TEA-sensitive potassium channels

    PubMed Central

    Reis, G M L; Duarte, I D G

    2006-01-01

    Background and Purpose: Central anti-nociceptive actions of baclofen involve activation of K+ channels. Here we assessed what types of K+ channel might participate in the peripheral anti-nociception induced by baclofen. Experimental approach: Nociceptive thresholds to mechanical stimulation in rat paws treated with intraplantar prostaglandin E2.(PGE2) to induce hyperalgesia were measured 3h after PGE2 injection. Other agents were also given by intraplantar injection Key results: Baclofen elicited a dose-dependent (15 - 240 μg per paw) anti-nociceptive effect. An intermediate dose of baclofen (60 μg) did not produce antinociception in the contralateral paw, showing its peripheral site of action. The GABAB receptor antagonist saclofen (12.5 - 100 μg per paw) antagonized, in a dose-dependent manner, peripheral antinociception induced by baclofen (60 μg), suggesting a specific effect. This antinociceptive action of baclofen was unaffected by bicuculline, GABAA receptor antagonist (80 μg per paw), or by (1,2,5,6 tetrahydropyridin-4-yl) methylphosphinic acid, GABAC receptor antagonist (20 μg per paw). The peripheral antinociception induced by baclofen (60 μg) was reversed, in a dose-dependent manner, by the voltage-dependent K+ channel blockers tetraethylammonium (7.5 - 30 μg per paw) and 4-aminopyridine (2.5 - 10 μg per paw). The blockers of other K+ channels, glibenclamide (160 μg), tolbutamide (320 μg), charybdotoxin (2 μg), dequalinium (50 μg) and caesium (500 μg) had no effect. Conclusions and Implications: This study provides evidence that the peripheral antinociceptive effect of the GABAB receptor agonist baclofen results from the activation of tetraethylammonium-sensitive K+ channels. Other K+ channels appear not to be involved. PMID:17016510

  18. Direct detection of calmodulin tuning by ryanodine receptor channel targets using a Ca2+-sensitive acrylodan-labeled calmodulin.

    PubMed

    Fruen, Bradley R; Balog, Edward M; Schafer, Janet; Nitu, Florentin R; Thomas, David D; Cornea, Razvan L

    2005-01-11

    Calmodulin (CaM) activates the skeletal muscle ryanodine receptor (RyR1) at nanomolar Ca(2+) concentrations but inhibits it at micromolar Ca(2+) concentrations, indicating that binding of Ca(2+) to CaM may provide a molecular switch for modulating RyR1 channel activity. To directly examine the Ca(2+) sensitivity of RyR1-complexed CaM, we used an environment-sensitive acrylodan adduct of CaM. The resulting (ACR)CaM probe displayed high-affinity binding to, and Ca(2+)-dependent regulation of, RyR1 similar to that of unlabeled wild-type (WT) CaM. Upon addition of Ca(2+), (ACR)CaM exhibited a substantial (>50%) decrease in fluorescence (K(Ca) = 2.7 +/- 0.8 microM). A peptide derived from the RyR1 CaM binding domain (RyR1(3614)(-)(43)) caused an even more pronounced Ca(2+)-dependent fluorescence decrease, and a >or=10-fold leftward shift in its K(Ca) (0.2 +/- 0.1 microM). In the presence of intact RyR1 channels in SR vesicles, (ACR)CaM fluorescence spectra were distinct from those in the presence of RyR1(3614)(-)(43), although a Ca(2+)-dependent decrease in fluorescence was still observed. The K(Ca) for (ACR)CaM fluorescence in the presence of SR (0.8 +/- 0.4 microM) was greater than in the presence of RyR1(3614)(-)(43) but was consistent with functional determinations showing the conversion of (ACR)CaM from channel activator (apoCaM) to inhibitor (Ca(2+)CaM) at Ca(2+) concentrations between 0.3 and 1 microM. These results indicate that binding to RyR1 targets evokes significant changes in the CaM structure and Ca(2+) sensitivity (i.e., CaM tuning). However, changes resulting from binding of CaM to the full-length, tetrameric channels are clearly distinct from changes caused by the RyR1-derived peptide. We suggest that the Ca(2+) sensitivity of CaM when in complex with full-length channels may be tuned to respond to physiologically relevant changes in Ca(2+).

  19. Phentolamine inhibits the pacemaker activity of mouse interstitial cells of Cajal by activating ATP-sensitive K+ channels.

    PubMed

    Ahn, Seung Whan; Kim, Sang Hun; Kim, Jin Ho; Choi, Seok; Yeum, Cheol Ho; Wie, Hee Wook; Sun, Jae Myeong; So, Insuk; Jun, Jae Yeoul

    2010-03-01

    The aim of this study was to clarify if phentolamine has proven effects on the pacemaker activities of interstitial cells of Cajal (ICC) from the mouse small intestine involving the ATPsensitive K(+) channels and adrenergic receptor. The actions of phentolamine on pacemaker activities were investigated using whole-cell patch-clamp technique and intracellular Ca(2+) analysis at 30 degrees C in cultured mouse intestinal ICC. ICC generated spontaneous pacemaker currents at a holding potential of -70 mV. Treatment with phentolamine reduced the frequency and amplitude of the pacemaker currents and increased the resting outward currents. Moreover, under current clamping (I = 0), phentolamine hyperpolarized the ICC membrane and decreased the amplitude of the pacemaker potentials. We also observed that phentolamine inhibited spontaneous [Ca(2+)](i) oscillations in ICC. The alpha-adrenergic drugs prazosin, yohimbine, methoxamine, and clonidine had no effect on ICC intestinal pacemaker activity and did not block phentolamine-induced effects. Phentolamine-induced effects on the pacemaker currents and the pacemaker potentials were significantly inhibited by ATP sensitive K(+) channel blocker glibenclamide, but not by TEA, apamin, or 4-aminopyridine. In addition, the NO synthase inhibitor, L-NAME and the guanylate cyclase inhibitor, ODQ were incapable of blocking the phentolamine-induced effects. These results demonstrate that phentolamine regulates the pacemaker activity of ICC via ATP-sensitive K(+) channel activation. Phentolamine could act through an adrenergic receptor- and also through NO-independent mechanism that involves intracellular Ca(2+) signaling.

  20. Reduced thermal sensitivity and Nav1.8 and TRPV1 channel expression in sensory neurons of aged mice

    PubMed Central

    Wang, Shuying; Davis, Brian M.; Zwick, Melissa; Waxman, Stephen G.; Albers, Kathryn M.

    2010-01-01

    Sensory neurons in aging mammals undergo changes in anatomy, physiology and gene expression that correlate with reduced sensory perception. In this study we compared young and aged mice to identify proteins that might contribute to this loss of sensation. We first show using behavioral testing that thermal sensitivity in aged male and female mice is reduced. Expression of sodium channel (Nav1.8 and Nav1.9) and transient receptor potential vanilloid (TRPV) channels in DRG and peripheral nerves of young and old male mice was then examined. Immunoblotting and RT-PCR assays showed reduced Nav1.8 levels in aged mice. No change was measured in TRPV1 mRNA levels in DRG though TRPV1 protein appeared reduced in the DRG and peripheral nerves. The GFRα3 receptor, which binds the growth factor artemin and is expressed by TRPV1-positive neurons, was also decreased in the DRG of aged animals. These findings indicate that loss of thermal sensitivity in aging animals may result from a decreased level of TRPV1 and Nav1.8 and decreased trophic support that inhibits efficient transport of channel proteins to peripheral afferents. PMID:15979214

  1. Predictors of insulin initiation in metformin and sulfonylurea users in primary care practices: the role of kidney function.

    PubMed

    Kostev, Karel; Dippel, Franz-Werner; Rathmann, Wolfgang

    2014-09-01

    The aims were to investigate predictors of insulin initiation in new users of metformin or sulfonylureas in primary care practices, in particular, its association with decreased renal function. Data from 9103 new metformin and 1120 sulfonylurea users with normal baseline glomerular filtration rate (eGFR) >90 ml/min/1.73 m(2) from 1072 practices were retrospectively analyzed (Disease Analyzer Germany: 01/2003-06/2012). Cox regression models and propensity score matching was used to adjust for confounders (age, sex, practice characteristics, comorbidity). Insulin treatment was started in 394 (4.3%) metformin and in 162 (14.5%) sulfonylurea users within 6 years (P < .001). Kaplan-Meier curves (propensity score matched patients) showed that the metformin group was at a lower risk of insulin initiation compared to sulfonylurea users throughout the study period. A substantial eGFR decline (category: 15-<30 ml/min/1.73 m(2)) was significantly associated with a higher likelihood to have insulin initiated (adjusted hazard ratio [HR]: 2.39; 95% CI: 1.09-5.23) in metformin but not in sulfonylurea (HR: 0.45; 95% CI: 0.16-1.30) users. New users of sulfonylurea monotherapy in primary care practices in Germany were about 3-fold more likely to start insulin therapy than those with metformin. Kidney function decline was associated with earlier insulin initiation in metformin but not in sulfonylurea users.

  2. Volume-sensitive outwardly rectifying chloride channel blockers protect against high glucose-induced apoptosis of cardiomyocytes via autophagy activation.

    PubMed

    Wang, Lin; Shen, Mingzhi; Guo, Xiaowang; Wang, Bo; Xia, Yuesheng; Wang, Ning; Zhang, Qian; Jia, Lintao; Wang, Xiaoming

    2017-03-16

    Hyperglycemia is a well-characterized contributing factor for cardiac dysfunction and heart failure among diabetic patients. Apoptosis of cardiomyocytes plays a major role during the onset and pathogenesis of diabetic cardiomyopathy (DCM). Nonetheless, the molecular machinery underlying hyperglycemia-induced cardiac damage and cell death remains elusive. In the present study, we found that chloride channel blockers, 4,4'-diisothiocya-natostilbene-2,2'- disulfonic acid (DIDS) and 4-(2-butyl-6,7-dichlor-2-cyclopentyl-indan-1-on-5-yl) oxybutyric acid (DCPIB), inhibited high glucose-activated volume-sensitive outwardly rectifying (VSOR) Cl(-) channel and improved the viability of cardiomyocytes. High glucose induced cardiomyocyte apoptosis by suppressing the autophagic stress, which can be reversed via blockade of VSOR Cl(-) channel. VSOR activation in high glucose-treated cardiomyocytes was attributed to increased intracellular levels of reactive oxygen species (ROS). Taken together, our study unraveled a role of VSOR chloride currents in impaired autophagy and increased apoptosis of high glucose-exposed cardiomyocyte, and has implications for a therapeutic potential of VSOR chloride channel blockers in DCM.

  3. A High-Throughput Functional Screen Identifies Small Molecule Regulators of Temperature- and Mechano-Sensitive K2P Channels

    PubMed Central

    2013-01-01

    K2P (KCNK) potassium channels generate “leak” potassium currents that strongly influence cellular excitability and contribute to pain, somatosensation, anesthesia, and mood. Despite their physiological importance, K2Ps lack specific pharmacology. Addressing this issue has been complicated by the challenges that the leak nature of K2P currents poses for electrophysiology-based high-throughput screening strategies. Here, we present a yeast-based high-throughput screening assay that avoids this problem. Using a simple growth-based functional readout, we screened a library of 106,281 small molecules and identified two new inhibitors and three new activators of the mammalian K2P channel K2P2.1 (KCNK2, TREK-1). By combining biophysical, structure–activity, and mechanistic analysis, we developed a dihydroacridine analogue, ML67-33, that acts as a low micromolar, selective activator of temperature- and mechano-sensitive K2P channels. Biophysical studies show that ML67-33 reversibly increases channel currents by activating the extracellular selectivity filter-based C-type gate that forms the core gating apparatus on which a variety of diverse modulatory inputs converge. The new K2P modulators presented here, together with the yeast-based assay, should enable both mechanistic and physiological studies of K2P activity and facilitate the discovery and development of other K2P small molecule modulators. PMID:23738709

  4. Volume-sensitive outwardly rectifying chloride channel blockers protect against high glucose-induced apoptosis of cardiomyocytes via autophagy activation

    PubMed Central

    Wang, Lin; Shen, Mingzhi; Guo, Xiaowang; Wang, Bo; Xia, Yuesheng; Wang, Ning; Zhang, Qian; Jia, Lintao; Wang, Xiaoming

    2017-01-01

    Hyperglycemia is a well-characterized contributing factor for cardiac dysfunction and heart failure among diabetic patients. Apoptosis of cardiomyocytes plays a major role during the onset and pathogenesis of diabetic cardiomyopathy (DCM). Nonetheless, the molecular machinery underlying hyperglycemia-induced cardiac damage and cell death remains elusive. In the present study, we found that chloride channel blockers, 4,4′-diisothiocya-natostilbene-2,2′- disulfonic acid (DIDS) and 4-(2-butyl-6,7-dichlor-2-cyclopentyl-indan-1-on-5-yl) oxybutyric acid (DCPIB), inhibited high glucose-activated volume-sensitive outwardly rectifying (VSOR) Cl− channel and improved the viability of cardiomyocytes. High glucose induced cardiomyocyte apoptosis by suppressing the autophagic stress, which can be reversed via blockade of VSOR Cl− channel. VSOR activation in high glucose-treated cardiomyocytes was attributed to increased intracellular levels of reactive oxygen species (ROS). Taken together, our study unraveled a role of VSOR chloride currents in impaired autophagy and increased apoptosis of high glucose-exposed cardiomyocyte, and has implications for a therapeutic potential of VSOR chloride channel blockers in DCM. PMID:28300155

  5. 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.

  6. 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 ...

  7. 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...

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

    PubMed

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

    2014-01-01

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

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

    PubMed Central

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

    2016-01-01

    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

  10. Unfolding of a temperature-sensitive domain controls voltage-gated channel activation

    PubMed Central

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

    2016-01-01

    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 (BacNaV) C-terminal cytoplasmic domain (CTD) affects function. Our studies show that the BacNaV 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 BacNaV 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 BacNaV voltage dependencies, and demonstrate that a discrete domain can encode the temperature dependent response of a channel. PMID:26919429

  11. 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.

  12. The ethanol withdrawal syndrome: A role for dihydropyridine-sensitive calcium channels in neuronal hyperexcitability states

    SciTech Connect

    Whittington, M.A.

    1990-01-01

    This project investigated the effects of dihydropyridine calcium channel blockers on behavioral and electrophysiological aspects of ethanol withdrawal. The effects of the dihydropyridine (+)-PN 200-110, on changes in neuronal function during ethanol withdrawal, were compared with effects on changes caused by the GABAergic convulsant drug bicuculline. Behavioral correlates of ethanol withdrawal were measured in two strains of mice using a rating of handling-induced convulsions. Concurrent chronic treatment with ethanol and the dihydropyridine calcium channel blockers ([plus minus])-nitrendipine, ([plus minus])-nimodipine or ([plus minus])-PN 200-110 prevented withdrawal-induced increased in convulsive behavior. This effect was dose dependent. The duration of chronic treatment with calcium channel blocker affected the degree of protection against increases in convulsive behavior seen during ethanol withdrawal. Concurrent chronic treatment with ethanol, and the mixed calcium channel activator/blocker ([plus minus])-BAY K 8644, prevented ethanol withdrawal-induced increases in convulsive behavior. Single acute injections of nitrendipine immediately on cessation of chronic treatment with ethanol, or 2h later, reduced withdrawal-induced increases in convulsive behavior in a dose-dependent manner throughout the 12h test period. Slices isolated from mice after chronic ethanol treatment showed a complex, time-dependent pattern of changes in the above measurements, culminating in epileptiform discharges seen from 4h to 7h into withdrawal.

  13. The effect of mitochondrial ATP-sensitive potassium channels on apoptosis of chick embryo cecal cells by Eimeria tenella.

    PubMed

    Yang, Sha-sha; Zheng, Ming-xue; Xu, Huan-cheng; Cui, Xiao-zhen; Zhang, Yan; Zhao, Wen-long; Bai, Rui

    2015-04-01

    The objective of this study was to investigate the effect of mitochondrial ATP-sensitive potassium (mitoKATP) channels on apoptosis induced by Eimeria tenella. At 24, 48, 72, 96 and 120 h after Eimeria tenella infection, TUNEL assays and translation of phosphatidyl serines to the host cell plasma membrane surface showed that diazoxide-treated chick embryo cecal cells underwent less apoptosis (P <0.05), while light microscopy showed that infection rates of treated cells were higher (P <0.01) than untreated cells. Caspase 9 and caspase 3 of infected cells were activated less (P <0.01) in diazoxide-treated cells than untreated cells. These results indicate that opening mitoKATP channels can protect chick embryo cecal cells from mitochondria-dependent apoptosis induced by Eimeria tenella by inhibiting activations of caspase 9 and caspase 3.

  14. Acid-sensitive TWIK and TASK Two-pore Domain Potassium Channels Change Ion Selectivity and Become Permeable to Sodium in Extracellular Acidification*

    PubMed Central

    Ma, Liqun; Zhang, Xuexin; Zhou, Min; Chen, Haijun

    2012-01-01

    Two-pore domain K+ channels (K2P) mediate background K+ conductance and play a key role in a variety of cellular functions. Among the 15 mammalian K2P isoforms, TWIK-1, TASK-1, and TASK-3 K+ channels are sensitive to extracellular acidification. Lowered or acidic extracellular pH (pHo) strongly inhibits outward currents through these K2P channels. However, the mechanism of how low pHo affects these acid-sensitive K2P channels is not well understood. Here we show that in Na+-based bath solutions with physiological K+ gradients, lowered pHo largely shifts the reversal potential of TWIK-1, TASK-1, and TASK-3 K+ channels, which are heterologously expressed in Chinese hamster ovary cells, into the depolarizing direction and significantly increases their Na+ to K+ relative permeability. Low pHo-induced inhibitions in these acid-sensitive K2P channels are more profound in Na+-based bath solutions than in channel-impermeable N-methyl-d-glucamine-based bath solutions, consistent with increases in the Na+ to K+ relative permeability and decreases in electrochemical driving forces of outward K+ currents of the channels. These findings indicate that TWIK-1, TASK-1, and TASK-3 K+ channels change ion selectivity in response to lowered pHo, provide insights on the understanding of how extracellular acidification modulates acid-sensitive K2P channels, and imply that these acid-sensitive K2P channels may regulate cellular function with dynamic changes in their ion selectivity. PMID:22948150

  15. Vildagliptin vs sulfonylurea in Indian Muslim diabetes patients fasting during Ramadan

    PubMed Central

    Shete, Abhijit; Shaikh, Aheson; Nayeem, K Javeed; Rodrigues, Lily; Ali, Mohamed Sheikamunadeen Sadiq; Shah, Parag; Khanna, Rajiv; Majid, Sarfaraj; Rasheed, Sabeer A; Shaikh, Shehla; Rahman, Tawfiqur

    2013-01-01

    AIM: To compare the use of vildagliptin and sulfonylurea with or without metformin in Indian Muslim patients with type 2 diabetes mellitus, fasting during Ramadan. METHODS: This was a 4-wk, multicenter, non-interventional, open-label, observational study. Incidence of hypoglycemic events (HEs), adverse events, and changes in glycosylated hemoglobin A1c (HbA1c), fasting plasma glucose, postprandial plasma glucose and body weight were measured pre- and post-Ramadan. RESULTS: Totally, 97 patients were recruited and all completed the study (vildagliptin group, n = 55; sulfonylurea group, n = 42). HEs were reported in low frequencies in both the vildagliptin and the sulfonylurea groups [0 vs 2 (4.8%) patients, respectively]. Interestingly, HbA1c reduced by -0.43% (-4.71 mmol/mol) in the vildagliptin group [8.75% (72.10 mmol/mol) to 8.32% (67.38 mmol/mol), P = 0.009] while in the sulfonylurea group there was a small increase by 0.01% [0.08 mmol/mol; 8.64% (70.92 mmol/mol) to 8.65% (71.00 mmol/mol), P = 0.958]. Higher percentage of vildagliptin-treated patients achieved HbA1c < 7.0% (< 53 mmol/mol) compared with sulfonylurea (16.4% vs 4.8%). Mean decrease in the body weight was 1.2 kg and 0.03 kg, respectively (P < 0.001). Both treatment groups were well tolerated during Ramadan. CONCLUSION: Vildagliptin is an attractive treatment option for Indian patients with type 2 diabetes mellitus who are fasting during Ramadan. PMID:24379927

  16. In vitro and in vivo characterization of a novel naphthylamide ATP-sensitive K+ channel opener, A-151892

    PubMed Central

    Gopalakrishnan, Murali; Buckner, Steven A; Shieh, Char-Chang; Fey, Thomas; Fabiyi, Adebola; Whiteaker, Kristi L; Davis-Taber, Rachel; Milicic, Ivan; Daza, Anthony V; Scott, Victoria E S; Castle, Neil A; Printzenhoff, David; London, Brecht; Turner, Sean C; Carroll, William A; Sullivan, James P; Coghlan, Michael J; Brune, Michael E

    2004-01-01

    Openers of ATP-sensitive K+ channels are of interest in several therapeutic indications including overactive bladder and other lower urinary tract disorders. This study reports on the in vitro and in vivo characterization of a structurally novel naphthylamide N-[2-(2,2,2-trifluoro-1-hydroxy-1-trifluoromethyl-ethyl)-naphthalen-1-yl]-acetamide (A-151892), as an opener of the ATP-sensitive potassium channels. A-151892 was found to be a potent and efficacious potassium channel opener (KCO) as assessed by glibenclamide-sensitive whole-cell current and fluorescence-based membrane potential responses (−log EC50=7.63) in guinea-pig bladder smooth muscle cells. Evidence for direct interaction with KCO binding sites was derived from displacement of binding of the 1,4-dihydropyridine opener [125I]A-312110. A-151892 displaced [125I]A-312110 binding to bladder membranes with a −log Ki value of 7.45, but lacked affinity against over 70 neurotransmitter receptor and ion channel binding sites. In pig bladder strips, A-151892 suppressed phasic, carbachol-evoked and electrical field stimulus-evoked contractility in a glibenclamide-reversible manner with −log IC50 values of 8.07, 7.33 and 7.02 respectively, comparable to that of the potencies of the prototypical cyanoguanidine KCO, P1075. The potencies to suppress contractions in thoracic aorta (−log IC50=7.81) and portal vein (−log IC50=7.98) were not substantially different from those observed for suppression of phasic contractility of the bladder smooth muscle. In vivo, A-151892 was found to potently suppress unstable bladder contractions in obstructed models of unstable contractions in both pigs and rats with pED35% values of 8.05 and 7.43, respectively. These results demonstrate that naphthylamide analogs exemplified by A-151892 are novel KATP channel openers and may serve as chemotypes to exploit additional analogs with potential for the treatment of overactive bladder and lower urinary tract symptoms. PMID:15302680

  17. A residue in the TRPM2 channel outer pore is crucial in determining species-dependent sensitivity to extracellular acidic pH.

    PubMed

    Zou, Jie; Yang, Wei; Beech, David J; Jiang, Lin-Hua

    2011-08-01

    Acidic pH is an important parameter regulating ion channel activity and its biological function. This study investigated inhibition of the hTRPM2 channels by extracellular acidic pH and compared the sensitivity of human (h) and mouse (m) TRPM2 channel to such an inhibition. The initial inhibition of hTRPM2 channel currents was substantially reversible, but the reversibility progressively diminished as the exposure to acidic pH was prolonged and it was essentially lost in the steady state, suggesting that extracellular acidic pH induces initial reversible inhibition and subsequent irreversible inactivation. Like the hTRPM2 channel, the mTRPM2 channel was sensitive to inhibition by pH 4.0-5.5, but the kinetics was significantly slower. Moreover, in contrast to the complete inhibition of the hTRPM2 channel, the mTRPM2 channel was insensitive to pH 6.0. Replacement of residue Gln(992) in the outer pore with the equivalent residue His(995) in the hTRPM2 channel resulted in a mutant mTRPM2 channel with the pH sensitivity and kinetics of inhibition of the wild-type hTRPM2 channel. Conversely, the reciprocal mutation H995Q in the hTRPM2 channel dramatically slowed down the kinetics of inhibition. Swapping other residues in the pore region failed to produce such opposing effects. Taken together, our results suggest a crucial role of residue His(995)/Gln(992) in the outer pore of TRPM2 channels in determining species-dependent effects of extracellular acidic pH.

  18. A novel high-throughput screening assay for HCN channel blocker using membrane potential-sensitive dye and FLIPR.

    PubMed

    Vasilyev, Dmitry V; Shan, Qin J; Lee, Yan T; Soloveva, Veronica; Nawoschik, Stanley P; Kaftan, Edward J; Dunlop, John; Mayer, Scott C; Bowlby, Mark R

    2009-10-01

    Hyperpolarization-activated cation nonselective (HCN) channels represent an interesting group of targets for drug development. In this study, the authors report the development of a novel membrane potential-sensitive dye (MPSD) assay for HCN channel modulators that has been miniaturized into 384-well fluorescent imaging plate reader (FLIPR) high-throughput screening (HTS) format. When optimized (by cell plating density, plate type, cell recovery from cryopreservation), the well-to-well signal variability was low, with a Z' = 0.73 and coefficient of variation = 6.4%, whereas the MPSD fluorescence signal amplitude was -23,700 +/- 1500 FLIPR(3) relative fluorescence units (a linear relationship was found between HCN1 MPSD fluorescence signal and the cell plating density) and was completely blocked by 30 microM ZD7288. The assay tolerated up to 1% DMSO, inclusion of which did not significantly change the signal kinetics or amplitude. A single-concentration screening of an ion channel-focused library composed of 4855 compounds resulted in 89 HCN1 blocker hits, 51 of which were subsequently analyzed with an 8-point concentration-response analysis on the IonWorks HT electrophysiology platform. The correlation between MPSD and the electrophysiology assay was moderate, as shown by the linear regression analysis (r(2) = 0.56) between the respective IC(50)s obtained using these 2 assays. The reported HTS-compatible HCN channel blocker assay can serve as a tool in drug discovery in the pursuit of HCN channel isoform-selective small molecules that could be used in the development of clinically relevant compounds.

  19. Phentolamine relaxes human corpus cavernosum by a nonadrenergic mechanism activating ATP-sensitive K+ channel.

    PubMed

    Silva, L F G; Nascimento, N R F; Fonteles, M C; de Nucci, G; Moraes, M E; Vasconcelos, P R L; Moraes, M O

    2005-01-01

    To investigate the pharmacodynamics of phentolamine in human corpus cavernosum (HCC) with special attention to the role of the K+ channels. Strips of HCC precontracted with nonadrenergic stimuli and kept in isometric organ bath immersed in a modified Krebs-Henseleit solution enriched with guanethidine and indomethacine were used in order to study the mechanism of the phentolamine-induced relaxation. Phentolamine caused relaxation (approximately 50%) in HCC strips precontracted with K+ 40 mM. This effect was not blocked by tetrodotoxin (1 microM) (54.6+/-4.6 vs 48.9+/-6.4%) or (atropine (10 microM) (52.7+/-6.5 vs 58.6+/-5.6%). However, this relaxation was significantly attenuated by L-NAME (100 microM) (59.7+/-5.8 vs 27.8+/-7.1%; P<0.05; n = 8) and ODQ (100 microM) (62.7+/-5.1 vs 26.8+/-3.9%; P<0.05; n = 8). Charybdotoxin and apamin (K(Ca)-channel blockers) did not affect the phentolamine relaxations (54.6+/-4.6 vs 59.3+/-5.2%). Glibenclamide (100 microM), an inhibitor of K(ATP)-channel, caused a significant inhibition (56.7+/-6.3 vs 11.3+/-2.3%; P<0.05; n = 8) of the phentolamine-induced relaxation. In addition, the association of glibenclamide and L-NAME almost abolished the phentolamine-mediated relaxation (54.6+/-5.6 vs 5.7+/-1.4%; P<0.05; n = 8). The results suggest that phentolamine relaxes HCC by a nonadrenergic-noncholinergic mechanism dependent on nitric oxide synthase activity and activation of K(ATP)-channel.

  20. Cardiac specific ATP-sensitive K+ channel (KATP) overexpression results in embryonic lethality.

    PubMed

    Toib, Amir; Zhang, Hai Xia; Broekelmann, Thomas J; Hyrc, Krzysztof L; Guo, Qiusha; Chen, Feng; Remedi, Maria S; Nichols, Colin G

    2012-09-01

    Transgenic mice overexpressing SUR1 and gain of function Kir6.2[∆N30, K185Q] K(ATP) channel subunits, under cardiac α-myosin heavy chain (αMHC) promoter control, demonstrate arrhythmia susceptibility and premature death. Pregnant mice, crossed to carry double transgenic progeny, which harbor high levels of both overexpressed subunits, exhibit the most extreme phenotype and do not deliver any double transgenic pups. To explore the fetal lethality and embryonic phenotype that result from K(ATP) overexpression, wild type (WT) and K(ATP) overexpressing embryonic cardiomyocytes were isolated, cultured and voltage-clamped using whole cell and excised patch clamp techniques. Whole mount embryonic imaging, Hematoxylin and Eosin (H&E) and α smooth muscle actin (αSMA) immunostaining were used to assess anatomy, histology and cardiac development in K(ATP) overexpressing and WT embryos. Double transgenic embryos developed in utero heart failure and 100% embryonic lethality by 11.5 days post conception (dpc). K(ATP) currents were detectable in both WT and K(ATP)-overexpressing embryonic cardiomyocytes, starting at early stages of cardiac development (9.5 dpc). In contrast to adult cardiomyocytes, WT and K(ATP)-overexpressing embryonic cardiomyocytes exhibit basal and spontaneous K(ATP) current, implying that these channels may be open and active under physiological conditions. At 9.5 dpc, live double transgenic embryos demonstrated normal looping pattern, although all cardiac structures were collapsed, probably representing failed, non-contractile chambers. In conclusion, K(ATP) channels are present and active in embryonic myocytes, and overexpression causes in utero heart failure and results in embryonic lethality. These results suggest that the K(ATP) channel may have an important physiological role during early cardiac development.

  1. Trafficking and assembly of the cold-sensitive TRPM8 channel.

    PubMed

    Erler, Isabell; Al-Ansary, Dalia M M; Wissenbach, Ulrich; Wagner, Thomas F J; Flockerzi, Veit; Niemeyer, Barbara A

    2006-12-15

    TRPM (transient receptor potential melastatin-like) channels are distinct from many other members of the transient receptor potential family in regard to their overall size (>1000 amino acids), the lack of N-terminal ankyrin-like repeats, and hydrophobicity predictions that may allow for more than six transmembrane regions. Common to each TRPM member is a prominent C-terminal coiled coil region. Here we have shown that TRPM8 channels assemble as multimers using the putative coiled coil region within the intracellular C terminus and that this assembly can be disturbed by a single point mutation within the coiled coil region. This mutant neither gives rise to functional channels nor do its subunits interact or form protein complexes that correspond to a multimer. However, they are still transported to the plasma membrane. Furthermore, wild-type currents can be suppressed by expressing the membrane-attached C-terminal region of TRPM8. To separate assembly from trafficking, we investigated the maturation of TRPM8 protein by identifying and mutating the relevant N-linked glycosylation site and showing that glycosylation is neither essential for multimerization nor for transport to the plasma membrane per se but appears to facilitate efficient multimerization and transport.

  2. Sensitivity of Δ G Through Open Heavy Quark Production using Electron Decay Channels at PHENIX

    NASA Astrophysics Data System (ADS)

    Xie, Wei

    2000-10-01

    The PHENIX experiment at Relativistic Heavy Ion Collider at Brookhaven National Laboratory is capable of directly measuring the portion of the proton spin carri ed by gluons spin(Δ G)in several channels including direct photon product ion, π production and heavy flavor production. The extraction of Δ G throu gh independent channels gives good control of systematic and theoretical uncerta inty. In this paper we report on PHENIX's Δ G measurement in open heavy q uark production using electron decay channels. Heavy flavor production, cbarc and bbarb, is dominated by gluon-gluon interactions and gives rise to a double spin asymmetry A_LL ~ fracΔ G(x_A)G(x_A)øtimes fracΔ G(x_B)G(x_B)øtimes a_LL^ggarrow qbarq , from which Δ G can be extracted. We will report a full detector simulation which includes the effects of electron trigger and dilutions due to the conversion in inner chamber and π^0 Dalitz decays.

  3. Tight genetic linkage between the kdr insecticide resistance trait and a voltage-sensitive sodium channel gene in the house fly.

    PubMed Central

    Knipple, D C; Doyle, K E; Marsella-Herrick, P A; Soderlund, D M

    1994-01-01

    The kdr insecticide resistance trait in the house fly, Musca domestica, confers resistance to the rapid paralysis (knockdown) and lethal effects of 1,1,1-trichloro-2,2-bis(p-chlorophenyl)ethane (DDT) and pyrethroids. Flies with the kdr trait exhibit reduced neuronal sensitivity to these compounds, which are known to act at voltage-sensitive sodium channels of nerve membranes. To test the hypothesis that a mutation in a voltage-sensitive sodium channel gene confers the kdr phenotype, we have cloned genomic DNA corresponding to a segment of the house fly homologue of the para sodium channel gene of Drosophila melanogaster, identified restriction-site polymorphisms within this segment between the kdr strain 538ge and an inbred insecticide-susceptible lab stain, and developed a sensitive polymerase chain reaction-based diagnostic procedure to determine the sodium channel genotype of individual flies. A genetic linkage analysis performed with these molecular markers shows that the kdr trait is tightly linked (within about 1 map unit) to the voltage-sensitive sodium channel gene segment exhibiting the DNA sequence polymorphism. These findings provide genetic evidence for a mutation at or near a voltage-sensitive sodium channel gene as the basis for kdr resistance. Images PMID:8146143

  4. Developmentally-regulated sodium channel subunits are differentially sensitive to α–cyano containing pyrethroids

    EPA Science Inventory

    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 ch...

