BK CHANNEL MODULATION BY BETA SUBUNITS

描述(申请人提供)：BK通道是大电导、电压和钙激活的K通道(MAXI-K，SLO)，由一个四聚体的α亚基和最多四个β亚基组成。β1是四种β亚基之一，存在于平滑肌中，调节BK通道的电压敏感性和钙敏感性，以及激活和失活的动力学。这项提案的总体目标是确定贝塔1调制阿尔法的结构基础，人们对此知之甚少。建议绘制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。方法是在每个跨膜片段(TM)的胞外侧翼区域的α和β区，连续突变四个半胱氨酸(Cys)，一次一个。α中有7个TM(SO-S6)，Beta1中有2个TM(TM1和TM2)。所有突变体都将在HEK-293细胞中表达，并对表达和功能进行筛选。所有的功能突变都将在完整的细胞中用一种新的膜不敏感的交联剂(0.5-1 nm跨度)和氧化剂(0.3 nm跨度)进行探测，这两种交联剂都是专用于交联半胱氨酸的。不同交联剂浓度和不同反应时间的完整细胞表面BK通道的交联度将通过定量Western blotting来确定。将计算每对α-半胱氨酸和β-半胱氨酸的相对速率常数，并将反映该对的接近程度。用交联剂处理的细胞内由内向外的斑块中的电生理效应将被显示为相邻的交联半胱氨酸的功能效应。如果αTM在门控期间正常运动，通过αSO-S6与β1 TM1或TM2的共价交联链，应该会深刻地影响功能，可能改变α-β相互作用。将在通道的打开状态下以外向外贴片确定用于改变功能的Cys对的交联的速率常数，并将其与在关闭状态下确定的速率常数进行比较。这两种状态下的速率常数的差异将指示在选通过程中，哪些αTM相对于Beta TM移动。BK通道在调节平滑肌收缩张力和神经元功能方面发挥重要作用，其病理生理学与中风、高血压和心血管疾病有关。更多地了解BK通道调节的分子机制将有助于改进治疗方法。

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.