Mechanisms of Ion Channel Activity

描述(由申请人提供)：离子通道是一种蛋白质，它通过打开和关闭(门控)细胞膜的孔来控制离子通过细胞膜的被动流量。这项提议寻求继续研究离子通道门的基本机制。目前的研究重点是大电导钙和电压激活的钾通道(BK)，它在许多生理过程中发挥着关键作用，包括控制肌肉收缩、调节突触传递和神经元的信息整合。尽管在理解钙离子和电压如何激活BK通道，以及辅助β1亚基如何调节这一活动方面已经取得了很大进展，但许多基本问题仍然存在。为了回答这些问题，克隆的BK通道将在非洲爪哇卵母细胞和HEK293细胞中表达，单通道电流将通过膜片钳技术记录下来，并将使用先进的技术进行分析以确定门控机制。需要检验的最初假设是，多种钙离子和电压依赖的调节机制共同作用于控制BK通道的开放-关闭转换(GATING)。为了验证这一假设，第一个具体目标将是：1)确定所提出的五种钙依赖调节机制中的每一种对BK通道门控的贡献，包括任何协同作用。通过单独和各种组合研究钙离子依赖的调节机制，合作将解决这一问题。第二个具体目标是：2)建立一个全面的BK通道动力学门控机制，包括所有已知的钙和电压依赖的调节机制及其协同作用。门控机制将按照一个大的多状态多层模型来制定，该模型指定状态、状态之间的转变、转变的速率常数、由钙和电压调制的速率常数以及在门控中涉及的任何合作作用。第三个具体目标是：3)确定β1亚基调节BK通道门控的机制。这将通过识别由β1亚基修饰的动力学门控机制中的特定步骤来完成。了解离子通道是如何关闭其毛孔的，这一研究的目标将有助于理解、诊断和治疗与离子通道缺陷相关的疾病(通道病)。

DESCRIPTION (provided by applicant):Ion channels are proteins that control the passive flux of ions through cell membranes by opening and closing (gating) their pores. This proposal seeks to continue work on investigating the fundamental mechanisms by which ion channels gate. The current focus is the large conductance Ca 2+-and voltage activated K+ (BK) channel, which plays a key role in many physiological processes, including control of muscle contraction, regulation of synaptic transmission, and integration of information in neurons. Although much progress has been made towards understanding how Ca 2+ and voltage activate BK channels, and how accessory beta1subunits modulate this activity, many basic questions remain. To work towards answering these questions, cloned BK channels will be expressed in Xenopus oocytes and HEK293 cells, currents will be recorded from single channels with the patch clamp technique, and the data will be analyzed with advanced techniques to determine gating mechanism. The initial hypothesis to be tested is that multiple Ca 2+- and voltage-dependent regulatory mechanisms act jointly to control the opening-closing transitions (gating) of BK channels. To test this hypothesis, the first specific aim will: 1) determine the contributions, including any cooperative interactions, of each of the five proposed Ca 2+-- dependent regulatory mechanisms to the gating of BK channels. Cooperatively it will be resolved by studying the Ca2+-dependent regulatory mechanisms in isolation and in various combinations. The second specific aim will: 2) develop a comprehensive kinetic gating mechanism for BK channels that incorporates all of the known Ca 2+and voltage-dependent regulatory mechanisms and their cooperative interactions. The gating mechanism will be formulated in terms of a large multi-state multi-tiered model that specifies the states, the transitions among the states, the rate constants for the transitions, modulation of the rate constants by Ca 2+ and voltage, and any cooperative interactions involved in the gating. The third specific aim will: 3) determine the mechanism by which beta1subunits modulate the gating of BK channels. This will be done by identifying the particular steps in the kinetic gating mechanism that are modified by beta1subunits. Understanding how ion channels gate their pores, the goal of this research, will facilitate the comprehension, diagnosis, and treatment of diseases associated with defective ion channels (channelopathies).