Ferenc Török, Sarah Salamon, Nadine J Ortner, Monica L Fernández-Quintero, Jan Matthes, Jörg Striessnig
{"title":"致病性 Cav1.3 L 型 Ca2+ 通道变体引起的失活会增强对二氢吡啶类 Ca2+ 通道阻滞剂的敏感性。","authors":"Ferenc Török, Sarah Salamon, Nadine J Ortner, Monica L Fernández-Quintero, Jan Matthes, Jörg Striessnig","doi":"10.1111/bph.17357","DOIUrl":null,"url":null,"abstract":"<p><strong>Background and purpose: </strong>Pathogenic gain-of-function mutations in Ca<sub>v</sub>1.3 L-type voltage-gated Ca<sup>2+</sup>-channels (CACNA1D) cause neurodevelopmental disorders with or without endocrine symptoms. We aimed to confirm a pathogenic gain-of function phenotype of CACNA1D de novo missense mutations A749T and L271H, and investigated the molecular mechanism causing their enhanced sensitivity for the Ca<sup>2+</sup>-channel blocker isradipine, a potential therapeutic for affected patients.</p><p><strong>Experimental approach: </strong>Wildtype and mutant channels were expressed in tsA-201 cells and their gating analysed using whole-cell and single-channel patch-clamp recordings. The voltage-dependence of isradipine action was quantified using protocols inducing variable fractions of inactivated channels. The molecular basis for altered channel gating in the mutants was investigated using in silico modelling and molecular dynamics simulations.</p><p><strong>Key results: </strong>Both mutations were confirmed pathogenic due to characteristic shifts of voltage-dependent activation and inactivation towards negative potentials (~20 mV). At negative holding potentials both mutations showed significantly higher isradipine sensitivity compared to wildtype. The affinity for wildtype and mutant channels increased with channel inactivation as predicted by the modulated receptor hypothesis (30- to 40-fold). The IC<sub>50</sub> was indistinguishable for wildtype and mutants when >50% of channels were inactivated.</p><p><strong>Conclusions and implications: </strong>Mutations A749T and L271H induce pathogenic gating changes. Like wildtype, isradipine inhibition is strongly voltage-dependent. Our data explains their apparent higher drug sensitivity at a given negative voltage by the availability of more inactivated channels due to their more negative inactivation voltage range. Low nanomolar isradipine concentrations will only inhibit Ca<sub>v</sub>1.3 channels in neurons during prolonged depolarized states without selectivity for mutant channels.</p>","PeriodicalId":9262,"journal":{"name":"British Journal of Pharmacology","volume":" ","pages":""},"PeriodicalIF":6.8000,"publicationDate":"2024-10-07","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"0","resultStr":"{\"title\":\"Inactivation induced by pathogenic Ca<sub>v</sub>1.3 L-type Ca<sup>2+</sup>-channel variants enhances sensitivity for dihydropyridine Ca<sup>2+</sup> channel blockers.\",\"authors\":\"Ferenc Török, Sarah Salamon, Nadine J Ortner, Monica L Fernández-Quintero, Jan Matthes, Jörg Striessnig\",\"doi\":\"10.1111/bph.17357\",\"DOIUrl\":null,\"url\":null,\"abstract\":\"<p><strong>Background and purpose: </strong>Pathogenic gain-of-function mutations in Ca<sub>v</sub>1.3 L-type voltage-gated Ca<sup>2+</sup>-channels (CACNA1D) cause neurodevelopmental disorders with or without endocrine symptoms. We aimed to confirm a pathogenic gain-of function phenotype of CACNA1D de novo missense mutations A749T and L271H, and investigated the molecular mechanism causing their enhanced sensitivity for the Ca<sup>2+</sup>-channel blocker isradipine, a potential therapeutic for affected patients.</p><p><strong>Experimental approach: </strong>Wildtype and mutant channels were expressed in tsA-201 cells and their gating analysed using whole-cell and single-channel patch-clamp recordings. The voltage-dependence of isradipine action was quantified using protocols inducing variable fractions of inactivated channels. The molecular basis for altered channel gating in the mutants was investigated using in silico modelling and molecular dynamics simulations.</p><p><strong>Key results: </strong>Both mutations were confirmed pathogenic due to characteristic shifts of voltage-dependent activation and inactivation towards negative potentials (~20 mV). At negative holding potentials both mutations showed significantly higher isradipine sensitivity compared to wildtype. The affinity for wildtype and mutant channels increased with channel inactivation as predicted by the modulated receptor hypothesis (30- to 40-fold). The IC<sub>50</sub> was indistinguishable for wildtype and mutants when >50% of channels were inactivated.