Voltage-driven Structural Transitions in Voltage-Gated Calcium Channels

描述(由申请人提供)：去极化引发的钙通过电压门控钙通道(Cav)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。膜去极化是通过四种跨膜结构--电压感应域(VSD)来感知的，VSD包围并控制着中央钙选择孔的激活、失活和失活特性，该孔控制着钙内流的数量和时间。与同源四聚体KV通道不同，Cav孔和四个VSD由一个长的多肽链(α1)编码。因此，每个VSD都有一个独特的初级氨基酸序列，这意味着不同的电压敏感特性。关键是，膜去极化与钙内流耦合的电压敏感过程仍然知之甚少，辅助亚基如β和α2Delta改变通道电压依赖性的分子机制仍需阐明。这种知识的缺乏持续存在的部分原因是离子和门控电流测量到目前为止还没有捕捉到CAV通道中单个VSD的特性。利用电压钳荧光法(VCF)，我们已经解决了人类CaV1.2的四个VSD的时间和电压依赖的特性，揭示了它们非常不同的功能特性。我们现在有了实验工具和理论公式来回答关于CaV1.2通道运行的关键悬而未决的问题，这些问题描述在四个具体的目标中：(1)建立单个电压敏感结构域对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)通过α2β亚基(2b)调节，(3)确定每个VSD在电压和钙依赖的失活中的作用，以及(4)建立一个CaV1.2模型，解释四个不同的VSD的操作和作用。在每个VSD上特别标记的CaV1.2通道将使用切开卵母细胞技术进行电压钳制，因此电压引起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电压依赖操作的所有实验解决的方面，包括控制激发诱发的钙内流的相互作用。这一建议的创新方面包括(1)实验方法，这在Cav超家族中是前所未有的；(2)VSD是调节亚基调节的目标的假设；(3)由引人注目的初步结果支持的前提，即CaV1.2 VSD是失活的驱动和调节因子；(4)理论方法提出了与Cav通道的分子结构和不对称性一致的第一个模型。最后，本研究将有助于理解CaV1.2电压依赖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.