Molecular Mechanism of Cation Channel Selectivity

描述（由申请人提供）：离子在生物膜上的转移是神经兴奋、肌肉细胞收缩、信号转导和激素分泌等生理过程的核心。离子通道起着至关重要的作用，它在膜内提供了一个通道，即离子传导孔，允许特定的离子沿着它们的电化学梯度穿过。在人类中，离子通道几乎存在于所有组织中，发挥着各种生理基本功能。由于它们在人体中的普遍性和重要性，通道的功能障碍通常是一系列人类疾病的罪魁祸首。选择特定离子种类的能力被称为离子选择性，是定义离子通道功能的基本性质。在四聚体阳离子通道中，包括最大的离子通道家族，包括K+， Ca2+， Na+和环核苷酸门控（CNG）通道，选择性通常是离子通道孔内部分独特结构和化学环境的直接结果，选择性过滤器在不同的通道中是不同的。随着离子通道的遗传、生化和电生理分析的进步，我们对这组通道中控制离子选择性的分子细节的理解已经取得了长足的进步，最近，从KcsA K+通道结构的开创性工作开始，对几个成员的结构表征以及随后对其他K+选择性通道的工作。尽管这些结构研究提供了K+通道选择性过滤器的直接可视化，但影响K+选择性的决定因素仍在激烈的争论中。此外，其他四聚体阳离子通道的结构信息很少，其离子选择性的分子基础仍不清楚。我们研究的总体目标是了解四聚体阳离子通道中离子选择性的结构基础。利用几种代表选择性和非选择性通道的离子传导孔的模型蛋白的极高分辨率晶体结构，我们旨在阐明两组生理上必需的阳离子通道中离子选择性的基本原理。一种是非选择性的、Ca2+可渗透的环核苷酸门控（CNG）通道，以蜡样芽孢杆菌的NaK及其模拟CNG的嵌合体为模型系统；另一种是K+选择性通道，以热自养甲烷菌的K+选择性NaK突变体（NaK2K）和MthK K+通道为模型系统。蛋白质晶体学、电生理学和蛋白质化学合成的结合方法将被用于拟议的研究

DESCRIPTION (provided by applicant): Transfer of ions across biological membranes is central to physiological processes like nerve excitation, muscle cell contraction, signal transduction and hormone secretion. Ion channels play a vital role by providing a passageway, the ion conduction pore, within membranes to allow specific ions to traverse down their electrochemical gradient. In humans, ion channels are found in nearly all tissues serving a variety of physiologically essential functions. Because of their prevalence and importance in the human body, dysfunction of a channel is often to blame for a wide range of human pathologies. The ability to select for specific ionic species is known as ion selectivity and is a fundamental property defining ion channel function. In tetrameric cation channels, which comprise the single largest family of ion channels including the K+, Ca2+, Na+, and cyclic nucleotide-gated (CNG) channels, selectivity is usually a direct consequence of the unique structural and chemical environment within part of the ion channel pore, the selectivity filter, which is distinct among different channels. Our understanding of the molecular details governing ion selectivity in this group of channels has come a long way with the advancement of genetic, biochemical, and electrophysiological analysis of ion channels and, more recently, the structural characterization of several members beginning with the ground breaking work of the KcsA K+ channel structure and subsequent work on other K+ selective channels. Although these structural studies offer a direct visualization of the selectivity filter of K+ channels, the determinant factors contributingto K+ selectivity are still under heated debate. Moreover, there is little structural information available for other tetrameric cation channels and the molecular basis for their ion selectivity remains unclear. The overall goal of our research is to understand the structural basis of ion selectivity in tetrameric cation channels. Taking advantage of the extremely high resolution crystal structures of several model proteins representing the ion conduction pores of both selective and non-selective channels, we aim to elucidate basic principles of ion selectivity in two groups of physiologically essential cation channels. One is the non-selective, Ca2+ permeable cyclic nucleotide-gated (CNG) channels, using NaK from Bacillus cereus and its CNG- mimicking chimeras as model systems; the other is K+ selective channels, using a K+ selective NaK mutant (NaK2K) and the MthK K+ channel from Methanobacterium thermoautotrophicum as model systems. A combined approach of protein crystallography, electrophysiology and protein chemical synthesis will be employed in the proposed studies