MECHANISMS OF IONIC CHANNEL ACTIVITY

离子通道是控制选定离子的被动通量的蛋白质。 通过打开和关闭(门控)毛孔来穿过细胞膜。离子 通道在许多生理过程中起着关键作用，包括 神经元中信息的整合，动作电位的传播 在神经和肌肉细胞中，突触传递和激素控制 分泌物。这项提案寻求继续调查 离子通道关闭其毛孔的基本机制。焦点 这项研究的重点是大电导钙激活的钾通道 来自神经和肌肉。尽管众所周知，BK频道是 由体内钙离子的增加和细胞的去极化激活 膜电位，这种激活发生的机制是 不清楚。为了回答这个问题，膜片钳 这项技术将被用来记录流经单个 培养的大鼠骨骼肌和克隆的BK的BK通道 非洲爪哇卵母细胞和人胚胎中表达的通道 肾)293细胞株。然后，单通道电流将被 用最先进的技术分析以确定动力学 通道的门控机构。这种门控机制将指定 在选通期间进入的状态数、转换路径 在这些状态中，跃迁的速率常数和 由钙离子、电压和β产生的速率常数的变化 BK通道的子单元。将实现四个具体目标，以 解决方案：1)通道在范围内选通的机制 很低的钙离子到很高的钙离子；2)建议的初级 和次要的钙结合位点到门控；3)通过 该电压激活通道；以及4)通过该机制 β亚基增加了钙离子敏感性，改变了门控。一个 了解离子通道如何关闭其毛孔的重要一步 以及确定离子通道对正常细胞的贡献 功能，以及识别疾病中的有缺陷的通道 进程，是为了建立动态门控机制 频道。这项提案将为BK频道实现这一目标而努力。

Ion channels are proteins that control the passive flux of selected ions through cell membranes by opening and closing (gating) their pores. Ion channels play a key role in many physiological processes, including integration of information in neurons, propagation of action potentials in nerve and muscle cells, synaptic transmission, and control of hormone secretion. This proposal seeks to continue work on investigating the fundamental mechanisms by which ion channels gate their pores. The focus of this research is on large conductance Ca2+-activated K+ (BK) channels from nerve and muscle. Although it is known that BK channels are activated by both increases in internal Ca2+ and depolarization of the membrane potential, the mechanisms by which this activation occurs is not clear. To work towards answering this question, the patch-clamp technique will be used to record ionic currents flowing through single BK channels in cultured rat skeletal muscle and also from cloned BK channels expressed in Xenopus oocytes and the HEK (human embryonic kidney) 293 cell line. The single-channel currents will then be analyzed with state-of-the-art techniques to determine the kinetic gating mechanisms of the channels. Such gating mechanisms will specify the numbers of states entered during gating, the transition pathways among the states, the rate constants for the transitions, and the changes in the rate constants produced by Ca2+i, voltage, and the beta subunit of the BK channel. Four specific aims will be carried out to resolve: 1) the mechanism by which the channels gate over the range of very low to very high Ca2+i; 2) the contributions of proposed primary and secondary Ca2+-binding sites to the gating; 3) the mechanism by which voltage activates the channels; and 4) the mechanism by which the beta subunit increases the Ca2+i sensitivity and alters the gating. An important step towards understanding how ion channels gate their pores and determining the contributions of ion channels to normal cellular function, as well as to identifying defective channels in disease processes, is to establish the kinetic gating mechanisms of the channels. This proposal will work towards this goal for BK channels.