Modular Design of Voltage-Gated Channel Proteins

描述（由申请人提供）：电压感知的基本原理，电可兴奋细胞的标志性特征，仍然是谜，是严格审查和争议的主题。这正是该计划打算填补的知识差距。这一奋进的最终目标是了解基于电压门控通道蛋白的模块化设计的电压感知机制。主要目标是：定义最适合于实现电压感测的关键功能的蛋白质折叠;描绘足以感测的决定因素的最小集合;揭示通用电压传感器设计的分子蓝图，对于该通用电压传感器设计，有限数量的指定扰动将使其适于感测宽范围的膜电位;并建立两个模块之间相互作用的表面相容性和从一个模块到另一个模块的变化传播，这在完整电压下产生孔对电压的精确灵敏度。门控通道我们建议通过过表达和重组成脂质双层和巨脂蛋白体来表征隔离的电压传感器模块（VSM）、孔模块（PM）和自组装的[VSM-PM]复合物的通道特性，旨在从其组成模块中概括完整的电压门控K+通道（Kv）的功能特征。我们建议通过生成和筛选VSM突变体的随机文库来探索电压传感器序列景观，旨在识别和展示具有新电压门控表型的未知通道。我们打算通过X射线晶体学确定KvLm及其模块的原子分辨率结构。令人兴奋的结果已经出现，为该计划的一个决定性的富有成效的阶段铺平了道路。总的来说，行程需要从模块到序列，到结构，再回到机制。这一重点突出、切合实际的计划概述了一种全新的电压检测思维方式。

DESCRIPTION (provided by applicant): The fundamental principles underlying voltage sensing, a hallmark feature of electrically excitable cells, are still enigmatic and the subject of intense scrutiny and controversy. This is precisely the gap in knowledge the program intends to fill. The ultimate goal of this endeavor is the understanding of the mechanism of voltage sensing based on the modular design of voltage-gated channel proteins. Major objectives are: to define the protein fold(s) best suited to fulfill the pivotal function of voltage sensing; to delineate a minimum set of determinants sufficient for sensing; to uncover a molecular blueprint for a versatile voltage sensor design for which a finite number of specified perturbations would adapt it to sense a wide range of membrane potential; and to establish the surface compatibility underlying the interaction between the two modules and the propagation of change from one module to the other that produces the exquisite sensitivity of the pore to voltage in intact voltage-gated channels. We propose to characterize the channel properties of the isolated voltage sensor module (VSM), the pore module (PM), and the self-assembled [VSM-PM] complex by overexpression and reconstitution into lipid bilayers and giant proteoliposomes, aiming to recapitulate the functional features of the intact voltage-gated K+ channel (Kv) from its component modules. We propose to explore the voltage sensor sequence landscape approached by generating and screening random libraries of VSM mutants aiming to identify and demonstrate unsuspected channels with new voltage-gating phenotypes. We intend to determine the atomic resolution-structures of KvLm and its modules by X-ray crystallography. Exciting results have already emerged which pave the way for a decidedly productive phase of the program. Overall, the itinerary entails going from modules to sequence, to structure and back to mechanism. This focused and realistic program outlines a novel way of thinking about voltage sensing.