Modular Design of Voltage-Gated Channel Proteins

描述（由申请人提供）：电压传感的基本原理，电兴奋细胞的标志性特征，仍然是一个谜，是严格审查和争议的主题。这正是该项目想要填补的知识空白。这项工作的最终目标是理解基于电压门控通道蛋白模块化设计的电压传感机制。主要目标是：确定最适合实现电压传感关键功能的蛋白质折叠；描述一组最小的决定因素，足以感知；揭示通用电压传感器设计的分子蓝图，有限数量的特定扰动将使其适应广泛的膜电位；并建立两个模块之间相互作用的表面相容性，以及从一个模块到另一个模块的变化传播，从而在完整的电压门控通道中产生对电压的精致灵敏度。我们建议通过过表达和重组成脂质双层和巨型蛋白脂质体来表征隔离电压传感器模块（VSM）、孔模块（PM）和自组装[VSM-PM]复合物的通道特性，旨在从其组成模块中概括完整电压门控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的电压门控表型的未知通道。我们打算用x射线晶体学来确定KvLm及其组件的原子分辨率结构。已经出现了令人兴奋的结果，这为该计划的决定性生产阶段铺平了道路。总的来说，这个过程需要从模块到序列，到结构，再到机制。这个重点突出和现实的程序概述了一种思考电压传感的新方法。

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.