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内皮素-1对牛脑血管平滑肌细胞ClC-3通道表达的影响 总被引:1,自引:0,他引:1
目的 研究内皮素 1(ET 1)对牛脑血管平滑肌细胞(CSMC)内源性ClC 3表达的影响。方法 采用培养的CSMC ,用免疫印迹 (westernblot)方法分析CSMCClC 3通道表达。结果 ①牛脑基底动脉、大脑中动脉、微动脉均有ClC 3蛋白表达 ,分子量约 10 5ku ,以微动脉表达最丰富 ,基底动脉表达最少 ;②培养的CSMC有内源性ClC 3蛋白表达 ,分子量约 95ku ;③ET 1能增加CSMCClC 3蛋白表达 ;④nifedipine、SK&F96 36 5能减少ET 1作用 ,环匹阿尼酸(cyclopiazonicacid ,CPA)浓度依赖性促进ClC 3蛋白表达 ,并能增强ET 1作用。结论 脑血管平滑肌细胞存在内源性ClC 3,ET 1可增强ClC 3表达 ,减少或增加胞浆内Ca2 + 浓度均可影响ET 1的作用 相似文献
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Eguchi H Tsujino A Kaibara M Hayashi H Shirabe S Taniyama K Eguchi K 《Muscle & nerve》2006,34(3):292-297
Acetazolamide, a carbonic anhydrase inhibitor, is used empirically in neuromuscular diseases with episodic ataxia, weakness, and myotonia, although not all of the mechanisms responsible for its therapeutic effects are understood. To elucidate whether acetazolamide acts directly on the human skeletal muscle voltage-gated chloride channel (ClC-1), which is associated with myotonia, we evaluated the effects of acetazolamide on ClC-1 expressed in cultured mammalian cells, using whole-cell recording. Acetazolamide significantly shifted the voltage dependency of the open probability (P(o)) toward negative potentials in a dose-dependent manner, resulting in an increase of chloride conductance at voltages near the resting membrane potential. This effect was attenuated when using a pipette solution containing 30 mmol/L Hepes. These results suggest that acetazolamide can influence the voltage-dependent opening gate of ClC-1 through a mechanism related to intracellular acidification by inhibiting carbonic anhydrase, and that the therapeutic effects of acetazolamide in neuromuscular diseases may be mediated by activation of ClC-1. 相似文献
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The analysis of myotonia congenita mutations discloses functional clusters of amino acids within the CBS2 domain and the C‐terminal peptide of the ClC‐1 channel 下载免费PDF全文
Concetta Altamura Sabrina Lucchiari Dalila Sahbani Gianna Ulzi Giacomo P. Comi Paola D'Ambrosio Roberta Petillo Luisa Politano Liliana Vercelli Tiziana Mongini Maria Teresa Dotti Rosanna Cardani Giovanni Meola Mauro Lo Monaco Emma Matthews Michael G. Hanna Maria Rosaria Carratù Diana Conte Paola Imbrici Jean‐François Desaphy 《Human mutation》2018,39(9):1273-1283
Myotonia congenita (MC) is a skeletal‐muscle hyperexcitability disorder caused by loss‐of‐function mutations in the ClC‐1 chloride channel. Mutations are scattered over the entire sequence of the channel protein, with more than 30 mutations located in the poorly characterized cytosolic C‐terminal domain. In this study, we characterized, through patch clamp, seven ClC‐1 mutations identified in patients affected by MC of various severities and located in the C‐terminal region. The p.Val829Met, p.Thr832Ile, p.Val851Met, p.Gly859Val, and p.Leu861Pro mutations reside in the CBS2 domain, while p.Pro883Thr and p.Val947Glu are in the C‐terminal peptide. We showed that the functional properties of mutant channels correlated with the clinical phenotypes of affected individuals. In addition, we defined clusters of ClC‐1 mutations within CBS2 and C‐terminal peptide subdomains that share the same functional defect: mutations between 829 and 835 residues and in residue 883 induced an alteration of voltage dependence, mutations between 851 and 859 residues, and in residue 947 induced a reduction of chloride currents, whereas mutations on 861 residue showed no obvious change in ClC‐1 function. This study improves our understanding of the mechanisms underlying MC, sheds light on the role of the C‐terminal region in ClC‐1 function, and provides information to develop new antimyotonic drugs. 相似文献
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Mathilde Keck Olga Andrini Olivier Lahuna Johanna Burgos L. Pablo Cid Francisco V. Sepúlveda Sébastien L‘Hoste Anne Blanchard Rosa Vargas‐Poussou Stéphane Lourdel Jacques Teulon 《Human mutation》2013,34(9):1269-1278
Mutations in the CLCNKB gene encoding the ClC‐Kb Cl? channel cause Bartter syndrome, which is a salt‐losing renal tubulopathy. Here, we investigate the functional consequences of seven mutations. When expressed in Xenopus laevis oocytes, four mutants carried no current (c.736G>C, p.Gly246Arg; c.1271G>A, p.Gly424Glu; c.1313G>A, p.Arg438His; c.1316T>C, p.Leu439Pro), whereas others displayed a 30%–60% reduction in conductance as compared with wild‐type ClC‐Kb (c.242T>C, p.Leu81Pro; c.274C>T, p.Arg92Trp; c.1052G>C, p.Arg351Pro). Anion selectivity and sensitivity to external Ca2+ and H+, typical of the ClC‐Kb channel, were not modified in the partially active mutants. In oocytes, we found that all the mutations reduced surface expression with a profile similar to that observed for currents. In HEK293 cells, the currents in the mutants had similar profiles to those obtained in oocytes, except for p.Leu81Pro, which produced no current. Furthermore, p.Arg92Trp and p.Arg351Pro mutations did not modify the unit‐conductance of closely related ClC‐K1. Western blot analysis in HEK293 cells showed that ClC‐Kb protein abundance was lower for the nonconducting mutants but similar to wild‐type for other mutants. Overall, two classes of mutants can be distinguished: nonconducting mutants associated with low total protein expression, and partially conducting mutants with unaltered channel properties and ClC‐Kb protein abundance. 相似文献
