Modular Design of Voltage-Gated Channel Proteins

描述(申请人提供)：电压传感的基本原理，这是电可兴奋细胞的一个显著特征，仍然是谜一样的，并受到严格审查和争议的主题。这正是该计划打算填补的知识空白。这项工作的最终目的是基于电压门控通道蛋白的模块化设计来理解电压传感的机制。主要目标是：定义最适合完成电压传感的关键功能的蛋白质折叠(S)；描绘足够传感的最小决定因素集；揭示多功能电压传感器设计的分子蓝图，对于该设计，有限数量的指定扰动将使其适合于感知广泛的膜电位；以及建立两个模块之间相互作用和从一个模块到另一个模块的变化传播的表面兼容性，该变化在完整的电压门控通道中产生对电压的精致敏感性。我们建议通过过度表达和重组成脂质双层和巨型蛋白脂质体来表征分离电压传感器模块(VSM)、孔道模块(PM)和自组装的[VSM-PM]复合体的通道特性，旨在概括完整的电压门控K+通道(Kv)的组成模块的功能特征。我们建议通过产生和筛选VSM突变体的随机文库来探索电压传感器序列图景，目的是识别和展示具有新的电压门控表型的意外通道。我们打算用X射线结晶学来确定KvLm及其模块的原子分辨结构。令人振奋的结果已经出现，这为该计划的一个绝对富有成效的阶段铺平了道路。总体而言，整个行程需要从模块到顺序，再到结构，再回到机制。这一重点和现实的计划概述了电压传感的一种新的思维方式。 与公共健康相关：离子通道是一类特殊的膜蛋白，允许离子选择性和有控制地在膜上扩散，是细胞功能和调节的基础。它们的设计是蛋白质化学和进化的奇迹，它们的功能障碍是人类毁灭性疾病(如癫痫和心律失常)的根源。电压传感器是唯一赋予Na+和K+通道的元件，它是神经动作电位的基础，对跨膜电压具有极高的敏感性，仍然是一个谜，需要进一步研究。我们计划的主要目的是建立结构-功能关系，重点是电压门控通道蛋白跨膜结构域的模块化设计。

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. PUBLIC HEALTH RELEVANCE: Ion channels, a special class of membrane proteins that allow the selective and regulated diffusion of ions across membranes, are fundamental for cell function and regulation. Their design is a marvel of protein chemistry and evolution, and their dysfunction is at the root of devastating human diseases such as epilepsy and arrhythmia. The voltage sensor, the unique element that endows Na+ and K+ channels, which underlie the nerve action potential with the exquisite sensitivity to transmembrane voltage, remains enigmatic and needs further study. The thrust of our program aims to establish structure-function relationships with a primary emphasis on the modular design of the transmembrane domain in voltage-gated channel proteins.