Mechanisms of Ion Channel Activity

描述（由申请人提供）：离子通道是通过打开和关闭（门控）毛孔来控制离子通过细胞膜的被动通量的蛋白质。本提案旨在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ca2 +和电压激活的K+ (BK)通道，它在许多生理过程中起着关键作用，包括控制肌肉收缩、调节突触传递和神经元信息整合。尽管在了解ca2 +和电压如何激活BK通道以及辅助β 1亚基如何调节这种活动方面取得了很大进展，但仍存在许多基本问题。为了解决这些问题，我们将克隆的BK通道在爪蟾卵母细胞和HEK293细胞中表达，利用膜片钳技术记录单个通道的电流，并利用先进的技术分析数据以确定门控机制。最初要测试的假设是多种ca2 +和电压依赖的调节机制共同作用来控制BK通道的开闭转换（门控）。为了验证这一假设，第一个具体目标将是：1)确定五种Ca 2+依赖性调节机制对BK通道门控的贡献，包括任何合作相互作用。合作，它将通过研究Ca2+依赖的调节机制在孤立和各种组合来解决。第二个具体目标是：2)为BK通道开发一个综合的动力学门控机制，该机制包含所有已知的ca2 +和电压依赖性调节机制及其合作相互作用。门控机制将根据一个大的多态多层模型来制定，该模型指定了状态，状态之间的转换，转换的速率常数，ca2 +和电压调制的速率常数，以及门控中涉及的任何合作相互作用。第三个具体目标是：3)确定β 1亚基调节BK通道门控的机制。这将通过识别由β 1亚基修饰的动力学门控机制中的特定步骤来完成。本研究的目标是了解离子通道如何控制它们的孔隙，这将有助于理解、诊断和治疗与缺陷离子通道（通道病）相关的疾病。

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).