Structure and Function of Potassium Channels

BK型钙激活钾通道作为胞浆钙离子检测器 能够快速响应和调节跨膜电压的物质。这些频道是 控制神经元和平滑肌动作电位放电的关键 收缩能力，因此，BK通道功能障碍与运动有关 人类的疾病和癫痫，以及小鼠模型的高血压。一个 因此，对BK通道中选通的结构基础的理解具有直接意义 导致人类疾病，并可能最终导致新的治疗干预措施。 在BK通道缺乏3-D结构的情况下，我们寻求获得结构性 钙离子依赖的通道激活机制的研究进展 原核生物钙门控钾通道MthK和TvoK的研究 服从于功能和结构的研究。BK、MthK和TvoK都包含RCK 紧跟在它们的毛孔衬里螺旋之后的区域。常见的分子 BK、MthK和TvoK共享的架构表明，理解 原核生物近亲的构象动力学将提供机械学上的洞察 与BK频道相关。我们获得这些新奇见解的方法将是 多学科，使用电生理学检测功能和核磁共振波谱 探头结构。我们寻求获得的具体见解涉及1)之间的关系 RCK亚基界面和通道门控上的钙依赖运动和2) RCK亚基中单个残基运动的[Ca~(2+)]依赖性。 我们建议未来两年的研究集中在两个目标上：1) 确定亚基间键对钙依赖的能量贡献 在MthK中选通。我们将测试盐桥和氢气的能量贡献 通过靶向单个侧链在RCK结构域亚基界面结合 这些相互作用的成分与突变，并分析突变的影响 关于使用单通道电生理学的门控。2)识别钙依赖 RCK结构域中的中间结构构象。在这里我们将分析 溶液原子尺度下结构运动与[Ca~(2+)]的关系 核磁共振光谱学，通过测量单个残基的化学位移扰动作为 [Ca2+]的功能。这些实验将实现构象步骤的解析 在钙离子依赖的门控通路中，这是电生理无法解决的 或者X射线实验。

BK-type calcium-activated potassium channels serve as cytoplasmic Ca2+ detectors that can rapidly respond to and modulate transmembrane voltage. These channels are critical in controlling action potential firing in neurons as well as smooth muscle contractility, and consequently, dysfunction of BK channels is associated with movement disorders and epilepsy in humans, and with hypertension in mouse models. An understanding of the structural basis for gating in BK channels thus has direct relevance to human disease, and may ultimately lead to novel therapeutic interventions. In the absence of a 3-D structure for the BK channel, we seek to gain structural insights toward mechanisms of Ca2+-dependent channel activation through an understanding of the prokaryotic Ca2+-gated K channels MthK and TvoK, which are amenable to both functional and structural study. BK, MthK, and TvoK all contain RCK domains that immediately follow their pore-lining helices. The common molecular architecture shared by BK, MthK, and TvoK suggests that understanding the conformational dynamics of the prokaryotic relatives will provide mechanistic insight relevant to BK channels. Our approach toward gaining these novel insights will be multidisciplinary, using electrophysiology to assay function and NMR spectroscopy to probe structure. The specific insights we seek to gain concern 1) the relation between Ca2+-dependent movements at the RCK subunit interfaces and channel gating and 2) the [Ca2+]-dependence of individual residue movements in RCK subunits. Our proposed research over the coming two-year period is focused on two aims: 1) To determine the energetic contributions of intersubunit bonds to Ca2+-dependent gating in MthK. We will test the energetic contributions of salt bridges and hydrogen bonds at RCK domain subunit interfaces by targeting the individual sidechain components of these interactions with mutagenesis, and assaying the mutation effects on gating using single-channel electrophysiology. 2) To identify Ca2+-dependent intermediate structural conformations in an RCK domain. Here we will analyze the relation between structural movements and [Ca2+] at the atomic scale using solution NMR spectroscopy, by measuring chemical shift perturbations of individual residues as a function of [Ca2+]. These experiments will enable the resolution of conformational steps in the Ca2+-dependent gating pathway that cannot be resolved using electrophysiological or X-ray experiments.