BK CHANNEL MODULATION BY BETA SUBUNITS

描述（由申请人提供）：BK通道是大电导、电压和Ca激活的K通道（maxi-K，K），由α亚基和多达四个β亚基的四聚体组成。β 1是四种β亚基之一，存在于平滑肌中，调节电压和Ca敏感性以及BK通道激活和失活的动力学。这项提议的总体目标是确定β 1对α的调制的结构基础，对此知之甚少。建议映射它们在通道的闭合状态和开放状态下的物理相互作用，并确定这些相互作用的功能后果。该方法是在α和β中的每个跨膜片段（TM）的细胞外侧翼区中，将四个连续残基突变为半胱氨酸（Cys），每次一个。alpha中有7个TM（SO-S6），beta 1中有2个TM（TM 1和TM 2）。所有突变体将在HEK-293细胞中表达，并筛选表达和功能。将在完整细胞中用新型膜不渗透交联剂（0.5 - 1 nm跨度）和氧化剂（0.3 nm跨度）探测所有功能突变体对，这两种交联剂都对Cys具有特异性。将通过定量Western印迹法测定在不同交联剂浓度和反应时间下完整细胞的细胞表面上BK通道的交联程度。将计算每对α Cys和β 1 Cys的相对速率常数，并反映该对的接近度。显示为邻居的Cys的交联对的功能效应将在来自用交联剂处理的细胞的由内而外的贴片中电生理地确定。如果α TM在门控期间正常移动，则通过α SO-S6与β 1 TM 1或TM 2的共价交联的束缚应深刻地影响功能，可能改变α-β相互作用。将在由外向外贴片中通道的开放状态下确定Cys对的功能改变交联的速率常数，并与在封闭状态下确定的速率常数进行比较。两种状态下速率常数的差异将指示在门控期间哪些α TM相对于β TM移动。BK通道在调节平滑肌和神经元功能的收缩张力中起主要作用，并且它们的病理生理学与中风、高血压和心血管疾病有关。对BK通道调节的分子机制的更深入理解将导致改进的治疗方法。

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.