Determinants of Sodium Channel Function: Ion Pair Interactions Across Domains

项目总结/摘要 本计画将探讨钠离子作用所产生的生物电讯息的基本机制 可兴奋细胞膜的通道蛋白。我们将把我们的注意力集中在电压敏感域的 人体骨骼肌纤维中发现的钠通道。这个结构域的突变导致了 遗传性疾病，称为钠通道病，包括肌肉强直和周期性麻痹。在 这项工作，我们将调查的分子手段，其中该域的部分，S1至S4，相互作用， 控制钠离子通道的激活、开放和快速失活的基本功能， 无法对膜电位的变化做出反应。我们的假设针对的是 片段S1至S3中的带负电荷的氨基酸，所谓的反电荷， 片段S4中的氨基酸。我们将使用电压钳电生理学来测试突变的影响 可以逆转带负电或带正电的氨基酸的电荷。这些电荷逆转突变将 比较对激活的影响和两种形式的快速失活。我们的目标是确定 与给定钠通道结构域的S4片段的反电荷相互作用，其决定了特定的 这是一个不对称的渠道。为了做到这一点，我们将量化所有重要突变对 使用IFM / QQQ失活缺陷背景，并使用门控电流， 直接测试电压传感器运动。比较电荷固定及其再活化将允许 在两种形式的快速灭活过程中，S1-S3与S4片段相互作用的类似可定量测量，以及 在恢复过程中。最后，我们将建立电压传感器域的模型，在这些模型中插入我们的突变， 模型，然后运行模型的计算机模拟，以响应膜电位的变化 增强了它们在肌肉纤维中的典型功能。我们的研究将进一步加深我们对 为钠通道电压敏感性的研究提供基础， 通过肌纤维的通道病突变。

Project Summary / Abstract This project will investigate basic mechanisms of bioelectric information as produced by the actions of sodium channel proteins of excitable cell membranes. We will focus our attention on the voltage-sensing domain of the sodium channel found in human skeletal muscle fibers. Mutations in this domain are responsible for a number of inherited diseases, called sodium channelopathies, and include muscle myotonia and periodic paralysis. In this work we will investigate the molecular means by which the segments of this domain, S1 to S4, interact to control basic sodium channel functions of activation, or opening, and fast inactivation, during which the channel is unable to respond to changes in membrane potential. Our hypotheses target putative interactions of negatively charged amino acids in segments S1 to S3, so-called countercharges, with positively charged amino acids in the segment S4. We will use voltage clamp electrophysiology to test the effects of mutations that reverse the charge of negatively, or positively charged amino acids. These charge-reversing mutations will be compared for effects on activation and for two forms of fast inactivation. Our goal is to identify countercharge interaction with the S4 segment of a given sodium channel domain, that determines a specific function of this asymmetric channel. To do this we will quantify the effects of all significant mutations on activation parameters using the IFM / QQQ inactivation deficient background, and using gating currents to directly test voltage sensor movement. Comparison of charge immobilization and its remobilization will allow a similar quantifiable measure of S1-S3 interaction with S4 segments during two forms of fast inactivation, and during recovery. Finally, we will build models of the voltage sensor domains, insert our mutations in these models, and then run computer simulations of the models in response to the change in membrane potential that elicits their typical function in muscle fibers. Our studies will further our understanding of the molecular basis of voltage-sensitivity in sodium channels and provide a foundation for studies on dysfunction produced by channelopathy mutations of muscle fibers.