Voltage-driven Structural Transitions in Voltage-Gated Calcium Channels

描述（由申请人提供）：通过电压门控Ca（CaV）通道的去极化启动的Ca内流引起过多的生理反应，如神经递质释放、肌肉收缩和基因表达。膜去极化是由四个跨膜结构，电压传感器域（VSD），它包围和控制的激活，失活和失活的中央钙选择性孔管理的量和时间的钙流入的属性。与同源四聚体KV通道相反，CaV孔和四个VSD由单个长多肽链（α 1）编码。因此，每个VSD具有独特的一级氨基酸序列，表明不同的电压敏感特性。重要的是，耦合膜去极化钙内流的电压敏感过程仍然知之甚少，辅助亚基，如β和α 2 δ，改变通道的电压依赖性的分子机制，仍然需要阐明。这种知识的缺乏持续存在，部分原因是离子和门控电流测量迄今尚未捕获CaV通道中单个VSD的特性。使用电压钳荧光法（VCF），我们已经解决了时间和电压依赖性的四个VSD的人CaV1.2揭示其高度不同的功能特性。我们现在有实验工具和理论公式来回答关于CaV1.2通道操作的关键未解决的问题，如在四个具体目标中所描述的：（1）建立单个电压感知域对CaV1.2通道激活的贡献。(2)建立辅助亚基调节CaV1.2通道电压依赖性激活的分子机制。(2a)α 2 δ亚单位的调节（2b）β亚单位的调节（3）确定每种VSD在电压和钙依赖性失活中的作用和（4）建立CaV1.2模型，解释四种不同VSD的操作和作用。CaV1.2通道在每个VSD处用小的、环境敏感的荧光团特异性标记，将使用切开卵母细胞技术进行电压钳位，使得电压诱发的荧光变化将反映局部构象重排。一系列与CaV1.2结构一致的物理相关模型，并解释了CaV1.2电压依赖性操作的所有实验解决方面，包括控制兴奋诱发Ca内流的相互作用。这一提议的创新之处包括：（1）实验方法，这在CaV超家族中是前所未有的;（2）VSD是调节亚基调节的靶点的假设;（3）由惊人的初步结果支持的前提，即CaV 1.2 VSD是失活的驱动因子和调节因子;（4）理论方法提出了第一个与CaV通道的分子结构和不对称性相一致的模型。最后，这项研究将有助于理解由CaV1.2电压依赖性改变引起的病理状态的分子机制，如Timothy综合征。

DESCRIPTION (provided by applicant): A depolarization-initiated influx of Ca through voltage-gated Ca (CaV) channels gives rise to a plethora of physiological responses such as neurotransmitter release, muscle contraction and gene expression. Membrane depolarization is sensed by four transmembrane structures, the voltage sensor domains (VSDs), which surround and control the activation, deactivation and inactivation properties of a central Ca-selective pore governing the amount and timing of Ca influx. In contrast to homotetrameric KV channels, the CaV pore and four VSDs are encoded by a single long polypeptide chain (alpha1). Thus, each VSD has a unique primary amino acid sequence, suggesting distinct voltage-sensing properties. Critically, the voltage-sensing processes coupling membrane depolarization to Ca influx are still poorly understood and the molecular mechanisms by which auxiliary subunits, such as beta and alpha2delta, alter the voltage dependence of the channel, still need to be elucidated. This lack of knowledge persists in part because ionic and gating current measurements have not thus far captured the properties of individual VSDs in CaV channels. Using Voltage Clamp Fluorometry (VCF), we have resolved that the time- and voltage-dependent properties of each of the four VSDs of human CaV1.2 revealing their highly distinct functional properties. We now have the experimental tools and theoretical formulation to answer key unresolved questions on the operation of CaV1.2 channels, as delineated in four specific aims: (1) To establish the contribution of individual voltage sensing domains to CaV1.2 channel activation. (2) To establish the molecular mechanism by which accessory subunits regulate voltage-dependent activation of CaV1.2 channels. (2a) regulation by alpha2delta subunits (2b) regulation by beta subunits (3) To determine the role of each VSD in Voltage- and Ca-dependent Inactivation and (4) To develop a CaV1.2 model accounting for the operation and role of the four distinct VSDs. CaV1.2 channels specifically labeled at each VSD with small, environment-sensitive fluorophores will be voltage-clamped using the cut-open oocyte technique, so that voltage-evoked fluorescence changes will reflect local conformational rearrangements. A series of physically-relevant models consistent with the CaV1.2 structure and accounting for all experimentally- resolved aspects of CaV1.2 voltage-dependent operation, including the interactions governing excitation- evoked Ca influx. The innovative aspects of this proposal include (1) the experimental approach, unprecedented for the CaV superfamily; (2) the hypothesis that VSDs are the targets of regulation by modulatory subunits; (3) the premise, supported by striking preliminary results, that CaV1.2 VSDs are drivers and regulators for inactivation; (4) the theoretical approach proposes the first model consistent with the molecular architecture and asymmetry of CaV channels. Finally, this study will contribute to the understanding of the molecular mechanisms of pathological states caused by altered CaV1.2 voltage dependence, such as Timothy Syndrome.