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

K+通道是神经系统、骨骼肌、平滑肌和心肌细胞兴奋性的关键调节因子 分泌腺。因此，K+通道功能障碍也就不足为奇了。 数不清的人类病理，如：神经系统疾病、心脏病和糖尿病。因为这个原因， 在原子水平上了解决定细胞的K+通道的性质是极其重要的 兴奋性。了解原子细节上的离子选择性、渗透和选通将使我们能够 找出高度特异的治疗剂，能够准确地识别特定的 需要调节以纠正给定的经络病变的通道的运动状态。由此可以得出结论 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，我们已经做出了 两个重要的科学贡献：对KCSA最小动力学循环的第一个原子分辨率描述 以及与每个动力学循环反应相关的能量学的量化。然而，重要的是 悬而未决的问题仍然存在，主要是因为我们无法同时进行结构性和功能性 研究了：1)通道的开放状态2)高度保守的甘氨酸残基突变体的选择性 过滤器，已知会影响关闭和打开状态下的失活门控、离子选择性和/或离子结合 通道的状态，3)串联-四聚体，用于分析离子通道功能的协作性，以及4)突变体，其 从一开始就精确解耦激活门，剖析KCSA的非导通开态 选择性过滤器上的离子渗透/失活。为此，我们提出了以下具体目标：1) 表征选择性过滤器、离子占有率和导电性之间的结构-功能相关性 KCSA在激活门打开时被捕获的性质2)以确定结构与功能的相关性 用串联异四聚体研究KCSA亚基协同作用3)了解KCSA变构的作用 对离子渗透开始、C型失活和离子选择性的耦合以及4)理解 甘氨酸残基在K+通道选择性过滤器中的结构和功能作用。我们的新奇之处 实验方法，再加上我们在离子通道方面的丰富经验，使我们完全有资格 执行提议的项目。最后，这个项目的完成将使我们更接近一个完全的原子论 了解离子通道的功能，可以让我们识别出更适合离子通道的动力学中间体 作为下一代更具体和更安全的治疗药物的药物靶标。

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.