  5. A Conserved Residue Cluster That Governs Kinetics of ATP-dependent Gating of Kir6.2 Potassium Channels.

    PubMed

    Zhang, Roger S; Wright, Jordan D; Pless, Stephan A; Nunez, John-Jose; Kim, Robin Y; Li, Jenny B W; Yang, Runying; Ahern, Christopher A; Kurata, Harley T

    2015-06-19

    ATP-sensitive potassium (KATP) channels are heteromultimeric complexes of an inwardly rectifying Kir channel (Kir6.x) and sulfonylurea receptors. Their regulation by intracellular ATP and ADP generates electrical signals in response to changes in cellular metabolism. We investigated channel elements that control the kinetics of ATP-dependent regulation of KATP (Kir6.2 + SUR1) channels using rapid concentration jumps. WT Kir6.2 channels re-open after rapid washout of ATP with a time constant of ∼60 ms. Extending similar kinetic measurements to numerous mutants revealed fairly modest effects on gating kinetics despite significant changes in ATP sensitivity and open probability. However, we identified a pair of highly conserved neighboring amino acids (Trp-68 and Lys-170) that control the rate of channel opening and inhibition in response to ATP. Paradoxically, mutations of Trp-68 or Lys-170 markedly slow the kinetics of channel opening (500 and 700 ms for W68L and K170N, respectively), while increasing channel open probability. Examining the functional effects of these residues using φ value analysis revealed a steep negative slope. This finding implies that these residues play a role in lowering the transition state energy barrier between open and closed channel states. Using unnatural amino acid incorporation, we demonstrate the requirement for a planar amino acid at Kir6.2 position 68 for normal channel gating, which is potentially necessary to localize the ϵ-amine of Lys-170 in the phosphatidylinositol 4,5-bisphosphate-binding site. Overall, our findings identify a discrete pair of highly conserved residues with an essential role for controlling gating kinetics of Kir channels.

  6. BK channels in rat and human pulmonary smooth muscle cells are BKα-β1 functional complexes lacking the oxygen-sensitive stress axis regulated exon insert

    PubMed Central

    Detweiler, Neil D.; Song, Li; McClenahan, Samantha J.; Versluis, Rachel J.; Kharade, Sujay V.; Kurten, Richard C.; Rhee, Sung W.

    2016-01-01

    Abstract A loss of K+ efflux in pulmonary arterial smooth muscle cells (PASMCs) contributes to abnormal vasoconstriction and PASMC proliferation during pulmonary hypertension (PH). Activation of high-conductance Ca2+-activated (BK) channels represents a therapeutic strategy to restore K+ efflux to the affected PASMCs. However, the properties of BK channels in PASMCs—including sensitivity to BK channel openers (BKCOs)—are poorly defined. The goal of this study was to compare the properties of BK channels between PASMCs of normoxic (N) and chronic hypoxic (CH) rats and then explore key findings in human PASMCs. Polymerase chain reaction results revealed that 94.3% of transcripts encoding BKα pore proteins in PASMCs from N rats represent splice variants lacking the stress axis regulated exon insert, which confers oxygen sensitivity. Subsequent patch-clamp recordings from inside-out (I-O) patches confirmed a dense population of BK channels insensitive to hypoxia. The BK channels were highly activated by intracellular Ca2+ and the BKCO lithocholate; these responses require BKα-β1 subunit coupling. PASMCs of CH rats with established PH exhibited a profound overabundance of the dominant oxygen-insensitive BKα variant. Importantly, human BK (hBK) channels in PASMCs from human donor lungs also represented the oxygen-insensitive BKα variant activated by BKCOs. The hBK channels showed significantly enhanced Ca2+ sensitivity compared with rat BK channels. We conclude that rat BK and hBK channels in PASMCs are oxygen-insensitive BKα-β1 complexes highly sensitive to Ca2+ and the BKCO lithocholate. BK channels are overexpressed in PASMCs of a rat model of PH and may provide an abundant target for BKCOs designed to restore K+ efflux. PMID:28090300

  7. Aerosol Retrievals over the Ocean using Channel 1 and 2 AVHRR Data: A Sensitivity Analysis and Preliminary Results

    NASA Technical Reports Server (NTRS)

    Mishchenko, Michael I.; Geogdzhayev, Igor V.; Cairns, Brian; Rossow, William B.; Lacis, Andrew A.

    1999-01-01

    This paper outlines the methodology of interpreting channel 1 and 2 AVHRR radiance data over the oceans and describes a detailed analysis of the sensitivity of monthly averages of retrieved aerosol parameters to the assumptions made in different retrieval algorithms. The analysis is based on using real AVHRR data and exploiting accurate numerical techniques for computing single and multiple scattering and spectral absorption of light in the vertically inhomogeneous atmosphere-ocean system. We show that two-channel algorithms can be expected to provide significantly more accurate and less biased retrievals of the aerosol optical thickness than one-channel algorithms and that imperfect cloud screening and calibration uncertainties are by far the largest sources of errors in the retrieved aerosol parameters. Both underestimating and overestimating aerosol absorption as well as the potentially strong variability of the real part of the aerosol refractive index may lead to regional and/or seasonal biases in optical thickness retrievals. The Angstrom exponent appears to be the most invariant aerosol size characteristic and should be retrieved along with optical thickness as the second aerosol parameter.

  8. Affinity purification of the voltage-sensitive sodium channel from electroplax with resins selective for sialic acid

    SciTech Connect

    James, W.M.; Emerick, M.C.; Agnew, W.S. )

    1989-07-11

    The voltage-sensitive sodium channel present in the eel (Electrophorus electricus) has an unusually high content of sialic acid, including {alpha}-(2{yields}8)-linked polysialic acid, not found in other electroplax membrane glycopeptides. Lectins from Limax flavus (LFA) and wheat germ (WGA) proved the most effective of 11 lectin resins tried. The most selective resin was prepared from IgM antibodies against Neisseria meningitidis {alpha}-(2{yields}8)-polysialic acid which were affinity purified and coupled to Sepharose 4B. The sodium channel was found to bind to WGA, LFA, and IgM resins and was readily eluted with the appropriate soluble carbohydrates. Experiments with LFA and IgM resins demonstrated binding and unbinding rates and displacement kinetics, which suggest highly specific binding at multiple sites on the sodium channel protein. In preparative-scale purification of protein previously fractionated by anion-exchange chromatography, without stabilizing TTX, high yields were reproducibly obtained. Further, when detergent extracts were prepared from electroplax membranes fractionated by low-speed sedimentation, a single step over the IgM resin provided a 70-fold purification, yielding specific activities of 3,200 pmol of ({sup 3}H)TTX-binding sites/mg of protein and a single polypeptide of {approximately}285,000 Da on SDS-acrylamide gels. No small peptides were observed after this 5-h isolation. The authors describe a cation-dependent stabilization with millimolar levels of monovalent and micromolar levels of divalent species.

  9. Affinity purification of the voltage-sensitive sodium channel from electroplax with resins selective for sialic acid.

    PubMed

    James, W M; Emerick, M C; Agnew, W S

    1989-07-11

    The voltage-sensitive sodium channel present in the eel (Electrophorus electricus) has an unusually high content of sialic acid, including alpha-(2----8)-linked polysialic acid, not found in other electroplax membrane glycopeptides. Lectins from Limax flavus (LFA) and wheat germ (WGA) proved the most effective of 11 lectin resins tried. The most selective resin was prepared from IgM antibodies against Neisseria meningitidis alpha-(2----8)-polysialic acid which were affinity purified and coupled to Sepharose 4B. The sodium channel was found to bind to WGA, LFA, and IgM resins and was readily eluted with the appropriate soluble carbohydrates. Experiments with LFA and IgM resins demonstrated binding and unbinding rates and displacement kinetics, which suggest highly specific binding at multiple sites on the sodium channel protein. In preparative-scale purification of protein previously fractionated by anion-exchange chromatography, without stabilizing TTX, high yields were reproducibly obtained. Further, when detergent extracts were prepared from electroplax membranes fractionated by low-speed sedimentation, a single step over the IgM resin provided a 70-fold purification, yielding specific activities of 3200 pmol of [3H]TTX-binding sites/mg of protein and a single polypeptide of approximately 285,000 Da on SDS-acrylamide gels. No small peptides were observed after this 5-h isolation. We further describe a cation-dependent stabilization with millimolar levels of monovalent and micromolar levels of divalent species.

  10. The canonical transient receptor potential 6 (TRPC6) channel is sensitive to extracellular pH in mouse platelets.

    PubMed

    Berna-Erro, Alejandro; Albarran, Letizia; Dionisio, Natalia; Redondo, Pedro C; Alonso, Nieves; Gomez, Luis J; Salido, Gines M; Rosado, Juan A

    2014-01-01

    The canonical transient receptor potential-6 (TRPC6) is a receptor-activated non-selective Ca(2+) channel regulated by a variety of modulators such as diacylglycerol, Ca(2+)/calmodulin or phosphorylation. The present study is aimed to investigate whether different situations, such as acidic pH, exposure to reactive oxygen species (ROS) or hypoxic-like conditions modulate TRPC6 channel function. Here we show normal aggregation and Ca(2+) mobilization stimulated by thrombin in TRPC6 KO platelets; however, OAG (1-oleoyl-2-acetyl-sn-glycerol)-evoked Ca(2+) entry was attenuated in the absence of TRPC6. Exposure of mouse platelets to acidic pH resulted in abolishment of thrombin-evoked aggregation and attenuated platelet aggregation induced by thapsigargin (TG) or OAG. Both OAG-induced Ca(2+) entry and platelet aggregation were greatly attenuated in cells expressing TRPC6 channels. Exposure of platelets to H2O2 or deferoxamine did not clearly alter thrombin, TG or OAG-induced platelet aggregation. Our results indicate that TRPC6 is sensitive to acidic pH but not to exposure to ROS or hypoxic-like conditions, which might be involved in the pathogenesis of the altered platelet responsiveness to DAG-generating agonists in disorders associated to acidic pH.

  11. Proton-sensitive cation channels and ion exchangers in ischemic brain injury: new therapeutic targets for stroke?

    PubMed Central

    Leng, Tiandong; Shi, Yejie; Xiong, Zhi-Gang; Sun, Dandan

    2014-01-01

    Ischemic brain injury results from complicated cellular mechanisms. The present therapy for acute ischemic stroke is limited to thrombolysis with the recombinant tissue plasminogen activator (rtPA) and mechanical recanalization. Therefore, a better understanding of ischemic brain injury is needed for the development of more effective therapies. Disruption of ionic homeostasis plays an important role in cell death following cerebral ischemia. Glutamate receptor-mediated ionic imbalance and neurotoxicity have been well established in cerebral ischemia after stroke. However, non-NMDA receptor-dependent mechanisms, involving acid-sensing ion channel 1a (ASIC1a), transient receptor potential melastatin 7 (TRPM7), and Na+/H+ exchanger isoform 1 (NHE1), have recently emerged as important players in the dysregulation of ionic homeostasis in the CNS under ischemic conditions. These H+-sensitive channels and/or exchangers are expressed in the majority of cell types of the neurovascular unit. Sustained activation of these proteins causes excessive influx of cations, such as Ca2+, Na+, and Zn2+, and leads to ischemic reperfusion brain injury. In this review, we summarize recent pre-clinical experimental research findings on how these channels/exchangers are regulated in both in vitro and in vivo models of cerebral ischemia. The blockade or transgenic knockdown of these proteins was shown to be neuroprotective in these ischemia models. Taken together, these non-NMDA receptor-dependent mechanisms may serve as novel therapeutic targets for stroke intervention. PMID:24467911

  12. Evaluation of the in vivo efficacy of novel monosubstituted sulfonylureas against H37Rv and extensively drug-resistant tuberculosis.

    PubMed

    Liu, Yu; Bao, Pengtao; Wang, Di; Li, Zhengming; Li, Yun; Tang, Liping; Zhou, Yi; Zhao, Weiguo

    2014-01-01

    Sulfonylureas have been regarded as potential drug candidates against tuberculosis (TB) because they can inhibit the biosynthesis of branched-chain amino acids by targeting acetohydroxyacid synthase (AHAS). We demonstrated previously that novel monosubstituted sulfonylureas showed potent in vitro activities against TB. In the current study, we further explored the anti-TB activity of monosubstituted sulfonylureas in a mouse model. Compounds 30 and 31 exhibited the most efficacy: a single intragastric administration at a dose of 250 mg/kg led to a reduced lung bacterial count, and the dose of 500 mg/kg achieved a >99% reduction in bacterial load for both H37Rv and extensively drug-resistant isolates. These results indicate that these compounds are more potent than commercial sulfonylureas in vivo and may provide insight into the potential implications for the design of novel drugs to combat TB by targeting AHAS.

  13. pH-sensitive K(+) channel TREK-1 is a novel target in pancreatic cancer.

    PubMed

    Sauter, Daniel R P; Sørensen, Christiane E; Rapedius, Markus; Brüggemann, Andrea; Novak, Ivana

    2016-10-01

    Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal cancers and new therapeutic targets are urgently needed. One of the hallmarks of cancer is changed pH-homeostasis and potentially pH-sensors may play an important role in cancer cell behavior. Two-pore potassium channels (K2P) are pH-regulated channels that conduct a background K(+) current, which is involved in setting the plasma membrane potential (Vm). Some members of the K2P superfamily were reported as crucial players in driving tumor progression. The aim of this study was to investigate pH-regulated K(+) currents in PDAC cells and determine possible effects on their pathological phenotype. Using a planar high-throughput patch-clamp system (SyncroPatch 384PE) we identified a pH-regulated K(+) current in the PDAC cell line BxPC-3. The current was inhibited by extracellular acidification and intracellular alkalization. Exposure to a set of different K(+) channel inhibitors, and the TREK-1 (K2P2.1)-specific activator BL1249, TREK-1 was identified as the main component of pH-regulated current. A voltage-sensor dye (VF2.1.Cl) was used to monitor effects of pH and BL1249 on Vm in more physiological conditions and TREK-1-mediated current was found as critical player in setting Vm. We assessed a possible role of TREK-1 in PDAC progression using cell proliferation and migration assays and observed similar trends with attenuated proliferation/migration rates in acidic (pH<7.0) and alkaline (pH>7.4) conditions. Notably, BL1249 inhibited both PDAC cell proliferation and migration indicating that hyperpolarization of Vm attenuates cancer cell behavior. TREK-1 may therefore be a promising novel target for PDAC therapy.

  14. Molecular size of different neurotoxin receptors on the voltage-sensitive Na+ channel.

    PubMed

    Barhanin, J; Schmid, A; Lombet, A; Wheeler, K P; Lazdunski, M; Ellory, J C

    1983-01-25

    Measurements were made of the molecular sizes of two distinct receptors on the Na+ channel in rat brain synaptosomes that are specific for different neurotoxins. Radiation inactivation of the binding of radiolabeled derivatives of the toxins was consistent with Mr = 260,000 for the tetrodotoxin receptor and Mr = 266,000 for the receptor specific for two scorpion toxins, toxin II from Centruroides suffusus suffusus and toxin gamma from Tityus serrulatus serrulatus. Covalent cross-linking of the latter to its receptor similarly indicated Mr = 270,000. It seems most likely that these two distinct receptors reside on the same molecule.

  15. Stimulation of neuronal KATP channels by cGMP-dependent protein kinase: involvement of ROS and 5-hydroxydecanoate-sensitive factors in signal transduction

    PubMed Central

    Chai, Yongping

    2010-01-01

    The ATP-sensitive potassium (KATP) channel couples intracellular metabolic state to membrane excitability. Recently, we demonstrated that neuronal KATP channels are functionally enhanced by activation of a nitric oxide (NO)/cGMP/cGMP-dependent protein kinase (PKG) signaling cascade. In this study, we further investigated the intracellular mechanism underlying PKG stimulation of neuronal KATP channels. By performing single-channel recordings in transfected HEK293 and neuroblastoma SH-SY5Y cells, we found that the increase of Kir6.2/SUR1 (i.e., the neuronal-type KATP) channel currents by PKG activation in cell-attached patches was diminished by 5-hydroxydecanoate (5-HD), an inhibitor of the putative mitochondrial KATP channel; N-(2-mercaptopropionyl)glycine, a reactive oxygen species (ROS) scavenger, and catalase, a hydrogen peroxide (H2O2)-decomposing enzyme. These reagents also ablated NO-induced KATP channel stimulation and prevented the shifts in the single-channel open- and closed-time distributions resulting from PKG activation and NO induction. Bath application of H2O2 reproduced PKG stimulation of Kir6.2/SUR1 but did not activate tetrameric Kir6.2LRKR368/369/370/371AAAA channels. Moreover, neither the PKG activator nor exogenous H2O2 was able to enhance the function of KATP channels in the presence of Ca2+ chelators and calmodulin antagonists, whereas the stimulatory effect of H2O2 was unaffected by 5-HD. Altogether, in this report we provide novel evidence that activation of PKG stimulates neuronal KATP channels by modulating intrinsic channel gating via a 5-HD-sensitive factor(s)/ROS/Ca2+/calmodulin signaling pathway that requires the presence of the SUR1 subunit. This signaling pathway may contribute to neuroprotection against ischemic injury and regulation of neuronal excitability and neurotransmitter release by modulating the function of neuronal KATP channels. PMID:20053925

  16. Transient neonatal diabetes due to a missense mutation (E227K) in the gene encoding the ATP-sensitive potassium channel (KCNJ11)

    PubMed Central

    Martins, Luísa; Lourenço, Rita; Maia, Ana Lúcia; Maciel, Paula; Monteiro, Maria Isabel; Pacheco, Lucinda; Anselmo, João; César, Rui; Gomes, Maria Fernanda

    2015-01-01

    Key Clinical Message Neonatal diabetes is a monogenic form of diabetes. Herein, we report on a newborn presenting diabetic ketoacidosis at 17 days of life. A KCNJ11 mutation was identified. In such cases, insulin can be replaced by sulfonylurea with a successful metabolic control, as an example of how molecular diagnosis may influence the clinical management of the disorder. PMID:26509005

  17. Activation of the mitochondrial ATP-sensitive K+ channel reduces apoptosis of spleen mononuclear cells induced by hyperlipidemia

    PubMed Central

    2013-01-01

    Background We have previously demonstrated that increased rates of superoxide generation by extra-mitochondrial enzymes induce the activation of the mitochondrial ATP-sensitive potassium channel (mitoKATP) in the livers of hypertriglyceridemic (HTG) mice. The resulting mild uncoupling mediated by mitoKATP protects mitochondria against oxidative damage. In this study, we investigate whether immune cells from HTG mice also present increased mitoKATP activity and evaluate the influence of this trait on cell redox state and viability. Methods Oxygen consumption (Clark-type electrode), reactive oxygen species production (dihydroethidium and H2-DCF-DA probes) and cell death (annexin V, cytocrome c release and Trypan blue exclusion) were determined in spleen mononuclear cells. Results HTG mice mononuclear cells displayed increased mitoKATP activity, as evidenced by higher resting respiration rates that were sensitive to mitoKATP antagonists. Whole cell superoxide production and apoptosis rates were increased in HTG cells. Inhibition of mitoKATP further increased the production of reactive oxygen species and apoptosis in these cells. Incubation with HTG serum induced apoptosis more strongly in WT cells than in HTG mononuclear cells. Cytochrome c release into the cytosol and caspase 8 activity were both increased in HTG cells, indicating that cell death signaling starts upstream of the mitochondria but does involve this organelle. Accordingly, a reduced number of blood circulating lymphocytes was found in HTG mice. Conclusions These results demonstrate that spleen mononuclear cells from hyperlipidemic mice have more active mitoKATP channels, which downregulate mitochondrial superoxide generation. The increased apoptosis rate observed in these cells is exacerbated by closing the mitoKATP channels. Thus, mitoKATP opening acts as a protective mechanism that reduces cell death induced by hyperlipidemia. PMID:23764148

  18. Activation by intracellular GDP, metabolic inhibition and pinacidil of a glibenclamide-sensitive K-channel in smooth muscle cells of rat mesenteric artery.

    PubMed Central

    Zhang, H; Bolton, T B

    1995-01-01

    1. Single-channel recordings were made from cell-attached and isolated patches, and whole-cell currents were recorded under voltage clamp from single smooth muscle cells obtained by enzymic digestion of a small branch of the rat mesenteric artery. 2. In single voltage-clamped cells 1 mM uridine diphosphate (UDP) or guanidine diphosphate (GDP) added to the pipette solution, or pinacidil (100 microM) a K-channel opener (KCO) applied in the bathing solution, evoked an outward current of up to 100pA which was blocked by glibenclamide (10 microM). In single cells from which recordings were made by the 'perforated patch' (nystatin pipette) technique, metabolic inhibition by 1 mM NaCN and 10 mM 2-deoxy-glucose also evoked a similar glibenclamide-sensitive current. 3. Single K-channel activity was observed in cell-attached patches only infrequently unless the metabolism of the cell was inhibited, whereupon channel activity blocked by glibenclamide was seen; pinacidil applied to the cell evoked similar glibenclamide-sensitive channel activity. If the patch was pulled off the cell to form an isolated inside-out patch, similar glibenclamide-sensitive single-channel currents were observed in the presence of UDP and/or pinacidil to those seen in cell-attached mode; channel conductance was 20 pS (60:130 K-gradient) and openings showed no voltage-dependence and noisy inward currents, typical of the nucleoside diphosphate (NDP) activated K-channel (KNDP) seen previously in rabbit portal vein. 4. Formation of an isolated inside-out patch into an ATP-free solution did not increase the probability of channel opening which declined with time even when some single-channel activity had occurred in the cell-attached mode before detachment. However, application of 1 mM UDP or GDP, but not ATP, to inside-out patches evoked single-channel activity. Application of ATP-free solution to isolated patches, previously exposed to ATP and in which channel activity had been seen, did not evoke

  19. A Nav1.7 channel mutation associated with hereditary erythromelalgia contributes to neuronal hyperexcitability and displays reduced lidocaine sensitivity.

    PubMed

    Sheets, Patrick L; Jackson, James O; Waxman, Stephen G; Dib-Hajj, Sulayman D; Cummins, Theodore R

    2007-06-15

    Mutations in the TTX-sensitive voltage-gated sodium channel subtype Nav1.7 have been implicated in the painful inherited neuropathy, hereditary erythromelalgia. Hereditary erythromelalgia can be difficult to treat and, although sodium channels are targeted by local anaesthetics such as lidocaine (lignocaine), some patients do not respond to treatment with local anaesthetics. This study examined electrophysiological differences in Nav1.7 caused by a hereditary erythromelalgia mutation (N395K) that lies within the local anaesthetic binding site of the channel. The N395K mutation produced a hyperpolarized voltage dependence of activation, slower kinetics of deactivation, and impaired steady-state slow inactivation. Computer simulations indicate that the shift in activation is the major determinant of the hyperexcitability induced by erythromelalgia mutations in sensory neurons, but that changes in slow inactivation can modulate the overall impact on excitability. This study also investigated lidocaine inhibition of the Nav1.7-N395K channel. We show that the N395K mutation attenuates the inhibitory effects of lidocaine on both resting and inactivated Nav1.7. The IC50 for lidocaine was estimated at 500 microM for inactivated wild-type Nav1.7 and 2.8 mM for inactivated Nav1.7-N395K. The N395K mutation also significantly reduced use-dependent inhibition of lidocaine on Nav1.7 current. In contrast, a different hereditary erythromelalgia mutation (F216S), not located in the local anaesthetic binding site, had no effect on lidocaine inhibition of Nav1.7 current. Our observation of reduced lidocaine inhibition on Nav1.7-N395K shows that the residue N395 is critical for lidocaine binding to Nav1.7 and suggests that the response of individuals with hereditary erythromelalgia to lidocaine treatment may be determined, at least in part, by their specific genotype.

  20. A Single Crossing-Over Event in Voltage-Sensitive Na+ Channel Genes May Cause Critical Failure of Dengue Mosquito Control by Insecticides

    PubMed Central

    Hirata, Koichi; Komagata, Osamu; Itokawa, Kentaro; Yamamoto, Atsushi; Tomita, Takashi; Kasai, Shinji

    2014-01-01

    The voltage-sensitive sodium (Na+) channel (Vssc) is the target site of pyrethroid insecticides. Pest insects develop resistance to this class of insecticide by acquisition of one or multiple amino acid substitution(s) in this channel. In Southeast Asia, two major Vssc types confer pyrethroid resistance in the dengue mosquito vector Aedes aegypti, namely, S989P+V1016G and F1534C. We expressed several types of Vssc in Xenopus oocytes and examined the effect of amino acid substitutions in Vssc on pyrethroid susceptibilities. S989P+V1016G and F1534C haplotypes reduced the channel sensitivity to permethrin by 100- and 25-fold, respectively, while S989P+V1016G+F1534C triple mutations reduced the channel sensitivity to permethrin by 1100-fold. S989P+V1016G and F1534C haplotypes reduced the channel sensitivity to deltamethrin by 10- and 1-fold (no reduction), respectively, but S989P+V1016G+F1534C triple mutations reduced the channel sensitivity to deltamethrin by 90-fold. These results imply that pyrethroid insecticides are highly likely to lose their effectiveness against A. aegypti if such a Vssc haplotype emerges as the result of a single crossing-over event; thus, this may cause failure to control this key mosquito vector. Here, we strongly emphasize the importance of monitoring the occurrence of triple mutations in Vssc in the field population of A. aegypti. PMID:25166902

  1. [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.

  2. 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.

  3. A novel exon in the human Ca2+-activated Cl- channel Ano1 imparts greater sensitivity to intracellular Ca2.

    PubMed

    Strege, Peter R; Bernard, Cheryl E; Mazzone, Amelia; Linden, David R; Beyder, Arthur; Gibbons, Simon J; Farrugia, Gianrico

    2015-11-01

    Anoctamin 1 (Ano1; TMEM16A) is a Ca(2+)-activated Cl(-) channel (CACC) expressed in interstitial cells of Cajal. The mechanisms by which Ca(2+) regulates Ano1 are incompletely understood. In the gastrointestinal tract, Ano1 is required for normal slow wave activity and is involved in regulating cell proliferation. Splice variants of Ano1 have varying electrophysiological properties and altered expression in disease states. Recently, we identified a transcript for human Ano1 containing a novel exon-"exon 0" upstream of and in frame with exon 1. The electrophysiological properties of this longer Ano1 isoform are unknown. Our aim was to determine the functional contribution of the newly identified exon to the Ca(2+) sensitivity and electrophysiological properties of Ano1. Constructs with [Ano1(+0)] or without [Ano1(-0)] the newly identified exon were transfected into human embryonic kidney-293 cells. Voltage-clamp electrophysiology was used to determine voltage- and time-dependent parameters of whole cell Cl(-) currents between isoforms with varying concentrations of intracellular Ca(2+), extracellular anions, or Cl(-) channel inhibitors. We found that exon 0 did not change voltage sensitivity and had no impact on the relative permeability of Ano1 to most anions. Ano1(+0) exhibited greater changes in current density but lesser changes in kinetics than Ano1(-0) in response to varying intracellular Ca(2+). The CACC inhibitor niflumic acid inhibited current with greater efficacy and higher potency against Ano1(+0) compared with Ano1(-0). Likewise, the Ano1 inhibitor T16Ainh-A01 reduced Ano1(+0) more than Ano1(-0). In conclusion, human Ano1 containing exon 0 imparts its Cl(-) current with greater sensitivity to intracellular Ca(2+) and CACC inhibitors.

  4. A novel exon in the human Ca2+-activated Cl− channel Ano1 imparts greater sensitivity to intracellular Ca2+

    PubMed Central

    Strege, Peter R.; Bernard, Cheryl E.; Mazzone, Amelia; Linden, David R.; Beyder, Arthur; Gibbons, Simon J.

    2015-01-01

    Anoctamin 1 (Ano1; TMEM16A) is a Ca2+-activated Cl− channel (CACC) expressed in interstitial cells of Cajal. The mechanisms by which Ca2+ regulates Ano1 are incompletely understood. In the gastrointestinal tract, Ano1 is required for normal slow wave activity and is involved in regulating cell proliferation. Splice variants of Ano1 have varying electrophysiological properties and altered expression in disease states. Recently, we identified a transcript for human Ano1 containing a novel exon-“exon 0” upstream of and in frame with exon 1. The electrophysiological properties of this longer Ano1 isoform are unknown. Our aim was to determine the functional contribution of the newly identified exon to the Ca2+ sensitivity and electrophysiological properties of Ano1. Constructs with [Ano1(+0)] or without [Ano1(−0)] the newly identified exon were transfected into human embryonic kidney-293 cells. Voltage-clamp electrophysiology was used to determine voltage- and time-dependent parameters of whole cell Cl− currents between isoforms with varying concentrations of intracellular Ca2+, extracellular anions, or Cl− channel inhibitors. We found that exon 0 did not change voltage sensitivity and had no impact on the relative permeability of Ano1 to most anions. Ano1(+0) exhibited greater changes in current density but lesser changes in kinetics than Ano1(−0) in response to varying intracellular Ca2+. The CACC inhibitor niflumic acid inhibited current with greater efficacy and higher potency against Ano1(+0) compared with Ano1(−0). Likewise, the Ano1 inhibitor T16Ainh-A01 reduced Ano1(+0) more than Ano1(−0). In conclusion, human Ano1 containing exon 0 imparts its Cl− current with greater sensitivity to intracellular Ca2+ and CACC inhibitors. PMID:26359375

  5. KCa3.1 channel inhibition sensitizes malignant gliomas to temozolomide treatment

    PubMed Central

    D'Alessandro, Giuseppina; Grimaldi, Alfonso; Chece, Giuseppina; Porzia, Alessandra; Esposito, Vincenzo; Santoro, Antonio; Salvati, Maurizio; Mainiero, Fabrizio; Ragozzino, Davide; Angelantonio, Silvia Di; Wulff, Heike; Catalano, Myriam; Limatola, Cristina

    2016-01-01

    Malignant gliomas are among the most frequent and aggressive cerebral tumors, characterized by high proliferative and invasive indexes. Standard therapy for patients, after surgery and radiotherapy, consists of temozolomide (TMZ), a methylating agent that blocks tumor cell proliferation. Currently, there are no therapies aimed at reducing tumor cell invasion. Ion channels are candidate molecular targets involved in glioma cell migration and infiltration into the brain parenchyma. In this paper we demonstrate that: i) blockade of the calcium-activated potassium channel KCa3.1 with TRAM-34 has co-adjuvant effects with TMZ, reducing GL261 glioma cell migration, invasion and colony forming activity, increasing apoptosis, and forcing cells to pass the G2/M cell cycle phase, likely through cdc2 de-phosphorylation; ii) KCa3.1 silencing potentiates the inhibitory effect of TMZ on glioma cell viability; iii) the combination of TMZ/TRAM-34 attenuates the toxic effects of glioma conditioned medium on neuronal cultures, through a microglia dependent mechanism since the effect is abolished by clodronate-induced microglia killing; iv) TMZ/TRAM-34 co-treatment increases the number of apoptotic tumor cells, and the mean survival time in a syngeneic mouse glioma model (C57BL6 mice implanted with GL261 cells); v) TMZ/TRAM-34 co-treatment reduces cell viability of GBM cells and cancer stem cells (CSC) freshly isolated from patients. Taken together, these data suggest a new therapeutic approach for malignant glioma, targeting both glioma cell proliferating and migration, and demonstrate that TMZ/TRAM-34 co-treatment affects both glioma cells and infiltrating microglia, resulting in an overall reduction of tumor cell progression. PMID:27096953

  6. Molecular Aspects of Structure, Gating, and Physiology of pH-Sensitive Background K2P and Kir K+-Transport Channels

    PubMed Central

    Sepúlveda, Francisco V.; Pablo Cid, L.; Teulon, Jacques; Niemeyer, María Isabel

    2015-01-01

    K+ channels fulfill roles spanning from the control of excitability to the regulation of transepithelial transport. Here we review two groups of K+ channels, pH-regulated K2P channels and the transport group of Kir channels. After considering advances in the molecular aspects of their gating based on structural and functional studies, we examine their participation in certain chosen physiological and pathophysiological scenarios. Crystal structures of K2P and Kir channels reveal rather unique features with important consequences for the gating mechanisms. Important tasks of these channels are discussed in kidney physiology and disease, K+ homeostasis in the brain by Kir channel-equipped glia, and central functions in the hearing mechanism in the inner ear and in acid secretion by parietal cells in the stomach. K2P channels fulfill a crucial part in central chemoreception probably by virtue of their pH sensitivity and are central to adrenal secretion of aldosterone. Finally, some unorthodox behaviors of the selectivity filters of K2P channels might explain their normal and pathological functions. Although a great deal has been learned about structure, molecular details of gating, and physiological functions of K2P and Kir K+-transport channels, this has been only scratching at the surface. More molecular and animal studies are clearly needed to deepen our knowledge. PMID:25540142

  7. Molecular aspects of structure, gating, and physiology of pH-sensitive background K2P and Kir K+-transport channels.

    PubMed

    Sepúlveda, Francisco V; Pablo Cid, L; Teulon, Jacques; Niemeyer, María Isabel

    2015-01-01

    K(+) channels fulfill roles spanning from the control of excitability to the regulation of transepithelial transport. Here we review two groups of K(+) channels, pH-regulated K2P channels and the transport group of Kir channels. After considering advances in the molecular aspects of their gating based on structural and functional studies, we examine their participation in certain chosen physiological and pathophysiological scenarios. Crystal structures of K2P and Kir channels reveal rather unique features with important consequences for the gating mechanisms. Important tasks of these channels are discussed in kidney physiology and disease, K(+) homeostasis in the brain by Kir channel-equipped glia, and central functions in the hearing mechanism in the inner ear and in acid secretion by parietal cells in the stomach. K2P channels fulfill a crucial part in central chemoreception probably by virtue of their pH sensitivity and are central to adrenal secretion of aldosterone. Finally, some unorthodox behaviors of the selectivity filters of K2P channels might explain their normal and pathological functions. Although a great deal has been learned about structure, molecular details of gating, and physiological functions of K2P and Kir K(+)-transport channels, this has been only scratching at the surface. More molecular and animal studies are clearly needed to deepen our knowledge.

