Gating Mechanisms of the Prokaryotic Calcium Activated Potassium channel MthK

原核生物钙激活钾通道 MthK 的门控机制

• 批准号: 7949975
• 负责人: David John Posson
• 金额: $ 5.58万
• 依托单位: WEILL MEDICAL COLL OF CORNELL UNIV
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-09-01 至 2011-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7949975
• 关键词: Accounting; Ammonium; Behavior; Binding; Biological Assay; Calcium; Calcium-Activated Potassium Channel; Crystallography; Data; Dependence; Electrons; Exhibits; Family; Future; Investigation; Ion Channel; Ion Channel Gating; Ions; Kinetics; Labyrinth; Lipid Bilayers; Location; Methanobacteria; Methanobacterium; Modeling; Molecular Conformation; Movement; Neurons; Organ; Pharmacologic Substance; Physiology; Positioning Attribute; Potassium; Potassium Channel; Probability; Resolution; Roentgen Rays; Sensory; Side; Signal Transduction; Structural Models; Structure; Techniques; Testing; Thallium; channel blockers; electron density; improved; mutant; prevent; response; tetrabutylammonium

DESCRIPTION (provided by candidate): Ion channel gating, the ability of channels to switch between an open conducting state and a closed non- conducting state, is the most fundamental mechanistic feature of channels throughout physiology. The overall aim of this proposal is to investigate the gating mechanisms of the MthK calcium-activated K+ ion channel. MthK is a prokaryotic K+ channel from Methanobacterium thermoautotrophicum that is representative of a family of channels activated by calcium. Eucharyotic channels of this type are known to be important for the firing behavior of neurons and the response of sensory organs such as in the inner ear. They are promising pharmaceutical targets and therefore are important topics of biophysical investigation. The principle techniques we propose using are electrophysiological recording of channels in artificial lipid bilayers and crystallography. We will test the widely-held assumption that the MthK pore-domain closes by a movement of inner-pore helices (TM2 helices) into a KcsA-like conformation, thereby preventing the entrance of K+ at the intracellular side of the pore. In the longer term, we will explore whether the K+ selective region, called the selectivity filter, may also serve as a permeation gate, alternating between open and closed configurations. Electrophysiological evidence will come from a study of the state-dependence and kinetics of block by intracellular blockers. Results from the KcsA channel will be compared with MthK in order to discern similar or unique gating mechanisms between these channels. Single channel recording in artificial bilayers will allow direct determination of the channel open probability, percent block, and gating kinetics in the presence of compounds such as tetrabutylammonium. Structural evidence for closed MthK conformations will come from x-ray crystallography. We have been optimizing crystal conditions for MthK in the absence of calcium which may reveal a closed channel conformation. We have already begun solving the structure of a mutant MthK (in the presence of calcium) that does not exhibit channel openings in the lipid bilayer and therefore may be a constitutively closed channel. These structural results will likely strengthen conclusions from the functional studies mentioned and establish the mechanism of MthK gating experimentally. Finally, in the future we will use mutational and structural studies to examine the possibility of selectivity filter gating within the MthK K+ channel.

描述（由候选人提供）：离子通道门控，通道在开放的传导状态和封闭的非传导状态之间切换的能力是通道的最基本机械特征。该提案的总体目的是研究MTHK钙激活的K+离子通道的门控机制。 MTHK是一种来自甲摩菌菌群的原用K+通道，它代表了钙激活的一系列通道家族。已知这种类型的舒适通道对于神经元的发射行为和感觉器官（例如内耳）的响应很重要。它们是有希望的药物靶标，因此是生物物理研究的重要主题。我们建议使用的原理技术是人工脂质双层和晶体学中通道的电生理记录。我们将测试广泛的假设，即MTHK孔隙域通过内部孔洞（TM2螺旋）移动到KCSA样构象中，从而阻止K+在孔内的细胞内侧进入KCSA样构象。从长远来看，我们将探讨K+选择性区域（称为选择性过滤器）是否也可以用作渗透门，在开放和封闭的配置之间交替。电生理证据将来自对细胞内阻滞剂的状态依赖性和动力学的研究。 KCSA通道的结果将与MTHK进行比较，以辨别这些通道之间的相似或独特的门控机制。人工双层中的单个通道记录将允许在存在诸如四丁丁基铵之类的化合物的情况下直接确定通道的开放概率，百分比和门控动力学。封闭的MTHK构象的结构证据将来自X射线晶体学。在没有钙的情况下，我们一直在优化MTHK的晶体条件，这可能显示出封闭的通道构象。我们已经开始求解突变体MTHK的结构（在存在钙的情况下），该结构未在脂质双层中表现出通道开口，因此可能是组成型封闭的通道。这些结构性结果可能会加强提到的功能研究的结论，并通过实验建立MTHK门控的机制。最后，将来我们将使用突变和结构研究来检查MTHK K+通道内选择性过滤门控的可能性。