BK CHANNEL MODULATION BY BETA SUBUNITS

BK通道是大电导、电压和钙激活的K通道(MAXI-K，SLO)，由 由α亚基和最多4个β亚基组成的四聚体。Beta“！是四种类型的Beta亚基之一，可在 并调节电压和钙敏感性以及激活和失活的动力学 BK频道。这项提案的总体目标是确定 贝塔尔对阿尔法的调制，人们对此知之甚少。建议将它们的物理相互作用映射到 通道的关闭状态和打开状态，并确定这些状态的功能后果 互动。方法是将半胱氨酸(Cys)连续突变为四个残基，一次一个，在 每个跨膜片段(TM)的胞外侧翼区(TM)，在α和β中。有7个TM (SO-S6)在阿尔法和2个TM(TM1和TM2)在贝塔尔。所有突变体都将在HEK-293细胞中表达 对表情和功能进行筛选。所有一对功能突变都将在完整的细胞中用一种新的 无膜交联剂(0.5-1 nm跨度)和氧化剂(0.3 nm跨度)，两者都是专用于 交联型半胱氨酸。完整细胞表面BK通道的交联度随时间的变化 交联剂浓度和反应时间将通过定量Western blotting来确定。相对的 将计算每对α半胱氨酸和贝塔半胱氨酸的速率常数，并将反映 这两个人。将确定显示为相邻的半胱氨酸的交联对的功能效应 经交联剂处理的细胞的电生理学内向外贴片。共价拴系法 如果αTMS正常，αSO-S6与βTM1或TM2的交联会深刻影响功能 在门控期间移动，可能会改变阿尔法-贝塔相互作用。函数变换的速率常数 将在通道的打开状态下以外向外贴片确定成对的Cys的交联度 与在闭合状态下确定的速率常数进行比较。两种状态下的速率常数的差异 将指示在选通期间，哪些阿尔法TM相对于Beta中的TM移动。BK渠道在以下方面发挥了重要作用 平滑肌收缩张力的调节和神经元功能及其病理生理学 与中风、高血压和心血管疾病有关。更好地理解分子 BK通道的调节机制将有助于改善治疗方法。

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.