  8. Chronic hypoxia-induced acid-sensitive ion channel expression in chemoafferent neurons contributes to chemoreceptor hypersensitivity

    PubMed Central

    Liu, X.; He, L.; Fidone, S. J.

    2011-01-01

    Previously we demonstrated that chronic hypoxia (CH) induces an inflammatory condition characterized by immune cell invasion and increased expression of inflammatory cytokines in rat carotid body. It is well established that chronic inflammatory pain induces the expression of acid-sensitive ion channels (ASIC) in primary sensory neurons, where they contribute to hyperalgesia and allodynia. The present study examines the effect of CH on ASIC expression in petrosal ganglion (PG), which contains chemoafferent neurons that innervate oxygen-sensitive type I cells in the carotid body. Five isoforms of ASIC transcript were increased ∼1.5–2.5-fold in PG following exposure of rats to 1, 3, or 7 days of hypobaric hypoxia (380 Torr). ASIC transcript was not increased in the sympathetic superior cervical ganglion (SCG). In the PG, CH also increased the expression of channel-interacting PDZ domain protein, a scaffolding protein known to enhance the surface expression and the low pH-induced current density mediated by ASIC3. Western immunoblot analysis showed that CH elevated ASIC3 protein in PG, but not in SCG or the (sensory) nodose ganglion. ASIC3 transcript was likewise elevated in PG neurons cultured in the presence of inflammatory cytokines. Increased ASIC expression was blocked in CH rats concurrently treated with the nonsteroidal anti-inflammatory drug ibuprofen (4 mg·kg−1·day−1). Electrophysiological recording of carotid sinus nerve (CSN) activity in vitro showed that the specific ASIC antagonist A-317567 (100 μM) did not significantly alter hypoxia-evoked activity in normal preparations but blocked ∼50% of the hypoxic response following CH. Likewise, a high concentration of ibuprofen, which is known to block ASIC1a, reduced hypoxia-evoked CSN activity by ∼50% in CH preparations. Our findings indicate that CH induces inflammation-dependent phenotypic adjustments in chemoafferent neurons. Following CH, ASIC are important participants in chemotransmission

  9. Regulation of myometrial contraction by ATP-sensitive potassium (KATP) channel via activation of SUR2B and Kir 6.2 in mouse

    PubMed Central

    HONG, Seung Hwa; KYEONG, Kyu-Sang; KIM, Chan Hyung; KIM, Young Chul; CHOI, Woong; YOO, Ra Young; KIM, Hun Sik; PARK, Yeon Jin; JI, Il Woon; JEONG, Eun-Hwan; KIM, Hak Soon; XU, Wen-Xie; LEE, Sang Jin

    2016-01-01

    ATP-sensitive potassium (KATP) channels are well characterized in cardiac, pancreatic and many other muscle cells. In the present study, functional expression of the KATP channel was examined in non-pregnant murine longitudinal myometrium. Isometric contraction measurements and Western blot were used. KATP channel openers (KCOs), such as pinacidil, cromakalim, diazoxide and nicorandil, inhibited spontaneous myometrial contractions in a reversible and glibenclamide-sensitive manner. KCOs inhibited oxytocin (OXT)- and prostaglandin F2α (PGF2α)-induced phasic contractions in a glibenclamide-sensitive manner. SUR2B and Kir6.2 were detected by Western blot, whereas SUR1, SUR2A and Kir6.1 were not. These results show that pinacidl, cromakalim, diazoxide and nicorandil-sensitive KATP channels exist in murine myometrium, which are composed of SUR2B and Kir6.2. Based on the modulatory effects of the KATP channel on spontaneous contraction, OXT- and PGF2α-induced contractions, KATP channels seem to play an essential role in murine myometrial motility via activation of SUR2B and Kir6.2. PMID:27086859

  10. Can water sensitive urban design systems help to preserve natural channel-forming flow regimes in an urbanised catchment?

    PubMed

    Wella-Hewage, Chathurika Subhashini; Alankarage Hewa, Guna; Pezzaniti, David

    2016-01-01

    Increased stormwater runoff and pollutant loads due to catchment urbanisation bring inevitable impacts on the physical and ecological conditions of environmentally sensitive urban streams. Water sensitive urban design (WSUD) has been recognised as a possible means to minimise these negative impacts. This paper reports on a study that investigated the ability of infiltration-based WSUD systems to replicate the predevelopment channel-forming flow (CFF) regime in urban catchments. Catchment models were developed for the 'pre-urban', 'urban' and 'managed' conditions of a case study catchment and the hydrological effect on CFF regime was investigated using a number of flow indices. The results clearly show that changes to flow regime are apparent under urban catchment conditions and are even more severe under highly urbanised conditions. The use of WSUD systems was found to result in the replication of predevelopment flow regimes, particularly at low levels of urbanisation. Under highly urbanised conditions (of managed catchments) overcontrol of the CFF indices was observed as indicated by flow statistics below their pre-urban values. The overall results suggest that WSUD systems are highly effective in replicating the predevelopment CFF regime in urban streams and could be used as a means to protect environmentally sensitive urban streams.

  11. Cooperation between cAMP signalling and sulfonylurea in insulin secretion.

    PubMed

    Shibasaki, T; Takahashi, T; Takahashi, H; Seino, S

    2014-09-01

    Although glucose is physiologically the most important regulator of insulin secretion, glucose-induced insulin secretion is modulated by hormonal and neural inputs to pancreatic β-cells. Most of the hormones and neurotransmitters evoke intracellular signals such as cAMP, Ca²⁺ , and phospholipid-derived molecules by activating G protein-coupled receptors (GPCRs). In particular, cAMP is a key second messenger that amplifies insulin secretion in a glucose concentration-dependent manner. The action of cAMP on insulin secretion is mediated by both protein kinase A (PKA)-dependent and Epac2A-dependent mechanisms. Many of the proteins expressed in β-cells are phosphorylated by PKA in vitro, but only a few proteins in which PKA phosphorylation directly affects insulin secretion have been identified. On the other hand, Epac2A activates the Ras-like small G protein Rap in a cAMP-dependent manner. Epac2A is also directly activated by various sulfonylureas, except for gliclazide. 8-pCPT-2'-O-Me-cAMP, an Epac-selective cAMP analogue, and glibenclamide, a sulfonylurea, synergistically activate Epac2A and Rap1, whereas adrenaline, which suppresses cAMP production in pancreatic β-cells, blocks activation of Epac2A and Rap1 by glibenclamide. Thus, cAMP signalling and sulfonylurea cooperatively activate Epac2A and Rap1. This interaction could account, at least in part, for the synergistic effects of incretin-related drugs and sulfonylureas in insulin secretion. Accordingly, clarification of the mechanism of Epac2A activation may provide therapeutic strategies to improve insulin secretion in diabetes.

  12. Large Conductance Ca2+-Activated K+ Channels Modulate Uterine α1-Adrenergic Sensitivity in Ovine Pregnancy

    PubMed Central

    Hynan, Linda S.; Liu, Xiao-tie; Roy, Timothy

    2014-01-01

    The uteroplacental vasculature is refractory to α-adrenergic stimulation, and large conductance Ca2+-activated K+ channels (BKCa) may contribute. We examined the effects of uterine artery (UA) BKCa inhibition with tetraethylammonium (TEA) on hemodynamic responses to phenylephrine (PE) at 101 to 117 days and 135 to 147 days of ovine gestation, obtaining dose responses for mean arterial pressure (MAP), heart rate (HR), and uteroplacental blood flow (UPBF) and vascular resistance (UPVR) before and during UA TEA infusions. The UA α1-adrenergic receptors (α1-ARs) were assessed. The PE increased MAP and UPVR and decreased HR and UPBF dose dependently at both gestations (P < .001, analysis of variance). The %▵MAP was less at 135 to 147 days before and during TEA infusions (P ≤ .008); however, responses during TEA were greater (P ≤ .002). The PE increased %▵UPVR>>%▵MAP, thus %▵UPBF fell. The TEA enhanced PE-mediated increases in %▵UPVR at 135 to 147 days (P ≤ .03). The UA α1-AR expression was unchanged in pregnancy. Uterine vascular responses to PE exceed systemic vascular responses throughout pregnancy and are attenuated by BKCa activation, suggesting BKCa protect UPBF. PMID:24026311

  13. The leak channel NALCN controls tonic firing and glycolytic sensitivity of substantia nigra pars reticulata neurons

    PubMed Central

    Lutas, Andrew; Lahmann, Carolina; Soumillon, Magali; Yellen, Gary

    2016-01-01

    Certain neuron types fire spontaneously at high rates, an ability that is crucial for their function in brain circuits. The spontaneously active GABAergic neurons of the substantia nigra pars reticulata (SNr), a major output of the basal ganglia, provide tonic inhibition of downstream brain areas. A depolarizing 'leak' current supports this firing pattern, but its molecular basis remains poorly understood. To understand how SNr neurons maintain tonic activity, we used single-cell RNA sequencing to determine the transcriptome of individual mouse SNr neurons. We discovered that SNr neurons express the sodium leak channel, NALCN, and that SNr neurons lacking NALCN have impaired spontaneous firing. In addition, NALCN is involved in the modulation of excitability by changes in glycolysis and by activation of muscarinic acetylcholine receptors. Our findings suggest that disruption of NALCN could impair the basal ganglia circuit, which may underlie the severe motor deficits in humans carrying mutations in NALCN. DOI: http://dx.doi.org/10.7554/eLife.15271.001 PMID:27177420

  14. Core-shell magnetic molecularly imprinted polymers as sorbent for sulfonylurea herbicide residues.

    PubMed

    Miao, Shan Shan; Wu, Mei Sheng; Zuo, Hai Gen; Jiang, Chen; Jin, She Feng; Lu, Yi Chen; Yang, Hong

    2015-04-15

    Sulfonylurea herbicides are widely used at lower dosage for controlling broad-leaf weeds and some grasses in cereals and economic crops. It is important to develop a highly efficient and selective pretreatment method for analyzing sulfonylurea herbicide residues in environments and samples from agricultural products based on magnetic molecularly imprinted polymers (MIPs). The MIPs were prepared by a surface molecular imprinting technique especially using the vinyl-modified Fe3O4@SiO2 nanoparticle as the supporting matrix, bensulfuron-methyl (BSM) as the template molecule, methacrylic acid (MAA) as a functional monomer, trimethylolpropane trimethacrylate (TRIM) as a cross-linker, and azodiisobutyronitrile (AIBN) as an initiator. The MIPs show high affinity, recognition specificity, fast mass transfer rate, and efficient adsorption performance toward BSM with the adsorption capacity reaching up to 37.32 mg g(-1). Furthermore, the MIPs also showed cross-selectivity for herbicides triasulfuron (TS), prosulfuron (PS), and pyrazosulfuron-ethyl (PSE). The MIP solid phase extraction (SPE) column was easier to operate, regenerate, and retrieve compared to those of C18 SPE column. The developed method showed highly selective separation and enrichment of sulfonylurea herbicide residues, which enable its application in the pretreatment of multisulfonylurea herbicide residues.

  15. Design, Synthesis, Activity and Docking Study of Sorafenib Analogs Bearing Sulfonylurea Unit.

    PubMed

    Wu, Chunjiang; Wang, Min; Tang, Qidong; Luo, Rong; Chen, Le; Zheng, Pengwu; Zhu, Wufu

    2015-10-23

    Two series of novel sorafenib analogs containing a sulfonylurea unit were synthesized and their chemical structures were confirmed by ¹H-NMR, ¹³C-NMR, MS spectrum and elemental analysis. The synthesized compounds were evaluated for the cytotoxicity against A549, Hela, MCF-7, and PC-3 cancer cell lines. Some of the compounds showed moderate cytotoxic activity, especially compounds 1-(2,4-difluorophenylsulfonyl)-3-(4-(2-(methylcarbamoyl)pyridin-4-yloxy)phenyl)urea (6c) and 1-(4-bromophenylsulfonyl)-3-(4-(2-(methylcarbamoyl)pyridin-4-yloxy)phenyl)urea (6f) with the IC50 values against four cancer cell lines ranging from 16.54±1.22 to 63.92±1.81 μM, respectively. Inhibitory rates against vascular endothelial growth factor receptor-2 (VEGFR2/KDR) kinase at 10 μM of target compounds were further carried out in this paper in order to investigate the target of these compounds. Structure-activity relationships (SARs) and docking studies indicated that the sulfonylurea unit was important to these kinds of compounds. None of the substitutions in the phenoxy group and small halogen atoms such as 2,4-difluoro substitution of the aryl group contributed to the activity. The results suggested that sulfonylurea sorafenib analogs are worthy of further study.

  16. Sulfonylureas have antifungal activity and are potent inhibitors of Candida albicans acetohydroxyacid synthase.

    PubMed

    Lee, Yu-Ting; Cui, Chang-Jun; Chow, Eve W L; Pue, Nason; Lonhienne, Thierry; Wang, Jian-Guo; Fraser, James A; Guddat, Luke W

    2013-01-10

    The sulfonylurea herbicides exert their activity by inhibiting plant acetohydroxyacid synthase (AHAS), the first enzyme in the branched-chain amino acid biosynthesis pathway. It has previously been shown that if the gene for AHAS is deleted in Candida albicans , attenuation of virulence is achieved, suggesting AHAS as an antifungal drug target. Herein, we have cloned, expressed, and purified C. albicans AHAS and shown that several sulfonylureas are inhibitors of this enzyme and possess antifungal activity. The most potent of these compounds is ethyl 2-(N-((4-iodo-6-methoxypyrimidin-2-yl)carbamoyl)sulfamoyl)benzoate (10c), which has a K(i) value of 3.8 nM for C. albicans AHAS and an MIC₉₀ of 0.7 μg/mL for this fungus in cell-based assays. For the sulfonylureas tested there was a strong correlation between inhibitory activity toward C. albicans AHAS and fungicidal activity, supporting the hypothesis that AHAS is the target for their inhibitory activity within the cell.

  17. P2X4 Activation Modulates Volume-sensitive Outwardly Rectifying Chloride Channels in Rat Hepatoma Cells*

    PubMed Central

    Varela, Diego; Penna, Antonello; Simon, Felipe; Eguiguren, Ana Luisa; Leiva-Salcedo, Elías; Cerda, Oscar; Sala, Francisco; Stutzin, Andrés

    2010-01-01

    Volume-sensitive outwardly rectifying (VSOR) Cl− channels are critical for the regulatory volume decrease (RVD) response triggered upon cell swelling. Recent evidence indicates that H2O2 plays an essential role in the activation of these channels and that H2O2 per se activates the channels under isotonic isovolumic conditions. However, a significant difference in the time course for current onset between H2O2-induced and hypotonicity-mediated VSOR Cl− activation is observed. In several cell types, cell swelling induced by hypotonic challenges triggers the release of ATP to the extracellular medium, which in turn, activates purinergic receptors and modulates cell volume regulation. In this study, we have addressed the effect of purinergic receptor activation on H2O2-induced and hypotonicity-mediated VSOR Cl− current activation. Here we show that rat hepatoma cells (HTC) exposed to a 33% hypotonic solution responded by rapidly activating VSOR Cl− current and releasing ATP to the extracellular medium. In contrast, cells exposed to 200 μm H2O2 VSOR Cl− current onset was significantly slower, and ATP release was not detected. In cells exposed to either 11% hypotonicity or 200 μm H2O2, exogenous addition of ATP in the presence of extracellular Ca2+ resulted in a decrease in the half-time for VSOR Cl− current onset. Conversely, in cells that overexpress a dominant-negative mutant of the ionotropic receptor P2X4 challenged with a 33% hypotonic solution, the half-time for VSOR Cl− current onset was significantly slowed down. Our results indicate that, at high hypotonic imbalances, swelling-induced ATP release activates the purinergic receptor P2X4, which in turn modulates the time course of VSOR Cl− current onset in a extracellular Ca2+-dependent manner. PMID:20056605

  18. Control of volume-sensitive chloride channel inactivation by the coupled action of intracellular chloride and extracellular protons.

    PubMed

    Hernández-Carballo, Carmen Y; De Santiago-Castillo, José A; Rosales-Saavedra, Teresa; Pérez-Cornejo, Patricia; Arreola, Jorge

    2010-08-01

    The volume-sensitive chloride current (I(ClVol)) exhibit a time-dependent decay presumably due to channel inactivation. In this work, we studied the effects of chloride ions (Cl(-)) and H(+) ions on I(ClVol) decay recorded in HEK-293 and HL-60 cells using the whole-cell patch clamp technique. Under control conditions ([Cl(-)](e) = [Cl(-)](i) = 140 mM and pH(i) = pH(e) = 7.3), I(ClVol) in HEK cells shows a large decay at positive voltages but in HL-60 cells I(ClVol) remained constant independently of time. In HEK-293 cells, simultaneously raising the [Cl(-)](e) and [Cl(-)](i) from 25 to 140 mM (with pH(e) = pH(i) = 7.3) increased the fraction of inactivated channels (FIC). This effect was reproduced by elevating [Cl(-)](i) while keeping the [Cl(-)](e) constant. Furthermore, a decrease in pH(e) from 7.3 to 5.5 accelerated current decay and increased FIC when [Cl(-)] was 140 mM but not 25 mM. In HL-60 cells, a slight I(ClVol) decay was seen when the pH(e) was reduced from 7.3 to 5.5. Our data show that inactivation of I(ClVol) can be controlled by changing either the Cl(-) or H(+) concentration or both. Based on our results and previously published data, we have built a model that explains VRAC inactivation. In the model the H(+) binding site is located outside the electrical field near the extracellular entry whilst the Cl(-) binding site is intracellular. The model depicts inactivation as a pore constriction that happens by simultaneous binding of H(+) and Cl(-) ions to the channel followed by a voltage-dependent conformational change that ultimately causes inactivation.

  19. Channeling of electron transport to improve collection efficiency in mesoporous titanium dioxide dye sensitized solar cell stacks

    SciTech Connect

    Fakharuddin, Azhar; Ahmed, Irfan; Yusoff, Mashitah M.; Jose, Rajan E-mail: joserajan@gmail.com; Khalidin, Zulkeflee

    2014-02-03

    Dye-sensitized solar cell (DSC) modules are generally made by interconnecting large photoelectrode strips with optimized thickness (∼14 μm) and show lower current density (J{sub SC}) compared with their single cells. We found out that the key to achieving higher J{sub SC} in large area devices is optimized photoelectrode volume (V{sub D}), viz., thickness and area which facilitate the electron channeling towards working electrode. By imposing constraints on electronic path in a DSC stack, we achieved >50% increased J{sub SC} and ∼60% increment in photoelectric conversion efficiency in photoelectrodes of similar V{sub D} (∼3.36 × 10{sup −4} cm{sup 3}) without using any metallic grid or a special interconnections.

  20. Purification of binding protein for Tityus gamma toxin identified with the gating component of the voltage-sensitive Na+ channel.

    PubMed Central

    Norman, R I; Schmid, A; Lombet, A; Barhanin, J; Lazdunski, M

    1983-01-01

    The gating component associated with the voltage-sensitive Na+ channel from electroplax membranes of Electrophorus electricus has been purified by using toxin gamma from the venom of the scorpion Tityus serrulatus serrulatus. The toxin-binding site was efficiently solubilized with Lubrol PX, resulting in an extract of high initial specific activity. Purification was achieved by adsorption of the toxin-binding component to DEAE-Sephadex A-25 followed by desorption at high ionic strength and chromatography on either wheat germ agglutinin-Ultrogel or Sepharose 6B. Maximal final specific activities were at least 42% of the specific activity expected for a pure toxin-binding component. The purified material exhibited a Stokes radius of 85 A, and sodium dodecyl sulfate/polyacrylamide gel electrophoresis demonstrated a single polypeptide component of Mr 270,000. Furthermore, tetrodotoxin binding activity and Tityus gamma toxin binding activity copurified, suggesting that the selectivity filter and the gating component of the Na+ channel are carried by the same polypeptide chain. Images PMID:6306665

  1. Reconnaissance survey of sulfonamide, sulfonylurea, and imidazolinone herbicides in surface streams and groundwater of the Midwestern United States

    USGS Publications Warehouse

    Steinheimer, T.R.; Pfeiffer, R.L.; Scoggin, K.D.; Battaglin, W.A.

    2000-01-01

    The study objective was to conduct a small scale synoptic survey of representative water resources draining agricultural land for occurrence of several herbicide residues. These new classes of herbicides are commonly applied pre-emergence or post-emergence in conservation tillage systems to control grasses and broadleaf weeds in cropped and noncropped areas. Both surface water and groundwater samples were collected from 44 midwestern locations during the summer of 1997, and analyzed for herbicide residues of 15 sulfonylurea and imidazolinone chemicals, and one sulfonamide. Each site was sampled between mid-June and late-October with several stream sites sampled twice. The method, developed jointly by the chemical manufacturer's and the U.S. Environmental Protection Agency, provides a 100 ng/L limit of quantitation in surface water for all analytes. Analytes were detected and identity confirmed in surface water at six sites and in ground water at two sites. The most frequently detected herbicides were imazaquin, imazethapyr, and nicosulfuron. For field studies in which the source of surface and ground water associated with the farming system on the agricultural landscape is known, the sensitivity of the method can be improved with only minor modifications in detection criteria.

  2. AHR-16303B, a novel antagonist of 5-HT2 receptors and voltage-sensitive calcium channels

    SciTech Connect

    Barrett, R.J.; Appell, K.C.; Kilpatrick, B.F.; Proakis, A.G.; Nolan, J.C.; Walsh, D.A. )

    1991-01-01

    In vivo and in vitro methods were used to characterize AHR-16303B, a novel compound with antagonistic action at 5-HT2 receptors and voltage-sensitive calcium channels. The 5-HT2 receptor-antagonistic properties of AHR-16303B were demonstrated by inhibition of (a) (3H)ketanserin binding to rat cerebral cortical membranes (IC50 = 165 nM); (b) 5-hydroxytryptamine (5-HT)-induced foot edema in rats (minimum effective dose, (MED) = 0.32 mg/kg orally, p.o.); (c) 5-HT-induced vasopressor responses in spontaneously hypertensive rats (SHR) (ID50 = 0.18 mg/kg intravenously (i.v.), 1.8 mg/kg p.o.), (d) 5-HT-induced antidiuresis in rats (MED = 1 mg/kg p.o.), and (e) platelet aggregation induced by 5-HT + ADP (IC50 = 1.5 mM). The calcium antagonist properties of AHR-16303B were demonstrated by inhibition of (a) (3H)nimodipine binding to voltage-sensitive calcium channels on rabbit skeletal muscle membranes (IC50 = 15 nM), (b) KCl-stimulated calcium flux into cultured PC12 cells (IC50 = 81 nM), and (c) CaCl2-induced contractions of rabbit thoracic aortic strips (pA2 = 8.84). AHR-16303B had little or no effect on binding of radioligands to dopamine2 (DA2) alpha 1, alpha 2, H1, 5-HT1 alpha, beta 2, muscarinic M1, or sigma opioid receptors; had no effect on 5-HT3 receptor-mediated vagal bradycardia; and had only minor negative inotropic, chronotropic, and dromotropic effects on isolated guinea pig atria. In conscious SHR, 30 mg/kg p.o. AHR-16303B completely prevented the vasopressor responses to i.v. 5-HT, and decreased blood pressure (BP) by 24% 3 h after dosing.

  3. Confidence Limits with Multiple Channels and Arbitrary Probability Distributions for Sensitivity and Expected Background

    NASA Astrophysics Data System (ADS)

    Perrotta, Andrea

    A MC method is proposed to compute upper limits, in a pure Bayesian approach, when the errors associated to the experimental sensitivity and to the expected background content are not Gaussian distributed or not small enough to apply the usual approximations. It is relatively easy to extend the procedure to the multichannel case (for instance when different decay branchings, or luminosities or experiments have to be combined). Some of the searches for supersymmetric particles performed in the DELPHI experiment at the LEP electron-positron collider use such a procedure to propagate the systematics into the calculation of the cross-section upper limits. One of these searches will be described as an example.

  4. Different Contribution of Redox-Sensitive Transient Receptor Potential Channels to Acetaminophen-Induced Death of Human Hepatoma Cell Line

    PubMed Central

    Badr, Heba; Kozai, Daisuke; Sakaguchi, Reiko; Numata, Tomohiro; Mori, Yasuo

    2016-01-01

    Acetaminophen (APAP) is a safe analgesic antipyretic drug at prescribed doses. Its overdose, however, can cause life-threatening liver damage. Though, involvement of oxidative stress is widely acknowledged in APAP-induced hepatocellular death, the mechanism of this increased oxidative stress and the associated alterations in Ca2+ homeostasis are still unclear. Among members of transient receptor potential (TRP) channels activated in response to oxidative stress, we here identify that redox-sensitive TRPV1, TRPC1, TRPM2, and TRPM7 channels underlie Ca2+ entry and downstream cellular damages induced by APAP in human hepatoma (HepG2) cells. Our data indicate that APAP treatment of HepG2 cells resulted in increased reactive oxygen species (ROS) production, glutathione (GSH) depletion, and Ca2+ entry leading to increased apoptotic cell death. These responses were significantly suppressed by pretreatment with the ROS scavengers N-acetyl-L-cysteine (NAC) and 4,5-dihydroxy-1,3-benzene disulfonic acid disodium salt monohydrate (Tiron), and also by preincubation of cells with the glutathione inducer Dimethylfumarate (DMF). TRP subtype-targeted pharmacological blockers and siRNAs strategy revealed that suppression of either TRPV1, TRPC1, TRPM2, or TRPM7 reduced APAP-induced ROS formation, Ca2+ influx, and cell death; the effects of suppression of TRPV1 or TRPC1, known to be activated by oxidative cysteine modifications, were stronger than those of TRPM2 or TRPM7. Interestingly, TRPV1 and TRPC1 were labeled by the cysteine-selective modification reagent, 5,5′-dithiobis (2-nitrobenzoic acid)-2biotin (DTNB-2Bio), and this was attenuated by pretreatment with APAP, suggesting that APAP and/or its oxidized metabolites act directly on the modification target cysteine residues of TRPV1 and TRPC1 proteins. In human liver tissue, TRPV1, TRPC1, TRPM2, and TRPM7 channels transcripts were localized mainly to hepatocytes and Kupffer cells. Our findings strongly suggest that APAP

  5. Sensitivity of cloned muscle, heart and neuronal voltage-gated sodium channels to block by polyamines: a possible basis for modulation of excitability in vivo.

    PubMed

    Fu, Li-Ying; Cummins, Theodore R; Moczydlowski, Edward G

    2012-01-01

    Spermidine and spermine, are endogenous polyamines (PAs) that regulate cell growth and modulate the activity of numerous ion channel proteins. In particular, intracellular PAs are potent blockers of many different cation channels and are responsible for strong suppression of outward K (+) current, a phenomenon known as inward rectification characteristic of a major class of KIR K (+) channels. We previously described block of heterologously expressed voltage-gated Na (+) channels (NaV) of rat muscle by intracellular PAs and PAs have recently been found to modulate excitability of brain neocortical neurons by blocking neuronal NaV channels. In this study, we compared the sensitivity of four different cloned mammalian NaV isoforms to PAs to investigate whether PA block is a common feature of NaV channel pharmacology. We find that outward Na (+) current of muscle (NaV 1.4), heart (NaV 1.5), and neuronal (NaV 1.2, NaV 1.7) NaV isoforms is blocked by PAs, suggesting that PA metabolism may be linked to modulation of action potential firing in numerous excitable tissues. Interestingly, the cardiac NaV 1.5 channel is more sensitive to PA block than other isoforms. Our results also indicate that rapid binding of PAs to blocking sites in the NaV 1.4 channel is restricted to access from the cytoplasmic side of the channel, but plasma membrane transport pathways for PA uptake may contribute to long-term NaV channel modulation. PAs may also play a role in drug interactions since spermine attenuates the use-dependent effect of the lidocaine, a typical local anesthetic and anti-arrhythmic drug.

  6. The β Subunit Increases the Ca2+ Sensitivity of Large Conductance Ca2+-activated Potassium Channels by Retaining the Gating in the Bursting States

    PubMed Central

    Nimigean, Crina M.; Magleby, Karl L.

    1999-01-01

    Coexpression of the β subunit (KV,Caβ) with the α subunit of mammalian large conductance Ca2+- activated K+ (BK) channels greatly increases the apparent Ca2+ sensitivity of the channel. Using single-channel analysis to investigate the mechanism for this increase, we found that the β subunit increased open probability (Po) by increasing burst duration 20–100-fold, while having little effect on the durations of the gaps (closed intervals) between bursts or on the numbers of detected open and closed states entered during gating. The effect of the β subunit was not equivalent to raising intracellular Ca2+ in the absence of the beta subunit, suggesting that the β subunit does not act by increasing all the Ca2+ binding rates proportionally. The β subunit also inhibited transitions to subconductance levels. It is the retention of the BK channel in the bursting states by the β subunit that increases the apparent Ca2+ sensitivity of the channel. In the presence of the β subunit, each burst of openings is greatly amplified in duration through increases in both the numbers of openings per burst and in the mean open times. Native BK channels from cultured rat skeletal muscle were found to have bursting kinetics similar to channels expressed from alpha subunits alone. PMID:10051518

  7. Intramolecular interaction of SUR2 subtypes for intracellular ADP-Induced differential control of K(ATP) channels.

    PubMed

    Matsushita, Kenji; Kinoshita, Kengo; Matsuoka, Tetsuro; Fujita, Akikazu; Fujikado, Takashi; Tano, Yasuo; Nakamura, Haruki; Kurachi, Yoshihisa

    2002-03-22

    ATP-sensitive K+ (K(ATP)) channels are composed of sulfonylurea receptors (SURs) and inwardly rectifying Kir6.2-channels. The C-terminal 42 amino acid residues (C42) of SURs are responsible for ADP-induced differential activation of K(ATP) channels in SUR-subtypes. By examining ADP-effect on K(ATP) channels containing various chimeras of SUR2A and SUR2B, we identified a segment of 7 residues at central portion of C42 critical for this phenomenon. A 3-D structure model of the region containing the second nucleotide-binding domain (NBD2) of SUR and C42 was developed based on the structure of HisP, a nucleotide-binding protein forming the bacterial Histidine transporter complex. In the model, the polar and charged residues in the critical segment located within a distance that allows their electrostatic interaction with Arg1344 at the Walker-A loop of NBD2. Therefore, the interaction might be involved in the control of ADP-induced differential activation of SUR2-subtype K(ATP) channels.

  8. Purified omega-conotoxin GVIA receptor of rat brain resembles a dihydropyridine-sensitive L-type calcium channel

    SciTech Connect

    McEnery, M.W.; Snowman, A.M.; Sharp, A.H.; Snyder, S.H. ); Adams, M.E. )

    1991-12-15

    The {omega}-conotoxin GVIA (CTX) receptor has been purified 1,900-fold to apparent homogeneity by monitoring both reversible binding of {sup 125}I-labeled CTX ({sup 125}I-CTX) and photoincorporation of N-hydroxysuccinimidyl-4-azidobenzoate-{sup 125}I-CTX (HSA-{sup 125}I-CTX). Photoincorporation of HSA-{sup 125}I-CTX into a 230-kDa protein exhibits a pharmacologic and chromatographic profile indicating that the 230-kDa protein is the CTX-binding subunit of the receptor. The pharmacologic specificity of {sup 125}I-CTX binding to the purified CTX receptor closely resembles that of the native membrane-bound form with respect to sensitivity towards CTX and other peptide toxin antagonists. The purified CTX receptor comprises the 230-kDa protein ({alpha}{sub 1}) and four additional proteins with apparent molecular masses of 140 ({alpha}{sub 2}), 110, 70 ({beta}{sub 2}), and 60({beta}{sub 1}) kDa. This subunit structure closely resembles that of the 1,4-dihydropyridine-sensitive L-type calcium channel.

