Structure and Function of Potassium Channels

BK型钙激活钾通道作为细胞质钙离子检测器 可以快速响应和调节跨膜电压。这些信道 在控制神经元和平滑肌的动作电位放电中至关重要 收缩性，因此，BK通道功能障碍与运动有关 疾病和癫痫，以及小鼠模型中的高血压。一个 因此，对BK通道门控的结构基础的理解， 人类疾病，并可能最终导致新的治疗干预措施。 在没有BK通道的3-D结构的情况下，我们寻求获得结构 对钙离子依赖性通道激活机制的见解，通过一个 了解原核细胞Ca 2+门控K通道MthK和TvoK，它们是 适合功能和结构研究。BK、MthK和TvoK都包含RCK 紧接着它们的孔衬螺旋的结构域。普通分子 BK、MthK和TvoK共享的架构表明， 原核亲属的构象动力学将提供机制的见解 关于BK频道我们获得这些新见解的方法将是 多学科，使用电生理学来测定功能和NMR光谱， 探针结构我们寻求获得的具体见解涉及：1） RCK亚基界面的Ca 2+依赖性运动和通道门控，以及2） RCK亚基中单个残基运动的[Ca 2 +]依赖性。 我们建议在未来两年进行的研究，主要集中于两个目的： 为了确定亚基间键对Ca 2+依赖的 门控在MthK。我们将测试盐桥和氢的能量贡献 通过靶向单个侧链， 这些相互作用与诱变的成分，并测定突变效应 单通道电生理学门控技术2)为了识别钙依赖性 RCK结构域中的中间结构构象。在这里，我们将分析 结构运动与[Ca 2 +]在原子尺度上的关系 NMR光谱法，通过测量单个残基的化学位移扰动， [Ca 2 +]的作用。这些实验将能够解析构象步骤 在Ca 2+依赖性门控通路中，无法使用电生理学方法解决 或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.