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Candiate提供)：离子通道门控，通道在开放的导电状态和闭合的非导电状态之间切换的能力，是贯穿生理学的通道最基本的机械特征。本研究的主要目的是研究MthK钙激活K+离子通道的门控机制。MthK是自养甲烷杆菌的一种原核K+通道，是钙激活的一类通道的代表。已知这种类型的真核通道对神经元的放电行为和内耳等感觉器官的反应很重要。它们是很有前途的药物靶标，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电生理记录人工脂双层中的通道和结晶学。我们将检验广泛存在的假设，即MthK孔区是通过孔内螺旋(TM2螺旋)进入KCSA样构象而关闭的，从而防止K+进入孔内一侧。从长远来看，我们将探索K+选择性区域，称为选择性过滤器，是否也可以作为渗透门，在开放和关闭构型之间交替。电生理学证据将来自对细胞内阻滞剂阻断状态依赖和动力学的研究。KCSA通道的结果将与MthK进行比较，以辨别这些通道之间相似或独特的门控机制。在人工双层中的单通道记录将允许在存在诸如四丁基铵的化合物的情况下直接确定通道开放概率、阻断百分比和门控动力学。闭合MthK构象的结构证据将来自X射线结晶学。我们一直在优化MthK在无钙条件下的结晶条件，这可能会显示出闭合的通道构象。我们已经开始解决突变的MthK的结构(在钙存在的情况下)，它在脂质双层中不显示通道开放，因此可能是一个结构性关闭的通道。这些结构结果可能会加强上述功能研究的结论，并从实验上建立MthK门控的机制。最后，在未来，我们将使用突变和结构研究来考察MthK K+通道内选择性过滤门控的可能性。