  9. Upregulation of swelling-activated Cl− channel sensitivity to cell volume by activation of EGF receptors in murine mammary cells

    PubMed Central

    Abdullaev, Iskandar F; Sabirov, Ravshan Z; Okada, Yasunobu

    2003-01-01

    Whole-cell recordings showed that, in mouse mammary C127 cells transfected with the full genome of the bovine papilloma virus (BPV), a hypotonic challenge induced the activation of outwardly rectifying Cl− currents with a peak amplitude 2.7 times greater than that in control C127 cells. Cell-attached single-channel recordings showed that BPV-induced augmentation of the peak amplitude of the whole-cell current could not chiefly be explained by a small increase (1.2 times) in unitary conductance. There was no difference between control and BPV-transfected cells in the osmotic cell swelling rate, and hence, osmotic water permeability. However, a plot of the whole-cell current density as a function of cell volume, which was measured simultaneously, showed that the BPV-transfected cells had a strikingly greater volume sensitivity than control cells. Since the E5 protein of BPV has been reported to induce constitutive activation of the epidermal growth factor (EGF) receptor and platelet-derived growth factor (PDGF) receptor in a variety of cell lines including C127 cells, effects of the growth factors on volume-sensitive outwardly rectifying (VSOR) Cl− currents were examined in C127 cells. Application of PDGF peptides failed to affect the Cl− currents in control and BPV-transfected cells, although C127 cells are known to endogenously express PDGF receptors. In contrast, EGF peptides significantly increased the VSOR Cl− current in control cells. However, they failed to induce further augmentation of the current in BPV-transfected cells. VSOR Cl− currents were inhibited by tyrphostin B46, an inhibitor of the EGF receptor tyrosine kinase, in both control and BPV-transfected cells. The IC50 value in BPV-transfected cells (12 μm) was lower than that in control cells (31 μm). However, the VSOR Cl− currents in both cell types were insensitive to tyrphostin AG1296, an inhibitor of the PDGF receptor tyrosine kinase. The rate of regulatory volume decrease (RVD) was

  10. ATP-sensitive potassium channel-mediated lactate effect on orexin neurons: implications for brain energetics during arousal.

    PubMed

    Parsons, Matthew P; Hirasawa, Michiru

    2010-06-16

    Active neurons have a high demand for energy substrate, which is thought to be mainly supplied as lactate by astrocytes. Heavy lactate dependence of neuronal activity suggests that there may be a mechanism that detects and controls lactate levels and/or gates brain activation accordingly. Here, we demonstrate that orexin neurons can behave as such lactate sensors. Using acute brain slice preparations and patch-clamp techniques, we show that the monocarboxylate transporter blocker alpha-cyano-4-hydroxycinnamate (4-CIN) inhibits the spontaneous activity of orexin neurons despite the presence of extracellular glucose. Furthermore, fluoroacetate, a glial toxin, inhibits orexin neurons in the presence of glucose but not lactate. Thus, orexin neurons specifically use astrocyte-derived lactate. The effect of lactate on firing activity is concentration dependent, an essential characteristic of lactate sensors. Furthermore, lactate disinhibits and sensitizes these neurons for subsequent excitation. 4-CIN has no effect on the activity of some arcuate neurons, indicating that lactate dependency is not universal. Orexin neurons show an indirect concentration-dependent sensitivity to glucose below 1 mm, responding by hyperpolarization, which is mediated by ATP-sensitive potassium channels composed of Kir6.1 and SUR1 subunits. In conclusion, our study suggests that lactate is a critical energy substrate and a regulator of the orexin system. Together with the known effects of orexins in inducing arousal, food intake, and hepatic glucose production, as well as lactate release from astrocytes in response to neuronal activity, our study suggests that orexin neurons play an integral part in balancing brain activity and energy supply.

  11. Pituitary adenylate cyclase activating polypeptide (PACAP) dilates cerebellar arteries through activation of large-conductance Ca(2+)-activated (BK) and ATP-sensitive (K ATP) K (+) channels.

    PubMed

    Koide, Masayo; Syed, Arsalan U; Braas, Karen M; May, Victor; Wellman, George C

    2014-11-01

    Pituitary adenylate cyclase activating polypeptide (PACAP) is a potent vasodilator of numerous vascular beds, including cerebral arteries. Although PACAP-induced cerebral artery dilation is suggested to be cyclic AMP (cAMP)-dependent, the downstream intracellular signaling pathways are still not fully understood. In this study, we examined the role of smooth muscle K(+) channels and hypothesized that PACAP-mediated increases in cAMP levels and protein kinase A (PKA) activity result in the coordinate activation of ATP-sensitive K(+) (KATP) and large-conductance Ca(2+)-activated K(+) (BK) channels for cerebral artery dilation. Using patch-clamp electrophysiology, we observed that PACAP enhanced whole-cell KATP channel activity and transient BK channel currents in freshly isolated rat cerebellar artery myocytes. The increased frequency of transient BK currents following PACAP treatment is indicative of increased intracellular Ca(2+) release events termed Ca(2+) sparks. Consistent with the electrophysiology data, the PACAP-induced vasodilations of cannulated cerebellar artery preparations were attenuated by approximately 50 % in the presence of glibenclamide (a KATP channel blocker) or paxilline (a BK channel blocker). Further, in the presence of both blockers, PACAP failed to cause vasodilation. In conclusion, our results indicate that PACAP causes cerebellar artery dilation through two mechanisms: (1) KATP channel activation and (2) enhanced BK channel activity, likely through increased Ca(2+) spark frequency.

  12. Sodium channel diversity in the vestibular ganglion: NaV1.5, NaV1.8, and tetrodotoxin-sensitive currents.

    PubMed

    Liu, Xiao-Ping; Wooltorton, Julian R A; Gaboyard-Niay, Sophie; Yang, Fu-Chia; Lysakowski, Anna; Eatock, Ruth Anne

    2016-05-01

    Firing patterns differ between subpopulations of vestibular primary afferent neurons. The role of sodium (NaV) channels in this diversity has not been investigated because NaV currents in rodent vestibular ganglion neurons (VGNs) were reported to be homogeneous, with the voltage dependence and tetrodotoxin (TTX) sensitivity of most neuronal NaV channels. RT-PCR experiments, however, indicated expression of diverse NaV channel subunits in the vestibular ganglion, motivating a closer look. Whole cell recordings from acutely dissociated postnatal VGNs confirmed that nearly all neurons expressed NaV currents that are TTX-sensitive and have activation midpoints between -30 and -40 mV. In addition, however, many VGNs expressed one of two other NaV currents. Some VGNs had a small current with properties consistent with NaV1.5 channels: low TTX sensitivity, sensitivity to divalent cation block, and a relatively negative voltage range, and some VGNs showed NaV1.5-like immunoreactivity. Other VGNs had a current with the properties of NaV1.8 channels: high TTX resistance, slow time course, and a relatively depolarized voltage range. In two NaV1.8 reporter lines, subsets of VGNs were labeled. VGNs with NaV1.8-like TTX-resistant current also differed from other VGNs in the voltage dependence of their TTX-sensitive currents and in the voltage threshold for spiking and action potential shape. Regulated expression of NaV channels in primary afferent neurons is likely to selectively affect firing properties that contribute to the encoding of vestibular stimuli.

  13. [The influence of activation of the ATP-sensitive potassium channels by flocalin on the function of the cardiovascular system].

    PubMed

    Strutyns'kyĭ, R B; Rovenets', R A; Strutyns'ka, N A; Neshcheret, O P; Moĭbenko, O O

    2013-01-01

    In experiments on the anaesthetized dogs the influence of a new fluorine-containing opener of ATP-sensitive potassium (K(ATP)) channels flocalin on the cardiohemodynamic of great animals in vivo was studied. Flocalin introduced intravenously in doses 0.01 - 1.5 mgs/kg. It is shown that it reduces in dose-dependent manner a system arterial pressure, perfusion pressure in coronary artery and general peripheral resistance of vessels with maximal effects on 56.8 +/- 2.7, 22.4 +/- 4.7 and 47.2% +/- 6.5% accordingly at most dose 1.5 mgs/kg. Flocalin causes development of cardiodepressive reactions in heart, that is exhibited in dose-dependent the decrease of pressure in the left ventricle, speed of growth (dP/dt(max)) and reduction (dP/dt(min)) in it's of pressure with maximal effects on 37.1 +/- 5.1, 51.2 +/- 9.4 and 55.6% +/- 6.9% accordingly at introduction of most dose of flocalin. Diminish of the cardiac out put and heart rate with a maximal effects on 23.1% +/-12.7% and 19.2% +/- 1.7% accordingly at a dose 1.0 mgs/kg was shown. It should be noted that considerable reduction of heart rate and general peripheral resistance of vessels takes place only at the large doses of flocalin - 1 and 1.5 mgs/kg. Thus, it is shown that activation of K(ATP) channels by flocalin causes the dose-dependent decrease of pressure in the system of circulation of blood and contraction activity of myocardium.

  14. Overexpressed transient receptor potential vanilloid 3 ion channels in skin keratinocytes modulate pain sensitivity via prostaglandin E2.

    PubMed

    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

    2008-12-17

    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 transient receptor potential vanilloid (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 with wild-type controls, keratinocytes overexpressing TRPV3 exhibited larger currents as well as augmented prostaglandin E(2) (PGE(2)) release in response to two TRPV3 agonists, 2-aminoethoxydiphenyl borate (2APB) and heat. Thermal selection behavior and heat-evoked withdrawal behavior of naive mice overexpressing TRPV3 were not consistently altered. Upon selective pharmacological inhibition of TRPV1 with JNJ-17203212 [corrected], however, the keratinocyte-specific TRPV3 transgenic mice showed increased escape responses to noxious heat relative to their wild-type littermates. Coadministration 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 PGE(2).

  15. Mutations in the house fly Vssc1 sodium channel gene associated with super-kdr resistance abolish the pyrethroid sensitivity of Vssc1/tipE sodium channels expressed in Xenopus oocytes.

    PubMed

    Lee, S H; Smith, T J; Knipple, D C; Soderlund, D M

    1999-02-01

    The super-kdr insecticide resistance trait of the house fly confers resistance to pyrethroids and DDT by reducing the sensitivity of the fly nervous system. The super-kdr genetic locus is tightly linked to the Vssc1 gene, which encodes a voltage-sensitive sodium channel alpha subunit that is the principal site of pyrethroid action. DNA sequence analysis of Vssc1 alleles from several independent super-kdr fly strains identified two amino acid substitutions associated with the super-kdr trait: replacement of leucine at position 1014 with phenylalanine (L1014F), which has been shown to cause the kdr resistance trait in this species, and replacement of methionine at position 918 with threonine (M918T). We examined the functional significance of these mutations by expressing house fly sodium channels containing them in Xenopus laevis oocytes and by characterizing the biophysical properties and pyrethroid sensitivities of the expressed channels using two-electrode voltage clamp. House fly sodium channels that were specifically modified by site-directed mutagenesis to contain the M918T/L1014F double mutation gave reduced levels of sodium current expression in oocytes but otherwise exhibited functional properties similar to those of wildtype channels and channels containing the L1014F substitution. However, M918T/L1014F channels were completely insensitive to high concentrations of the pyrethroids cismethrin and cypermethrin. House fly sodium channels specifically modified to contain the M918T single mutation, which is not known to exist in nature except in association with the L1014F mutation, gave very small sodium currents in oocytes. Assays of these currents in the presence of high concentrations of cismethrin suggest that this mutation alone is sufficient to abolish the pyrethroid sensitivity of house fly sodium channels. These results define the functional significance of the Vssc1 mutations associated with the super-kdr trait of the house fly and are consistent with

  16. Phosphorylation regulates the sensitivity of voltage‐gated Kv7.2 channels towards phosphatidylinositol‐4,5‐bisphosphate

    PubMed Central

    Salzer, Isabella; Erdem, Fatma Asli; Chen, Wei‐Qiang; Heo, Seok; Koenig, Xaver; Schicker, Klaus W.; Kubista, Helmut; Lubec, Gert; Boehm, Stefan

    2016-01-01

    Key points Phosphatidylinositol‐4,5‐bisphosphate (PIP2) is a key regulator of many membrane proteins, including voltage‐gated Kv7.2 channels.In this study, we identified the residues in five phosphorylation sites and their corresponding protein kinases, the former being clustered within one of four putative PIP2‐binding domains in Kv7.2.Dephosphorylation of these residues reduced the sensitivity of Kv7.2 channels towards PIP2.Dephosphorylation of Kv7.2 affected channel inhibition via M1 muscarinic receptors, but not via bradykinin receptors.Our data indicated that phosphorylation of the Kv7.2 channel was necessary to maintain its low affinity for PIP2, thereby ensuring the tight regulation of the channel via G protein‐coupled receptors. Abstract The function of numerous ion channels is tightly controlled by G protein‐coupled receptors (GPCRs). The underlying signalling mechanisms may involve phosphorylation of channel proteins and participation of phosphatidylinositol‐4,5‐bisphosphate (PIP2). Although the roles of both mechanisms have been investigated extensively, thus far only little has been reported on their interaction in channel modulation. GPCRs govern Kv7 channels, the latter playing a major role in the regulation of neuronal excitability by determining the levels of PIP2 and through phosphorylation. Using liquid chromatography‐coupled mass spectrometry for Kv7.2 immunoprecipitates of rat brain membranes and transfected cells, we mapped a cluster of five phosphorylation sites in one of the PIP2‐binding domains. To evaluate the effect of phosphorylation on PIP2‐mediated Kv7.2 channel regulation, a quintuple alanine mutant of these serines (S427/S436/S438/S446/S455; A5 mutant) was generated to mimic the dephosphorylated state. Currents passing through these mutated channels were less sensitive towards PIP2 depletion via the voltage‐sensitive phosphatase Dr‐VSP than were wild‐type channels. In vitro phosphorylation assays with the

  17. Calcium-dependent glutamate release during neuronal development and synaptogenesis: different involvement of omega-agatoxin IVA- and omega-conotoxin GVIA-sensitive channels.

    PubMed Central

    Verderio, C; Coco, S; Fumagalli, G; Matteoli, M

    1995-01-01

    Hippocampal neurons maintained in primary culture recycle synaptic vesicles and express functional glutamate receptors since early stages of neuronal development. By analyzing glutamate-induced cytosolic calcium changes to sense presynaptically released neurotransmitter, we demonstrate that the ability of neurons to release glutamate in the extracellular space is temporally coincident with the property of synaptic vesicles to undergo exocytotic-endocytotic recycling. Neuronal differentiation and maturation of synaptic contacts coincide with a change in the subtype of calcium channels primarily involved in controlling neurosecretion. Whereas omega-agatoxin IVA-sensitive channels play a role in controlling neurotransmitter secretion at all stages of neuronal differentiation, omega-conotoxin GVIA-sensitive channels are primarily involved in mediating glutamate release at early developmental stages only. PMID:7604011

  18. EFFECTS OF PYRETHROIDS ON VOLTAGE-SENSITIVE CALCIUM CHANNELS: A CRITICAL EVALUATION OF STRENGTHS, WEAKNESSES, DATA NEEDS, AND RELATIONSHIP TO ASSESSMENT OF CUMULATIVE NEUROTOXICITY.

    EPA Science Inventory

    A recently published review (Soderlund et al., 2002, Toxicology 171, 3-59.) of the mechanisms of acute neurotoxicity of pyrethroid compounds postulated that voltage-sensitive calcium channels (VSCC) may be a target of some pyrethroid compounds and that effects on VSCC may contrib...

  19. How important is the synclinal conformation of sulfonylureas to explain the inhibition of AHAS: a theoretical study.

    PubMed

    Jaña, Gonzalo A; Delgado, Eduardo J; Medina, Fabiola E

    2014-03-24

    The inhibitory activity of 15 sulfonylureas on acetohydroxyacid synthase (AHAS) is addressed theoretically in order to stress how important the conformation is to explain their differences as AHAS inhibitors. The study includes calculations in gas phase, solution, and in the enzymatic environment. The results suggest that both the activation Gibbs free energy and Gibbs free energy change associated with the conformational change in solution allow for determining if sulfonylureas should have high or low inhibition activity. QM/MM calculations were also carried out in order to identify the role of the amino acid residues and the effects involved in the stabilization of the active conformation in the binding pocket. On the other hand, the analysis of the frontier molecular orbitals of the sulfonylureas in the binding pocket allowed us to explain the inhibitory activity in terms of the reactivity of the carbonyl carbon.

  20. Sigma-1 receptor alters the kinetics of Kv1.3 voltage gated potassium channels but not the sensitivity to receptor ligands.

    PubMed

    Kinoshita, Maho; Matsuoka, Yoshikazu; Suzuki, Takeshi; Mirrielees, Jennifer; Yang, Jay

    2012-05-03

    Sigma1 receptors (Sigma1R) are intracellular chaperone proteins that bind psychotropic drugs and also clinically used drugs such as ketamine and haloperidol. Co-expression of the Sigma1R has been reported to enhance the sensitivity of several voltage-gated ion channels to Sigma1R ligands. Kv1.3 is the predominant voltage-gated potassium channel expressed in T lymphocytes with a documented role in immune activation. To gain a better understanding of Sigma1R modulation of Kv ion channels, we investigated the effects of Sigma1R co-expression on Kv1.3 physiology and pharmacology in ion channels expressed in Xenopus oocytes. We also explored the protein domains of Kv1.3 necessary for protein:protein interaction between Kv1.3 and Sigma1R through co-immunoprecipitation studies. Slowly inactivating outward-going currents consistent with Kv1.3 expression were elicited on step depolarizations. The current characterized by E(rev), V(1/2), and slope factor remained unchanged when co-expressed with Sigma1R. Analysis of inactivation time constant revealed a faster Kv1.3 current decay when co-expressed with Sigma1R. However the sensitivity to Sigma1R ligands remained unaltered when co-expressed with the Sigma1R in contrast to the previously reported modulation of ligand sensitivity in closely related Kv1.4 and Kv1.5 voltage gated potassium channels. Co-immunoprecipitation assays of various Kv1.3 truncation constructs indicated that the transmembrane domain of the Kv1.3 protein was responsible for the protein:protein interaction with the Sigma1R. Sigma1R likely interacts with different domains of Kv ion channel family proteins resulting in distinct modulation of different channels.

  1. Substitutions in the Domain III Voltage-sensing Module Enhance the Sensitivity of an Insect Sodium Channel to a Scorpion β-Toxin*

    PubMed Central

    Song, Weizhong; Du, Yuzhe; Liu, Zhiqi; Luo, Ningguang; Turkov, Michael; Gordon, Dalia; Gurevitz, Michael; Goldin, Alan L.; Dong, Ke

    2011-01-01

    Scorpion β-toxins bind to the extracellular regions of the voltage-sensing module of domain II and to the pore module of domain III in voltage-gated sodium channels and enhance channel activation by trapping and stabilizing the voltage sensor of domain II in its activated state. We investigated the interaction of a highly potent insect-selective scorpion depressant β-toxin, Lqh-dprIT3, from Leiurus quinquestriatus hebraeus with insect sodium channels from Blattella germanica (BgNav). Like other scorpion β-toxins, Lqh-dprIT3 shifts the voltage dependence of activation of BgNav channels expressed in Xenopus oocytes to more negative membrane potentials but only after strong depolarizing prepulses. Notably, among 10 BgNav splice variants tested for their sensitivity to the toxin, only BgNav1-1 was hypersensitive due to an L1285P substitution in IIIS1 resulting from a U-to-C RNA-editing event. Furthermore, charge reversal of a negatively charged residue (E1290K) at the extracellular end of IIIS1 and the two innermost positively charged residues (R4E and R5E) in IIIS4 also increased the channel sensitivity to Lqh-dprIT3. Besides enhancement of toxin sensitivity, the R4E substitution caused an additional 20-mV negative shift in the voltage dependence of activation of toxin-modified channels, inducing a unique toxin-modified state. Our findings provide the first direct evidence for the involvement of the domain III voltage-sensing module in the action of scorpion β-toxins. This hypersensitivity most likely reflects an increase in IIS4 trapping via allosteric mechanisms, suggesting coupling between the voltage sensors in neighboring domains during channel activation. PMID:21454658

  2. Activation of cGMP-Dependent Protein Kinase Stimulates Cardiac ATP-Sensitive Potassium Channels via a ROS/Calmodulin/CaMKII Signaling Cascade

    PubMed Central

    Chai, Yongping; Zhang, Dai-Min; Lin, Yu-Fung

    2011-01-01

    Background Cyclic GMP (cGMP)-dependent protein kinase (PKG) is recognized as an important signaling component in diverse cell types. PKG may influence the function of cardiac ATP-sensitive potassium (KATP) channels, an ion channel critical for stress adaptation in the heart; however, the underlying mechanism remains largely unknown. The present study was designed to address this issue. Methods and Findings Single-channel recordings of cardiac KATP channels were performed in both cell-attached and inside-out patch configurations using transfected human embryonic kidney (HEK)293 cells and rabbit ventricular cardiomyocytes. We found that Kir6.2/SUR2A (the cardiac-type KATP) channels were activated by cGMP-selective phosphodiesterase inhibitor zaprinast in a concentration-dependent manner in cell-attached patches obtained from HEK293 cells, an effect mimicked by the membrane-permeable cGMP analog 8-bromo-cGMP whereas abolished by selective PKG inhibitors. Intriguingly, direct application of PKG moderately reduced rather than augmented Kir6.2/SUR2A single-channel currents in excised, inside-out patches. Moreover, PKG stimulation of Kir6.2/SUR2A channels in intact cells was abrogated by ROS/H2O2 scavenging, antagonism of calmodulin, and blockade of calcium/calmodulin-dependent protein kinase II (CaMKII), respectively. Exogenous H2O2 also concentration-dependently stimulated Kir6.2/SUR2A channels in intact cells, and its effect was prevented by inhibition of calmodulin or CaMKII. PKG stimulation of KATP channels was confirmed in intact ventricular cardiomyocytes, which was ROS- and CaMKII-dependent. Kinetically, PKG appeared to stimulate these channels by destabilizing the longest closed state while stabilizing the long open state and facilitating opening transitions. Conclusion The present study provides novel evidence that PKG exerts dual regulation of cardiac KATP channels, including marked stimulation resulting from intracellular signaling mediated by ROS (H2O2 in

  3. [Determination of 32 sulfonylurea herbicide residues in sweet corns and green soybeans by QuEChERS-liquid chromatography-tandem mass spectrometry].

    PubMed

    Wang, Lianzhu; Huang, Xiaoyan; Wang, Dengfei; Chen, Yong; Xu, Dunming; Zhou, Yu

    2015-05-01

    A fast method based on QuEChERS methodology for the simultaneous determination of 32 sulfonylurea herbicide residues in sweet corns and green soybeans was developed using liquid chromatography-tandem mass spectrometry (LC-MS/MS). The clean-up effects of three dispersive sorbents were evaluated in terms of the residue mass for extracts after evaporation and recoveries. The three sorbents were C18, a mixture of two sorbents--silica coated with zirconium dioxide (Z-Sep) and C18, a bonded C18 zirconia-coated silica (Z-Sep+). As a result, the best effects were obtained from using Z-Sep/C18 sorbents. The samples were extracted with acetonitrile, and salted out with anhydrous magnesium sulphate and sodium chloride. The extracts were cleaned up with dispersive solid phase extraction using Z-Sep/C18 sorbents. Chromatographic analysis was carried out using a CSH C18 column with gradient elution. The pesticides were analyzed by negative electrospray ionization tandem mass spectrometry under scheduled multiple reaction monitoring mode. The quantification was achieved using matrix-matched standard calibrations as the external standard. The recoveries at fortification levels of 10, 20, 100 µg/kg in sweet corns and green beans ranged from 80.0% to 108.2% with the relative standard deviations of 1.2%-13.0%. The limits of quantification (S/N ≥ 10) were 0.2-5.0 µg/kg. The method has been proven to be simple, sensitive, environmental, and thus suitable for the determination of the 32 sulfonylurea herbicide residues in sweet corns and green soy- beans.

  4. Sevoflurane postconditioning affects post-ischaemic myocardial mitochondrial ATP-sensitive potassium channel function and apoptosis in ageing rats.

    PubMed

    Jiang, Jing-Jing; Li, Chao; Li, Heng; Zhang, Lei; Lin, Zong-Hang; Fu, Bao-Jun; Zeng, Yin-Ming

    2016-05-01

    This study investigated the effect of sevoflurane postconditioning on post-ischaemic cardiac function, infarct size, myocardial mitochondrial ATP-sensitive potassium channel (mitoKATP) function and apoptosis in ageing rats to determine the possible mechanism underlying the cardioprotective property of sevoflurane. Ageing rat hearts were isolated and attached to a Langendorff apparatus. The hearts were then exposed or not to sevoflurane postconditioning in the presence or absence of 100 μmol/L 5-hydroxydecanoate (5-HD), a selective mitoKATP inhibitor. The infarct size was measured by triphenyltetrazolium chloride (TTC) staining. Mitochondrial morphology was observed by electron microscopy and scored using FlaMeng semiquantitative analysis. In addition, the expression levels of Bax, Bcl-2, and cytochrome-C (Cyt-C) were determined by Western blot analysis at the end of reperfusion. Sevoflurane postconditioning increased coronary flow, improved functional recovery, reduced Bax/Bcl-2 and Cyt-C phosphorylation levels, and decreased mitochondrial lesion severity and the extent of apoptosis. The protective effects of sevoflurane postconditioning were prevented by the mitoKATP inhibitor 5-HD. Sevoflurane postconditioning significantly protected the function of ageing hearts that were subjected to ischaemia and reperfusion, and these protective effects were mediated by mitoKATP opening.

  5. Voltage-gated Na+ channel II immunoreactivity is selectively up-regulated in hippocampal interneurons of seizure sensitive gerbils.

    PubMed

    Kim, Ji-Eun; Kwak, Sung-Eun; Choi, Hui-Chul; Song, Hong-Ki; Kim, Yeong-In; Jo, Seung-Mook; Kang, Tae-Cheon

    2008-06-27

    In the present study, we investigated the distribution of voltage-gated Na(+) channels (VGSCs) in the normal and epileptic hippocampus of gerbils (a genetic epilepsy model) in order to confirm the relationship between VGSC and seizure activity in these animals. There was no difference of VGSC I immunoreactivity in the hippocampus between seizure-resistant (SR) and seizure sensitive (SS) gerbils. VGSC II immunoreactivity was rarely detected in the perikarya of principal neurons and interneurons in the SR gerbil hippocampus. However, in the SS gerbil hippocampus, VGSC II immunoreactivity was densely observed in the somata of interneurons located in the stratum radiatum and stratum lacunosum-moleculare. Double immunofluorescent study showed immunoreactivity for calretinin (approximately 80% in VGSC II-positive neurons) or calbindin D-28k (approximately 20% in VGSC II-positive neurons) in VGSC II-immunoreactive neurons. VGSC II-immunoreactive neurons did not show parvalbumin immunoreactivity. These findings suggest that seizure activity in SS gerbils may be related to the selective hyperactivation of interneurons in stratum lacunosum-moleculare via the up-regulation of VGSC II expression, which leads to the disinhibition of CA1 pyramidal cells.

  6. A genome-wide study of panic disorder suggests the amiloride-sensitive cation channel 1 as a candidate gene.

    PubMed

    Gregersen, Noomi; Dahl, Hans A; Buttenschøn, Henriette N; Nyegaard, Mette; Hedemand, Anne; Als, Thomas D; Wang, August G; Joensen, Sofus; Woldbye, David Pd; Koefoed, Pernille; Kristensen, Ann S; Kruse, Torben A; Børglum, Anders D; Mors, Ole

    2012-01-01

    Panic disorder (PD) is a mental disorder with recurrent panic attacks that occur spontaneously and are not associated to any particular object or situation. There is no consensus on what causes PD. However, it is recognized that PD is influenced by environmental factors, as well as genetic factors. Despite a significant hereditary component, genetic studies have only been modestly successful in identifying genes of importance for the development of PD. In this study, we conducted a genome-wide scan using microsatellite markers and PD patients and control individuals from the isolated population of the Faroe Islands. Subsequently, we conducted a fine mapping, which revealed the amiloride-sensitive cation channel 1 (ACCN1) located on chromosome 17q11.2-q12 as a potential candidate gene for PD. The further analyses of the ACCN1 gene using single-nucleotide polymorphisms (SNPs) revealed significant association with PD in an extended Faroese case-control sample. However, analyses of a larger independent Danish case-control sample yielded no substantial significant association. This suggests that the possible risk alleles associated in the isolated population are not those involved in the development of PD in a larger outbred population.

  7. Actions of ethanol on voltage-sensitive sodium channels: effects on neurotoxin-stimulated sodium uptake in synaptosomes.

    PubMed

    Mullin, M J; Hunt, W A

    1985-02-01

    Exposure of rat brain synaptosomes to ethanol in vitro reduced the neurotoxin-stimulated uptake of 22Na+. This effect of ethanol was concentration-dependent, occurred with concentrations of ethanol achieved in vivo and was fully reversible. The inhibitory effect of ethanol on neurotoxin-stimulated sodium uptake was due to a decrease in the maximal effect of the neurotoxins. Ethanol reduced the rate of batrachotoxin-stimulated sodium uptake when measured at 3, 5 and 7 but not 10 or 20 sec after the addition of 22Na+. In a series of aliphatic alcohols, there was a good correlation between potency for inhibition of batrachotoxin-stimulated 22Na+ uptake and the membrane/buffer partition coefficient, suggesting that a hydrophobic site in the membrane was involved in the action of the alcohols. Ethanol did not affect the scorpion venom-induced enhancement of batrachotoxin-stimulated sodium uptake. The inhibitory potency of tetrodotoxin was also unaffected by ethanol. These results demonstrate that ethanol has an inhibitory effect on neurotoxin-stimulated sodium influx occurring in voltage-sensitive sodium channels of brain tissue.

  8. On-channel base stacking in microchip capillary gel electrophoresis for high-sensitivity DNA fragment analysis.

    PubMed

    Kim, Dae-Kwang; Kang, Seong Ho

    2005-01-28

    We evaluated a novel strategy for high-sensitivity DNA fragment analysis in a conventional glass double-T microfluidic chip. The microchip allows for a DNA on-channel concentration based on base stacking (BS) with a microchip capillary gel electrophoretic (MCGE) separation step in a poly(vinylpyrrolidone) (PVP) sieving matrix. Depending if low conductivity caused a neutralization reaction between the hydroxide ions and the run buffer component Tris+, the stacking of DNA fragments were processed in the microchip. Compared to a conventional MCGE separation with a normal electrokinetic injection, the peak heights of 50-2650-base pair (bp) DNA fragments on the MCGE-BS separation were increased 3.9-8.0-fold. When we applied the MCGE-BS method to the analysis of a clinical sample of bovine theileria after PCR reaction, the peak height intensity of the amplified 816-bp DNA fragment from the 18S rRNA of T. buffeli was enhanced 7.0-fold compared to that of the normal injection method.

  9. Channelized debris flow hazard mitigation through the use of flexible barriers: a simplified computational approach for a sensitivity analysis.

    NASA Astrophysics Data System (ADS)

    Segalini, Andrea; Ferrero, Anna Maria; Brighenti, Roberto

    2013-04-01

    A channelized debris flow is usually represented by a mixture of solid particles of various sizes and water, flowing along a laterally confined inclined channel-shaped region up to an unconfined area where it slow down its motion and spreads out into a flat-shaped mass. The study of these phenomena is very difficult due to their short duration and unpredictability, lack of historical data for a given basin and complexity of the involved mechanical phenomena. The post event surveys allow for the identification of some depositional features and provide indication about the maximum flow height; however they lack information about development of the phenomena with time. For this purpose the monitoring of recursive events has been carried out by several Authors. Most of the studies, aimed at the determination of the characteristic features of a debris flow, were carried out in artificial channels, where the main involved variables were measured and other where controlled during the tests; however, some uncertainties remained and other scaled models where developed to simulate the deposition mechanics as well as to analyze the transportation mechanics and the energy dissipation. The assessment of the mechanical behavior of the protection structures upon impact with the flow as well as the energy associated to it are necessary for the proper design of such structures that, in densely populated area, can avoid victims and limit the destructive effects of such a phenomenon. In this work a simplified structural model, developed by the Authors for the safety assessment of retention barrier against channelized debris flow, is presented and some parametric cases are interpreted through the proposed approach; this model is developed as a simplified and efficient tool to be used for the verification of the supporting cables and foundations of a flexible debris flow barrier. The present analytical and numerical-based approach has a different aim of a FEM model. The computational

  10. Slo1 Tail Domains, but Not the Ca2+ Bowl, Are Required for the β1 Subunit to Increase the Apparent Ca2+ Sensitivity of BK Channels

    PubMed Central

    Qian, Xiang; Nimigean, Crina M.; Niu, Xiaowei; Moss, Brenda L.; Magleby, Karl L.

    2002-01-01

    Functional large-conductance Ca2+- and voltage-activated K+ (BK) channels can be assembled from four α subunits (Slo1) alone, or together with four auxiliary β1 subunits to greatly increase the apparent Ca2+ sensitivity of the channel. We examined the structural features involved in this modulation with two types of experiments. In the first, the tail domain of the α subunit, which includes the RCK2 (regulator of K+ conductance) domain and Ca2+ bowl, was replaced with the tail domain of Slo3, a BK-related channel that lacks both a Ca2+ bowl and high affinity Ca2+ sensitivity. In the second, the Ca2+ bowl was disrupted by mutations that greatly reduce the apparent Ca2+ sensitivity. We found that the β1 subunit increased the apparent Ca2+ sensitivity of Slo1 channels, independently of whether the α subunits were expressed as separate cores (S0-S8) and tails (S9-S10) or full length, and this increase was still observed after the Ca2+ bowl was mutated. In contrast, β1 subunits no longer increased Ca2+ sensitivity when Slo1 tails were replaced by Slo3 tails. The β1 subunits were still functionally coupled to channels with Slo3 tails, as DHS-I and 17 β-estradiol activated these channels in the presence of β1 subunits, but not in their absence. These findings indicate that the increase in apparent Ca2+ sensitivity induced by the β1 subunit does not require either the Ca2+ bowl or the linker between the RCK1 and RCK2 domains, and that Slo3 tails cannot substitute for Slo1 tails. The β1 subunit also induced a decrease in voltage sensitivity that occurred with either Slo1 or Slo3 tails. In contrast, the β1 subunit–induced increase in apparent Ca2+ sensitivity required Slo1 tails. This suggests that the allosteric activation pathways for these two types of actions of the β1 subunit may be different. PMID:12451052

  11. A novel substituted aminoquinoline selectively targets voltage-sensitive sodium channel isoforms and NMDA receptor subtypes and alleviates chronic inflammatory and neuropathic pain.

