ABSTRACT

-->Low voltage-activated Cav1.3 L-type Ca²⁺-channels are key regulators of neuronal excitability controlling neuronal development and different types of learning and memory. Their physiological functions are enabled by their negative activation voltage-range, which allows Cav1.3 to be active at subthreshold voltages. Alternative splicing in the C-terminus of their pore-forming α1-subunits gives rise to C-terminal long (Cav1.3_L) and short (Cav1.3_S) splice variants allowing Cav1.3_S to activate at even more negative voltages than Cav1.3_L. We discovered that inclusion of exons 8b, 11, and 32 in Cav1.3_S further shifts activation (−3 to −4 mV) and inactivation (−4 to −6 mV) to more negative voltages as revealed by functional characterization in tsA-201 cells. We found transcripts of these exons in mouse chromaffin cells, the cochlea, and the brain. Our data further suggest that Cav1.3-containing exons 11 and 32 constitute a significant part of native channels in the brain. We therefore investigated the effect of these splice variants on human disease variants. Splicing did not prevent the gating defects of the previously reported human pathogenic variant S652L, which further shifted the voltage-dependence of activation of exon 11-containing channels by more than −12 mV. In contrast, we found no evidence for gating changes of the CACNA1D missense variant R498L, located in exon 11, which has recently been identified in a patient with an epileptic syndrome.

Our data demonstrate that alternative splicing outside the C-terminus involving exons 11 and 32 contributes to channel fine-tuning by stabilizing negative activation and inactivation gating properties of wild-type and mutant Cav1.3 channels.

 抽象的

-->低电压激活的 Cav1.3 L 型 Ca ²⁺通道是控制神经元发育以及不同类型学习和记忆的神经元兴奋性的关键调节因子。它们的生理功能是通过负激活电压范围实现的，这使得 Cav1.3 在亚阈值电压下激活。其成孔 α1 亚基 C 端的选择性剪接产生 C 端长 (Cav1.3 _L ) 和短 (Cav1.3 _S ) 剪接变体，使 Cav1.3 _S在更负的电压下激活比 Cav1.3 _L 。我们发现，Cav1.3 _S中包含外显子 8b、11 和 32 进一步将激活（-3 至 -4 mV）和失活（-4 至 -6 mV）转变为更负的电压，如 tsA- 中的功能表征所揭示的201个细胞。我们在小鼠嗜铬细胞、耳蜗和大脑中发现了这些外显子的转录本。我们的数据进一步表明，含有 Cav1.3 的外显子 11 和 32 构成了大脑中天然通道的重要部分。因此，我们研究了这些剪接变体对人类疾病变体的影响。剪接并不能阻止先前报道的人类致病性变异 S652L 的门控缺陷，该缺陷进一步将含有外显子 11 的通道激活的电压依赖性改变了超过 -12 mV。相比之下，我们没有发现位于外显子 11 的CACNA1D错义变异 R498L 的门控变化的证据，该变异最近在一名癫痫综合征患者中被发现。

我们的数据表明，C 末端外部涉及外显子 11 和 32 的选择性剪接通过稳定野生型和突变型 Cav1.3 通道的负激活和失活门控特性，有助于通道微调。