Molecular mechanisms of potassium channel permeation and gating

描述（由申请人提供）：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。K+通道有两个主要的机械特征：1）渗透，包括通过其孔的离子流的大小和孔的选择性（离子类型之间的急性区分），和2）门控，打开和关闭离子流的孔的过程。上述任何一个方面的故障都可能产生灾难性的生理后果。该基金的总体目标是通过采用基于结构的功能分析来了解K+通道中的离子传导，选择性和门控机制。为了实现这一目标，将使用模型细菌K+通道，参与兴奋性的真核K+通道的同源物。细菌通道更容易进行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离子通道渗透的各个方面。目的1.1将使用多学科三管齐下的方法研究KcsA选择性：1）使用单通道电流记录分析的不渗透离子对KcsA阻断的电生理学，2）阻断的KcsA通道的X射线晶体学，以及3）分子动力学，以计算离子通过KcsA孔时的能量景观。虽然已经提出了几种不同的机制K+通道的选择性，我们新兴的功能，结构和分子动力学研究可能会导致一个新的图片。目的1.2将确定通过两个结构相似的钾通道孔的不均匀传导的分子机制。带负电荷的氨基酸，位于MthK和KcsA的细胞外孔的嘴，在调谐离子传导中发挥的作用，使用KcsA和MthK突变体的电生理和X-射线晶体学分析，将进行研究。我们的第二个主要目标是研究K+通道的门控方面。目的2.1探讨KcsA中质子活化的机制。在通道束交叉处的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相互作用或偶联以打开具有质子的通道。为了进一步深入了解机制，将结晶和解决具有改变的门控的KcsA突变体的结构。目的2.2将通过获得具有闭合构象的野生型和突变的MthK的X射线晶体学结构，并通过确定细胞内阻断剂进入通道孔是否取决于通道是打开还是关闭，来研究MthK通道激活门的位置。将MthK的通道阻断与KcsA的类似实验进行比较，KcsA是一种已知以交叉收缩门控的通道。所提出的目标将提供重要的新的见解钾通道特性的基本性质，许多信息可能很容易适用于我们的理解更广泛的家庭的离子通道。

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.