    PubMed

    Tabakoff, Boris; Ren, Wenhua; Vanderlinden, Lauren; Snell, Lawrence D; Matheson, Christopher J; Wang, Ze-Jun; Levinson, Rock; Smothers, C Thetford; Woodward, John J; Honse, Yumiko; Lovinger, David; Rush, Anthony M; Sather, William A; Gustafson, Daniel L; Hoffman, Paula L

    2016-08-05

    Recent understanding of the systems that mediate complex disease states, has generated a search for molecules that simultaneously modulate more than one component of a pathologic pathway. Chronic pain syndromes are etiologically connected to functional changes (sensitization) in both peripheral sensory neurons and in the central nervous system (CNS). These functional changes involve modifications of a significant number of components of signal generating, signal transducing and signal propagating pathways. Our analysis of disease-related changes which take place in sensory neurons during sensitization led to the design of a molecule that would simultaneously inhibit peripheral NMDA receptors and voltage sensitive sodium channels. In the current report, we detail the selectivity of N,N-(diphenyl)-4-ureido-5,7-dichloro-2-carboxy-quinoline (DCUKA) for action at NMDA receptors composed of different subunit combinations and voltage sensitive sodium channels having different α subunits. We show that DCUKA is restricted to the periphery after oral administration, and that circulating blood levels are compatible with its necessary concentrations for effects at the peripheral cognate receptors/channels that were assayed in vitro. Our results demonstrate that DCUKA, at concentrations circulating in the blood after oral administration, can modulate systems which are upregulated during peripheral sensitization, and are important for generating and conducting pain information to the CNS. Furthermore, we demonstrate that DCUKA ameliorates the hyperalgesia of chronic pain without affecting normal pain responses in neuropathic and inflammation-induced chronic pain models.

  12. Assessment of channeling bias among initiators of glucose-lowering drugs: A UK cohort study

    PubMed Central

    Ankarfeldt, Mikkel Z; Thorsted, Brian L; Groenwold, Rolf HH; Adalsteinsson, Erpur; Ali, M Sanni; Klungel, Olaf H

    2017-01-01

    Background Channeling bias may occur when a newly marketed drug and an established drug, despite similar indications, are prescribed to patients with different prognostic characteristics (ie, confounding). Aim To investigate channeling bias and its impact on relative effectiveness of glucagon-like peptide-1 (GLP-1) analogs versus basal insulin and dipeptidyl peptidase-4 inhibitors (DPP-4i) versus sulfonylurea. Methods In the UK Clinical Practice Research Datalink, patients with type 2 diabetes initiating treatment between 2006 and 2015 were included. Analyses were stratified by years since first prescription of GLP-1 and DPP-4i, respectively. The characteristics of GLP-1 versus insulin and DPP-4i versus sulfonylurea initiators were compared over time. After propensity score matching, the relative effectiveness regarding 6-month changes in glycated hemoglobin (HbA1c) and body weight was estimated. Results In total, 8,398 GLP-1, 14,807 insulin, 24,481 DPP-4i, and 33,505 sulfonylurea initiators were identified. No major channeling was observed. Considerable overlap in distributions of characteristics allowed for propensity score-matched analyses. Relative effectiveness was similar across time. The overall relative effect of GLP-1 versus insulin showed no difference for HbA1c and relative increase in body weight (3.57 kg [95% confidence interval {CI}: 3.21, 3.92]) for insulin. The overall relative effect of DPP-4i versus sulfonylurea showed relative decrease in HbA1c (−0.34% [95% CI: −0.38, −0.30]) and increase in body weight (1.58 kg [95% CI: 1.38, 1.78]) for sulfonylurea. Conclusion No major channeling was identified in the investigated glucose-lowering drugs. Relative effectiveness could be estimated already in the first year after launch and was consistent in the years thereafter. PMID:28176886

  13. Gating of a G protein-sensitive mammalian Kir3.1 prokaryotic Kir channel chimera in planar lipid bilayers.

    PubMed

    Leal-Pinto, Edgar; Gómez-Llorente, Yacob; Sundaram, Shobana; Tang, Qiong-Yao; Ivanova-Nikolova, Tatyana; Mahajan, Rahul; Baki, Lia; Zhang, Zhe; Chavez, Jose; Ubarretxena-Belandia, Iban; Logothetis, Diomedes E

    2010-12-17

    Kir3 channels control heart rate and neuronal excitability through GTP-binding (G) protein and phosphoinositide signaling pathways. These channels were the first characterized effectors of the βγ subunits of G proteins. Because we currently lack structures of complexes between G proteins and Kir3 channels, their interactions leading to modulation of channel function are not well understood. The recent crystal structure of a chimera between the cytosolic domain of a mammalian Kir3.1 and the transmembrane region of a prokaryotic KirBac1.3 (Kir3.1 chimera) has provided invaluable structural insight. However, it was not known whether this chimera could form functional K(+) channels. Here, we achieved the functional reconstitution of purified Kir3.1 chimera in planar lipid bilayers. The chimera behaved like a bona fide Kir channel displaying an absolute requirement for PIP(2) and Mg(2+)-dependent inward rectification. The channel could also be blocked by external tertiapin Q. The three-dimensional reconstruction of the chimera by single particle electron microscopy revealed a structure consistent with the crystal structure. Channel activity could be stimulated by ethanol and activated G proteins. Remarkably, the presence of both activated Gα and Gβγ subunits was required for gating of the channel. These results confirm the Kir3.1 chimera as a valid structural and functional model of Kir3 channels.

  14. Chick RGS2L demonstrates concentration-dependent selectivity for pertussis toxin-sensitive and -insensitive pathways that inhibit L-type Ca2+ channels.

    PubMed

    Tosetti, Patrizia; Parente, Valeria; Taglietti, Vanni; Dunlap, Kathleen; Toselli, Mauro

    2003-05-15

    In neuronal cells, the influx of Ca2+ ions through voltage-dependent L-type calcium (L) channels couples excitation to multiple cellular functions. In addition to voltage, several neurotransmitters, hormones and cytokines regulate L channel gating via binding to G-protein-coupled receptors. Intracellular molecules that modify G-protein activity - such as regulator of G-protein-signalling (RGS) proteins - are therefore potential candidates for regulating Ca2+ influx through L channels. Here we show that a novel RGS2 splice variant from chick dorsal root ganglion (DRG) neurons, RGS2L, reduces bradykinin (BK)-mediated inhibition of neuronal L channels and accelerates recovery from inhibition. Chick RGS2 reduces the inhibition mediated by both the pertussis toxin (PTX)-sensitive (Gi/o-coupled) and the PTX-insensitive (presumably Gq/11-coupled) pathways. However, we demonstrate for the first time in a living cell that the extent of coupling to each pathway varies with RGS2L concentration. A low concentration of recombinant chick RGS2L (10 nM) preferentially reduces the inhibition mediated by the PTX-insensitive pathway, whereas a 100-fold higher concentration attenuates both PTX-sensitive- and PTX-insensitive-mediated components equally. Our data suggest that factors promoting RGS2L gene induction may regulate Ca2+ influx through L channels by recruiting low-affinity interactions with Gi/o that are absent at basal RGS2L levels.

  15. Effects of in vitro lead exposure on voltage-sensitive calcium channels differ among cell types in central neurons of Lymnaea stagnalis.

    PubMed

    Audesirk, G; Audesirk, T

    1989-01-01

    The effects of acute in vitro lead exposure on slowly inactivating voltage-sensitive calcium channels in central neurons of the freshwater pond snail Lymnaea stagnalis were studied under voltage clamp. Three physiologically distinct cell types were used: two subsets of the B cell cluster (Bpos and Bneg) and the pedal giant neuron (RPeD1). In Bpos neurons, 5 nM free Pb2+ irreversibly inhibited current flow through calcium channels by 38 +/- 10%. In Bneg neurons, 5 nM free Pb2+ slightly inhibited inward currents (12 +/- 6%) and may have shifted their voltage dependence to more depolarized voltages. The inhibition and voltage shift were irreversible. In RPeD1 neurons, Pb2+ caused a small, statistically insignificant inhibition of inward current (5 nM free Pb2+; 18 +/- 19%; 30 nM free Pb2+: 31 +/- 23%). The effects of Pb2+ were fully reversible. These data indicate that (1) voltage-sensitive calcium channels in Lymnaea neurons are inhibited by nanomolar concentrations of free Pb2+; (2) there are multiple types of calcium channels in Lymnaea neurons; and (3) the effects of in vitro lead exposure differ qualitatively among channel types.

  16. Chick RGS2L demonstrates concentration-dependent selectivity for pertussis toxin-sensitive and -insensitive pathways that inhibit L-type Ca2+ channels

    PubMed Central

    Tosetti, Patrizia; Parente, Valeria; Taglietti, Vanni; Dunlap, Kathleen; Toselli, Mauro

    2003-01-01

    In neuronal cells, the influx of Ca2+ ions through voltage-dependent L-type calcium (L) channels couples excitation to multiple cellular functions. In addition to voltage, several neurotransmitters, hormones and cytokines regulate L channel gating via binding to G-protein-coupled receptors. Intracellular molecules that modify G-protein activity – such as regulator of G-protein-signalling (RGS) proteins – are therefore potential candidates for regulating Ca2+ influx through L channels. Here we show that a novel RGS2 splice variant from chick dorsal root ganglion (DRG) neurons, RGS2L, reduces bradykinin (BK)-mediated inhibition of neuronal L channels and accelerates recovery from inhibition. Chick RGS2 reduces the inhibition mediated by both the pertussis toxin (PTX)-sensitive (Gi/o-coupled) and the PTX-insensitive (presumably Gq/11-coupled) pathways. However, we demonstrate for the first time in a living cell that the extent of coupling to each pathway varies with RGS2L concentration. A low concentration of recombinant chick RGS2L (10 nm) preferentially reduces the inhibition mediated by the PTX-insensitive pathway, whereas a 100-fold higher concentration attenuates both PTX-sensitive- and PTX-insensitive-mediated components equally. Our data suggest that factors promoting RGS2L gene induction may regulate Ca2+ influx through L channels by recruiting low-affinity interactions with Gi/o that are absent at basal RGS2L levels. PMID:12651916

  17. T-type calcium channel Cav3.2 deficient mice show elevated anxiety, impaired memory and reduced sensitivity to psychostimulants

    PubMed Central

    Gangarossa, Giuseppe; Laffray, Sophie; Bourinet, Emmanuel; Valjent, Emmanuel

    2014-01-01

    The fine-tuning of neuronal excitability relies on a tight control of Ca2+ homeostasis. The low voltage-activated (LVA) T-type calcium channels (Cav3.1, Cav3.2 and Cav3.3 isoforms) play a critical role in regulating these processes. Despite their wide expression throughout the central nervous system, the implication of T-type Cav3.2 isoform in brain functions is still poorly characterized. Here, we investigate the effect of genetic ablation of this isoform in affective disorders, including anxiety, cognitive functions as well as sensitivity to drugs of abuse. Using a wide range of behavioral assays we show that genetic ablation of the cacna1h gene results in an anxiety-like phenotype, whereas novelty-induced locomotor activity is unaffected. Deletion of the T-type channel Cav3.2 also triggers impairment of hippocampus-dependent recognition memories. Acute and sensitized hyperlocomotion induced by d-amphetamine and cocaine are dramatically reduced in T-type Cav3.2 deficient mice. In addition, the administration of the T-type blocker TTA-A2 prevented the expression of locomotor sensitization observed in wildtype mice. In conclusion, our data reveal that physiological activity of this specific Ca2+ channel is required for affective and cognitive behaviors. Moreover, our work highlights the interest of T-type channel blockers as therapeutic strategies to reverse drug-associated alterations. PMID:24672455

  18. ZD0947, a sulphonylurea receptor modulator, detects functional sulphonylurea receptor subunits in murine vascular smooth muscle ATP-sensitive K(+) channels.

    PubMed

    Yamamoto, Tadashi; Takahara, Kohei; Uchida, Keiichiro; Teramoto, Noriyoshi

    2017-02-16

    In order to identify functional sulphonylurea receptor (SUR.x) subunits of native ATP-sensitive K(+) channels (KATP channels) in mouse portal vein, the effects of ZD0947, a SUR.x modulator, were investigated on spontaneous portal vein contractions, macroscopic membrane currents and unitary currents recorded (using patch-clamp techniques) in freshly dispersed mouse portal vein myocytes. Spontaneous contractions in mouse portal vein were reversibly reduced by ZD0947 in a concentration-dependent manner (Ki =293nM). The relaxation elicited by 3µM ZD0947 was antagonized by the additional application of glibenclamide (300nM), but not gliclazide (100-300nM). In the conventional whole-cell configuration, 100µM ZD0947 elicited inward glibenclamide-sensitive currents at a holding potential of -60mV that demonstrated selectivity for K(+)(i.e. KATP currents). The peak amplitude of the membrane current elicited by 30µM or 100µM ZD0947 was smaller than that elicited by 100µM pinacidil at -60mV. In the cell-attached mode, 100µM ZD0947 activated glibenclamide-sensitive K(+) channels with a conductance (35 pS) similar to that of recombinant Kir6.1/SUR2B channels that were expressed in HEK293 cells and activated by 100µM ZD0947. These results demonstrate that ZD0947 caused a significant vascular relaxation through the activation of KATP channels and that SUR2B may be the major functional subunit of SUR.x in mouse portal vein KATP channels, based on its pharmacological selectivity.

  19. Tetrodotoxin-sensitive α-subunits of voltage-gated sodium channels are relevant for inhibition of cardiac sodium currents by local anesthetics.

    PubMed

    Stoetzer, C; Doll, T; Stueber, T; Herzog, C; Echtermeyer, F; Greulich, F; Rudat, C; Kispert, A; Wegner, F; Leffler, A

    2016-06-01

    The sodium channel α-subunit (Nav) Nav1.5 is regarded as the most prevalent cardiac sodium channel required for generation of action potentials in cardiomyocytes. Accordingly, Nav1.5 seems to be the main target molecule for local anesthetic (LA)-induced cardiotoxicity. However, recent reports demonstrated functional expression of several "neuronal" Nav's in cardiomyocytes being involved in cardiac contractility and rhythmogenesis. In this study, we examined the relevance of neuronal tetrodotoxin (TTX)-sensitive Nav's for inhibition of cardiac sodium channels by the cardiotoxic LAs ropivacaine and bupivacaine. Effects of LAs on recombinant Nav1.2, 1.3, 1.4, and 1.5 expressed in human embryonic kidney cell line 293 (HEK-293) cells, and on sodium currents in murine, cardiomyocytes were investigated by whole-cell patch clamp recordings. Expression analyses were performed by reverse transcription PCR (RT-PCR). Cultured cardiomyocytes from neonatal mice express messenger RNA (mRNA) for Nav1.2, 1.3, 1.5, 1.8, and 1.9 and generate TTX-sensitive sodium currents. Tonic and use-dependent block of sodium currents in cardiomyocytes by ropivacaine and bupivacaine were enhanced by 200 nM TTX. Inhibition of recombinant Nav1.5 channels was similar to that of TTX-resistant currents in cardiomyocytes but stronger as compared to inhibition of total sodium current in cardiomyocytes. Recombinant Nav1.2, 1.3, 1.4, and 1.5 channels displayed significant differences in regard to use-dependent block by ropivacaine. Finally, bupivacaine blocked sodium currents in cardiomyocytes as well as recombinant Nav1.5 currents significantly stronger in comparison to ropivacaine. Our data demonstrate for the first time that cardiac TTX-sensitive sodium channels are relevant for inhibition of cardiac sodium currents by LAs.

  20. Bidirectional Modulation of Thermal and Chemical Sensitivity of TRPM8 Channels by the Initial Region of the N-terminal Domain*

    PubMed Central

    Pertusa, María; González, Alejandro; Hardy, Paulina; Madrid, Rodolfo; Viana, Félix

    2014-01-01

    TRPM8, a nonselective cation channel activated by cold, voltage, and cooling compounds such as menthol, is the principal molecular detector of cold temperatures in primary sensory neurons of the somatosensory system. The N-terminal domain of TRPM8 consists of 693 amino acids, but little is known about its contribution to channel function. Here, we identified two distinct regions within the initial N terminus of TRPM8 that contribute differentially to channel activity and proper folding and assembly. Deletion or substitution of the first 40 residues yielded channels with augmented responses to cold and menthol. The thermal threshold of activation of these mutants was shifted 2 °C to higher temperatures, and the menthol dose-response curve was displaced to lower concentrations. Site-directed mutagenesis screening revealed that single point mutations at positions Ser-26 or Ser-27 by proline caused a comparable increase in the responses to cold and menthol. Electrophysiological analysis of the S27P mutant revealed that the enhanced sensitivity to agonists is related to a leftward shift in the voltage dependence of activation, increasing the probability of channel openings at physiological membrane potentials. In addition, we found that the region encompassing positions 40–60 is a key element in the proper folding and assembly of TRPM8. Different deletions and mutations within this region rendered channels with an impaired function that are retained within the endoplasmic reticulum. Our results suggest a critical contribution of the initial region of the N-terminal domain of TRPM8 to thermal and chemical sensitivity and the proper biogenesis of this polymodal ion channel. PMID:24917670

  1. Bidirectional modulation of thermal and chemical sensitivity of TRPM8 channels by the initial region of the N-terminal domain.

    PubMed

    Pertusa, María; González, Alejandro; Hardy, Paulina; Madrid, Rodolfo; Viana, Félix

    2014-08-08

    TRPM8, a nonselective cation channel activated by cold, voltage, and cooling compounds such as menthol, is the principal molecular detector of cold temperatures in primary sensory neurons of the somatosensory system. The N-terminal domain of TRPM8 consists of 693 amino acids, but little is known about its contribution to channel function. Here, we identified two distinct regions within the initial N terminus of TRPM8 that contribute differentially to channel activity and proper folding and assembly. Deletion or substitution of the first 40 residues yielded channels with augmented responses to cold and menthol. The thermal threshold of activation of these mutants was shifted 2 °C to higher temperatures, and the menthol dose-response curve was displaced to lower concentrations. Site-directed mutagenesis screening revealed that single point mutations at positions Ser-26 or Ser-27 by proline caused a comparable increase in the responses to cold and menthol. Electrophysiological analysis of the S27P mutant revealed that the enhanced sensitivity to agonists is related to a leftward shift in the voltage dependence of activation, increasing the probability of channel openings at physiological membrane potentials. In addition, we found that the region encompassing positions 40-60 is a key element in the proper folding and assembly of TRPM8. Different deletions and mutations within this region rendered channels with an impaired function that are retained within the endoplasmic reticulum. Our results suggest a critical contribution of the initial region of the N-terminal domain of TRPM8 to thermal and chemical sensitivity and the proper biogenesis of this polymodal ion channel.

  2. In-Depth Study of the Interaction, Sensitivity, and Gating Modulation by PUFAs on K+ Channels; Interaction and New Targets

    PubMed Central

    Moreno, Cristina; de la Cruz, Alicia; Valenzuela, Carmen

    2016-01-01

    Voltage gated potassium channels (KV) are membrane proteins that allow selective flow of K+ ions in a voltage-dependent manner. These channels play an important role in several excitable cells as neurons, cardiomyocytes, and vascular smooth muscle. Over the last 20 years, it has been shown that omega-3 polyunsaturated fatty acids (PUFAs) enhance or decrease the activity of several cardiac KV channels. PUFAs-dependent modulation of potassium ion channels has been reported to be cardioprotective. However, the precise cellular mechanism underlying the cardiovascular benefits remained unclear in part because new PUFAs targets and signaling pathways continue being discovered. In this review, we will focus on recent data available concerning the following aspects of the KV channel modulation by PUFAs: (i) the exact residues involved in PUFAs-KV channels interaction; (ii) the structural PUFAs determinants important for their effects on KV channels; (iii) the mechanism of the gating modulation of KV channels and, finally, (iv) the PUFAs modulation of a few new targets present in smooth muscle cells (SMC), KCa1.1, K2P, and KATP channels, involved in vascular relaxation. PMID:27933000

  3. Occurrence of sulfonylurea, sulfonamide, imidazolinone, and other herbicides in rivers, reservoirs and ground water in the Midwestern United States, 1998

    USGS Publications Warehouse

    Battaglin, W.A.; Furlong, E.T.; Burkhardt, M.R.; Peter, C.J.

    2000-01-01

    Sulfonylurea (SU), sulfonamide (SA), and imidazolinone (IMI) herbicides are relatively new classes of chemical compounds that function by inhibiting the action of a plant enzyme, stopping plant growth, and eventually killing the plant. These compounds generally have low mammalian toxicity, but plants demonstrate a wide range in sensitivity to SUs, SAs, and IMIs with over a 10000-fold difference in observed toxicity levels for some compounds. SUs, SAs, and IMIs are applied either pre- or post-emergence to crops commonly at 1/50th or less of the rate of other herbicides. Little is known about their occurrence, fate, or transport in surface water or ground water in the USA. To obtain information on the occurrence of SU, SA, and IMI herbicides in the Midwestern United States, 212 water samples were collected from 75 surface-water and 25 ground-water sites in 1998. These samples were analyzed for 16 SU, SA and IMI herbicides by USGS Methods Research and Development Program staff using high-performance liquid chromatography/mass spectrometry. Samples were also analyzed for 47 pesticides or pesticide degradation products. At least one of the 16 SUs, SAs or IMIs was detected above the method reporting limit (MRL) of 0.01 ??g/l in 83% of 130 stream samples. Imazethapyr was detected most frequently (71% of samples) followed by flumetsulam (63% of samples) and nicosulfuron (52% of samples). The sum of SU, SA and IMI concentrations exceeded 0.5 ??g/l in less than 10% of stream samples. Acetochlor, alachlor, atrazine, cyanazine and metolachlor were all detected in 90% or more of 129 stream samples. The sum of the concentration of these five herbicides exceeded 50 ??g/l in approximately 10% of stream samples. At least one SU, SA, or IMI herbicide was detected above the MRL in 24% of 25 ground-water samples and 86% of seven reservoir samples. Copyright (C) 2000 Elsevier Science B.V.

  4. Cross-Resistance to Short Residual Sulfonylurea Herbicides in Transgenic Tobacco Plants 1

    PubMed Central

    Gabard, Jerome M.; Charest, Pierre J.; Iyer, V. N.; Miki, Brian L.

    1989-01-01

    Transgenic Nicotiana tabacum plants, produced by Agrobacterium tumefaciens-mediated transformation with a mutant gene (csr1-1) coding for acetohydroxyacid synthase (AHAS) from a chlorsulfuron resistant Arabidopsis thaliana line GH50 (GW Haughn et al. [1988] Mol Gen Genet 211: 266-271; GW Haughn, C Somerville [1986] Mol Gen Genet 204: 430-434), were selected directly on 80 micrograms per liter (225 nanomolar) chlorsulfuron. The expression of csr-1 in two separate transgenic lines CHL-1 and CHL-2 was confirmed by biochemical and genetic analyses. The AHAS activity of GH50 and the equivalent component of AHAS activity in CHL-2 was resistant to three short residual sulfonylurea herbicides, DPX-M6316, DPX-A7881, and DPX-L5300, in addition to chlorsulfuron but not to the sulfonylurea CGA 131′036. Cross-resistance to the imidazolinones AC 263, 499, AC 252, 214, and AC 243,997 was not observed. Parallel observations were made on the inhibition of seedling growth in soil or on culture medium. The relevance of these findings for the application of transgenic plants in agriculture is discussed. Images Figure 1 PMID:16667071

  5. Cell signalling in insulin secretion: the molecular targets of ATP, cAMP and sulfonylurea.

    PubMed

    Seino, S

    2012-08-01

    Clarification of the molecular mechanisms of insulin secretion is crucial for understanding the pathogenesis and pathophysiology of diabetes and for development of novel therapeutic strategies for the disease. Insulin secretion is regulated by various intracellular signals generated by nutrients and hormonal and neural inputs. In addition, a variety of glucose-lowering drugs including sulfonylureas, glinide-derivatives, and incretin-related drugs such as dipeptidyl peptidase IV (DPP-4) inhibitors and glucagon-like peptide 1 (GLP-1) receptor agonists are used for glycaemic control by targeting beta cell signalling for improved insulin secretion. There has been a remarkable increase in our understanding of the basis of beta cell signalling over the past two decades following the application of molecular biology, gene technology, electrophysiology and bioimaging to beta cell research. This review discusses cell signalling in insulin secretion, focusing on the molecular targets of ATP, cAMP and sulfonylurea, an essential metabolic signal in glucose-induced insulin secretion (GIIS), a critical signal in the potentiation of GIIS, and the commonly used glucose-lowering drug, respectively.

  6. Conjugates of degraded and oxidized hydroxyethyl starch and sulfonylureas: synthesis, characterization, and in vivo antidiabetic activity.

    PubMed

    Abbas, Muhammad Azhar; Hameed, Shahid; Farman, Muhammad; Kressler, Jörg; Mahmood, Nasir

    2015-01-21

    Orally administered drugs usually face the problem of low water solubility, low permeability, and less retention in bloodstream leading to unsatisfactory pharmacokinetic profile of drugs. Polymer conjugation has attracted increasing interest in the pharmaceutical industry for delivering such low molecular weight (Mw) drugs as well as some complex compounds. In the present work, degraded and oxidized hydroxyethyl starch (HES), a highly biocompatible semisynthetic biopolymer, was used as a drug carrier to overcome the solubility and permeability problems. The HES was coupled with synthesized N-arylsulfonylbenzimidazolones, a class of sulfonylurea derivatives, by creating an amide linkage between the two species. The coupled products were characterized using GPC, FT-IR, (1)H NMR, and (13)C NMR spectroscopy. The experiments established the viability of covalent coupling between the biopolymer and N-arylsulfonylbenzimidazolones. The coupled products were screened for their in vivo antidiabetic potential on male albino rats. The coupling of sulfonylurea derivatives with HES resulted in a marked increase of the hypoglycemic activity of all the compounds. 2,3-Dihydro-3-(4-nitrobenzensulfonyl)-2-oxo-1H-benzimidazole coupled to HES10100 was found most potent with a 67% reduction in blood glucose level of the rats as compared to 41% reduction produced by tolbutamide and 38% by metformin.

  7. Sex differences in the contribution of ATP-sensitive K+ channels in trigeminal ganglia under an acute muscle pain condition

    PubMed Central

    Niu, Katelyn; Saloman, Jami L.; Zhang, Youping; Ro, Jin Y.

    2011-01-01

    In this study, we examined whether functional subunits of the ATP-dependent K+ channel (KATP) are expressed in trigeminal ganglia (TG), which contains sensory neurons that innervate oral and facial structures. We also investigated whether direct activation of the KATP effectively attenuates mechanical hypersensitivity in the context of an acute orofacial muscle pain condition. The KATP expression in TG and behavioral studies were conducted in age matched male and female Sprague Dawley rats. RT-PCR experiments showed that the mRNAs for the inwardly rectifying pore-forming subunits, Kir6.1 and Kir6.2, as well as the regulatory sulphonylurea subunits, SUR1 and SUR2, were reliably detected in TG. Subsequent western blot analysis confirmed that proteins for all 4 subunits are expressed in TG, and showed that Kir6.2 is expressed at a significantly higher level in male TG compared to that of female rats. This observation was confirmed by the immunohistochemical demonstration of higher percentages of Kir6 positive masseter afferents in female rats. Masseteric injection of capsaicin evokes a time dependent increase in masseter sensitivity to noxious mechanical stimulation. A specific KATP agonist, pinacidil, dose-dependently attenuated the capsaicin-induced mechanical hypersensitivity in male rats. The dose of pinacidil (20µg) that completely blocked the capsaicin responses in male rats was ineffective in female rats regardless of their estrus phases. Only at the highest dose (300µg) we used, pinacidil was partially effective in female rats. Similarly, another KATP agonist, diazoxide which targets different KATP subunits also showed sex specific responses in attenuating capsaicin-induced masseter hypersensitivity. These data suggested that sex differences in functional KATP expression in TG may underlie sex specific responses to KATP agonists. The present study provided novel information on sex differences in KATP expression in TG and its contribution under an orofacial

  8. Analysis on mechanism of ATP-sensitive K+ channel opener natakalim improving congestive heart failure after myocardial infarction

    PubMed Central

    Jin, Feng

    2016-01-01

    The action mechanism of natakalim, a novel ATP-sensitive potassium channel opener, was studied in ameliorating the congestive heart failure (CHF) after myocardial infarction. A total of 25 healthy Wistar male rats (age, 10 weeks; average weight, 300 g) were selected, and the CHF models after acute myocardial infarction (AMI) were prepared by ligation of left anterior descending branch. They were randomly divided into the sham operation group, the model group and the groups of 1, 3 and 9 mg/kg/day natakalims. Each group had 5 mice that were sacrificed after 8 weeks. We compared left ventricular end-diastolic diameter (LVEDD), left ventricular ejection fraction (LVEF), N-terminal prohormone of brain natriuretic peptide (NT-proBNP), left ventricular mass index, myocardial cell cross-sectional area, myocardial collagen content, plasma endothelin-1 (ET-1) and endothelial nitric oxide synthase (eNOS) levels. Compared with the sham operation, the LVEDD and NT-proBNP in the model group and each natakalim group were elevated. LVEF decreased significantly, while the left ventricular mass index, myocardial cell cross-sectional area, myocardial collagen content, plasma ET-1 and eNOS levels increased. Natakalim intervention improved the above changes and the improvement effect of 3 mg/kg/day group was the highest. The mechanism of natakalim against the endothelin system can be explained by the fact that inhibiting ET-1 synthesis can reduce the ET-1 levels in circulation leading to the release of NO and PGI2. Inhibition of the vasoconstriction effect of ET-1 can improve the hemodynamics of high-load status and ameliorate the cardiac systolic and diastolic functions. In conclusion, natakalim can improve the ventricular remodeling of CHF after AMI, and 3 mg/kg/day was the most effective dose. PMID:28101177

  9. Hydrostatic pressure activates ATP-sensitive K+ channels in lung epithelium by ATP release through pannexin and connexin hemichannels.

    PubMed

    Richter, Katrin; Kiefer, Kevin P; Grzesik, Benno A; Clauss, Wolfgang G; Fronius, Martin

    2014-01-01

    Lungs of air-breathing vertebrates are constantly exposed to mechanical forces and therefore are suitable for investigation of mechanotransduction processes in nonexcitable cells and tissues. Freshly dissected Xenopus laevis lungs were used for transepithelial short-circuit current (ISC) recordings and were exposed to increased hydrostatic pressure (HP; 5 cm fluid column, modified Ussing chamber). I(SC) values obtained under HP (I(5cm)) were normalized to values before HP (I(0cm)) application (I(5cm)/I(0cm)). Under control conditions, HP decreased I(SC) (I(5cm)/I(0cm)=0.84; n=68; P<0.0001). This effect was reversible and repeatable ≥30 times. Preincubation with ATP-sensitive K(+) channel (K(ATP)) inhibitors (HMR1098 and glibenclamide) prevented the decrease in I(SC) (I(5cm)/I(0cm): HMR1098=1.19, P<0.0001; glibenclamide=1.11, P<0.0001). Similar effects were observed with hemichannel inhibitors (I(5cm)/I(0cm): meclofenamic acid=1.09, P<0.0001; probenecid=1.0, P<0.0001). The HP effect was accompanied by release of ATP (P<0.05), determined by luciferin-luciferase luminescence in perfusion solution from the luminal side of an Ussing chamber. ATP release was abrogated by both meclofenamic acid and probenecid. RT-PCR experiments revealed the expression of pannexin and connexin hemichannels and KATP subunit transcripts in X. laevis lung. These data show an activation of KATP in pulmonary epithelial cells in response to HP that is induced by ATP release through mechanosensitive pannexin and connexin hemichannels. These findings represent a novel mechanism of mechanotransduction in nonexcitable cells.

  10. Effects of cyclooxygenase-2 inhibitor and adenosine triphosphate-sensitive potassium channel opener in syngeneic mouse islet transplantation.

