Voltage-driven Structural Transitions in Voltage-Gated Calcium Channels

电压门控钙通道中电压驱动的结构转变

• 批准号: 9178075
• 负责人: Riccardo Olcese
• 金额: $ 32.5万
• 依托单位: UNIVERSITY OF CALIFORNIA LOS ANGELES
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-12-01 至 2018-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9178075
• 关键词: Accounting; Address; Amino Acid Sequence; Architecture; Arrhythmia; Attenuated; Binding Sites; Blood Vessels; Calcium Channel; Calmodulin; Cells; Charge; Coupling; Data; Data Set; Dependence; Disease; Drug usage; Employee Strikes; Environment; Exhibits; Fluorescence; Fluorometry; Formulation; Gene Expression; Health; Human; Hypertension; Immobilization; Individual; Ion Channel; Ions; Kinetics; Knowledge; L-Type Calcium Channels; Label; Left; Measurement; Membrane; Methodology; Modeling; Molecular; Molecular Conformation; Movement; Muscle Contraction; Mutagenesis; Myocardial Contraction; Nerve; Oocytes; Optics; Pathologic; Periodicity; Physiological; Physiological Processes; Process; Property; Proteins; Regulation; Research Proposals; Role; Scheme; Series; Signal Transduction; Structure; Techniques; Thermodynamics; Time; Timothy syndrome; Ursidae Family; Voltage-Clamp Technics; Work; analytical tool; conformational conversion; design; fluorophore; innovation; insight; mutant; neurotransmitter release; novel therapeutics; operation; polypeptide; public health relevance; response; sensor; tool; voltage; voltage clamp

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.

描述（由申请人提供）：通过电压门控CA（CAV）通道的去极化引起的CA涌入产生了很多生理反应，例如神经递质释放，肌肉收缩和基因表达。通过四个跨膜结构，即电压传感器结构域（VSD）感测膜去极化，它们围绕和控制了中央CA选择性孔的激活，失活和失活的特性，该孔控制了CA涌入的量和时间。与同型KV通道相反，CAV孔和四个VSD由单个长多肽链（alpha1）编码。因此，每个VSD都有一个独特的原代氨基酸序列，表明了不同的电压感应特性。至关重要的是，耦合膜去极化与CA涌入的电压感应过程仍然很少了解，并且辅助亚基（例如beta和alpha2delta）的分子机制会改变通道的电压依赖性，但仍需要阐明。缺乏知识仍然存在部分是因为离子和门控电流测量尚未捕获CAV通道中各个VSD的特性。使用电压夹具荧光测定法（VCF），我们解决了人cav1.2的四个VSD的时间和电压依赖性特性，揭示了它们高度不同的功能性能。现在，我们拥有实验工具和理论公式，可以回答有关CAV1.2通道操作的关键未解决的问题，如四个特定目的所述：（1）确定单个电压传感域对CAV1.2 CAV1.2通道激活的贡献。 （2）建立分子机制，辅助亚基调节CAV1.2通道的电压依赖性激活。 （2a）α2delta亚基（2B）对β亚基的调节（2B）（3）确定每个VSD在电压和CA依赖性失活中的作用，以及（4）开发CAV1.2 cav1.2模型占四种不同VSD的操作和作用的模型。 CAV1.2使用切开的卵母细胞技术在每个VSD上专门在每个VSD上标记为小，环境敏感的荧光团，因此电压偏置的荧光变化将反映出局部构象的重排。一系列与CAV1.2结构一致的物理相关模型，并考虑了CAV1.2电压依赖性操作的所有实验解析方面的结构，包括控制激发的相互作用 - 引起了CA的涌入。该提案的创新方面包括（1）实验方法，为CAV超家族史无前例； （2）假设VSD是调节亚基调节的目标； （3）在引人注目的初步结果的支持下，CAV1.2 VSD是失活的驱动因素和调节器； （4）理论方法提出的第一个模型与CAV通道的分子结构和不对称性一致。最后，这项研究将有助于理解由CAV1.2电压依赖性改变（例如蒂莫西综合征）引起的病理状态的分子机制。