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Extracellular magnesium and calcium reduce myotonia in isolated ClC‐1 chloride channel‐inhibited human muscle 下载免费PDF全文
Martin Skov MSc Frank Vincenzo De Paoli MD PhD Jesper Lausten MD Ole Baekgaard Nielsen PhD Thomas Holm Pedersen PhD 《Muscle & nerve》2015,51(1):65-71
Introduction: Experimental myotonia induced in rat muscle by ClC‐1 chloride channel‐inhibited has been shown to be related inversely to extracellular concentrations of Mg2+ and Ca2+ ([Mg2+]o and [Ca2+]o) within physiological ranges. Because this implicates a role for [Mg2+]o and [Ca2+]o in the variability of symptoms among myotonia congenita patients, we searched for similar effects of [Mg2+]o and [Ca2+]o on myotonia in human muscle. Methods: Bundles of muscle fibers were isolated from abdominal rectus in patients undergoing abdominal surgery. Myotonia was induced by ClC‐1 inhibition using 9‐anthracene carboxylic acid (9‐AC) and was assessed from integrals of force induced by 5‐Hz stimulation for 2 seconds. Results: Myotonia disappeared gradually when [Mg2+]o or [Ca2+]o were elevated throughout their physiological ranges. These effects of [Mg2+]o and [Ca2+]o were additive and interchangeable. Conclusions: These findings suggest that variations in symptoms in myotonia congenita patients may arise from physiological variations in serum Mg2+ and Ca2+. Muscle Nerve 51 : 65–71, 2015 相似文献
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Expression of novel isoforms of the CIC-1 chloride channel in astrocytic glial cells in vitro 总被引:1,自引:0,他引:1
Zhang XD Morishima S Ando-Akatsuka Y Takahashi N Nabekura T Inoue H Shimizu T Okada Y 《Glia》2004,47(1):46-57
Chloride channels play an important role in glial astrocyte function. However, in astrocytes, no chloride channels besides the gamma-aminobutyric acid (GABA)A receptor, glycine receptor, and ClC-2 chloride channels have been molecularly identified. In this study, we examined the expression of the ClC-1 chloride channel in rat astrocytic glioma C6 cells and rat primary astrocytes. Five isoforms of ClC-1, but not skeletal muscle ClC-1 (SM ClC-1), were found to be expressed in C6 cells. Comparison with rat SM ClC-1 showed that common features shared by these isoforms are a short 3' end with a deletion of the nucleotides from 3115 to 3197 and a substitution of T by C at nucleotides 480 and 1733. Three of the five isoforms, M1, M2, and M3, were produced by partial deletion of ClC-1 exon 7, partial insertion of ClC-1 exon 7a, and a TAG insertion at nucleotide 858, respectively. One of the two remaining isoforms, M4, was produced by partial deletion of ClC-1 exon 8 at nucleotide 937; the other, M5, was the same as SM ClC-1 except for the short 3' end and substitutions at the two positions. Only the M5 isoform could be expressed as a functional channel in Xenopus oocytes. This glial isoform exhibited less dependence on voltage and extracellular Cl- than rat SM ClC-1. However, the anion selectivity sequence and the anthracene-9-carboxylic acid (9-AC) sensitivity of this channel were the same as for SM ClC-1. Since whole-cell recordings failed to detect ClC-1-like Cl- currents in C6 cells, it appears that the ClC-1 isoform is functioning in intracellular organelles. In rat primary astrocytes, we found that the M2 isoform as well as two additional distinct isoforms were expressed. The present study showed that astrocytic glial cells express multiple isoforms of the ClC-1 chloride channel, which has been thought to be expressed almost exclusively in the skeletal muscle. 相似文献
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Grunnet M Jespersen T Colding-Jørgensen E Schwartz M Klaerke DA Vissing J Olesen SP Dunø M 《Muscle & nerve》2003,28(6):722-732
Voltage-gated ClC-1 chloride channels encoded by the CLCN1 gene have a major role in setting the membrane potential in skeletal muscle. More than 60 CLCN1 mutations have been associated with myotonia congenita. These mutations are traditionally classified as recessive (Becker's disease) or dominant (Thomsen's disease). In this study, we have electrophysiologically characterized two new dominant ClC-1 mutations, thereby elucidating the observed phenotype in patients. The two ClC-1 mutants M128V and E193K were identified, and the DNA was isolated from patients and subsequently expressed in Xenopus laevis oocytes for electrophysiological characterization. Both ClC-1 mutants, M128V and E193K, showed a large rightward shift in the current-voltage relationship. In addition, the activation kinetics were slowed in the ClC-1 M128V mutant, as compared to the wild-type ClC-1. Interestingly, ClC-1 E193K revealed a change in reversal potential compared to wild-type channels. This finding supports the notion that the E193 amino acid is an important determinant in the selectivity filter of the human ClC-1 channel. The electrophysiological behavior of both mutants demonstrates a severe reduction in ClC-1 channel conductance under physiologically relevant membrane potentials. These studies thereby explain the molecular background for the observed myotonia in patients. 相似文献