MECHANISMS OF IONIC CHANNEL ACTIVITY

离子通道是一种蛋白质，它控制选定离子的被动通量 通过打开和关闭（门控）细胞膜的孔。 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 分泌物这项建议旨在继续开展调查工作， 离子通道门控孔隙的基本机制。重点 本研究的重点是大电导Ca ~（2+）激活的K ~+（BK）通道 从神经和肌肉。 虽然已知BK通道是 激活的同时增加内部钙离子和去极化的 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 技术将被用来记录离子电流流过单 培养大鼠骨骼肌及克隆BK的BK通道 在非洲爪蟾卵母细胞和HEK（人胚胎干细胞）中表达的通道 肾）293细胞系。 然后，单通道电流将为 用最先进的技术进行分析， 通道的门控机制。 这种门控机制将指定 门控期间进入的状态数， 在状态之间，转变的速率常数，以及 由Ca 2 +i、电压和β产生的速率常数的变化 BK通道的亚基。 将实现四个具体目标， 解决：1）通道在范围内选通的机制 非常低到非常高的Ca 2 +i; 2）建议的主要贡献 和次级Ca 2+结合位点的门控; 3）机制， 该电压激活通道;以及4）该电压激活通道的机制。 β亚基增加Ca ~（2+）敏感性并改变门控。 一个 了解离子通道如何控制其孔的重要一步 并确定离子通道对正常细胞的贡献， 功能，以及识别疾病中有缺陷的通道 过程，是建立动力学门控机制， 渠道 本提案将努力实现BK渠道的这一目标。

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.