    PubMed

    Juang, J-H; Kuo, C-H

    2010-12-01

    In the initial days after transplantation, islet grafts may be attacked by cytokines via cyclooxygenase-2 (COX-2), producing primary nonfunction. In addition, chronic overstimulation of β-cells may impair insulin secretion. To enhance the function of transplanted islets, the present study investigated the effects of rofecoxib, a COX-2 inhibitor, and NN414 (6-chloro-3-[1-methylcyclopropyl]amino-4H-thieno[3,2-e]-1,2,4-thiadiazine 1,1-dioxide), an adenosine triphosphate-sensitive potassium channel opener, on islet transplantation. Male inbred C57BL/6 mice were used as donors and recipients. One hundred fifty islets were isolated via collagenase digestion and density gradient, and syngeneically transplanted under the kidney capsule in mice with streptozotocin-induced diabetes. Recipients were treated with or without rofecoxib, 10 mg/kg/d orally, or with or without NN414, 3 mg/kg/d orally, for 4 weeks. After transplantation, recipient body weight, blood glucose concentration, and intraperitoneal glucose tolerance were measured. The grafted kidney was extracted for determination of insulin content at 4 weeks. In the rofecoxib-treated and NN414-treated groups and both control groups, body weight remained stable, and the blood glucose concentration decreased progressively. However, at 4 weeks after transplantation in the groups treated or not treated with rofecoxib or NN414, no significant difference was observed in recipient body weight, blood glucose concentration, and glucose tolerance or in insulin content of the graft. These data indicate that posttransplantation treatment with rofecoxib or NN414 has no beneficial effect on transplantation outcome in diabetic mouse recipients engrafted with a marginal islet mass.

  11. Dihydropyridine-sensitive calcium channels in cardiac and skeletal muscle membranes: studies with antibodies against the. cap alpha. subunits

    SciTech Connect

    Takahashi, M.; Catterall, W.A.

    1987-08-25

    Polyclonal antibodies (PAC-2) against the purified skeletal muscle calcium channel were prepared and shown to be directed against ..cap alpha.. subunits of this protein by immunoblotting and immunoprecipitation. These polypeptides have an apparent molecular weight of 162,000 without reduction of disulfide bonds. Under conditions where the functional properties of the purified skeletal muscle calcium channel are retained, ..beta.. subunits (M/sub r/ 50,000) and lambda subunits (M/sub r/ 33,000) are coprecipitated, demonstrating specific noncovalent association of these three polypeptides in the purified skeletal muscle channel. PAC-2 immunoprecipitated cardiac calcium channels labeled with (/sup 3/H)isopropyl 4-(2,1,3-benzoxadiazol-4-yl)-1,4-dihydro-2,6-dimethyl-5-(methoxycarbonyl)pyridine-3-carboxylate ((/sup 3/H)PN200-110) at a 3-fold higher concentration than skeletal muscle channels. Preincubation with cardiac calcium channels blocked only 49% of the immunoreactivity of PAC-2 toward skeletal muscle channels, indicating that these two proteins have both homologous and distinct epitopes. The immunoreactive component of the cardiac calcium channel was identified by immunoprecipitation and polyacrylamide gel electrophoresis as a polypeptide with an apparent molecular weight of 170,000 before reduction of disulfide bonds and 141,000 after reduction, in close analogy with the properties of the ..cap alpha../sub 2/ subunits of the skeletal muscle channel. The calcium channels were radiolabeled with /sup 32/P and /sup 125/I. It is concluded that these two calcium channels have a homologous, but distinct, ..cap alpha.. subunit as a major polypeptide component.

  12. Preparation of environmental samples for determination of sulfonylurea herbicides by solid-phase extraction using a polymeric sorbent.

    PubMed

    Young, M S

    1998-01-01

    An analytical procedure was developed for rapid determination of sulfonylurea herbicides in water or soil. Samples were prepared by solid-phase extraction using polymeric sorbents in cartridges and analyzed by liquid chromatography with photodiode array detection. Recoveries were consistent for all sample matrixes tested and were generally > 85%, except for tribenuron methyl (75%). Detection limits were < 100 ng/L for water samples.

  13. Upregulation of apical sodium-chloride cotransporter and basolateral chloride channels is responsible for the maintenance of salt-sensitive hypertension.

    PubMed

    Capasso, Giovambattista; Rizzo, Maria; Garavaglia, Maria Lisa; Trepiccione, Francesco; Zacchia, Miriam; Mugione, Alessandra; Ferrari, Patrizia; Paulmichl, Markus; Lang, Florian; Loffing, Johannes; Carrel, Monique; Damiano, Sara; Wagner, Carsten A; Bianchi, Giuseppe; Meyer, Giuliano

    2008-08-01

    We investigated which of the NaCl transporters are involved in the maintenance of salt-sensitive hypertension. Milan hypertensive (MHS) rats were studied 3 mo after birth. In MHS, compared with normotensive strain (MNS), mRNA abundance, quantified by competitive PCR on isolated tubules, was unchanged, both for Na+/H+ isoform 3 (NHE3) and Na+-K+-2Cl- (NKCC2), but higher (119%, n = 5, P < 0.005) for Na+-Cl- (NCC) in distal convoluted tubules (DCT). These results were confirmed by Western blots, which revealed: 1) unchanged NHE3 in the cortex and NKCC2 in the outer medulla; 2) a significant increase (52%, n = 6, P < 0.001) of NCC in the cortex; 3) alpha- and beta-sodium channels [epithelial Na+ channel (ENaC)] unaffected in renal cortex and slightly reduced in the outer medulla, while gamma-ENaC remained unchanged. Pendrin protein expression was unaffected. The role of NCC was reinforced by immunocytochemical studies showing increased NCC on the apical membrane of DCT cells of MHS animals, and by clearance experiments demonstrating a larger sensitivity (P < 0.001) to bendroflumethiazide in MHS rats. Kidney-specific chloride channels (ClC-K) were studied by Western blot experiments on renal cortex and by patch-clamp studies on primary culture of DCT dissected from MNS and MHS animals. Electrophysiological characteristics of ClC-K channels were unchanged in MHS rats, but the number of active channels in a patch was 0.60 +/- 0.21 (n = 35) in MNS rats and 2.17 +/- 0.59 (n = 23) in MHS rats (P < 0.05). The data indicate that, in salt-sensitive hypertension, there is a strong upregulation, both of NCC and ClC-K along the DCT, which explains the persistence of hypertension.

  14. Magnetic field sensitivity at 7-T using dual-helmholtz transmit-only coil and 12-channel receive-only bended coil.

    PubMed

    Kim, Kyoung-Nam; Ryu, Yeunchul; Seo, Jeung-Hoon; Kim, Young-Bo

    2016-11-01

    The purpose of this study was to combine a dual-Helmholtz (DH) transmit (Tx)-only coil and 12-channel receive (Rx)-only bended phased array (PA) coil to improve the magnetic flux (|B1 |) sensitivity in the superior-to-inferior (S-I) direction during human brain magnetic resonance imaging (MRI) at 7-T. The proposed coil combination was primarily implemented by electromagnetic (EM) simulation and compared with the 16-leg birdcage coil and 8-channel PA coil, which are generally used for the Tx- and Rx-only modes, respectively. The optimal coil combinations for the proposed structure were determined by |B1 | field calculations using the |BT(+) | and |BR(-) | fields, which are respectively the transmit and receive components of the |B1 | field. The coil performance was then evaluated by a bench test and 7-T MRI experiment. The results of the computational calculations indicated that the |BT(+) | field of the DH coil was distributed similarly to that of the 16-leg birdcage coil despite the fewer conducting legs of the former. However, the 12-channel Rx-only bended PA coil had clearly higher |BR(-) | profiles compared to the 8-channel PA coil. The results of the 7-T in vivo experiment showed that the proposed combination of the DH Tx-only coil and 12-channel Rx-only bended PA coil had better |B1 | field homogeneity in the sagittal slice as well as higher |B1 | field sensitivity during human brain MRI compared to an 8-channel Rx-only PA coil. SCANNING 38:515-524, © 2015 Wiley Periodicals, Inc.

  15. A contrast-sensitive channelized-Hotelling observer to predict human performance in a detection task using lumpy backgrounds and Gaussian signals

    NASA Astrophysics Data System (ADS)

    Park, Subok; Badano, Aldo; Gallas, Brandon D.; Myers, Kyle J.

    2007-03-01

    Previously, a non-prewhitening matched filter (NPWMF) incorporating a model for the contrast sensitivity of the human visual system was introduced for modeling human performance in detection tasks with different viewing angles and white-noise backgrounds by Badano et al. But NPWMF observers do not perform well detection tasks involving complex backgrounds since they do not account for random backgrounds. A channelized-Hotelling observer (CHO) using difference-of-Gaussians (DOG) channels has been shown to track human performance well in detection tasks using lumpy backgrounds. In this work, a CHO with DOG channels, incorporating the model of the human contrast sensitivity, was developed similarly. We call this new observer a contrast-sensitive CHO (CS-CHO). The Barten model was the basis of our human contrast sensitivity model. A scalar was multiplied to the Barten model and varied to control the thresholding effect of the contrast sensitivity on luminance-valued images and hence the performance-prediction ability of the CS-CHO. The performance of the CS-CHO was compared to the average human performance from the psychophysical study by Park et al., where the task was to detect a known Gaussian signal in non-Gaussian distributed lumpy backgrounds. Six different signal-intensity values were used in this study. We chose the free parameter of our model to match the mean human performance in the detection experiment at the strongest signal intensity. Then we compared the model to the human at five different signal-intensity values in order to see if the performance of the CS-CHO matched human performance. Our results indicate that the CS-CHO with the chosen scalar for the contrast sensitivity predicts human performance closely as a function of signal intensity.

  16. Impact of sulfonylurea receptor 1 genetic variability on non-insulin-dependent diabetes mellitus prevalence and treatment: a population study.

    PubMed

    Meirhaeghe, A; Helbecque, N; Cottel, D; Arveiler, D; Ruidavets, J B; Haas, B; Ferrières, J; Tauber, J P; Bingham, A; Amouyel, P

    2001-06-01

    The high affinity sulfonylurea receptor 1 (SUR1) is involved in the metabolism of glucose in pancreatic beta-cells. We investigated the impact of the SUR1 intron 16-3t-->c polymorphism on non-insulin-dependent diabetes mellitus (NIDDM) prevalence in a large representative sample of French men and women, 35-64 years old, and explored potential relationships between the SUR1 intron 16 -t-->c polymorphism and sulfonylurea therapy efficiency. This study took place in Lille (northern), Strasbourg (eastern), and Toulouse (southern France). One hundred and twenty-two subjects with NIDDM were registered. We stratified NIDDM subjects according to their medical treatment: sulfonylureas (n = 70) versus other treatments (n = 50). From the three populations, a control group was selected (n = 1,250). Subjects carrying the cc intron 16 genotype had an increased risk of NIDDM [odds ratio (OR) = 1.76, 95% confidence interval (CI) 1.10-2.80; P = 0.017]. Subjects bearing at least one -3c allele and treated with sulfonylurea agents had fasting plasma triglyceride concentrations 35% lower than subjects that were tt homozygous (P = 0.026), whereas no difference could be detected between genotypes in NIDDM subjects treated with other treatments. The SUR1 intron 16 -3t-->c polymorphism was associated with an increased susceptibility to NIDDM in this population study, and seems to modulate the sulfonylurea therapy efficiency on hypertriglyceridemia reduction. This observation may help to better target the various therapies available for treatment of NIDDM.

  17. Quantitative autoradiography of the binding sites for ( sup 125 I) iodoglyburide, a novel high-affinity ligand for ATP-sensitive potassium channels in rat brain

    SciTech Connect

    Gehlert, D.R.; Gackenheimer, S.L.; Mais, D.E.; Robertson, D.W. )

    1991-05-01

    We have developed a high specific activity ligand for localization of ATP-sensitive potassium channels in the brain. When brain sections were incubated with ({sup 125}I)iodoglyburide (N-(2-((((cyclohexylamino)carbonyl)amino)sulfonyl)ethyl)-5-{sup 125}I-2- methoxybenzamide), the ligand bound to a single site with a KD of 495 pM and a maximum binding site density of 176 fmol/mg of tissue. Glyburide was the most potent inhibitor of specific ({sup 125}I)iodoglyburide binding to rat forebrain sections whereas iodoglyburide and glipizide were slightly less potent. The binding was also sensitive to ATP which completely inhibited binding at concentrations of 10 mM. Autoradiographic localization of ({sup 125}I)iodoglyburide binding indicated a broad distribution of the ATP-sensitive potassium channel in the brain. The highest levels of binding were seen in the globus pallidus and ventral pallidum followed by the septohippocampal nucleus, anterior pituitary, the CA2 and CA3 region of the hippocampus, ventral pallidum, the molecular layer of the cerebellum and substantia nigra zona reticulata. The hilus and dorsal subiculum of the hippocampus, molecular layer of the dentate gyrus, cerebral cortex, lateral olfactory tract nucleus, olfactory tubercle and the zona incerta contained relatively high levels of binding. A lower level of binding (approximately 3- to 4-fold) was found throughout the remainder of the brain. These results indicate that the ATP-sensitive potassium channel has a broad presence in the rat brain and that a few select brain regions are enriched in this subtype of neuronal potassium channels.

  18. Two distinct voltage-sensing domains control voltage sensitivity and kinetics of current activation in CaV1.1 calcium channels.

    PubMed

    Tuluc, Petronel; Benedetti, Bruno; Coste de Bagneaux, Pierre; Grabner, Manfred; Flucher, Bernhard E

    2016-06-01

    Alternative splicing of the skeletal muscle CaV1.1 voltage-gated calcium channel gives rise to two channel variants with very different gating properties. The currents of both channels activate slowly; however, insertion of exon 29 in the adult splice variant CaV1.1a causes an ∼30-mV right shift in the voltage dependence of activation. Existing evidence suggests that the S3-S4 linker in repeat IV (containing exon 29) regulates voltage sensitivity in this voltage-sensing domain (VSD) by modulating interactions between the adjacent transmembrane segments IVS3 and IVS4. However, activation kinetics are thought to be determined by corresponding structures in repeat I. Here, we use patch-clamp analysis of dysgenic (CaV1.1 null) myotubes reconstituted with CaV1.1 mutants and chimeras to identify the specific roles of these regions in regulating channel gating properties. Using site-directed mutagenesis, we demonstrate that the structure and/or hydrophobicity of the IVS3-S4 linker is critical for regulating voltage sensitivity in the IV VSD, but by itself cannot modulate voltage sensitivity in the I VSD. Swapping sequence domains between the I and the IV VSDs reveals that IVS4 plus the IVS3-S4 linker is sufficient to confer CaV1.1a-like voltage dependence to the I VSD and that the IS3-S4 linker plus IS4 is sufficient to transfer CaV1.1e-like voltage dependence to the IV VSD. Any mismatch of transmembrane helices S3 and S4 from the I and IV VSDs causes a right shift of voltage sensitivity, indicating that regulation of voltage sensitivity by the IVS3-S4 linker requires specific interaction of IVS4 with its corresponding IVS3 segment. In contrast, slow current kinetics are perturbed by any heterologous sequences inserted into the I VSD and cannot be transferred by moving VSD I sequences to VSD IV. Thus, CaV1.1 calcium channels are organized in a modular manner, and control of voltage sensitivity and activation kinetics is accomplished by specific molecular mechanisms

  19. Identifying Candidate Genes that Underlie Cellular pH Sensitivity in Serotonin Neurons Using Transcriptomics: A Potential Role for Kir5.1 Channels

    PubMed Central

    Puissant, Madeleine M.; Mouradian, Gary C.; Liu, Pengyuan; Hodges, Matthew R.

    2017-01-01

    Ventilation is continuously adjusted by a neural network to maintain blood gases and pH. Acute CO2 and/or pH regulation requires neural feedback from brainstem cells that encode CO2/pH to modulate ventilation, including but not limited to brainstem serotonin (5-HT) neurons. Brainstem 5-HT neurons modulate ventilation and are stimulated by hypercapnic acidosis, the sensitivity of which increases with increasing postnatal age. The proper function of brainstem 5-HT neurons, particularly during post-natal development is critical given that multiple abnormalities in the 5-HT system have been identified in victims of Sudden Infant Death Syndrome. Here, we tested the hypothesis that there are age-dependent increases in expression of pH-sensitive ion channels in brainstem 5-HT neurons, which may underlie their cellular CO2/pH sensitivity. Midline raphe neurons were acutely dissociated from neonatal and mature transgenic SSePet-eGFP rats [which have enhanced green fluorescent protein (eGFP) expression in all 5-HT neurons] and sorted with fluorescence-activated cell sorting (FACS) into 5-HT-enriched and non-5-HT cell pools for subsequent RNA extraction, cDNA library preparation and RNA sequencing. Overlapping differential expression analyses pointed to age-dependent shifts in multiple ion channels, including but not limited to the pH-sensitive potassium ion (K+) channel genes kcnj10 (Kir4.1), kcnj16 (Kir5.1), kcnk1 (TWIK-1), kcnk3 (TASK-1) and kcnk9 (TASK-3). Intracellular contents isolated from single adult eGFP+ 5-HT neurons confirmed gene expression of Kir4.1, Kir5.1 and other K+ channels, but also showed heterogeneity in the expression of multiple genes. 5-HT neuron-enriched cell pools from selected post-natal ages showed increases in Kir4.1, Kir5.1, and TWIK-1, fitting with age-dependent increases in Kir4.1 and Kir5.1 protein expression in raphe tissue samples. Immunofluorescence imaging confirmed Kir5.1 protein was co-localized to brainstem neurons and glia including 5

  20. Activation of ATP-sensitive potassium channels enhances DMT1-mediated iron uptake in SK-N-SH cells in vitro

    PubMed Central

    Du, Xixun; Xu, Huamin; Shi, Limin; Jiang, Zhifeng; Song, Ning; Jiang, Hong; Xie, Junxia

    2016-01-01

    Iron importer divalent metal transporter 1 (DMT1) plays a crucial role in the nigal iron accumulation in Parkinson’s disease (PD). Membrane hyperpolarization is one of the factors that could affect its iron transport function. Besides iron, selective activation of the ATP-sensitive potassium (KATP) channels also contributes to the vulnerability of dopaminergic neurons in PD. Interestingly, activation of KATP channels could induce membrane hyperpolarization. Therefore, it is of vital importance to study the effects of activation of KATP channels on DMT1-mediated iron uptake function. In the present study, activation of KATP channels by diazoxide resulted in the hyperpolarization of the membrane potential and increased DMT1-mediated iron uptake in SK-N-SH cells. This led to an increase in intracellular iron levels and a subsequent decrease in the mitochondrial membrane potential and an increase in ROS production. Delayed inactivation of the Fe2+-evoked currents by diazoxide was recorded by patch clamp in HEK293 cells, which demonstrated that diazoxide could prolonged DMT1-facilitated iron transport. While inhibition of KATP channels by glibenclamide could block ferrous iron influx and the subsequent cell damage. Overexpression of Kir6.2/SUR1 resulted in an increase in iron influx and intracellular iron levels, which was markedly increased after diazoxide treatment. PMID:27646472

  1. Co-expression of wild-type and mutant olfactory cyclic nucleotide-gated channels: restoration of the native sensitivity to Ca(2+) and Mg(2+) blockage.

    PubMed

    Picco, C; Gavazzo, P; Menini, A

    2001-08-08

    In the pore of homomeric cyclic nucleotide-gated (CNG) channels, Ca(2+) and Mg(2+) bind to a set of glutamate residues, which in the bovine olfactory CNG channel are located at position 340. However, native CNG channels from olfactory sensory neurons are composed by the assembly of three different types of subunits, each having a different residue -- glutamate, aspartate or glycine -- at the position corresponding to the binding site for external Ca(2+) and Mg(2+). We co-expressed the wild-type principal alpha subunit with its mutants E340G and E340D in different combinations in Xenopus laevis oocytes, and measured Ca(2+) and Mg(2+) blockage in excised outside-out membrane patches. The comparison between our results and data from native olfactory CNG channels indicates that the presence of all three residues -- glutamate, aspartate and glycine -- in the different subunits, is necessary to restore the sensitivity to external Ca(2+) and Mg(2+) measured in native channels.

  2. Detection of calcium activity in human monocytes by the fura-2 fluorescence method: in vitro differentiation sensitizes cells to dihydropyridine calcium channel modulators

    NASA Astrophysics Data System (ADS)

    Oraevsky, Alexander A.; Cabello, Olga A.; Shan, Qin; Tittel, Frank K.; Henry, Philip D.

    1994-07-01

    Dihydropyridine (DHP) calcium channel blockers have been shown to suppress atherogenesis in various species and controlled angiographic trials suggest that these drugs may retard the progression of occlusive coronary disease in humans. Because mononuclear leukocytes play a key role in the formation of early and advanced atheromatous lesions, we determined effects of DHP calcium channel modulators on calcium uptake by cells of the monocytic lineage. Human peripheral blood monocytes were evaluated before and after undergoing in vitro differentiation induced by two days of culture with fetal calf serum and FMLP. Changes in intracellular calcium activity were estimated with fura-2, a fluorescent calcium indicator. Freshly isolated (unactivated) monocytes were insensitive to DHP drugs both in the presence and absence of high potassium membrane depolarization. In contrast, nisoldipine, a DHP calcium channel blocker, and BAY K 8644, a DHP calcium channel activator, decreased and increased calcium uptake by KC1-depolarized differentiated monocytes. Results suggest that differentiation of monocytes to macrophages may involve a change in the expression and/or regulation of DHP- sensitive calcium channels.

  3. The Methanolic Extract from Murraya koenigii L. Inhibits Glutamate-Induced Pain and Involves ATP-Sensitive K+ Channel as Antinociceptive Mechanism

    PubMed Central

    Sharmin Ani, Nushrat; Chakraborty, Sudip

    2016-01-01

    Murraya koenigii L. is a perennial shrub, belonging to the family Rutaceae. Traditionally, the leaves of this plant are extensively used in treatment of a wide range of diseases and disorders including pain and inflammation. Although researchers have revealed the antinociceptive effects of this plant's leaves during past few years, the mechanisms underlying these effects are still unknown. Therefore, the present study evaluated some antinociceptive mechanisms of the methanolic extract of M. koenigii (MEMK) leaves along with its antinociceptive potential using several animal models. The antinociceptive effects of MEMK were evaluated using formalin-induced licking and acetic acid-induced writhing tests at the doses of 50, 100, and 200 mg/kg. In addition, we also justified the possible participations of glutamatergic system and ATP-sensitive potassium channels in the observed activities. Our results demonstrated that MEMK significantly (p < 0.01) inhibited the pain thresholds induced by formalin and acetic acid in a dose-dependent manner. MEMK also significantly (p < 0.01) suppressed glutamate-induced pain. Moreover, pretreatment with glibenclamide (an ATP-sensitive potassium channel blocker) at 10 mg/kg significantly (p < 0.05) reversed the MEMK-mediated antinociception. These revealed that MEMK might have the potential to interact with glutamatergic system and the ATP-sensitive potassium channels to exhibit its antinociceptive activities. Therefore, our results strongly support the antinociceptive effects of M. koenigii leaves and provide scientific basis of their analgesic uses in the traditional medicine. PMID:27812367

  4. Involvement of nitric oxide and ATP-sensitive potassium channels in the peripheral antinoceptive action of a tramadol-dexketoprofen combination in the formalin test.

    PubMed

    Isiordia-Espinoza, Mario A; Pozos-Guillén, Amaury; Pérez-Urizar, José; Chavarría-Bolaños, Daniel

    2014-11-01

    Systemic coadministration of tramadol and dexketoprofen can produce antinociceptive synergism in animals. There has been only limited evaluation of this drug combination in the peripheral nervous system in terms of the antinociceptive interaction and its mechanisms. The aim of the present study was to evaluate the peripheral antinociceptive interaction between tramadol and dexketoprofen in the formalin test and the involvement of the nitric oxide (NO)-cyclic guanosine monophosphate pathway and ATP-sensitive K(+) channels. Different doses of tramadol or dexketoprofen were administered locally to the formalin-injured mouse paw and the antinociceptive effect evaluated. ED50 values were calculated for both drugs alone and in combination. Coadministration of tramadol and dexketoprofen produced an antinociceptive synergistic interaction during the second phase of the formalin test. Pretreatment with NO antagonists, including l-NG-nitroarginine methyl ester and 1H-[1,2,4]-oxadiazolo-[4,3-a]-quinoxalin-1-one, or the ATP-sensitive K(+) channel antagonist glibenclamide reversed the antinociceptive synergistic effect of the tramadol-dexketoprofen combination, suggesting that NO and ATP-sensitive K(+) channels were involved.

  5. The residue I257 at S4–S5 linker in KCNQ1 determines KCNQ1/KCNE1 channel sensitivity to 1-alkanols

    PubMed Central

    Xie, Chang; Liu, Hao-wen; Pan, Na; Ding, Jiu-ping; Yao, Jing

    2016-01-01

    Aim: KCNQ1 and KCNE1 form a complex in human ventricular cardiomyocytes, which are important in maintaining a normal heart rhythm. In the present study we investigated the effects of a homologous series of 1-alkanols on KCNQ1/KCNE1 channels expressed in Xenopus oocytes. Methods: ECG recording was made in rats injected with ethanol-containing solution (0.3 mL, ip). Human KCNQ1 channel and its auxiliary subunit KCNE1 were heterologously coexpressed in Xenopus oocytes, which were superfused with ND96 solution; 1-alkanols (ethanol, 1-butanol and 1-hexanol) were delivered through a gravity-driven perfusion device. The slow-delayed rectifier potassium currents IKs (KCNQ1/KCNE1 currents) were recorded using a two-electrode voltage clamp method. Site-directed mutations (I257A) were made in KCNQ1. Results: In ECG recordings, a low concentration of ethanol (3%, v/v) slightly increased the heart rate of rats, whereas the higher concentrations of ethanol (10%, 50%, v/v) markedly reduced it. In oocytes coexpressing KCNQ1/KCNE1 channels, ethanol, 1-butanol and 1-hexanol dose-dependently inhibited IKs currents with IC50 values of 80, 11 and 2.7 mmol/L, respectively. Furthermore, the 1-alkanols blocked the KCNQ1 channel in both open and closed states, and a four-state model could adequately explain the effects of 1-alkanols on the closed-state channel block. Moreover, the mutation of I257A at the intracellular loop between S4 and S5 in KCNQ1 greatly decreased the sensitivity to 1-alkanols; and the IC50 values of ethanol, 1-butanol and 1-hexanol were increased to 634, 414 and 7.4 mmol/L, respectively. The mutation also caused the ablation of closed-state channel block. Conclusion: These findings provide new insight into the intricate mechanisms of the blocking effects of ethanol on the KCNQ1 channel. PMID:26725740

  6. Dihydropyridine-sensitive Ca/sup 2 +/ channels in mammalian skeletal muscle cells in culture: electrophysiological properties and interactions with Ca/sup 2 +/ channel activator (Bay K8644) and inhibitor (PN 200-110)

    SciTech Connect

    Cognard, C.; Romey, G.; Galizzi, J.P.; Fosset, M.; Lazdunski, M.

    1986-03-01

    The whole-cell patch-clamp technique has been used to analyze the properties of the dihydropyridine-sensitive Ca/sup 2 +/ channel in rat skeletal muscle cells (myoballs) in culture. The potential dependence of Ca/sup 2 +/ -channel activation is similar to that observed in cardiac cells. However, the skeletal muscle Ca/sup 2 +/ channel is activated more slowly. The voltage dependence of Ca/sup 2 +/-channel inactivation indicates a half-maximal inactivation (V/sub h0.5/) at -72 mV as compared to V/sub h0.5/ = -35 mV for cardiac cells. Blockade of the skeletal muscle Ca/sup 2 +/ channel by the dihydrophyridine (+)-PN 200-110 is voltage dependent, with a half-maximal effect of 13 nM for an application of the drug to the myoball membrane held at -90 mV and of 0.15 nM for an application at a potential of -65 mV. The 100-fold difference in apparent affinity is interpreted as a preferential association of PN 200-110 with the inactivated form of the Ca/sup 2 +/ channel. The K/sub 0.5/ value found from electrophysiological experiments for the binding to the inactivated state is nearly identical to the equilibrium dissociation constant found from binding experiments with (+)-(/sup 3/H)PN 200-110 using transverse-tubular membranes. The dihydropyridine activator Bay K8644 acts by increasing Ca/sup 2 +/ current amplitude and by slowing down deactivation.

  7. ATP-sensitive K+ channels mediate alpha 2D-adrenergic receptor contraction of arteriolar smooth muscle and reversal of contraction by hypoxia.

    PubMed

    Tateishi, J; Faber, J E

    1995-01-01

    Evidence in rat skeletal muscle suggests that local metabolic control of blood flow is facilitated by the reliance on alpha 2D-adrenergic receptors (ARs) for constriction of arterioles, together with the strong sensitivity of this constriction to inhibition by hypoxia. The present study examined the role of ATP-sensitive K+ (KATP) channels in the selective interaction between alpha 2D-ARs and hypoxia. Arterioles from rat cremaster muscle that possess both alpha 1D (alpha 1A/D)- and alpha 2D-AR subtypes were microcannulated, pressurized, and isolated in a tissue bath for measurement of changes in lumen diameter. Three studies first examined whether stimulation of alpha 2D- and alpha 1D-ARs involves inhibition of the KATP channel. Concentration-dependent constriction by the KATP antagonists glibenclamide (GLB, 0.01 to 10 mumol/L) and disopyramide (0.001 to 1 mmol/L) were abolished during alpha 2D stimulation but unaffected during alpha 1D stimulation. Activation of the KATP channel by cromakalim inhibited alpha 2D constriction with greater potency than alpha 1D (EC50, 7.0 +/- 0.2 versus 6.3 +/- 0.1). Finally, GLB (0.5 mumol/L) abolished dose-dependent alpha 2D constriction, whereas alpha 1D was unaffected. These data suggest that alpha 2D but not alpha 1D stimulation is "coupled" with closure of the KATP channel, leading to depolarization and contraction of vascular smooth muscle. In a second series, hypoxic (PO2, 6 mm Hg) inhibition of intrinsic smooth muscle tone was completely reversed by 0.1 mumol/L GLB, concentration-dependent GLB constriction was enhanced during hypoxia, and hypoxia reversed GLB constriction. These data confirm reports by others that hypoxia potentiates the activation of KATP channels, leading to hyperpolarization and relaxation. Finally, GLB constriction, which was abolished by concomitant alpha 2D stimulation, was completely restored by simultaneous activation of KATP channels with hypoxia. These findings suggest that the sensitivity of alpha

  8. Serotonin facilitates peripheral pain sensitivity in a manner that depends on the nonproton ligand sensing domain of ASIC3 channel.

    PubMed

    Wang, Xiang; Li, Wei-Guang; Yu, Ye; Xiao, Xian; Cheng, Jin; Zeng, Wei-Zheng; Peng, Zhong; Xi Zhu, Michael; Xu, Tian-Le

    2013-03-06

    Tissue acidosis and inflammatory mediators play critical roles in inflammatory pain. Extracellular acidosis activates acid-sensing ion channels (ASICs), which have emerged as key sensors for extracellular protons in the central and peripheral nervous systems and play key roles in pain sensation and transmission. Additionally, inflammatory mediators, such as serotonin (5-HT), are known to enhance pain sensation. However, functional interactions among protons, inflammatory mediators, and ASICs in pain sensation are poorly understood. In the present study, we show that 5-HT, a classical pro-inflammatory mediator, specifically enhances the proton-evoked sustained, but not transient, currents mediated by homomeric ASIC3 channels and heteromeric ASIC3/1a and ASIC3/1b channels. Unexpectedly, the effect of 5-HT on ASIC3 channels does not involve activation of 5-HT receptors, but is mediated via a functional interaction between 5-HT and ASIC3 channels. We further show that the effect of 5-HT on ASIC3 channels depends on the newly identified nonproton ligand sensing domain. Finally, coapplication of 5-HT and acid significantly increased pain-related behaviors as assayed by the paw-licking test in mice, which was largely attenuated in ASIC3 knock-out mice, and inhibited by the nonselective ASIC inhibitor amiloride. Together, these data identify ASIC3 channels as an unexpected molecular target for acute actions of 5-HT in inflammatory pain sensation and reveal an important role of ASIC3 channels in regulating inflammatory pain via coincident detection of extracellular protons and inflammatory mediators.

  9. Multilevel fitting of {sup 235}U resonance data sensitive to Bohr-and Brosa-fission channels

    SciTech Connect

    Moore, M.S.

    1995-05-01

    The recent determination of the K, J dependence of the neutron induced fission cross section of {sup 235}U by the Dubna group has led to a renewed interest in the mechanism of fission from saddle to scission. The K quantum numbers designate the so-called Bohr fission channels, which describe the fission properties at the saddle point. Certain other fission properties, e.g., the fragment mass and kinetic-energy distribution, are related to the properties of the scission point. The neutron energy dependence of the fragment kinetic energies has been measured by Hambsch et al., who analyzed their data according to a channel description of Brosa et al. How these two channel descriptions, the saddle-point Bohr channels and the scission-point Brosa channels, relate to one another is an open question, and is the subject matter of the present paper. We use the correlation coefficient between various data sets, in which variations are reported from resonance to resonance, as a measure of both-the statistical reliability of the data and of the degree to which different scission variables relate to different Bohr channels. We have carried out an adjustment of the ENDF/B-VI multilevel evaluation of the fission cross section of {sup 235}U, one that provides a reasonably good fit to the energy dependence of the fission, capture, and total cross sections below 100 eV, and to the Bohr-channel structure deduced from an earlier measurement by Pattenden and Postma. We have also further explored the possibility of describing the data of Hambsch et al. in the Brosa-channel framework with the same set of fission-width vectors, only in a different reference system. While this approach shows promise, it is clear that better data are also needed for the neutron energy variation of the scission-point variables.

  10. Expression and function of K(ATP) channels in normal and osteoarthritic human chondrocytes: possible role in glucose sensing.

