Molecular mechanisms of potassium channel permeation and gating

描述（申请人提供）：钾离子通道是细胞兴奋性的主要决定因素，在许多生理过程中起着至关重要的作用。K+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量大小和孔的选择性（对离子类型的急性区分）；2)门控，即打开和关闭孔的离子流动过程。上述任何一种功能出现故障都会造成灾难性的生理后果。这项资助的总体目标是通过基于结构的功能分析来了解离子传导、选择性和K+通道中的门控机制。为了实现这一目标，将使用模型细菌K+通道，真核生物K+通道参与兴奋性的同源物。细菌通道更容易进行生化和结构研究，以详细研究这些机制。我们的第一个主要目的是了解钾通道渗透的各个方面。Aim 1.1将使用多学科的三管齐下的方法来研究KcsA的选择性：1)通过分析单通道电流记录的非均匀离子阻断KcsA的电生理学，2)阻断KcsA通道的x射线晶体学，以及3)计算离子通过KcsA孔时的能量景观的分子动力学。虽然已经提出了几种不同的K+通道选择性机制，但我们新兴的功能，结构和分子动力学研究可能会导致一个新的画面。Aim 1.2将确定两个结构相似的钾通道孔不均匀传导的分子机制。利用KcsA和MthK突变体的电生理和x射线晶体学分析，将研究位于MthK和KcsA细胞外孔口的带负电荷氨基酸在调节离子传导中的作用。我们的第二个主要目的是研究K+通道中的门控方面。目的2.1将探讨KcsA中质子活化的机制。对通道束交叉处残基单突变和成对突变的功能和热力学结果的分析将阐明哪些残基直接相互作用或偶联以与质子打开通道。为了进一步了解其机制，将对门控改变的KcsA突变体的结构进行结晶和求解。Aim 2.2将通过获得野生型和突变型封闭构象的MthK的x射线晶体学结构，并确定细胞内阻滞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。MthK的通道阻塞将与KcsA的类似实验进行比较，KcsA是一种已知具有束交叉收缩的通道。提出的目标将为钾通道性质的基本性质提供重要的新见解，并且许多信息可能很容易适用于我们对更广泛的离子通道家族的理解。

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.