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）其孔来控制离子通过细胞膜的被动通量的蛋白质。这项建议旨在继续研究离子通道门控的基本机制。大电导钙离子和电压激活的钾离子通道（BK）在肌肉收缩、突触传递和神经元信息整合等生理过程中起着重要作用，是目前研究的热点。虽然在理解Ca 2+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性方面取得了很大进展，但许多基本问题仍然存在。为了回答这些问题，克隆的BK通道将在非洲爪蟾卵母细胞和HEK 293细胞中表达，电流将用膜片钳技术从单通道记录，数据将用先进技术分析以确定门控机制。最初的假设是要测试的是，多个Ca 2+和电压依赖性调节机制共同作用，以控制BK通道的开放-关闭转换（门控）。为了验证这一假设，第一个具体目标是：1）确定所提出的5种Ca 2+依赖性调节机制对BK通道门控的贡献，包括任何合作的相互作用。合作，它将通过研究钙离子依赖的监管机制，在隔离和各种组合解决。第二个具体目标是：（2）建立一个完整的BK通道动力学门控机制，包括所有已知的Ca 2+和电压依赖性调节机制及其协同作用。门控机制将制定一个大型的多状态多层模型，指定的状态，状态之间的转换，转换的速率常数，调制的速率常数的Ca 2+和电压，以及任何合作的相互作用中涉及的门控。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。这将通过确定β 1亚基修饰的动力学门控机制中的特定步骤来完成。了解离子通道如何门控它们的孔，这项研究的目标，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).