Structural Changes in BKCa Channels During Gating

描述(申请人提供)：大电导电压和钙依赖的钾离子通道(BK)是普遍存在的膜蛋白，选择性地传导K+离子，在血液流动、利尿、免疫和神经传递等多种生理过程中发挥重要作用。最近，人类BK基因缺陷与各种形式的全身性癫痫有关(Du W.等人，2005年)，这是一种影响全球4000多万人的疾病。BK通道有两个显著特征：1)它们可以被膜去极化和细胞内钙离子激活；2)它们具有独特的渗透特性，允许单通道电导为200-300ps，同时保持严格的K+选择性。这项提议将集中在人类BK频道(HSlo)的这两个方面。目前，钙依赖激活的分子事件和BK通道异常大电导的基础尚不清楚。我们建议通过结合各种强大的研究工具来解决这些问题，包括电生理学、分子生物学、生物化学和荧光光谱学。具体目标如下：特异性目标1：研究钙离子对BK通道跨膜区构象的影响。我们计划利用定点荧光标记和电压钳荧光技术来揭示钙激活过程中BK通道电压敏感区发生的结构变化。具体目的2：研究BK通道钙依赖激活的细胞内分子事件。钙离子被认为结合在细胞内C末端的多个位置，其中调节K+电导的两个功能结构域(RCK1和RCK2)有望在BK钙激活中发挥主要作用。我们设计了实验来鉴定和表征通道钙传感器，并阐明了在钙离子结合后导致通道开放的分子事件链。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差异，作为评估渗透差异的一种手段。这些研究将有助于了解BK通道的运行机制，特别是其钙依赖的分子基础和异常大电导的未知原因。与公共健康相关：大电导电压和钙依赖的钾离子通道(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础作用。细胞内钙浓度的升高激活了这一通道。这项建议的主要目的是研究在钙与细胞内结构结合后，导致允许钾离子流动的通道开放的分子事件。

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.