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 (Cav1.3L) and short (Cav1.3S) splice variants allowing Cav1.3S to activate at even more negative voltages than Cav1.3L. We discovered that inclusion of exons 8b, 11, and 32 in Cav1.3S 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.
摘要:
->低电压激活的Cav1.3L型Ca2-通道是神经元兴奋性控制神经元发育以及不同类型的学习和记忆的关键调节剂。它们的生理功能是通过它们的负激活电压范围来实现的,这允许Cav1.3在亚阈值电压下处于活动状态。它们的成孔α1亚基的C末端中的选择性剪接产生C末端长(Cav1.3L)和短(Cav1.3S)剪接变体,使Cav1.3S在比Cav1.3更高的负电压下激活。我们发现包含外显子8b,Cav1.3S中的图11和32进一步将活化(-3至-4mV)和失活(-4至-6mV)移位至更负电压,如通过tsA-201细胞中的功能表征所揭示。我们在小鼠嗜铬细胞中发现了这些外显子的转录本,耳蜗,还有大脑.我们的数据进一步表明,含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。剪接并不能防止先前报道的人类致病变体S652L的门控缺陷,这进一步改变了含外显子11通道的激活的电压依赖性超过-12mV。相比之下,我们没有发现CACNA1D错义变体R498L的门控变化的证据,位于外显子11,最近在患有癫痫综合征的患者中被发现。我们的数据表明,涉及外显子11和32的C末端外部的可变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而有助于通道微调。