    PubMed

    Rufino, Ana T; Rosa, Susana C; Judas, Fernando; Mobasheri, Ali; Lopes, M Celeste; Mendes, Alexandrina F

    2013-08-01

    ATP-sensitive potassium [K(ATP)] channels sense intracellular ATP/ADP levels, being essential components of a glucose-sensing apparatus in various cells that couples glucose metabolism, intracellular ATP/ADP levels and membrane potential. These channels are present in human chondrocytes, but their subunit composition and functions are unknown. This study aimed at elucidating the subunit composition of K(ATP) channels expressed in human chondrocytes and determining whether they play a role in regulating the abundance of major glucose transporters, GLUT-1 and GLUT-3, and glucose transport capacity. The results obtained show that human chondrocytes express the pore forming subunits, Kir6.1 and Kir6.2, at the mRNA and protein levels and the regulatory sulfonylurea receptor (SUR) subunits, SUR2A and SUR2B, but not SUR1. The expression of these subunits was no affected by culture under hyperglycemia-like conditions. Functional impairment of the channel activity, using a SUR blocker (glibenclamide 10 or 20 nM), reduced the protein levels of GLUT-1 and GLUT-3 by approximately 30% in normal chondrocytes, while in cells from cartilage with increasing osteoarthritic (OA) grade no changes were observed. Glucose transport capacity, however, was not affected in normal or OA chondrocytes. These results show that K(ATP) channel activity regulates the abundance of GLUT-1 and GLUT-3, although other mechanisms are involved in regulating the overall glucose transport capacity of human chondrocytes. Therefore, K(ATP) channels are potential components of a broad glucose sensing apparatus that modulates glucose transporters and allows human chondrocytes to adjust to varying extracellular glucose concentrations. This function of K(ATP) channels seems to be impaired in OA chondrocytes.

  11. Specificity for block by saxitoxin and divalent cations at a residue which determines sensitivity of sodium channel subtypes to guanidinium toxins

    PubMed Central

    1995-01-01

    bTyrosine 401 of the skeletal muscle isoform (mu 1) of the rat muscle Na channel is an important determinant of high affinity block by tetrodotoxin (TTX) and saxitoxin (STX) in Na-channel isoforms. In mammalian heart Na channels, this residue is substituted by cysteine, which results in low affinity for TTX/STX and enhanced sensitivity to block by Zn2+ and Cd2+. In this study, we investigated the molecular basis for high affinity block of Na channels by STX and divalent cations by measuring inhibition of macroscopic Na+ current for a series of point mutations at residue Tyr401 of the rat mu 1 Na channel expressed in Xenopus oocytes. Substitution of Tyr401 by Gly, Ala, Ser, Cys, Asp, His, Trp, and Phe produced functional Na+ currents without major perturbation of gating or ionic selectivity. High affinity block by STX and neosaxitoxin (NEO) with Ki values in the range of 2.6-18 nM required Tyr, Phe, or Trp, suggestive of an interaction between an aromatic ring and a guanidinium group of the toxin. The Cys mutation resulted in a 7- and 23-fold enhancement of the dissociation rate of STX and NEO, respectively, corresponding to rapid toxin dissociation rates of cardiac Na channels. High affinity block by Zn2+ (Ki = 8-23 microM) required Cys, His, or Asp, three residues commonly found to coordinate directly with Zn2+ in metalloproteins. For the Cys mutant of mu 1 and also for the cardiac isoform Na channel (rh1) expressed in the L6 rat muscle cell line, inhibition of macroscopic Na+ conductance by Zn2+ reached a plateau at 85-90% inhibition, suggesting the presence of a substate current. The Asp mutant also displayed enhanced affinity for inhibition of conductance by Ca2+ (Ki = 0.3 mM vs approximately 40 mM in wild type), but block by Ca2+ was incomplete, saturating at approximately 69% inhibition. In contrast, Cd2+ completely blocked macroscopic current in the Cys mutant and the L6 cell line. These results imply that the magnitude of substate current depends on the

  12. Autocrine insulin increases plasma membrane K(ATP) channel via PI3K-VAMP2 pathway in MIN6 cells.

    PubMed

    Xu, Shanhua; Kim, Ji-Hee; Hwang, Kyu-Hee; Das, Ranjan; Quan, Xianglan; Nguyen, Tuyet Thi; Kim, Soo-Jin; Cha, Seung-Kuy; Park, Kyu-Sang

    2015-12-25

    Regulation of ATP-sensitive inwardly rectifying potassium (KATP) channel plays a critical role in metabolism-secretion coupling of pancreatic β-cells. Released insulin from β-cells inhibits insulin and glucagon secretion with autocrine and paracrine modes. However, molecular mechanism by which insulin inhibits hormone secretion remains elusive. Here, we investigated the effect of autocrine insulin on surface abundance of KATP channel in mouse clonal β-cell line, MIN6. High glucose increased plasmalemmal sulfonylurea receptor 1 (SUR1), a component of KATP channel as well as exogenous insulin treatment. SUR1 trafficking by high glucose or insulin was blocked by inhibition of phosphoinositide 3-kinase (PI3K) with wortmannin. Pretreatment with brefeldin A or silencing of vesicle-associated membrane protein 2 (VAMP2) abolished insulin-mediated upregulation of surface SUR1. Functionally, glucose-stimulated cytosolic Ca(2+) ([Ca(2+)]i) increase was blunted by insulin or diazoxide, a KATP channel opener. Insulin-induced suppression of [Ca(2+)]i oscillation was prevented by an insulin receptor blocker. These results provide a novel molecular mechanism for autocrine negative feedback regulation of insulin secretion.

  13. Histamine Increases Neuronal Excitability and Sensitivity of the Lateral Vestibular Nucleus and Promotes Motor Behaviors via HCN Channel Coupled to H2 Receptor

    PubMed Central

    Li, Bin; Zhang, Xiao-Yang; Yang, Ai-Hong; Peng, Xiao-Chun; Chen, Zhang-Peng; Zhou, Jia-Yuan; Chan, Ying-Shing; Wang, Jian-Jun; Zhu, Jing-Ning

    2017-01-01

    Histamine and histamine receptors in the central nervous system actively participate in the modulation of motor control. In clinic, histamine-related agents have traditionally been used to treat vestibular disorders. Immunohistochemical studies have revealed a distribution of histaminergic afferents in the brainstem vestibular nuclei, including the lateral vestibular nucleus (LVN), which is critical for adjustment of muscle tone and vestibular reflexes. However, the mechanisms underlying the effect of histamine on LVN neurons and the role of histamine and histaminergic afferents in the LVN in motor control are still largely unknown. Here, we show that histamine, in cellular and molecular levels, elicits the LVN neurons of rats an excitatory response, which is co-mediated by the hyperpolarization-activated cyclic nucleotide-gated (HCN) channels and K+ channels linked to H2 receptors. Blockage of HCN channels coupled to H2 receptors decreases LVN neuronal sensitivity and changes their dynamic properties. Furthermore, in behavioral level, microinjection of histamine into bilateral LVNs significantly promotes motor performances of rats on both accelerating rota-rod and balance beam. This promotion is mimicked by selective H2 receptor agonist dimaprit, and blocked by selective H2 receptor antagonist ranitidine. More importantly, blockage of HCN channels to suppress endogenous histaminergic inputs in the LVN considerably attenuates motor balance and coordination, indicating a promotion role of hypothalamo-vestibular histaminergic circuit in motor control. All these results demonstrate that histamine H2 receptors and their coupled HCN channels mediate the histamine-induced increase in excitability and sensitivity of LVN neurons and contribute to the histaminergic improvement of the LVN-related motor behaviors. The findings suggest that histamine and the histaminergic afferents may directly modulate LVN neurons and play a critical role in the central vestibular

  14. Histamine Increases Neuronal Excitability and Sensitivity of the Lateral Vestibular Nucleus and Promotes Motor Behaviors via HCN Channel Coupled to H2 Receptor.

    PubMed

    Li, Bin; Zhang, Xiao-Yang; Yang, Ai-Hong; Peng, Xiao-Chun; Chen, Zhang-Peng; Zhou, Jia-Yuan; Chan, Ying-Shing; Wang, Jian-Jun; Zhu, Jing-Ning

    2016-01-01

    Histamine and histamine receptors in the central nervous system actively participate in the modulation of motor control. In clinic, histamine-related agents have traditionally been used to treat vestibular disorders. Immunohistochemical studies have revealed a distribution of histaminergic afferents in the brainstem vestibular nuclei, including the lateral vestibular nucleus (LVN), which is critical for adjustment of muscle tone and vestibular reflexes. However, the mechanisms underlying the effect of histamine on LVN neurons and the role of histamine and histaminergic afferents in the LVN in motor control are still largely unknown. Here, we show that histamine, in cellular and molecular levels, elicits the LVN neurons of rats an excitatory response, which is co-mediated by the hyperpolarization-activated cyclic nucleotide-gated (HCN) channels and K(+) channels linked to H2 receptors. Blockage of HCN channels coupled to H2 receptors decreases LVN neuronal sensitivity and changes their dynamic properties. Furthermore, in behavioral level, microinjection of histamine into bilateral LVNs significantly promotes motor performances of rats on both accelerating rota-rod and balance beam. This promotion is mimicked by selective H2 receptor agonist dimaprit, and blocked by selective H2 receptor antagonist ranitidine. More importantly, blockage of HCN channels to suppress endogenous histaminergic inputs in the LVN considerably attenuates motor balance and coordination, indicating a promotion role of hypothalamo-vestibular histaminergic circuit in motor control. All these results demonstrate that histamine H2 receptors and their coupled HCN channels mediate the histamine-induced increase in excitability and sensitivity of LVN neurons and contribute to the histaminergic improvement of the LVN-related motor behaviors. The findings suggest that histamine and the histaminergic afferents may directly modulate LVN neurons and play a critical role in the central vestibular

  15. Early-onset epileptic encephalopathy caused by a reduced sensitivity of Kv7.2 potassium channels to phosphatidylinositol 4,5-bisphosphate

    PubMed Central

    Soldovieri, Maria Virginia; Ambrosino, Paolo; Mosca, Ilaria; De Maria, Michela; Moretto, Edoardo; Miceli, Francesco; Alaimo, Alessandro; Iraci, Nunzio; Manocchio, Laura; Medoro, Alessandro; Passafaro, Maria; Taglialatela, Maurizio

    2016-01-01

    Kv7.2 and Kv7.3 subunits underlie the M-current, a neuronal K+ current characterized by an absolute functional requirement for phosphatidylinositol 4,5-bisphosphate (PIP2). Kv7.2 gene mutations cause early-onset neonatal seizures with heterogeneous clinical outcomes, ranging from self-limiting benign familial neonatal seizures to severe early-onset epileptic encephalopathy (Kv7.2-EE). In this study, the biochemical and functional consequences prompted by a recurrent variant (R325G) found independently in four individuals with severe forms of neonatal-onset EE have been investigated. Upon heterologous expression, homomeric Kv7.2 R325G channels were non-functional, despite biotin-capture in Western blots revealed normal plasma membrane subunit expression. Mutant subunits exerted dominant-negative effects when incorporated into heteromeric channels with Kv7.2 and/or Kv7.3 subunits. Increasing cellular PIP2 levels by co-expression of type 1γ PI(4)P5-kinase (PIP5K) partially recovered homomeric Kv7.2 R325G channel function. Currents carried by heteromeric channels incorporating Kv7.2 R325G subunits were more readily inhibited than wild-type channels upon activation of a voltage-sensitive phosphatase (VSP), and recovered more slowly upon VSP switch-off. These results reveal for the first time that a mutation-induced decrease in current sensitivity to PIP2 is the primary molecular defect responsible for Kv7.2-EE in individuals carrying the R325G variant, further expanding the range of pathogenetic mechanisms exploitable for personalized treatment of Kv7.2-related epilepsies. PMID:27905566

  16. Design, synthesis and cytotoxic activity of novel sulfonylurea derivatives of podophyllotoxin.

    PubMed

    Zhang, Zhi-Jun; Tian, Jing; Wang, Li-Ting; Wang, Mei-Juan; Nan, Xiang; Yang, Liu; Liu, Ying-Qian; Morris-Natschke, Susan L; Lee, Kuo-Hsiung

    2014-01-01

    Three series of novel sulfonylurea podophyllotoxin derivatives were designed, synthesized, and evaluated for in vitro cytotoxicity against four tumor cell lines (A-549, DU-145, KB and KBvin). Compounds 14c (IC₅₀: 1.41-1.76 μM) and 14e (IC₅₀: 1.72-2.01 μM) showed superior cytotoxic activity compared with etoposide (IC₅₀: 2.03 to >20 μM), a clinically available anticancer drug. Significantly, most of the compounds exhibited comparable cytotoxicity against the drug-resistant tumor cell line KBvin, while etoposide lost activity completely. Preliminary structure-activity relationship (SAR) correlations indicated that the 4'-O-methyl functionality in podophyllotoxin analogues may be essential to maintain cytotoxic activity, while an arylsulfonylurea side chain at podophyllotoxin's 4β position can significantly improve cytotoxic activity.

  17. Acid sensitive background potassium channels K2P3.1 and K2P9.1 undergo rapid dynamin-dependent endocytosis

    PubMed Central

    Mant, Alexandra; Williams, Sarah E; O'Kelly, Ita

    2013-01-01

    Acid-sensitive, two-pore domain potassium channels, K2P3.1 and K2P9.1, are implicated in cardiac and nervous tissue responses to hormones, neurotransmitters and drugs. K2P3.1 and K2P9.1 leak potassium from the cell at rest and directly impact membrane potential. Hence altering channel number on the cell surface drives changes in cellular electrical properties. The rate of K2P3.1 and K2P9.1 delivery to and recovery from the plasma membrane determines both channel number at the cell surface and potassium leak from cells. This study examines the endocytosis of K2P3.1 and K2P9.1. Plasma membrane biotinylation was used to follow the fate of internalized GFP-tagged rat K2P3.1 and K2P9.1 transiently expressed in HeLa cells. Confocal fluorescence images were analyzed using Imaris software, which revealed that both channels are endocytosed by a dynamin-dependent mechanism and over the course of 60 min, move progressively toward the nucleus. Endogenous endocytosis of human K2P3.1 and K2P9.1 was examined in the lung carcinoma cell line, A549. Endogenous channels are endocytosed over a similar time-scale to the channels expressed transiently in HeLa cells. These findings both validate the use of recombinant systems and identify an endogenous model system in which K2P3.1 and K2P9.1 trafficking can be further studied. PMID:23807092

  18. Acid sensitive background potassium channels K2P3.1 and K2P9.1 undergo rapid dynamin-dependent endocytosis.

    PubMed

    Mant, Alexandra; Williams, Sarah; O'Kelly, Ita

    2013-01-01

    Acid-sensitive, two-pore domain potassium channels, K(2P)3.1 and K(2P)9.1, are implicated in cardiac and nervous tissue responses to hormones, neurotransmitters and drugs. K(2P)3.1 and K(2P)9.1 leak potassium from the cell at rest and directly impact membrane potential. Hence altering channel number on the cell surface drives changes in cellular electrical properties. The rate of K(2P)3.1 and K(2P)9.1 delivery to and recovery from the plasma membrane determines both channel number at the cell surface and potassium leak from cells. This study examines the endocytosis of K(2P)3.1 and K(2P)9.1. Plasma membrane biotinylation was used to follow the fate of internalized GFP-tagged rat K(2P)3.1 and K(2P)9.1 transiently expressed in HeLa cells. Confocal fluorescence images were analyzed using Imaris software, which revealed that both channels are endocytosed by a dynamin-dependent mechanism and over the course of 60 min, move progressively toward the nucleus. Endogenous endocytosis of human K(2P)3.1 and K(2P)9.1 was examined in the lung carcinoma cell line, A549. Endogenous channels are endocytosed over a similar time-scale to the channels expressed transiently in HeLa cells. These findings both validate the use of recombinant systems and identify an endogenous model system in which K(2P)3.1 and K(2P)9.1 trafficking can be further studied.

  19. Wanderlust kinetics and variable Ca(2+)-sensitivity of dSlo [correction of Drosophila], a large conductance CA(2+)-activated K+ channel, expressed in oocytes.

    PubMed Central

    Silberberg, S D; Lagrutta, A; Adelman, J P; Magleby, K L

    1996-01-01

    Cloned large conductance Ca2+-activated K+ channels (BK or maxi-K+ channels) from Drosophila (dSlo) were expressed in Xenopus oocytes and studied in excised membrane patches with the patch-clamp technique. Both a natural variant and a mutant that eliminated a putative cyclic AMP-dependent protein kinase phosphorylation site exhibited large, slow fluctuations in open probability with time. These fluctuations, termed "wanderlust kinetics," occurred with a time course of tens of seconds to minutes and had kinetic properties inconsistent with simple gating models. Wanderlust kinetics was still observed in the presence of 5mM caffeine or 50 nM thapsigargin, or when the Ca2+ buffering capacity of the solution was increased by the addition of 5 mM HEDTA, suggesting that the wanderlust kinetics did not arise from Ca2+ release from caffeine and thapsigargin sensitive internal stores in the excised patch. The slow changes in kinetics associated with wanderlust kinetics could be generated with a discrete-state Markov model with transitions among three or more kinetic modes with different levels of open probability. To average out the wanderlust kinetics, large amounts of data were analyzed and demonstrated up to a threefold difference in the [Ca2+]i required for an open probability of 0.5 among channels expressed from the same injected mRNA. These findings indicate that cloned dSlo channels in excised patches from Xenopus oocytes can exhibit large variability in gating properties, both within a single channel and among channels. PMID:9643949

  20. Isoform-specific modulation of the chemical sensitivity of conserved TRPA1 channel in the major honeybee ectoparasitic mite, Tropilaelaps mercedesae

    PubMed Central

    Dong, Xiaofeng; Kashio, Makiko; Peng, Guangda; Wang, Xinyue; Tominaga, Makoto

    2016-01-01

    We identified and characterized the TRPA1 channel of Tropilaelaps mercedesae (TmTRPA1), one of two major species of honeybee ectoparasitic mite. Three TmTRPA1 isoforms with unique N-terminal sequences were activated by heat, and the isoform highly expressed in the mite's front legs, TmTRPA1b, was also activated by 27 plant-derived compounds including electrophiles. This suggests that the heat- and electrophile-dependent gating mechanisms as nocisensitive TRPA1 channel are well conserved between arthropod species. Intriguingly, one TmTRPA1 isoform, TmTRPA1a, was activated by only six compounds compared with two other isoforms, demonstrating that the N-terminal sequences are critical determinants for the chemical sensitivity. This is the first example of isoform-specific modulation of chemical sensitivity of TRPA1 channel in one species. α-terpineol showed repellent activity towards T. mercedesae in a laboratory assay and repressed T. mercedesae entry for reproduction into the brood cells with fifth instar larvae in hives. Thus, α-terpineol could be used as the potential compound to control two major honeybee ectoparasitic mites, T. mercedesae and Varroa destructor, in the apiculture industry. PMID:27307515

  1. Sensitivity and specificity of simultaneously acquired (dual channel) radiogallium and Tc-99m-HDP in painful hip and knee prosthetic joints

    SciTech Connect

    Skarzynski, J.J.; Sziklas, J.J.; Rosenberg, R.J.; Rich, D.A.; Spencer, R.P.

    1985-05-01

    Differentiation of prosthetic loosening from infection, by use of sequential bone and radiogallium imaging, has been discussed in the literature. The authors investigated simultaneous (2 channel) imaging of Ga-67 and Tc-99m-HDP in multiviews, in order to assess the parameter of Tc-99m-Ga-67 incongruity. Acquisition of data was carried out 2 days after 5 mCi of Ga-67 citrate IV and 2 hours after 8 mCi of Tc-99m-HDP. Dual data channels were used to insure perfect superimposition of the images and to reduce total imaging time. Normalized bone images were taken, then subtracted from those of Ga-67, by means of progressive weighting factors. A total of 68 studies were carried out on 43 patients. Exams involved both knee and hip prostheses, in population with 63% of the patients over age 60 years. Time from placement of the prosthesis to the dual radionuclide exam was within 2 years in 48% and within 5 years in 78%. Sensitivity was 0.88 and specificity 0.89. Using information on the follow-up dual channel studies, 40/43 cases were correctly identified (93%). Dual channel radionuclide imaging offers a readily available and accurate means of differentiating infection from loosening of hip or knee prostheses.

  2. Fasiglifam/TAK-875, a Selective GPR40 Agonist, Improves Hyperglycemia in Rats Unresponsive to Sulfonylureas and Acts Additively with Sulfonylureas.

    PubMed

    Ito, Ryo; Tsujihata, Yoshiyuki; Suzuki, Masami; Miyawaki, Kazumasa; Matsuda, Kae; Takeuchi, Koji

    2016-04-01

    Sulfonylureas (SUs) are widely used insulin secretagogues, but they have adverse effects including hypoglycemia and secondary failure. Fasiglifam/TAK-875, a selective GPR40 agonist, enhances glucose-stimulated insulin secretion and improves hyperglycemia. In the present study, we compared the in vivo glucose-lowering effects of fasiglifam with SUs. The risk of secondary failure of fasiglifam and the efficacy in rats desensitized to SUs were also evaluated. Moreover, we assessed whether fasiglifam was effective when combined with SUs. In diabetic neonatally streptozotocin-induced rats 1.5 days after birth (N-STZ-1.5), oral administrations of fasiglifam (3-30 mg/kg) dose dependently improved glucose tolerance; the effect was greater than that of glibenclamide at maximal effective doses (glucose AUC: fasiglifam, -37.6%; glibenclamide, -12.3%). Although the glucose-lowering effects of glibenclamide (10 mg/kg/day) were completely diminished in N-STZ-1.5 rats after 4 weeks of treatment, effects were maintained in rats receiving fasiglifam (10 mg/kg/day), even after 15 weeks. Fasiglifam (3-10 mg/kg) was still effective in two models desensitized to SUs: 15-week glibenclamide-treated N-STZ-1.5 rats and aged Zucker diabetic fatty (ZDF) rats. Acute administration of fasiglifam (3 mg/kg) and glimepiride (10 mg/kg) in combination additively decreased glucose AUC (fasiglifam, -25.3%; glimepiride, -20.0%; combination, -43.1%). Although glimepiride (10 mg/kg) decreased plasma glucose below normal in nonfasted control rats, fasiglifam (3 mg/kg) maintained normoglycemia, and no further exaggeration of hypoglycemia was observed with combination treatment. These results indicate that GPR40 agonists could be more effective and durable than SUs. Our results also provide new insights into GPR40 pharmacology and rationale for the use of GPR40 agonists in diabetic patients with SU failure.

  3. Low-Timing-Jitter Near-Infrared Single-Photon-Sensitive 16-Channel Intensified-Photodiode Detector

    NASA Technical Reports Server (NTRS)

    Krainak, Michael A.; Lu, Wei; Yang, Guangning; Sun, Xiaoli; Sykora, Derek; Jurkovic, Mike; Aebi, Verle; Costello, Ken; Burns, Richard

    2011-01-01

    We developed a 16-channel InGaAsP photocathode intensified-photodiode (IPD) detector with 78 ps (1-sigma) timing-jitter, less than 500 ps FWHM impulse response, greater than 15% quantum efficiency at 1064 nm wavelength with 131 kcps dark counts at 15 C.

  4. Inhibition of insulin release induced by galanin in the dog is not exclusively mediated by activation of ATP-sensitive K+ channels.

    PubMed

    Lorrain, J; Angel, I; Eon, M T; Oblin, A; Langer, S Z

    1993-01-01

    Exogenous galanin (9 pmol/min during 20 min i.a.) decreased insulin levels in the plasma sampled from the pancreatic vein of the blood-perfused pancreas of dogs in which ganglionic transmission and beta-adrenoceptors had been blocked. This effect was not modified by idazoxan (1 mg/kg followed by 0.02 mg/kg/min i.v.) but was partially reduced (50%) by glibenclamide (1 mg/kg followed by 0.02 mg/kg/min i.v.). This dose of glibenclamide blocked entirely the hypoinsulinemic activity of diazoxide, an activator of pancreatic ATP-modulated K+ channels, whereas the dose of idazoxan prevented the effect of the alpha 2-adrenoceptor agonist UK-14,304. Therefore, in dogs, the decrease in insulin secretion produced by exogenous galanin is only partially mediated by activation of glibenclamide-sensitive ATP-gated K+ channels and is independent of alpha 2-adrenoceptor stimulation.

  5. Kinetics of xylem loading, membrane potential maintenance, and sensitivity of K(+) -permeable channels to reactive oxygen species: physiological traits that differentiate salinity tolerance between pea and barley.

    PubMed

    Bose, Jayakumar; Shabala, Lana; Pottosin, Igor; Zeng, Fanrong; Velarde-Buendía, Ana-Maria; Massart, Amandine; Poschenrieder, Charlotte; Hariadi, Yuda; Shabala, Sergey

    2014-03-01

    Salt sensitive (pea) and salt tolerant (barley) species were used to understand the physiological basis of differential salinity tolerance in crops. Pea plants were much more efficient in restoring otherwise depolarized membrane potential thereby effectively decreasing K(+) efflux through depolarization-activated outward rectifying potassium channels. At the same time, pea root apex was 10-fold more sensitive to physiologically relevant H2 O2 concentration and accumulated larger amounts of H2 O2 under saline conditions. This resulted in a rapid loss of cell viability in the pea root apex. Barley plants rapidly loaded Na(+) into the xylem; this increase was only transient, and xylem and leaf Na(+) concentration remained at a steady level for weeks. On the contrary, pea plants restricted xylem Na(+) loading during the first few days of treatment but failed to prevent shoot Na(+) elevation in the long term. It is concluded that superior salinity tolerance of barley plants compared with pea is conferred by at least three different mechanisms: (1) efficient control of xylem Na(+) loading; (2) efficient control of H2 O2 accumulation and reduced sensitivity of non-selective cation channels to H2 O2 in the root apex; and (3) higher energy saving efficiency, with less ATP spent to maintain membrane potential under saline conditions.

  6. Binary Channel SAW Mustard Gas Sensor Based on PdPc0.3PANI0.7 hybrid Sensitive Film

    NASA Astrophysics Data System (ADS)

    Shi, Y. B.; Xiang, J. J.; Feng, Q. H.; Hu, Z. P.; Zhang, H. Q.; Guo, J. Y.

    2006-10-01

    This paper discussed the working principle of binary channel surface acoustic wave (SAW) lithium niobate piezoelectric chip detecting mustard, established the mathematic model of beat frequency output Δf and the mustard gas density δ. The MEMS craft solved the parameters of the binary channel SAW chip such as its interdigital electrode number was 15~25 couple, width and spacing were both 25µm, degree of overlapping was 2mm, fundamental frequency was 10~35MHz, frequency-domain width was 5~20Hz, and its back pt hot film's. According to TG-DSC thermal analysis, vacuum coating craft was adopted to solve the hybrid sensitive film forming craft parameter of PdPc0.3PANI0.7(phthalocyanine palladium0.3Poiyaniline0.7). The micro-appearance of sensitive film was analyzed through SEM. The sensor's sensitivity and response characteristic were tested and analyzed: appear linear change, its response time is less than 5min while its recovery time is less than 8min.

  7. Α-Dendrotoxin-sensitive Kv1 channels contribute to conduction failure of polymodal nociceptive C-fibers from rat coccygeal nerve.

    PubMed

    Wang, Xiu-Chao; Wang, Shan; Zhang, Ming; Gao, Fang; Yin, Chun; Li, Hao; Zhang, Ying; Hu, San-Jue; Duan, Jian-Hong

    2016-02-01

    It is known that some patients with diabetic neuropathy are usually accompanied by abnormal painful sensations. Evidence has accumulated that diabetic neuropathic pain is associated with the hyperexcitability of peripheral nociceptors. Previously, we demonstrated that reduced conduction failure of polymodal nociceptive C-fibers and enhanced voltage-dependent sodium currents of small dorsal root ganglion (DRG) neurons contribute to diabetic hyperalgesia. To further investigate whether and how potassium channels are involved in the conduction failure, α-dendrotoxin (α-DTX), a selective blocker of the low-threshold sustained Kv1 channel, was chosen to examine its functional capability in modulating the conduction properties of polymodal nociceptive C-fibers and the excitability of sensory neurons. We found that α-DTX reduced the conduction failure of C-fibers from coccygeal nerve in vivo accompanied by an increased initial conduction velocity but a decreased activity-dependent slowing of conduction velocity. In addition, the number of APs evoked by step currents was significantly enhanced after the treatment with α-DTX in small-diameter sensory neurons. Further study of the mechanism indicates α-DTX-sensitive K(+) current significantly reduced and the activation of this current in peak and steady state shifted to depolarization for diabetic neurons. Expression of Kv channel subunits Kv1.2 and Kv1.6 was downregulated in both small dorsal root ganglion neurons and peripheral C-fibers. Taken together, these results suggest that α-DTX-sensitive Kv1 channels might play an important role in regulating the conduction properties of polymodal nociceptive C-fibers and firing properties of sensory neurons.

  8. Use of octreotide to treat prolonged sulfonylurea-induced hypoglycemia in a patient with chronic renal failure.

    PubMed

    Nzerue, C M; Thomas, J; Volcy, J; Edeki, T

    2003-01-01

    A diabetic patient with chronic renal failure who developed recurrent and prolonged episodes of hypoglycemia associated with use of sulfonylurea agent is presented here. This patient was hospitalized with neuroglycopenic symptoms of hypoglycemia that persisted in spite of large doses of parenteral glucose replacement. On administration of somatostatin analogue octreotide, hypoglycemia resolved and, blood glucose levels were maintained even after cessation of parenteral glucose. The patient received 2 subcutaneous doses of octreotide 12 hours apart, and made a complete recovery. Our experience suggests that use of octerotide to treat refractory or prolonged sulfonylurea-included hypoglycemia in renal failure patients is safe and effective; large prospective studies would be needed to validate these findings.

  9. Niflumic acid-sensitive ion channels play an important role in the induction of glucose-stimulated insulin secretion by cyclic AMP in mice

    PubMed Central

    Fujimoto, W.; Miki, T.; Ogura, T.; Zhang, M.; Seino, Y.; Satin, L. S.; Nakaya, H.

    2015-01-01

    Aims/hypothesis We have previously reported that glucose-stimulated insulin secretion (GSIS) is induced by glucagon-like peptide-1 (GLP-1) in mice lacking ATP-sensitive K+ (KATP) channels (Kir6.2−/− mice [up-to-date symbol for Kir6.2 gene is Kcnj11]), in which glucose alone does not trigger insulin secretion. This study aimed to clarify the mechanism involved in the induction of GSIS by GLP-1. Methods Pancreas perfusion experiments were performed using wild-type (Kir6.2+/+) or Kir6.2−/− mice. Glucose concentrations were either changed abruptly from 2.8 to 16.7 mmol/l or increased stepwise (1.4 mmol/l per step) from 2.8 to 12.5 mmol/l. Electrophysiological experiments were performed using pancreatic beta cells isolated from Kir6.2−/− mice or clonal pancreatic beta cells (MIN6 cells) after pharmacologically inhibiting their KATP channels with glibenclamide. Results The combination of cyclic AMP plus 16.7 mmol/l glucose evoked insulin secretion in Kir6.2−/− pancreases where glucose alone was ineffective as a secretagogue. The secretion was blocked by the application of niflumic acid. In KATP channel-inactivated MIN6 cells, niflumic acid similarly inhibited the membrane depolarisation caused by cAMP plus glucose. Surprisingly, stepwise increases of glucose concentration triggered insulin secretion only in the presence of cAMP or GLP-1 in Kir6.2+/+, as in Kir6.2−/− pancreases. Conclusions/interpretation Niflumic acid-sensitive ion channels participate in the induction of GSIS by cyclic AMP in Kir6.2−/− beta cells. Cyclic AMP thus not only acts as a potentiator of insulin secretion, but appears to be permissive for GSIS via novel, niflumic acid-sensitive ion channels. This mechanism may be physiologically important for triggering insulin secretion when the plasma glucose concentration increases gradually rather than abruptly. PMID:19266181

  10. Retina derived relaxation is mediated by K(ir) channels and the inhibition of Ca(2+) sensitization in isolated bovine retinal arteries.

    PubMed

    Takır, Selçuk; Uydeş-Doğan, B Sönmez; Özdemir, Osman

    2015-03-01

    Retinal relaxing factor (RRF) has recently been identified as a novel paracrine regulator of retinal circulation acting differently from well known mediators of the endothelium and the retina. Herein, we aimed to characterize the relaxing mechanism of the retina, i.e. RRF, by evaluating the role of Ca(+2)-dependent and -independent signaling mechanisms as well as inward rectifier K(+) (Kir) channels. Retinal relaxation was determined by placing a piece of retinal tissue just on top of the precontracted bovine retinal arteries mounted in a wire myograph. The retina produced a complete relaxation response, which display a biphasic character, in depolarized arteries contracted by L-type Ca(2+) channel agonist, Bay k 8644. Blockade of L-type Ca(2+) channel by nifedipine, inhibition of sarcoplasmic reticulum Ca(2+)-ATPase by cyclopiazonic acid or removal of extracellular Ca(2+) did not influence the prominent relaxation to the retina. Originally, retinal relaxation was found to be unaffected from the inhibition of myosin light chain kinase by ML7, whereas, completely abolished in the presence of myosin light chain phosphatase (MLCP) inhibitor, Calyculin A. Moreover, the inhibition of Rho kinase by its putative inhibitor, Y-27632 displayed comparable relaxant effects to RRF in retinal arteries precontracted either by prostaglandin F2α or K(+), and augmented the moderate response to the retina in K(+) precontracted arteries. In addition, retinal relaxation was significantly inhibited and lost its biphasic character in the presence of Kir channel blocker, Ba(2+). Our results suggested that inhibition of Ca(2+) sensitization through the activation of MLCP, possibly via interfering with Rho kinase, and the opening of Kir channels are likely to be involved in the inhibitory influence of RRF on the retinal arteries.