</p><p><strong>Conclusions and implications: </strong>Mutations A749T and L271H induce pathogenic gating changes. Like wildtype, isradipine inhibition is strongly voltage-dependent. Our data explains their apparent higher drug sensitivity at a given negative voltage by the availability of more inactivated channels due to their more negative inactivation voltage range. Low nanomolar isradipine concentrations will only inhibit Ca<sub>v</sub>1.3 channels in neurons during prolonged depolarized states without selectivity for mutant channels.</p>\",\"PeriodicalId\":9262,\"journal\":{\"name\":\"British Journal of Pharmacology\",\"volume\":\" \",\"pages\":\"\"},\"PeriodicalIF\":6.8000,\"publicationDate\":\"2024-10-07\",\"publicationTypes\":\"Journal Article\",\"fieldsOfStudy\":null,\"isOpenAccess\":false,\"openAccessPdf\":\"\",\"citationCount\":\"0\",\"resultStr\":null,\"platform\":\"Semanticscholar\",\"paperid\":null,\"PeriodicalName\":\"British Journal of Pharmacology\",\"FirstCategoryId\":\"3\",\"ListUrlMain\":\"https://doi.org/10.1111/bph.17357\",\"RegionNum\":2,\"RegionCategory\":\"医学\",\"ArticlePicture\":[],\"TitleCN\":null,\"AbstractTextCN\":null,\"PMCID\":null,\"EPubDate\":\"\",\"PubModel\":\"\",\"JCR\":\"Q1\",\"JCRName\":\"PHARMACOLOGY & PHARMACY\",\"Score\":null,\"Total\":0}","platform":"Semanticscholar","paperid":null,"PeriodicalName":"British Journal of Pharmacology","FirstCategoryId":"3","ListUrlMain":"https://doi.org/10.1111/bph.17357","RegionNum":2,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q1","JCRName":"PHARMACOLOGY & PHARMACY","Score":null,"Total":0}
Inactivation induced by pathogenic Cav1.3 L-type Ca2+-channel variants enhances sensitivity for dihydropyridine Ca2+ channel blockers.
Background and purpose: Pathogenic gain-of-function mutations in Cav1.3 L-type voltage-gated Ca2+-channels (CACNA1D) cause neurodevelopmental disorders with or without endocrine symptoms. We aimed to confirm a pathogenic gain-of function phenotype of CACNA1D de novo missense mutations A749T and L271H, and investigated the molecular mechanism causing their enhanced sensitivity for the Ca2+-channel blocker isradipine, a potential therapeutic for affected patients.
Experimental approach: Wildtype and mutant channels were expressed in tsA-201 cells and their gating analysed using whole-cell and single-channel patch-clamp recordings. The voltage-dependence of isradipine action was quantified using protocols inducing variable fractions of inactivated channels. The molecular basis for altered channel gating in the mutants was investigated using in silico modelling and molecular dynamics simulations.
Key results: Both mutations were confirmed pathogenic due to characteristic shifts of voltage-dependent activation and inactivation towards negative potentials (~20 mV). At negative holding potentials both mutations showed significantly higher isradipine sensitivity compared to wildtype. The affinity for wildtype and mutant channels increased with channel inactivation as predicted by the modulated receptor hypothesis (30- to 40-fold). The IC50 was indistinguishable for wildtype and mutants when >50% of channels were inactivated.
Conclusions and implications: Mutations A749T and L271H induce pathogenic gating changes. Like wildtype, isradipine inhibition is strongly voltage-dependent. Our data explains their apparent higher drug sensitivity at a given negative voltage by the availability of more inactivated channels due to their more negative inactivation voltage range. Low nanomolar isradipine concentrations will only inhibit Cav1.3 channels in neurons during prolonged depolarized states without selectivity for mutant channels.
期刊介绍:
The British Journal of Pharmacology (BJP) is a biomedical science journal offering comprehensive international coverage of experimental and translational pharmacology. It publishes original research, authoritative reviews, mini reviews, systematic reviews, meta-analyses, databases, letters to the Editor, and commentaries.
Review articles, databases, systematic reviews, and meta-analyses are typically commissioned, but unsolicited contributions are also considered, either as standalone papers or part of themed issues.
In addition to basic science research, BJP features translational pharmacology research, including proof-of-concept and early mechanistic studies in humans. While it generally does not publish first-in-man phase I studies or phase IIb, III, or IV studies, exceptions may be made under certain circumstances, particularly if results are combined with preclinical studies.