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We aimed to confirm a pathogenic gain\u2010of function phenotype of <jats:italic>CACNA1D de novo<\/jats:italic> missense mutations A749T and L271H, and investigated the molecular mechanism causing their enhanced sensitivity for the Ca<jats:sup>2+<\/jats:sup>\u2010channel blocker isradipine, a potential therapeutic for affected patients.<\/jats:p><\/jats:sec><jats:sec><jats:title>Experimental Approach<\/jats:title><jats:p>Wildtype and mutant channels were expressed in tsA\u2010201 cells and their gating analysed using whole\u2010cell and single\u2010channel patch\u2010clamp recordings. The voltage\u2010dependence 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 <jats:italic>in silico<\/jats:italic> modelling and molecular dynamics simulations.<\/jats:p><\/jats:sec><jats:sec><jats:title>Key Results<\/jats:title><jats:p>Both mutations were confirmed pathogenic due to characteristic shifts of voltage\u2010dependent activation and inactivation towards negative potentials (~20\u2009mV). 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\u2010 to 40\u2010fold). The IC<jats:sub>50<\/jats:sub> was indistinguishable for wildtype and mutants when &gt;50% of channels were inactivated.<\/jats:p><\/jats:sec><jats:sec><jats:title>Conclusions and Implications<\/jats:title><jats:p>Mutations A749T and L271H induce pathogenic gating changes. Like wildtype, isradipine inhibition is strongly voltage\u2010dependent. 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<jats:sub>v<\/jats:sub>1.3 channels in neurons during prolonged depolarized states without selectivity for mutant channels.<\/jats:p><\/jats:sec>","DOI":"10.1111\/bph.17357","type":"journal-article","created":{"date-parts":[[2024,10,7]],"date-time":"2024-10-07T08:59:25Z","timestamp":1728291565000},"page":"181-197","update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":3,"title":["Inactivation induced by pathogenic Ca<sub>v<\/sub>1.3 L\u2010type Ca<sup>2+<\/sup>\u2010channel variants enhances sensitivity for dihydropyridine Ca<sup>2+<\/sup> channel blockers"],"prefix":"10.1111","volume":"182","author":[{"given":"Ferenc","family":"T\u00f6r\u00f6k","sequence":"first","affiliation":[{"name":"Department of Pharmacology and Toxicology, Center for Molecular Biosciences Innsbruck University of Innsbruck  Innsbruck Austria"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Sarah","family":"Salamon","sequence":"additional","affiliation":[{"name":"Center of Pharmacology, Institute II University of Cologne  Cologne Germany"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Nadine J.","family":"Ortner","sequence":"additional","affiliation":[{"name":"Department of Pharmacology and Toxicology, Center for Molecular Biosciences Innsbruck University of Innsbruck  Innsbruck Austria"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Monica L.","family":"Fern\u00e1ndez\u2010Quintero","sequence":"additional","affiliation":[{"name":"Department of General, Inorganic and Theoretical Chemistry, Center for Molecular Biosciences Innsbruck University of Innsbruck  Innsbruck Austria"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-2754-1555","authenticated-orcid":false,"given":"Jan","family":"Matthes","sequence":"additional","affiliation":[{"name":"Center of Pharmacology, Institute II University of Cologne  Cologne Germany"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-9406-7120","authenticated-orcid":false,"given":"J\u00f6rg","family":"Striessnig","sequence":"additional","affiliation":[{"name":"Department of Pharmacology and Toxicology, Center for Molecular Biosciences Innsbruck University of Innsbruck  Innsbruck Austria"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"311","published-online":{"date-parts":[[2024,10,7]]},"reference":[{"key":"e_1_2_12_2_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.softx.2015.06.001"},{"key":"e_1_2_12_3_1","doi-asserted-by":"publisher","DOI":"10.1111\/bph.16176"},{"key":"e_1_2_12_4_1","doi-asserted-by":"publisher","DOI":"10.1111\/bph.16178"},{"key":"e_1_2_12_5_1","doi-asserted-by":"publisher","DOI":"10.1111\/bph.14112"},{"key":"e_1_2_12_6_1","doi-asserted-by":"publisher","DOI":"10.1073\/pnas.81.20.6388"},{"key":"e_1_2_12_7_1","doi-asserted-by":"publisher","DOI":"10.1074\/jbc.M111.269951"},{"key":"e_1_2_12_8_1","doi-asserted-by":"publisher","DOI":"10.1016\/S0079-6107(98)00002-9"},{"key":"e_1_2_12_9_1","doi-asserted-by":"publisher","DOI":"10.1111\/bph.15868"},{"key":"e_1_2_12_10_1","doi-asserted-by":"publisher","DOI":"10.1063\/1.464397"},{"key":"e_1_2_12_11_1","doi-asserted-by":"publisher","DOI":"10.1080\/19336950.2020.1761171"},{"key":"e_1_2_12_12_1","doi-asserted-by":"publisher","DOI":"10.1063\/1.449071"},{"key":"e_1_2_12_13_1","doi-asserted-by":"publisher","DOI":"10.3758\/BF03193146"},{"key":"e_1_2_12_14_1","doi-asserted-by":"publisher","DOI":"10.1111\/bph.16060"},{"key":"e_1_2_12_15_1","unstructured":"Group MOEMCC. 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