Mechanism of Gating and Permeation in The TRKH K+ Channels

描述(申请人提供)：K+集中在所有细胞中，是许多生理过程所必需的。在细菌、真菌、植物和原生生物中，K+转运体超家族的膜蛋白或SKT蛋白介导K+的动态平衡。目前的研究重点是SKT家族成员TrkH，它与其密切相关的同源物TrkG和KtrB一起构成了SKT蛋白的最大亚家族。TrkH/TrkG/KtrB蛋白是细胞生长所必需的，也是抗生素的潜在靶点。该项目的长期目标是了解SKT蛋白的运行机制。以前，我的实验室从副溶血性弧菌中分离得到TrkH的结构，并与TrkA形成复合体，TrkA是一种胞质蛋白，包含两个调节K+电导的结构域，即RCK结构域。我们还测量了TrkH-TrkA复合体的第一个单通道电流，并证明了与TrkA结合的核苷酸调节通道的功能。基于这些令人兴奋的结果，我提出了以下两个具体目标：解开TrkA控制TrkH门控的机制；以及研究TrkH中离子选择性和导电性的结构基础。我们还将评估对TrkH门控、离子选择性和传导的扰动如何影响其在低K+浓度下拯救大肠杆菌的能力。综合起来，这些实验的结果将为TrkH中离子渗透和门控的结构基础提供新的知识，并最终将使我们更好地理解SKT蛋白质的基本机制。目的1.探讨TrkH-TrkA复合体的门控机制。目的2.研究TrkH的离子选择性和渗透机制。

DESCRIPTION (provided by applicant): K+ is concentrated in all cells and essential for many physiological processes. In bacteria, fungi, plants and protists, membrane proteins from the Superfamily of K+ Transporters, or SKT proteins, mediate homeostasis of K+. The focus of the current proposal is the SKT family member TrkH, which, together with its closely related homologs TrkG and KtrB, forms the largest sub-family of SKT proteins. TrkH/TrkG/KtrB proteins are required for cell growth and are potential targets for antibiotics. The long-term goal of the project is to understand mechanisms of operation for the SKT proteins. Previously, my lab obtained structures of TrkH from Vibrio parahaemolyticus in isolation and in complex with TrkA, a cytosolic protein comprising two Regulate- Conductance-of-K+, or RCK domains. We also measured the first single-channel currents from the TrkH-TrkA complex, and demonstrated that nucleotides binding to TrkA regulate the function of the channel. Based on these exciting results, I propose the following two Specific Aims: to unravel the mechanism of TrkA-controlled gating of TrkH; and to examine the structural basis of ion selectivity and conduction in TrkH. We will also assess how perturbations to TrkH gating, ion selectivity and conduction affect its abilit to rescue E. coli in low K+ concentrations. Combined, results from these experiments will provide new knowledge of structural underpinnings of ion permeation and gating in TrkH, and will eventually lead us to a better understanding of basic mechanisms of SKT proteins. Aim 1. To investigate the mechanism of gating in the TrkH-TrkA complex. Aim 2. To examine the mechanism of ion selectivity and permeation in TrkH.