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: 7677686
• 负责人: David John Posson
• 金额: $ 5.34万
• 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/7677686
• 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，即通道在开放导电状态和封闭非导电状态之间切换的能力，是整个生理通道最基本的机制特征。本提案的总体目的是研究MthK钙活化的K+离子通道的门控机制。MthK是来自热自养甲烷菌的原核K+通道，是钙激活通道家族的代表。这种类型的真核通道对于神经元的放电行为和感觉器官（如内耳）的反应是重要的。它们是有前途的药物靶点，因此是生物物理研究的重要课题。我们建议使用的主要技术是人工脂质双层通道的电生理记录和晶体学。我们将测试广泛持有的假设，即MthK孔域通过内孔螺旋（TM2螺旋）运动成ksa样构象而关闭，从而阻止K+进入孔的细胞内侧。从长远来看，我们将探索K+选择性区域，称为选择性滤波器，是否也可以作为一个渗透门，在开放和封闭构型之间交替。电生理学证据将来自于细胞内阻滞剂阻滞的状态依赖性和动力学研究。KcsA通道的结果将与MthK进行比较，以辨别这些通道之间相似或独特的门控机制。人工双层中的单通道记录将允许在四丁基铵等化合物存在的情况下直接确定通道打开概率，百分比阻塞和门控动力学。闭合MthK构象的结构证据将来自x射线晶体学。我们一直在优化MthK的晶体条件，在缺乏钙的情况下，可能会显示封闭的通道构象。我们已经开始解决突变MthK的结构（在钙存在的情况下），它在脂质双分子层中没有通道开口，因此可能是一个本构封闭通道。这些结构结果可能会加强上述功能研究的结论，并在实验上建立MthK门控的机制。最后，在未来，我们将使用突变和结构研究来检查MthK K+通道内选择性滤波门控的可能性。