Molecular mechanisms of potassium channel permeation and gating

描述(申请人提供)：钾(K)通道是细胞兴奋性的主要决定因素，在许多生理过程中发挥关键作用。钾通道有两个主要的机制特征：1)渗透性，包括通过其孔的离子流动的大小和孔的选择性(离子类型之间的尖锐区分)；2)门控，即对离子流开放和关闭孔的过程。以上两种情况中的任何一种都可能产生灾难性的生理后果。这项资助的总体目标是通过基于结构的功能分析来了解K通道中离子传导、选择性和门控的机制。为了实现这一目标，将使用细菌K通道的模型，即参与兴奋性的真核K通道的同系物。细菌通道更容易用于详细研究这些机制所需的生化和结构研究。我们的第一个主要目标是了解钾通道渗透的各个方面。AIM 1.1将用多学科三管齐下的方法研究KCSA的选择性：1)通过分析单通道电流记录分析无意义离子阻断KCSA的电生理学；2)阻断KCSA通道的X射线结晶学；以及3)分子动力学计算离子通过KCSA孔道时的能量景观。尽管已经提出了几种不同的K通道选择性机制，但我们新出现的功能、结构和分子动力学研究可能会导致一幅新的图景。AIM 1.2将确定通过两个结构相似的钾通道孔不均匀传导的分子机制。通过对KCSA和MthK突变体的电生理和X射线结晶学分析，将研究位于MthK和KCSA胞外孔口的带负电荷的氨基酸在调节离子传导中所起的作用。我们的第二个主要目标是研究在K频道中选通的各个方面。AIM 2.1将探索KCSA中质子活化的机制。对通道束交叉处残基的单个和成对突变的功能和热力学后果的分析将阐明哪些残基直接与质子相互作用或耦合打开通道。为了更深入地了解这一机制，我们将对门控改变的KCSA突变体的结构进行结晶和解决。Aim 2.2将通过获得闭合构象中野生型和突变的MthK的X射线结晶学结构，并通过确定细胞内阻滞剂对通道孔的访问是否依赖于通道的开放或关闭来研究MthK通道激活门的位置。MthK的通道阻断将与KCSA的类似实验进行比较，KCSA是一种已知的具有跨束收缩的通道。提出的目标将提供重要的新见解的基本性质的钾通道的性质，许多信息可能很容易适用于我们的理解更广泛的离子通道家族。

DESCRIPTION (provided by applicant): Potassium (K+) channels are major determinants of cell excitability and play crucial roles in many physiological processes. There are two major mechanistic features of K+ channels: 1) permeation, which comprises both the magnitude of ionic flow through their pores and the selectivity of the pore (acute discrimination between ion types), and 2) gating, the process of opening and closing the pore to ionic flow. Malfunction in either of the above can have disastrous physiological consequences. The overall objective of this grant is to understand mechanisms of ion conduction, selectivity and gating in K+ channels by employing structure-based functional analysis. To accomplish this goal, model bacterial K+ channels, homologues of the eukaryotic K+ channels involved in excitability, will be used. Bacterial channels lend themselves more readily to the biochemical and structural studies necessary to investigate these mechanisms in detail. Our first major aim is to understand aspects of permeation in potassium channels. Aim 1.1 will investigate KcsA selectivity using a multidisciplinary three-pronged approach: 1) electrophysiology of KcsA block by impermeant ions using analysis of single- channel current recordings, 2) X-ray crystallography of the blocked KcsA channels, and 3) molecular dynamics to calculate energetic landscapes as ions negotiate the KcsA pore. Although several different mechanisms have already been proposed for K+ channel selectivity, our emerging functional, structural and molecular dynamics studies may lead towards a novel picture. Aim 1.2 will identify the molecular mechanism for uneven conduction through two structurally similar potassium channel pores. The role that negatively charged amino acids, located by the extracellular pore mouths of both MthK and KcsA, play in tuning ion conduction using electrophysiological and X-ray crystallographic analysis of KcsA and MthK mutants, will be investigated. Our second major aim investigates aspects of gating in K+ channels. Aim 2.1 will probe the mechanism of proton activation in KcsA. Analysis of the functional and thermodynamic consequences of single and pair-wise mutation of residues at the channel bundle crossing will elucidate which residues directly interact or couple to open the channel with protons. In order to gain further insight into the mechanism, the structures of KcsA mutants with altered gating will be crystallized and solved. Aim 2.2 will investigate the location of the MthK channel activation gate by obtaining structures with X-ray crystallography of wildtype and mutated MthK in the closed conformation, and by determining whether the access of intracellular blockers to the channel pore is dependent on whether the channel is open or closed. Channel block for MthK will be compared to similar experiments for KcsA, a channel known to gate with a bundle-crossing constriction. The proposed aims will provide important new insights into the fundamental nature of potassium channel properties, and much of the information may be readily applicable to our understanding of the broader family of ion channels.