Török Ferenc, Salamon Sarah, Ortner Nadine J, Fernández-Quintero Monica L, Matthes Jan, Striessnig Jörg
Department of Pharmacology and Toxicology, Center for Molecular Biosciences Innsbruck, University of Innsbruck, Innsbruck, Austria.
Center of Pharmacology, Institute II, University of Cologne, Cologne, Germany.
Br J Pharmacol. 2025 Jan;182(1):181-197. doi: 10.1111/bph.17357. Epub 2024 Oct 7.
Pathogenic gain-of-function mutations in Ca1.3 L-type voltage-gated Ca-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-channel blocker isradipine, a potential therapeutic for affected patients.
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.
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 was indistinguishable for wildtype and mutants when >50% of channels were inactivated.
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 Ca1.3 channels in neurons during prolonged depolarized states without selectivity for mutant channels.
Ca1.3 L型电压门控钙通道（CACNA1D）中的致病性功能获得性突变会导致伴有或不伴有内分泌症状的神经发育障碍。我们旨在确认CACNA1D新发错义突变A749T和L271H的致病性功能获得性表型，并研究导致它们对钙通道阻滞剂伊拉地平敏感性增强的分子机制，伊拉地平是一种可能用于治疗受影响患者的药物。
野生型和突变型通道在tsA - 201细胞中表达，并使用全细胞和单通道膜片钳记录分析其门控特性。使用诱导不同比例失活通道的方案对伊拉地平作用的电压依赖性进行定量。利用计算机模拟和分子动力学模拟研究突变体中通道门控改变的分子基础。
由于电压依赖性激活和失活向负电位（约20 mV）的特征性偏移，这两种突变均被确认为致病性突变。在负的钳制电位下，与野生型相比，这两种突变均表现出显著更高的伊拉地平敏感性。如调制受体假说所预测，野生型和突变型通道的亲和力随通道失活而增加（30至40倍）。当超过50%的通道失活时，野生型和突变体的半数抑制浓度无法区分。
突变A749T和L271H诱导致病性门控变化。与野生型一样，伊拉地平抑制具有强烈的电压依赖性。我们的数据表明，在给定的负电压下，由于其更负的失活电压范围导致更多失活通道的存在，从而解释了它们明显更高药物敏感性的原因。低纳摩尔浓度的伊拉地平仅在长时间去极化状态下抑制神经元中的Ca1.3通道，且对突变型通道无选择性。
