High-resolution crystallographic and functional studies of K+ channel function.

K+通道是神经系统、骨骼肌、平滑肌和心肌中细胞兴奋性的关键调节剂， 分泌腺因此，K+通道的功能障碍是导致糖尿病的根本原因并不奇怪。 无数的人类疾病，如：神经系统疾病，心脏病和糖尿病。基于这个理由， 在原子水平上理解K+通道的特性是非常重要的， 兴奋性了解离子选择性，渗透和门控在原子细节将使我们能够 为了鉴定高度特异性的治疗剂， 通道的动力学状态，需要进行调节，以纠正给定的通道病。它遵循 二十年来，对KcsA封闭结构进行的功能、结构和计算研究， 提高了我们对结构如何定义K+通道功能的理解。最近，我们 两个重要的科学贡献：第一个原子分辨率的描述KcsA的最小动力学循环 以及与每个动力学循环反应相关的能量学的量化。但重要 尚未回答的问题仍然存在，主要是由于我们无法同时进行结构和功能 研究：1）通道的开放状态2）选择性中高度保守的甘氨酸残基的突变体 过滤器，已知其在关闭和打开时影响灭活门控、离子选择性和/或离子结合 3）串联四聚体，以剖析离子通道功能的协同性，和4）突变体， 通过从KcsA的起始精确地解耦激活门打开来剖析KcsA的非导电打开状态， 离子渗透/失活的选择性过滤器。因此，我们提出以下具体目标：1） 表征选择性过滤器、离子占有率和传导之间的结构-功能相关性 2）为了确定KcsA的结构-功能相关性， 使用串联异源四聚体的KcsA亚基协同性3）为了理解KcsA的变构作用， 耦合的离子渗透，C型失活和离子选择性的开始和4）为了了解 甘氨酸残基在K+通道选择性过滤器中的结构和功能作用。我们的新奇 实验方法，加上我们在离子通道方面的丰富经验，使我们完全有资格 执行拟议的项目。最后，这个项目的完成将使我们更接近一个完整的原子论 了解离子通道的功能，使我们能够确定离子通道动力学中间体更适合 作为下一代更特异、更安全的治疗药物的药物靶点。

K+ channels are key regulators of cell excitability in the nervous system, skeletal, smooth and cardiac muscle and secretory glands. Therefore, it is not surprising that dysfunction of K+ channels are the underlie cause of uncountable human pathologies, such as: neurological disorders, cardiac diseases and diabetes. For this reason, it is extremely important to understand at the atomic level the properties of K+ channels that determine cell excitability. Understanding ion selectivity, permeation and gating at atomic detail will allow us to identify highly-specific therapeutic agents that can recognize with precision a specific channel's kinetic state that need to be regulated to correct a given channelopathy. It follows that for two decades, functional, structural and computational studies, performed on the KcsA-closed structure, have improved our understanding of how the structure defines the function of K+ channels. Recently, we have made two important scientific contributions: the first atomic-resolution description of KcsA's minimal kinetic cycle and the quantification of the energetics associated with each kinetic cycle reaction. However, important unanswered questions remain, mostly due to our inability to conduct simultaneous structural and functional studies in: 1 ) the open-state of the channel 2) mutants of the highly conserved glycine residues in the selectivity filter, which are known to affect inactivation gating, ion selectivity and/or ion binding in the closed and open states of the channel, 3) tandem-tetramers to dissect cooperativity of ion channel function, and 4) mutants that dissect the non-conductive open states of KcsA by precisely uncoupling activation-gate opening from the onset of ion permeation/inactivation at the selectivity filter. Consequently, we propose the following Specific Aims: 1) To characterize the structure-function correlations between the selectivity filter, ion occupancy and conduction properties of KcsA “trapped” with its activation gate open 2) To determine the structure-function correlations of KcsA subunit cooperativity using tandem hetero-tetramers 3) To understand the role of KcsA's allosteric coupling on the onset of ion permeation, C-type inactivation and ion selectivity and 4) To understand the structural and functional roles of the glycine residues within the K+ channel selectivity filter. The novelty of our experimental approaches, together with our vast experience working with ion channels, fully qualifies us to perform the proposed project. Finally, the completion of this project will bring us closer to a complete atomistic understanding of ion-channel function, allowing us to identify ion-channels kinetic intermediates more suitable as pharmaceutical targets for the next generation of more specific and safer therapeutic drugs.