BK CHANNEL MODULATION BY BETA SUBUNITS

通过 Beta 子单元进行 BK 通道调制

基本信息

批准号：
7208171
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
项目类别：
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

项目摘要

DESCRIPTION (provided by applicant): 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. Beta1, 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 beta1, 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 beta1. 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 beta1 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 beta1 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 improved therapeutics.

描述（由申请人提供）：BK通道是大传导，电压和CA激活的K通道（Maxi-K，SLO），由Alpha亚基的四聚体和多达四个Beta亚基组成。 Beta1是四种类型的β亚基之一，在平滑肌中发现，调节BK通道的激活和失活的电压和CA敏感性和动力学。该提案的总体目标是确定Beta1对Alpha调制的结构基础，这一点鲜为人知。提议在通道的封闭状态和开放状态中绘制其物理相互作用，并确定这些相互作用的功能后果。该方法是在每个跨膜段（TM）和beta中的每个跨膜段（TM）的细胞外侧翼区域中一次突变到半胱氨酸（Cys）。 Alpha中有7个TMS（SO-S6），Beta1中有2个TMS（TM1和TM2）。所有突变体将在HEK-293细胞中表达，并筛选以表达和功能。所有功能突变体将在完整的细胞中探测具有新型的膜覆盖交联（0.5 -1 nm跨度）和氧化剂（0.3 nm跨度），均针对交联CYS（0.3 nm跨度）。 BK通道在完整细胞的细胞表面上具有不同交联浓度和反应时间的交联范围将由定量蛋白质印迹确定。将计算每对alpha cys和beta1 cys的相对速率常数，并将反映这对的接近度。证明是邻居的Cys的交联对的功能效应将在用交联处理的细胞中的内而外的斑块中确定。如果α1或TM2的alpha so-s6共价交联的束缚，则如果αTMS通常在门控过程中移动，可能会改变α-beta的相互作用，将对功能产生深远影响。 Cys对的改变功能的交联的速率常数将在外部外部贴片中以通道的开放状态确定，并将其与在封闭状态下确定的速率常数进行比较。两种状态中速率常数的差异将表明α的TMS相对于门控过程中的beta中的TMS移动。 BK通道在平滑肌和神经元功能的收缩张力调节中起主要作用，其病理生理学与中风，高血压和心血管疾病有关。对BK通道调节的分子机制的更多了解将改善治疗疗法。