Alternative Splicing at N Terminus and Domain I Modulates Cav1.2 Inactivation and Surface Expression
Alternative Splicing at N Terminus and Domain I Modulates Cav1.2 Inactivation and Surface Expression
复制标题
DOI：
10.1016/j.bpj.2018.03.029
复制
发表时间：
2018-05-08
影响因子：
3.4
通讯作者：
Soong, Tuck Wah
中科院分区：
文献类型：
--
作者：
Bartels, Peter;Yu, Dejie;Soong, Tuck Wah
Ca(v)1.2 L 型钙通道是 Ca2+ 流入启动兴奋-收缩耦合的关键管道，从而导致心脏收缩和动脉血管收缩，并改变神经元的膜兴奋性。已知其 α(1c) 成孔亚基会经历广泛的选择性剪接，产生许多电生理学和药理学特性不同的 Ca(v)1.2 亚型。在这里，我们检查了人 Ca(v)1.2 在包含或排除 N 末端外显子 1/1a 和 IS6 片段外显子 8/8a 的互斥外显子方面的结构-功能关系。这些外显子的表达模式显示出组织选择性：心脏变异体 1a/8a、一种平滑肌变异体 1/8 和一种脑亚型 1/8a。总体而言，与其他三种剪接变体相比，1/8a 在与 Ca-v beta(2a) 共表达时，在电压依赖性激活和失活以及失活动力学方面表现出显着且明显的变化。进一步分析表明，含有外显子 1 的 Ca(v)1 .2 通道的 V-1/2inact 超极化位移与 8a 的组合具有明显的相加效应。然而，对于 V-1/2act，这种相加效应不太明显。然而，当比较 Ca-v beta(2a) 与 Ca-v beta 3 共表达时，测量到的效果是 β 亚基依赖性的。值得注意的是，与含有外显子 1a 的 Ca(v)1.2 通道相比，含有外显子 1 的 Ca(v)1.2 通道中通过钙调蛋白 N 叶局部 Ca2+ 感应介导的钙依赖性失活显着增强。在细胞水平上，当与 Ca-v beta(2a) 或 Ca-v beta(3) 亚基共表达时，1/8a 或 1/8 变体的电流密度显着大于 1a/8a 和 1a/8 变体。这一发现与外显子 1-Ca(v)1.2 同工型的较高通道表面表达密切相关，我们通过蛋白质表面表达水平或门控电流对其进行了定量。我们的数据还通过直接比较单一单通道事件与宏观全细胞电流，提供了对选择性剪接的人类 Ca(v)1.2 通道改变的生物物理特性的更深入的分子理解。
The Ca(v)1.2 L-type calcium channel is a key conduit for Ca2+ influx to initiate excitation-contraction coupling for contraction of the heart and vasoconstriction of the arteries and for altering membrane excitability in neurons. Its alpha(1c) pore-forming subunit is known to undergo extensive alternative splicing to produce many Ca(v)1.2 isoforms that differ in their electrophysiological and pharmacological properties. Here, we examined the structure-function relationship of human Ca(v)1.2 with respect to the inclusion or exclusion of mutually exclusive exons of the N-terminus exons 1/1 a and IS6 segment exons 8/8a. These exons showed tissue selectivity in their expression patterns: heart variant 1a/8a, one smooth-muscle variant 1/8, and a brain isoform 1/8a. Overall, the 1/8a, when coexpressed with Ca-v beta(2a), displayed a significant and distinct shift in voltage-dependent activation and inactivation and inactivation kinetics as compared to the other three splice variants. Further analysis showed a clear additive effect of the hyperpolarization shift in V-1/2inact of Ca(v)1 .2 channels containing exon 1 in combination with 8a. However, this additive effect was less distinct for V-1/2act. However, the measured effects were beta-subunit-dependent when comparing Ca-v beta(2a) with Ca-v beta 3 coexpression. Notably, calcium-dependent inactivation mediated by local Ca2+-sensing via the N-lobe of calmodulin was significantly enhanced in exon-1-containing Ca(v)1.2 as compared to exon-1 a-containing Ca(v)1.2 channels. At the cellular level, the current densities of the 1/8a or 1/8 variants were significantly larger than the 1a/8a and 1a/8 variants when coexpressed either with Ca-v beta(2a) or Ca-v beta(3) subunit. This finding correlated well with a higher channel surface expression for the exon 1-Ca(v)1.2 isoform that we quantified by protein surface-expression levels or by gating currents. Our data also provided a deeper molecular understanding of the altered biophysical properties of alternatively spliced human Ca(v)1.2 channels by directly comparing unitary single-channel events with macroscopic whole-cell currents.