BK CHANNEL MODULATION BY BETA SUBUNITS

BK通道是大电导、电压和Ca激活的K通道（maxi-K，K），由一个 α亚基和多达四个β亚基的四聚体。贝塔”！，四种β亚基之一，存在于 平滑肌并调节电压和Ca敏感性以及激活和失活的动力学 BK频道本提案的总体目标是确定 α被β 1调制，对此知之甚少。建议将它们的物理相互作用绘制在 通道的关闭状态和打开状态，并确定这些状态的功能结果 交互.该方法是将突变为半胱氨酸（Cys）的四个连续残基，一次一个，在 每个跨膜区段（TM）的细胞外侧翼区以α和β表示。有7个TM （S 0-S6）和2个TM（TM 1和TM 2）。所有突变体将在HEK-293细胞中表达， 筛选表达和功能。所有功能突变体对将在完整细胞中用一种新的 膜不渗透性交联剂（0.5 - 1 nm跨度）和氧化剂（0.3 nm跨度），两者都对 交联Cys.在不同的浓度下，完整细胞表面BK通道的交联程度不同。 交联剂浓度和反应时间将通过定量蛋白质印迹来确定。相对 将计算每对α Cys和β Cys的速率常数，并将反映α Cys和β Cys的接近度。 一对。将确定显示为邻居的Cys的交联对的功能效应 在电生理学上，来自用交联剂处理的细胞的由内而外的贴片。通过共价键连接 如果α-TM正常地与β-TM 1或TM 2交联，则α-SO-S6与β-TM 1或TM 2的交联将深刻地影响功能。 在门控期间移动，可能改变α-β相互作用。功能改变的速率常数 Cys对的交联将在外向贴片中通道的开放状态下确定， 与在闭合状态下确定的速率常数相比。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 平滑肌和神经元功能中收缩张力的调节及其病理生理学， 与中风高血压和心血管疾病有关更好地理解分子 BK通道调节机制将导致改进的治疗方法。

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.