BK CHANNEL MODULATION BY BETA SUBUNITS

通过 Beta 子单元进行 BK 通道调制

• 批准号: 7536010
• 负责人: Arthur Karlin
• 金额: $ 35.22万
• 依托单位: COLUMBIA UNIVERSITY HEALTH SCIENCES
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-03-05 至 2010-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7536010
• 关键词: Affect; Biological Assay; Calcium-Activated Potassium Channel; Cardiovascular Diseases; Cell surface; Cells; Charybdotoxin; Crosslinker; Cysteine; Equilibrium; Functional disorder; Goals; Hypertension; Individual; Kinetics; Lead; Maps; Mediating; Membrane; Membrane Potentials; Molecular; Mono-S; Mutate; Neurons; Oxidants; Play; Predisposition; Reaction; Reaction Time; Regulation; Relative (related person); Role; Side; Smooth Muscle; Stroke; Suspension substance; Suspensions; Time; Toxin; Western Blotting; base; crosslink; extracellular; iberiotoxin; large-conductance calcium-activated potassium channels; mutant; novel; voltage

BK channels are large -conductance, voltage-and-Ca-activated K channels (maxi-K, slo), consisting of a tetramer of alpha subunits and up to four beta subunits. Beta"!, one of four types of beta subunits, is found in smooth muscle and modulates the voltage and Ca sensitivities and the kinetics of activation and deactivation of BK channels. The overall goal of this proposal is the determination of the structural basis for the modulation of alpha by betal, about which little is known. It is proposed to map their physical interactions in both the closed state and open state of the channel and to determine the functional consequences of these interactions. The approach is to mutate to cysteine (Cys) four consecutive residues, one at a time, in the extracellular flanking region of each transmembrane segment (TM) in alpha and in beta. There are 7 TMs (SO-S6) in alpha and 2 TMs (TM1 and TM2) in betal. All mutants will be expressed in HEK-293 cells and screened for expression and function. All pairs of functional mutants will be probed in intact cells with a novel membrane-impermeant crosslinker (0.5 -1 nm span) and an oxidizing agent (0.3 nm span), both specific for crosslinking Cys. The extents of crosslinking of BK channel on the cell surface of intact cells with varying crosslinker concentrations and reaction times will be determined by quantitative Western blotting. Relative rate constants of each pair of an alpha Cys and a betal Cys will be calculated and will reflect the proximity of the pair. The functional effects of crosslinking pairs of Cys shown to be neighbors will be determined electrophysiologically in inside-out patches from cells treated with crosslinkers. The tethering by covalent crosslinking of alpha SO-S6 to betal TM1 or TM2 should profoundly affect function if the alpha TMs normally move during gating, possibly altering alpha -beta interactions. The rate constants for function-altering crosslinking of pairs of Cys will be determined in the open state of the channel in outside-out patches and compared to the rate constants determined in the closed state. Differences in rate constants in the two states will indicate which TMs of alpha move relative to TMs in beta during gating. BK channels play a major role in the regulation of contractile tone in smooth muscle and neuronal function, and their pathophysiology is implicated in stroke, hypertension, and cardiovascular disease. Greater understanding of the molecular mechanisms of BK channel regulation will lead to improvedtherapeutics.

BK通道是大型传统，电压和CA激活的K通道（Maxi-K，SLO），由A组成 Alpha亚基的四聚体和多达四个Beta亚基。 beta“！，是四种类型的beta亚基之一，在 平滑肌并调节电压和Ca敏感性以及激活和停用的动力学 BK频道。该提案的总体目标是确定结构基础 Betal对Alpha的调制，几乎没有知道。建议在 封闭状态和通道的开放状态都确定这些功能后果 互动。该方法是突变到半胱氨酸（CYS）的四个连续残基，一次是在 α和β中每个跨膜段（TM）的细胞外侧翼区域。有7个TMS （SO-S6）在betal中的α和2个TMS（TM1和TM2）中。所有突变体将在HEK-293细胞中表达，并且 筛选表达和功能。所有功能突变体将在完整的细胞中使用新颖的细胞进行探测 膜 - 覆盖交联（0.5 -1 nm跨度）和氧化剂（0.3 nm跨度），均针对 交联的cys。 BK通道在完整细胞的细胞表面上的交联范围，变化 交联剂浓度和反应时间将通过定量蛋白质印迹确定。相对的 将计算每对alpha cys和betal cys的速率常数，并将反映 两人。将确定证明是邻居的Cys的交联成对的功能效应 在用交联链链接处理的细胞中进行电生理的内而外的斑块。共价束缚 如果alpha tms通常，alpha so-s6 to betal tm1或tm2的交联应深刻影响功能 在门控过程中移动，可能会改变α -beta相互作用。改变功能的速率常数 将在外部贴片中以通道的开放状态确定成对的Cys对，并确定 与在封闭状态下确定的速率常数相比。两种状态的速率常数差异 将指示门控过程中α的α的TMS相对于beta中的TMS移动。 BK频道在 平滑肌和神经元功能中收缩性张力的调节及其病理生理学是 与中风，高血压和心血管疾病有关。对分子的更多了解 BK通道调节的机制将导致改善治疗药物。