  11. Fine Tuning of CaV1.3 Ca2+ Channel Properties in Adult Inner Hair Cells Positioned in the Most Sensitive Region of the Gerbil Cochlea

    PubMed Central

    Zampini, Valeria; Johnson, Stuart L.; Franz, Christoph; Knipper, Marlies; Holley, Matthew C.; Magistretti, Jacopo; Russo, Giancarlo; Marcotti, Walter; Masetto, Sergio

    2014-01-01

    Hearing relies on faithful signal transmission by cochlear inner hair cells (IHCs) onto auditory fibres over a wide frequency and intensity range. Exocytosis at IHC ribbon synapses is triggered by Ca2+ inflow through CaV1.3 (L-type) Ca2+ channels. We investigated the macroscopic (whole-cell) and elementary (cell-attached) properties of Ca2+ currents in IHCs positioned at the middle turn (frequency ∼2 kHz) of the adult gerbil cochlea, which is their most sensitive hearing region. Using near physiological recordings conditions (body temperature and a Na+ based extracellular solution), we found that the macroscopic Ca2+ current activates and deactivates very rapidly (time constant below 1 ms) and inactivates slowly and only partially. Single-channel recordings showed an elementary conductance of 15 pS, a sub-ms latency to first opening, and a very low steady-state open probability (Po: 0.024 in response to 500-ms depolarizing steps at ∼−18 mV). The value of Po was significantly larger (0.06) in the first 40 ms of membrane depolarization, which corresponds to the time when most Ca2+ channel openings occurred clustered in bursts (mean burst duration: 19 ms). Both the Po and the mean burst duration were smaller than those previously reported in high-frequency basal IHCs. Finally, we found that middle turn IHCs are likely to express about 4 times more Ca2+ channels per ribbon than basal cells. We propose that middle-turn IHCs finely-tune CaV1.3 Ca2+ channel gating in order to provide reliable information upon timing and intensity of lower-frequency sounds. PMID:25409445

  12. Structure and function of hainantoxin-III, a selective antagonist of neuronal tetrodotoxin-sensitive voltage-gated sodium channels isolated from the Chinese bird spider Ornithoctonus hainana.

    PubMed

    Liu, Zhonghua; Cai, Tianfu; Zhu, Qi; Deng, Meichun; Li, Jiayan; Zhou, Xi; Zhang, Fan; Li, Dan; Li, Jing; Liu, Yu; Hu, Weijun; Liang, Songping

    2013-07-12

    In the present study, we investigated the structure and function of hainantoxin-III (HNTX-III), a 33-residue polypeptide from the venom of the spider Ornithoctonus hainana. It is a selective antagonist of neuronal tetrodotoxin-sensitive voltage-gated sodium channels. HNTX-III suppressed Nav1.7 current amplitude without significantly altering the activation, inactivation, and repriming kinetics. Short extreme depolarizations partially activated the toxin-bound channel, indicating voltage-dependent inhibition of HNTX-III. HNTX-III increased the deactivation of the Nav1.7 current after extreme depolarizations. The HNTX-III·Nav1.7 complex was gradually dissociated upon prolonged strong depolarizations in a voltage-dependent manner, and the unbound toxin rebound to Nav1.7 after a long repolarization. Moreover, analysis of chimeric channels showed that the DIIS3-S4 linker was critical for HNTX-III binding to Nav1.7. These data are consistent with HNTX-III interacting with Nav1.7 site 4 and trapping the domain II voltage sensor in the closed state. The solution structure of HNTX-III was determined by two-dimensional NMR and shown to possess an inhibitor cystine knot motif. Structural analysis indicated that certain basic, hydrophobic, and aromatic residues mainly localized in the C terminus may constitute an amphiphilic surface potentially involved in HNTX-III binding to Nav1.7. Taken together, our results show that HNTX-III is distinct from β-scorpion toxins and other β-spider toxins in its mechanism of action and binding specificity and affinity. The present findings contribute to our understanding of the mechanism of toxin-sodium channel interaction and provide a useful tool for the investigation of the structure and function of sodium channel isoforms and for the development of analgesics.

  13. Inhibitory action of insulin-sensitizing agents on calcium channels in smooth muscle cells from resistance arteries of guinea-pig

    PubMed Central

    Nakamura, Yoshito; Ohya, Yusuke; Onaka, Uran; Fujii, Koji; Abe, Isao; Fujishima, Masatoshi

    1998-01-01

    The actions of troglitazone, pioglitazone, metformin and bezafibrate, agents that improve insulin-resistance, on voltage-dependent Ca2+ channels in arterial smooth muscle cells were examined by use of the conventional and nystatin-perforated whole-cell clamp methods. Single cells were freshly isolated from resistance mesenteric arteries of guinea-pigs. The actions of these agents on 77 mM K+-induced contraction of the isolated arteries were also examined with the use of isometric tension recording. The thiazolidinedione derivatives, troglitazone and pioglitazone, inhibited whole-cell Ca2+ currents in a dose-dependent manner with dissociation constants of 3.0 μM and 44.9 μM and Hill coefficients of 0.61 and 0.68, respectively. These two agents inhibited the 77 mM K+-induced contraction with similar potencies as those inhibiting the Ca2+ currents. Metformin and bezafibrate had no apparent effects on the Ca2+ current or high K+-induced contraction. The inhibitory action of troglitazone on Ca2+ currents was not affected by the command potential, the holding potential, or the stimulation frequency, suggesting that its mode of the action differs from that of known organic Ca2+ channel antagonists. The inhibitory action of troglitazone on Ca2+ currents was not affected by the addition of insulin to, or the removal of glucose from, the solutions. In conclusion, the thiazolidinedione derivatives directly inhibited the voltage-dependent Ca2+ channels in a different manner from that of organic Ca2+ channel antagonists. This inhibitory action on Ca2+ channels was not a common feature of insulin-sensitizing agents. PMID:9517387

  14. Fine Tuning of CaV1.3 Ca2+ channel properties in adult inner hair cells positioned in the most sensitive region of the Gerbil Cochlea.

    PubMed

    Zampini, Valeria; Johnson, Stuart L; Franz, Christoph; Knipper, Marlies; Holley, Matthew C; Magistretti, Jacopo; Russo, Giancarlo; Marcotti, Walter; Masetto, Sergio

    2014-01-01

    Hearing relies on faithful signal transmission by cochlear inner hair cells (IHCs) onto auditory fibres over a wide frequency and intensity range. Exocytosis at IHC ribbon synapses is triggered by Ca(2+) inflow through Ca(V)1.3 (L-type) Ca(2+) channels. We investigated the macroscopic (whole-cell) and elementary (cell-attached) properties of Ca(2+) currents in IHCs positioned at the middle turn (frequency ∼ 2 kHz) of the adult gerbil cochlea, which is their most sensitive hearing region. Using near physiological recordings conditions (body temperature and a Na(+) based extracellular solution), we found that the macroscopic Ca(2+) current activates and deactivates very rapidly (time constant below 1 ms) and inactivates slowly and only partially. Single-channel recordings showed an elementary conductance of 15 pS, a sub-ms latency to first opening, and a very low steady-state open probability (Po: 0.024 in response to 500-ms depolarizing steps at ∼-18 mV). The value of Po was significantly larger (0.06) in the first 40 ms of membrane depolarization, which corresponds to the time when most Ca(2+) channel openings occurred clustered in bursts (mean burst duration: 19 ms). Both the Po and the mean burst duration were smaller than those previously reported in high-frequency basal IHCs. Finally, we found that middle turn IHCs are likely to express about 4 times more Ca(2+) channels per ribbon than basal cells. We propose that middle-turn IHCs finely-tune Ca(V)1.3 Ca(2+) channel gating in order to provide reliable information upon timing and intensity of lower-frequency sounds.

  15. Impaired activity of volume-sensitive anion channel during lactacidosis-induced swelling in neuronally differentiated NG108-15 cells.

    PubMed

    Mori, Shin-ichiro; Morishima, Shigeru; Takasaki, Mayumi; Okada, Yasunobu

    2002-12-06

    Acidosis coupled to lactate accumulation, called lactacidosis, occurs in cerebral ischemia or trauma and is known to cause persistent swelling in neuronal and glial cells. It is therefore possible that mechanisms of cell volume regulation are impaired during lactacidosis. Here we tested this possibility using neuronally differentiated NG108-15 cells. These cells responded to a hypotonic challenge with osmotic swelling followed by a regulatory volume decrease (RVD) under physiological pH conditions in the absence of lactate. Under normotonic conditions, sustained cell swelling without subsequent RVD was induced by exposure to lactate-containing solution with acidic pH (6.4 or 6.2), but not with physiological pH (7.4). Under whole-cell patch-clamp, osmotic swelling was found to activate outwardly rectifying Cl(-) currents in cells exposed to control hypotonic solution. A Cl(-) channel blocker, NPPB, inhibited both RVD and the swelling-activated Cl(-) current. RVD and the volume-sensitive Cl(-) current were also markedly inhibited by lactacidosis (pH 6.4 or 6.2), but neither by application of lactate with physiological pH (7.4) nor by acidification without lactate (pH 6.2). RT-PCR analysis showed mRNA expression of two isoforms of proton-coupled monocarboxylate transporters, MCT1 and MCT8, in differentiated NG108-15 cells. Thus, we conclude that persistence of neuronal cell swelling under lactacidosis is coupled to an impairment of the activity of the volume-sensitive Cl(-) channel and to dysfunction of RVD. It is also suggested that the volume-sensitive Cl(-) channel is inhibited by intracellular acidification induced by MCT-mediated proton influx under lactacidosis.

  16. Synergistic interaction between metformin and sulfonylureas on diclofenac-induced antinociception measured using the formalin test in rats

    PubMed Central

    Ortiz, Mario I

    2013-01-01

    BACKGROUND There is evidence that biguanides and sulfonylureas block diclofenac-induced antinociception (DIA) in rat models. However, little is known about the interaction between these hypoglycemics with respect to DIA. OBJECTIVE: To determine whether metformin-sulfonylurea combinations affect DIA during the formalin test. METHODS: Rats received the appropriate vehicle or diclofenac before 1% formaldehyde was injected into the paw. Rats were also pretreated with vehicle, glibenclamide, glipizide, metformin or glibenclamide/metformin and glipizide/metformin combinations before the diclofenac and formaldehyde injections, and the effect on antinociception was assessed. Isobolograms of the combinations were constructed to test for a synergistic interaction. RESULTS: Systemic injection of diclofenac resulted in antinociception during the second phase of the test. Systemic pretreatment with the combinations of glibenclamide (0.56 mg/kg to 10 mg/kg)/metformin (10 mg/kg to 180 mg/kg) and glipizide (0.56 mg/kg to10 mg/kg)/metformin (10 mg/kg to 180 mg/kg) blocked DIA. The derived theoretical effective doses for 50% of subjects (ED50) for the glibenclamide/metformin and glipizide/metformin combinations were 32.52 mg/kg and 32.42 mg/kg, respectively, and were significantly higher than the actual observed experimental ED50 values (7.57 mg/kg and 8.43 mg/kg, respectively). CONCLUSION: Pretreatment with glibenclamide, glipizide or metformin blocked DIA in a dose-dependent manner, and combining either sulfonylurea with metformin produced even greater effects. The observed ED50s for the combinations were approximately fourfold lower than the calculated additive effects. These data indicate that sulfonylureas interact to produce antagonism of DIA. Combination therapy is a common second-line treatment for patients with diabetes and metabolic syndrome, a group that experiences pain from multiple sources. The results suggest that at least some anti-inflammatory agents may not be

  17. Three-dimensional network electrolytes with highly efficient ion-transporting channels for quasi-solid-state dye-sensitized solar cells

    NASA Astrophysics Data System (ADS)

    Shi, Jifu; Chen, Jiubin; Li, Yujian; Zhu, Yanqing; Xu, Gang; Xu, Jiayou

    2015-05-01

    Quasi-solid-state dye-sensitized solar cells (DSCs) are fabricated with three-dimensional network poly(adipic acid pentaerythritol ester) (PAAPE). The PAAPEs are prepared by esterification of pentaerythritol and adipic acid. This three-dimensional structure provides highly efficient ion-transporting channels for iodide/triiodide (I-/I3-) transport in the gel electrolyte. The optimized gel electrolyte shows conductivity of 4.03 mS cm-1 at 25 °C. The fabricated DSC obtains a photoenergy conversion efficiency of 6.81% under AM 1.5 irradiation.

  18. Chronic fluoxetine treatment increases NO bioavailability and calcium-sensitive potassium channels activation in rat mesenteric resistance arteries.

    PubMed

    Pereira, Camila A; Ferreira, Nathanne S; Mestriner, Fabiola L; Antunes-Rodrigues, José; Evora, Paulo R B; Resstel, Leonardo B M; Carneiro, Fernando S; Tostes, Rita C

    2015-10-15

    Fluoxetine, a selective serotonin reuptake inhibitor (SSRI), has effects beyond its antidepressant properties, altering, e.g., mechanisms involved in blood pressure and vasomotor tone control. Although many studies have addressed the acute impact of fluoxetine on the cardiovascular system, there is a paucity of information on the chronic vascular effects of this SSRI. We tested the hypothesis that chronic fluoxetine treatment enhances the vascular reactivity to vasodilator stimuli by increasing nitric oxide (NO) signaling and activation of potassium (K+) channels. Wistar rats were divided into two groups: (I) vehicle (water for 21 days) or (II) chronic fluoxetine (10 mg/kg/day in the drinking water for 21 days). Fluoxetine treatment increased endothelium-dependent and independent vasorelaxation (analyzed by mesenteric resistance arteries reactivity) as well as constitutive NO synthase (NOS) activity, phosphorylation of eNOS at Serine1177 and NO production, determined by western blot and fluorescence. On the other hand, fluoxetine treatment did not alter vascular expression of neuronal and inducible NOS or guanylyl cyclase (GC). Arteries from fluoxetine-treated rats exhibited increased relaxation to pinacidil. Increased acetylcholine vasorelaxation was abolished by a calcium-activated K+ channel (KCa) blocker, but not by an inhibitor of KATP channels. On the other hand, vascular responses to Bay 41-2272 and 8-bromo-cGMP were similar between the groups. In conclusion, chronic fluoxetine treatment increases endothelium-dependent and independent relaxation of mesenteric resistance arteries by mechanisms that involve increased eNOS activity, NO generation, and KCa channels activation. These effects may contribute to the cardiovascular effects associated with chronic fluoxetine treatment.

  19. Empagliflozin added to metformin and sulfonylurea therapy in patients with sub-optimally controlled type 2 diabetes mellitus.

    PubMed

    Lewin, Andrew J; Frías, Juan P

    2015-04-01

    The combination of metformin and a sulfonylurea is commonly used in type 2 diabetes mellitus. Many patients on this combination therapy do not achieve or maintain glycemic targets and require the addition of a third antihyperglycemic agent. Among the options are the sodium glucose cotransporter 2 (SGLT2) inhibitors, a recently developed class of medications that effectively improve glycemic control and are associated with reduction in body weight and blood pressure. This article evaluates a 24-week, randomized, placebo-controlled study of the SGLT2 inhibitor empagliflozin, added to metformin plus sulfonylurea regimens. Empagliflozin led to significant reductions in glycated hemoglobin and fasting plasma glucose, as well as body weight and systolic blood pressure. Adverse events typically recorded with SGLT2 inhibitors were observed; notably, genital infections occurred in more patients on empagliflozin than placebo. Overall, empagliflozin was well tolerated. These results indicate that SGLT2 inhibitors can be successfully added to metformin plus sulfonylurea regimens. SGLT2 inhibitors are not the only therapeutic option in this clinical situation; however, based on the secondary effects observed in this and other studies, they appear to be of particular value for patients who are obese or overweight.

  20. Large-scale pharmacogenomic study of sulfonylureas and the QT, JT and QRS intervals: CHARGE Pharmacogenomics Working Group.

    PubMed

    Floyd, J S; Sitlani, C M; Avery, C L; Noordam, R; Li, X; Smith, A V; Gogarten, S M; Li, J; Broer, L; Evans, D S; Trompet, S; Brody, J A; Stewart, J D; Eicher, J D; Seyerle, A A; Roach, J; Lange, L A; Lin, H J; Kors, J A; Harris, T B; Li-Gao, R; Sattar, N; Cummings, S R; Wiggins, K L; Napier, M D; Stürmer, T; Bis, J C; Kerr, K F; Uitterlinden, A G; Taylor, K D; Stott, D J; de Mutsert, R; Launer, L J; Busch, E L; Méndez-Giráldez, R; Sotoodehnia, N; Soliman, E Z; Li, Y; Duan, Q; Rosendaal, F R; Slagboom, P E; Wilhelmsen, K C; Reiner, A P; Chen, Y-DI; Heckbert, S R; Kaplan, R C; Rice, K M; Jukema, J W; Johnson, A D; Liu, Y; Mook-Kanamori, D O; Gudnason, V; Wilson, J G; Rotter, J I; Laurie, C C; Psaty, B M; Whitsel, E A; Cupples, L A; Stricker, B H

    2016-12-13

    Sulfonylureas, a commonly used class of medication used to treat type 2 diabetes, have been associated with an increased risk of cardiovascular disease. Their effects on QT interval duration and related electrocardiographic phenotypes are potential mechanisms for this adverse effect. In 11 ethnically diverse cohorts that included 71 857 European, African-American and Hispanic/Latino ancestry individuals with repeated measures of medication use and electrocardiogram (ECG) measurements, we conducted a pharmacogenomic genome-wide association study of sulfonylurea use and three ECG phenotypes: QT, JT and QRS intervals. In ancestry-specific meta-analyses, eight novel pharmacogenomic loci met the threshold for genome-wide significance (P<5 × 10(-8)), and a pharmacokinetic variant in CYP2C9 (rs1057910) that has been associated with sulfonylurea-related treatment effects and other adverse drug reactions in previous studies was replicated. Additional research is needed to replicate the novel findings and to understand their biological basis.The Pharmacogenomics Journal advance online publication, 13 December 2016; doi:10.1038/tpj.2016.90.

  1. Induction of therapeutic hypothermia by pharmacological modulation of temperature-sensitive TRP channels: theoretical framework and practical considerations.

    PubMed

    Feketa, Viktor V; Marrelli, Sean P

    2015-01-01

    Therapeutic hypothermia has emerged as a remarkably effective method of neuroprotection from ischemia and is being increasingly used in clinics. Accordingly, it is also a subject of considerable attention from a basic scientific research perspective. One of the fundamental problems, with which current studies are concerned, is the optimal method of inducing hypothermia. This review seeks to provide a broad theoretical framework for approaching this problem, and to discuss how a novel promising strategy of pharmacological modulation of the thermosensitive ion channels fits into this framework. Various physical, anatomical, physiological and molecular aspects of thermoregulation, which provide the foundation for this text, have been comprehensively reviewed and will not be discussed exhaustively here. Instead, the first part of the current review, which may be helpful for a broader readership outside of thermoregulation research, will build on this existing knowledge to outline possible opportunities and research directions aimed at controlling body temperature. The second part, aimed at a more specialist audience, will highlight the conceptual advantages and practical limitations of novel molecular agents targeting thermosensitive Transient Receptor Potential (TRP) channels in achieving this goal. Two particularly promising members of this channel family, namely TRP melastatin 8 (TRPM8) and TRP vanilloid 1 (TRPV1), will be discussed in greater detail.

  2. The role of the capsaicin receptor TRPV1 and acid-sensing ion channels (ASICS) in proton sensitivity of subpopulations of primary nociceptive neurons in rats and mice.

    PubMed

    Leffler, A; Mönter, B; Koltzenburg, M

    2006-05-12

    A local elevation of H+-ion concentrations often occurs in inflammation and usually evokes pain by excitation of primary nociceptive neurons. Expression patterns and functional properties of the capsaicin receptor and acid-sensing ion channels suggest that they may be the main molecular substrates underlying this proton sensitivity. Here, we asked how the capsaicin receptor TRPV1 and acid-sensing ion channels (ASICS) contribute to the proton response in subpopulations of nociceptive neurons from adult rats and mice (wildtype C57/Bl6, Balb/C and TRPV1-null). In cultured dorsal root ganglion neurons, whole cell patch clamp recordings showed that the majority of capsaicin-sensitive rat dorsal root ganglion neurons displayed large proton-evoked inward currents with transient ASIC-like properties. In contrast, the prevalence of ASIC-like currents was smaller in both mouse wildtype strains and more frequent in capsaicin-insensitive neurons. Transient ASIC-like currents were more frequent in both species among isolectin B4-negative neurons. A significantly reduced proton response was observed for dissociated dorsal root ganglion neurons in TRPV1 deficient mice. Unmyelinated, but not thin myelinated nociceptors recorded extracellularly from TRPV1-null mutants showed a profound reduction of proton sensitivity. Together these findings indicate that there are significant differences between rat and mouse in the contribution of TRPV1 and ASIC subunits to proton sensitivity of sensory neurons. In both species ASIC subunits are more prevalent in the isolectin B4-negative neurons, some of which may represent thin myelinated nociceptors. However, the main acid-sensor in isolectin B4-positive and isolectin B4-negative unmyelinated nociceptors in mice is TRPV1.

  3. Sorption, desorption and leaching potential of sulfonylurea herbicides in Argentinean soils.

    PubMed

    Azcarate, Mariela P; Montoya, Jorgelina C; Koskinen, William C

    2015-01-01

    The sulfonylurea (SUs) herbicides are used to control broadleaf weeds and some grasses in a variety of crops. They have become popular because of their low application rates, low mammalian toxicity and an outstanding herbicidal activity. Sorption is a major process influencing the fate of pesticides in soil. The objective of this study was to characterize sorption-desorption of four sulfonylurea herbicides: metsulfuron-methyl (methyl 2-[(4-methoxy-6-methyl-1,3,5-triazin-2-yl)carbamoylsulfamoyl)]benzoate), sulfometuron-methyl (methyl 2-[(4,6-dimethylpyrimidin-2-yl)carbamoylsulfamoyl]benzoate), rimsulfuron (1-(4,6-dimethoxypyrimidin-2-yl)-3-(3-ethylsulfonyl-2-pyridylsulfonyl)urea) and nicosulfuron (2-[(4,6-dimethoxypyrimidin-2-yl)carbamoylsulfamoyl]-N,N-dimethylnicotinamide) from different soil horizons of different landscape positions. Sorption was studied in the laboratory by batch equilibration method. Sorption coefficients (K(d-SE)) showed that rimsulfuron (K(d-SE) = 1.18 to 2.08 L kg(-1)) and nicosulfuron (K(d-SE) = 0.02 to 0.47 L kg(-1)) were more highly sorbed than metsulfuron-methyl (K(d-SE) = 0.00 to 0.05 L kg(-1)) and sulfometuron-methyl (K(d-SE) = 0.00 to 0.05 L kg(-1)). Sorption coefficients (K(d-SE)) were correlated with pH and organic carbon content. All four herbicides exhibited desorption hysteresis where the desorption coefficients (K(d-D)) > K(d-SE). To estimate the leaching potential, K(oc) and ground-water ubiquity score (GUS) were used to calculate the half-life (t1/2) required to be classified as "leacher" or "nonleacher". According to the results, rimsulfuron and nicosulfuron herbicides would be classified as leachers, but factors such as landscape position, soil depth and the rate of decomposition in surface and subsurface soils could change the classification. In contrast, these factors do not affect classification of sulfometuron-methyl and metsulfuron-methyl; they would rank as leachers.

  4. Kinetics of PIP2 metabolism and KCNQ2/3 channel regulation studied with a voltage-sensitive phosphatase in living cells

    PubMed Central

    Falkenburger, Björn H.; Jensen, Jill B.

    2010-01-01

    The signaling phosphoinositide phosphatidylinositol 4,5-bisphosphate (PIP2) is synthesized in two steps from phosphatidylinositol by lipid kinases. It then interacts with KCNQ channels and with pleckstrin homology (PH) domains among many other physiological protein targets. We measured and developed a quantitative description of these metabolic and protein interaction steps by perturbing the PIP2 pool with a voltage-sensitive phosphatase (VSP). VSP can remove the 5-phosphate of PIP2 with a time constant of τ <300 ms and fully inhibits KCNQ currents in a similar time. PIP2 was then resynthesized from phosphatidylinositol 4-phosphate (PIP) quickly, τ = 11 s. In contrast, resynthesis of PIP2 after activation of phospholipase C by muscarinic receptors took ∼130 s. These kinetic experiments showed that (1) PIP2 activation of KCNQ channels obeys a cooperative square law, (2) the PIP2 residence time on channels is <10 ms and the exchange time on PH domains is similarly fast, and (3) the step synthesizing PIP2 by PIP 5-kinase is fast and limited primarily by a step(s) that replenishes the pool of plasma membrane PI(4)P. We extend the kinetic model for signaling from M1 muscarinic receptors, presented in our companion paper in this issue (Falkenburger et al. 2010. J. Gen. Physiol. doi:10.1085/jgp.200910344), with this new information on PIP2 synthesis and KCNQ interaction. PMID:20100891

  5. σ-1 Receptor Inhibition of ASIC1a Channels is Dependent on a Pertussis Toxin-Sensitive G-Protein and an AKAP150/Calcineurin Complex.

    PubMed

    Mari, Yelenis; Katnik, Christopher; Cuevas, Javier

    2015-10-01

    ASIC1a channels play a major role in various pathophysiological conditions including depression, anxiety, epilepsy, and neurodegeneration following ischemic stroke. Sigma-1 (σ-1) receptor stimulation depresses the activity of ASIC1a channels in cortical neurons, but the mechanism(s) by which σ-1 receptors exert their influence on ASIC1a remains unknown. Experiments were undertaken to elucidate the signaling cascade linking σ-1 receptors to ASIC1a channels. Immunohistochemical studies showed that σ-1 receptors, ASIC1a and A-kinase anchoring peptide 150 colocalize in the plasma membrane of the cell body and processes of cortical neurons. Fluorometric Ca(2+) imaging experiments showed that disruption of the macromolecular complexes containing AKAP150 diminished the effects of the σ-1 on ASIC1a, as did application of the calcineurin inhibitors, cyclosporin A and FK-506. Moreover, whole-cell patch clamp experiments showed that σ-1 receptors were less effective at decreasing ASIC1a-mediated currents in the presence of the VIVIT peptide, which binds to calcineurin and prevents cellular effects dependent on AKAP150/calcineurin interaction. The coupling of σ-1 to ASIC1a was also disrupted by preincubation of the neurons in the G-protein inhibitor, pertussis toxin (PTX). Taken together, our data reveal that σ-1 receptor block of ASIC1a function is dependent on activation of a PTX-sensitive G-protein and stimulation of AKAP150 bound calcineurin.

  6. The Association between Sulfonylurea Use and All-Cause and Cardiovascular Mortality: A Meta-Analysis with Trial Sequential Analysis of Randomized Clinical Trials

    PubMed Central

    Varvaki Rados, Dimitris; Catani Pinto, Lana; Reck Remonti, Luciana; Bauermann Leitão, Cristiane; Gross, Jorge Luiz

    2016-01-01

    Background Sulfonylureas are an effective and inexpensive treatment for type 2 diabetes. There is conflicting data about the safety of these drugs regarding mortality and cardiovascular outcomes. The objective of the present study was to evaluate the safety of the sulfonylureas most frequently used and to use trial sequential analysis (TSA) to analyze whether the available sample was powered enough to support the results. Methods and Findings Electronic databases were reviewed from 1946 (Embase) or 1966 (MEDLINE) up to 31 December 2014. Randomized clinical trials (RCTs) of at least 52 wk in duration evaluating second- or third-generation sulfonylureas in the treatment of adults with type 2 diabetes and reporting outcomes of interest were included. Primary outcomes were all-cause and cardiovascular mortality. Additionally, myocardial infarction and stroke events were evaluated. Data were summarized with Peto odds ratios (ORs), and the reliability of the results was evaluated with TSA. Forty-seven RCTs with 37,650 patients and 890 deaths in total were included. Sulfonylureas were not associated with all-cause (OR 1.12 [95% CI 0.96 to 1.30]) or cardiovascular mortality (OR 1.12 [95% CI 0.87 to 1.42]). Sulfonylureas were also not associated with increased risk of myocardial infarction (OR 0.92 [95% CI 0.76 to 1.12]) or stroke (OR 1.16 [95% CI 0.81 to 1.66]). TSA could discard an absolute difference of 0.5% between the treatments, which was considered the minimal clinically significant difference. The major limitation of this review was the inclusion of studies not designed to evaluate safety outcomes. Conclusions Sulfonylureas are not associated with increased risk for all-cause mortality, cardiovascular mortality, myocardial infarction, or stroke. Current evidence supports the safety of sulfonylureas; an absolute risk of 0.5% could be firmly discarded. Review registration PROSPERO CRD42014004330 PMID:27071029

  7. Kidney function decline in metformin versus sulfonylurea initiators: assessment of time-dependent contribution of weight, blood pressure and glycemic control

    PubMed Central

    Hung, Adriana M.; Roumie, Christianne L.; Greevy, Robert A.; Liu, Xulei; Grijalva, Carlos G.; Murff, Harvey J.; Griffin, Marie R.

    2016-01-01

    Background and objective We recently reported that kidney function declined faster among initiators of sulfonylureas compared to metformin; however, sulfonylurea compared to metformin use was also associated with increases in body mass index (BMI) and systolic blood pressure (SBP). We sought to determine if differences between sulfonylureas and metformin on kidney function decline were mediated by differential effects on BMI, SBP, or glucose control. Methods We identified 13238 veterans who initiated sulfonylurea or metformin treatment (2000–2007) with a baseline estimated glomerular filtration rate (eGFR) >60 ml/min, and followed them until a study event occurred, non-persistence on treatment, loss of follow-up or end of the study. The composite outcome was a sustained decline from baseline eGFR of ≥25%, end stage renal disease, or death. We estimated the association of cumulative measurements of potential mediators including BMI, SBP and glycated hemoglobin on the study outcome. We determined if controlling for these time-varying covariates accounted for the differences in outcome between sulfonylurea and metformin initiators. Results Compared to sulfonylurea use, metformin use was associated with a lower risk for renal function decline or death [adjusted hazard ratio (aHR) 0.82, 95% confidence interval 0.70, 0.97]. This protective association remained significant [aHR 0.83 (0.70–0.98)] when accounting for the cumulative time varying measurements of the three mediators of interest. Conclusion Metformin initiation was associated with a lower risk of kidney function decline or death compared to sulfonylureas which appeared to be independent of changes in BMI, SBP and glycated hemoglobin over time. PMID:23592561

  8. Cytochrome P450 2C9 *2 and *3 polymorphisms and the dose and effect of sulfonylurea in type II diabetes mellitus.

    PubMed

    Becker, M L; Visser, L E; Trienekens, P H; Hofman, A; van Schaik, R H N; Stricker, B H Ch

    2008-02-01

    Sulfonylurea hypoglycemics are mainly metabolized by the cytochrome P450 2C9 (CYP2C9) enzyme. The CYP2C9*2 and *3 polymorphisms encode proteins with less enzymatic activity and are correlated with elevated serum levels of sulfonylurea, as demonstrated in healthy volunteers. In this study, the effect of these variants is described for patients with diabetes mellitus treated with sulfonylurea. Associations between CYP2C9 polymorphisms, prescribed doses of sulfonylurea, and change in glucose levels after the start of sulfonylurea therapy were assessed in all patients with incident diabetes mellitus starting on sulfonylurea therapy in the Rotterdam Study, a population-based cohort study of 7,983 elderly people. In CYP2C9*3 allele carriers using tolbutamide, the prescribed dose was lower compared to patients with the wild-type CYP2C9 genotype. No differences in the prescribed dose were found in tolbutamide users with the CYP2C9*1/*2 or CYP2C9*2/*2 genotype compared to wild-type patients or in patients using other sulfonylurea. In CYP2C9*3 allele carriers, the mean decrease in fasting serum glucose levels after the start of tolbutamide therapy was larger than in patients with the wild-type genotype, although not statistically significant. Patients with diabetes mellitus who are carriers of a CYP2C9*3 allele require lower doses of tolbutamide to regulate their serum glucose levels compared to patients with the wild-type genotype.

  9. Exchange protein activated by cAMP (Epac) induces vascular relaxation by activating Ca2+-sensitive K+ channels in rat mesenteric artery.

    PubMed

    Roberts, Owain Llŷr; Kamishima, Tomoko; Barrett-Jolley, Richard; Quayle, John M; Dart, Caroline

    2013-10-15

    Vasodilator-induced elevation of intracellular cyclic AMP (cAMP) is a central mechanism governing arterial relaxation but is incompletely understood due to the diversity of cAMP effectors. Here we investigate the role of the novel cAMP effector exchange protein directly activated by cAMP (Epac) in mediating vasorelaxation in rat mesenteric arteries. In myography experiments, the Epac-selective cAMP analogue 8-pCPT-2-O-Me-cAMP-AM (5 μM, subsequently referred to as 8-pCPT-AM) elici