Structural Changes in BKCa Channels During Gating

描述（由申请人提供）：大电导电压和Ca2+依赖的K+通道（BK）是普遍存在的膜蛋白，选择性地传导K+离子，在多种生理过程中发挥基础作用，包括血液流动，尿，免疫和神经传递。最近，人类BK基因缺陷已被证实与各种形式的全身性癫痫有关（Du W. et al., 2005），这种疾病影响着全世界超过4000万人。BK通道具有两个显著特征：1)它们可以被膜去极化和细胞内Ca2+激活；2)它们具有独特的渗透特性，允许200-300 pS的单通道电导，同时保持严格的K+选择性。该提案将重点关注人类BK通道（hSlo）的这两个方面。目前，Ca2+依赖性激活的分子事件和BK通道异常大电导的基础仍不清楚。我们建议结合各种强大的研究工具，包括电生理学、分子生物学、生物化学和荧光光谱，来解决这些问题。具体目的1：研究Ca2+诱导的BK通道跨膜区构象变化。我们计划揭示Ca2+激活过程中BK通道电压感应区发生的结构变化，使用位点定向荧光标记和电压钳荧光测定法。特异性目的2：研究BK通道Ca2+依赖性激活的细胞内分子事件。Ca2+被认为在细胞内c端的多个位置结合，其中两个调节K+电导的功能域（RCK1和RCK2）预计在BK Ca2+激活中起主要作用。我们设计了实验来识别和表征通道Ca传感器，并揭示Ca2+结合后导致通道打开的分子事件链。在具体目标3中，我们将研究BK通道中高电导的能量学。我们将使用温度和D2O溶剂效应来研究BK和低电导通道之间的区别，作为评估渗透差异的手段。这些研究将有助于了解BK通道的运作机制，特别是其Ca2+依赖性的分子基础和异常大电导的未知原因。公共卫生相关性：大电导电压和Ca2+依赖性K+通道（BK）是普遍存在的细胞膜蛋白，在控制血压和神经元兴奋性方面发挥基本作用。细胞内钙浓度的升高激活了这一通道。本提案的主要目的是研究钙与细胞内结构结合后导致通道打开允许钾通量的分子事件。

DESCRIPTION (provided by applicant): The large conductance Voltage- and Ca2+ dependent K+ channels (BK) are ubiquitous membrane proteins that selectively conduct K+ ions, playing a fundamental role in a multitude of physiological processes including blood flow, uresis, immunity and neurotransmission. Very recently, defects in human BK gene have been associated to forms of generalized epilepsy (Du W. et al., 2005), a disease that affects more than 40 million people worldwide. Two striking features characterize BK channels: 1) They can be activated by both membrane depolarization and intracellular Ca2+ and 2) they possess unique permeation properties, which allow a single channel conductance of 200-300 pS while maintaining a strict K+ selectivity. This proposal will focus on both these aspects of the human BK channel (hSlo). At present, the molecular events underlying Ca2+ dependent activation and the basis for the unusually large conductance in BK channel remain unknown. We propose to address these questions by combining a variety of powerful investigative tools including electrophysiology, molecular biology, and biochemistry and fluorescence spectroscopy. The three specific aims are as follows: Specific Aim 1: To investigate the Ca2+ induced conformational changes of BK channel transmembrane regions. We plan to unravel the structural changes that are taking place in BK channel voltage sensing regions during Ca2+ activation, using site directed fluorescence labeling and voltage clamp fluorometry. Specific Aim 2: To investigate the intracellular molecular events underlying BK channel Ca2+ dependent activation. Ca2+ is believed to bind at multiple locations in the intracellular C-terminus where two functional domains that regulate the K+ conductance (RCK1 and RCK2) are expected to play a major role in BK Ca2+ activation. We have designed experiments to identify and characterize the channel Ca sensors and to shed light on the chain of molecular events that, following Ca2+ binding, lead to the opening of the channel. In Specific Aim 3 we will investigate the energetics of high conductance in BK channels. We will use temperature and D2O solvent effects to investigate the distinctions between BK and lower conductance channel as a means for evaluating differences in permeation. These studies will help to understand the mechanism of operation of BK channels, particularly the molecular basis of their Ca2+ dependence and the unknown causes for the unusual large conductance. PUBLIC HEALTH RELEVANCE: The large conductance Voltage- and Ca2+-dependent K+ channels (BK) are ubiquitous cell membrane proteins that play fundamental roles in controlling blood pressure and neuronal excitability. Elevation of the intracellular Calcium concentration activates this channel. The main objective of this proposal is to investigate the molecular events that, following the binding of Calcium to intracellular structures, lead to channel opening allowing potassium flux.