Gating Mechanisms of the Prokaryotic Calcium Activated Potassium channel MthK

描述（由候选人提供）：离子通道门控，通道在开放传导状态和闭合非传导状态之间切换的能力，是整个生理学中通道的最基本机械特征。本提案的总体目标是研究MthK钙激活K+离子通道的门控机制。MthK是来自热自养甲烷杆菌的原核K+通道，其代表由钙激活的通道家族。已知这种类型的真核细胞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和晶体学通道的电生理记录。我们将测试广泛持有的假设，即MthK孔结构域通过孔内螺旋（TM 2螺旋）移动成KcsA样构象而关闭，从而防止K+在孔的细胞内侧进入。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，在开放和关闭配置之间交替。电生理学证据将来自于对细胞内阻滞剂阻滞的状态依赖性和动力学的研究。将KcsA通道的结果与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化合物如四丁基铵存在下直接测定通道开放概率、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射线晶体学。我们一直在优化MthK的晶体条件下，钙的情况下，这可能揭示了一个封闭的通道构象。我们已经开始解决突变MthK的结构（在钙的存在下），不表现出在脂质双层通道开口，因此可能是一个组成性封闭的通道。这些结构的结果可能会加强上述功能研究的结论，并建立实验MthK门控的机制。最后，在未来，我们将使用突变和结构的研究，以检查选择性过滤门控内的MthK钾通道的可能性。

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.