CA2+DEPENDENT K+CHANNELS: ALLOSTERIC GATING

描述(申请人提供)：我们的长期目标是了解BK通道激活的分子机制。电压、细胞内钙离子和镁离子激活BK型钾通道。这些通道在调节肌肉收缩、神经传递和昼夜节律起搏器输出方面非常重要。最近，BK通道中的电压感受器、钙和镁结合部位已被确定。然而，位于不同结构域的传感器和激活门之间耦合的结构基础仍然难以捉摸。在BK通道门控这一关键步骤中的一个中心问题是，这些不同的结构域如何相互作用，以调节传感器和激活门之间的耦合。很明显，这个问题带来的知识差距是理解BK通道激活的关键障碍。最近的研究和我们的初步结果导致了一个普遍的假设来回答这个问题：电压传感器结构域(VSD)和胞浆结构域(CTD)之间的相互作用，这种界面排列将CTD上的配体结合位置耦合到激活门的打开。我们提出以下具体目标来检验这一假说的三个关键方面。1.论证了静电相互作用对VSD-CTD排列的影响。2.证明VSD-CTD对准耦合到激活栅极。3.证明VSD-CTD相互作用介导了钙离子结合与门控开放的偶联。我们开发了创新的方法来测量VSD-CTD的对准以及这种对准与激活门的耦合。这项研究将确定对BK通道激活至关重要的氨基酸和结构基序，并揭示这一分子过程中结构域之间相互作用的本质。一种流行的细胞内配体激活离子通道的模型是，配体结合改变胞浆结构域的构象，从而拉动多肽连接物打开激活门。我们的研究结果表明，在BK通道中，镁和钙也可能通过涉及不同结构域的残基的静电相互作用来推动电压传感器激活通道，这提供了一种新的配体依赖门控机制，这可能与许多其他离子通道相同。BK通道被认为是治疗神经元缺血、创伤和认知功能下降的靶点，最近的研究表明，BK通道与高血压、精神分裂症、癫痫和阵发性运动障碍有关。在这项研究中，对BK通道门控过程中分子事件的剖析，除了提供对离子通道门控原理的洞察外，还将有助于确定治疗学发展的特定靶点。

DESCRIPTION (provided by applicant): Our long-term goal is to understand the molecular mechanisms of BK channel activation. BK-type K+ channels are activated by voltage, intracellular Ca2+ and Mg2+. These channels are important in modulating muscle contraction, neural transmission and circadian pacemaker output. Recently, the voltage sensor, Ca2+ and Mg2+ binding sites in BK channels have been identified. However, the structural basis for the coupling between sensors and the activation gate, which are located in different structural domains, still remains elusive. A central question in this crucial step of BK channel gating is how these different structural domains interact with one another to mediate the coupling between the sensors and the activation gate. It has become apparent that a knowledge gap presented by this question is a critical barrier for understanding BK channel activation. Recent studies and our preliminary results lead to a general hypothesis for answering this question: interactions between the voltage sensor domain (VSD) and the cytosolic domain (CTD), and this interfacial alignment couples the ligand binding site on the CTD to the opening of the activation gate. We propose the following specific aims to examine three key aspects of this hypothesis. 1. To demonstrate that the electrostatic interactions affect the VSD-CTD alignment. 2. To demonstrate that the VSD-CTD alignment is coupled to the activation gate. 3. To show that the VSD-CTD interactions mediate coupling of Ca2+ binding to gate opening. We have developed innovative methods to measure VSD-CTD alignment and the coupling of this alignment to the activation gate. This study will identify amino acids and structural motifs important for BK channel activation and reveal the nature of the interactions among structural domains during this molecular process. A prevalent model proposed for ion channel activation by intracellular ligands is that ligand binding alters the conformation of the cytosolic domain, which pulls a peptide linker to open the activation gate. The results of our proposed study will show that in BK channels Mg2+ and Ca2+ may also activate the channel by pushing the voltage sensor via an electrostatic interaction involving the residues in different structural domains, which provides a novel mechanism of ligand dependent gating that may be shared by many other ion channels. BK channels are being pursued as therapeutic targets for neuronal ischemia, trauma and cognitive decline, and recent studies show that BK channels are associated with hypertension, schizophrenia, epilepsy and paroxysmal dyskinesia. The dissection of the molecular events during BK channel gating in this study will help identify specific targets for the development of therapeutics in addition to providing insights into the principles of ion channel gating.