Mechanisms of Ion Channel Activity

描述（由申请人提供）：本研究的长期目标是了解离子通道控制其孔隙的机制。本提案的研究重点是大电导Ca2+和电压激活（BK）通道，它在许多生理功能中起关键作用，包括控制肌肉收缩、调节神经元兴奋性和控制递质释放。BK通道是四聚体，具有一个电压传感器，两个高亲和Ca2+传感器，和一个低亲和Ca2+传感器在每四个亚基。尽管在了解这些不同传感器在激活通道中的作用方面已经取得了很大的进展，但在单通道水平上描述门控的综合动力学机制尚不可用。为了开发这一机制，通道将在HEK 293细胞和爪蟾卵母细胞中表达，并使用膜片钳技术记录切除膜斑块中单个BK通道的电流。然后，通过同时拟合在宽Ca2+和电压范围内获得的相邻打开和关闭间隔持续时间的二维驻留时间分布来分析单通道数据，以确定潜在的门控机制。目的1将分离和表征三种类型的Ca2+传感器对门控的贡献。这将在电压传感器存在的情况下完成，以表征各种传感器之间可能的相互作用。待测试的假设是，BK通道的门控被修改为每个亚基具有一种Ca2+传感器和一个电压传感器，将通过两层50状态变构门控机制来描述。Aim 2将使用Aim 1中获得的信息以及额外的实验信息来开发野生型BK通道的综合动力学门控机制，其中每个亚基有一个电压传感器和三个不同的Ca2+传感器。要测试的假设是，Ca2+和电压依赖性门控的BK通道是一致的大双层，递归，1250状态变构门控机制。待开发的动力学门控机制将指定通道在门控过程中进入的状态数，状态之间的转换途径，转换的速率常数，速率常数的电压和Ca2+依赖性，每个激活传感器的打开和关闭速率的变弹性变化，以及各种传感器之间的相互作用。该模型描述门控的能力将在单通道电流和已公布的宏观离子和门控电流中进行测试。BK通道缺陷与高血压、膀胱疾病、癫痫、阵发性运动障碍、自闭症和智力低下有关。获得有关门控机制的信息应该有助于识别和理解与BK通道相关的疾病过程，并设计治疗干预措施以恢复被破坏的生理功能。公共卫生相关性：大电导钙和电压活化钾（BK）通道参与许多关键的生理过程，包括控制骨骼肌和平滑肌收缩，调节神经细胞的兴奋性，调节激素和递质释放。BK通道缺陷或缺失与高血压、膀胱疾病、癫痫、发作性运动障碍、自闭症和智力低下有关。拟议的研究将深入了解BK通道的功能，这将有助于理解与BK通道相关的疾病过程，并有助于开发治疗干预措施以恢复被破坏的生理功能。

DESCRIPTION (provided by applicant): The long term objectives of this research are to understand the mechanisms by which ion channels gate their pores. Research in this proposal focuses on the large conductance Ca2+ and voltage-activated (BK) channel, which plays a key role in many physiological functions, including control of muscle contraction, regulation of neuronal excitability, and control of transmitter release. BK channels are tetramers, with one voltage sensor, two high affinity Ca2+ sensors, and one low affinity Ca2+ sensor on each of the four subunits. Although much progress has been made towards understanding the contributions of these different sensors in activating the channel, a comprehensive kinetic mechanism to describe gating at the single-channel level is not yet available. To develop this mechanism, channels will be expressed in HEK 293 cells and Xenopus oocytes, and currents will be recorded from single BK channels in excised patches of membrane using the patch clamp technique. The single-channel data will then be analyzed by simultaneously fitting two-dimensional dwell-time distributions of adjacent open and closed interval durations obtained over wide ranges of Ca2+ and voltage to determine the underlying gating mechanism. Aim 1 will isolate and characterize the contribution of each of the three types of Ca2+ sensors to the gating. This will be done in the presence of the voltage sensors to characterize possible interactions among the various sensors. The hypothesis to be tested is that the gating of BK channels modified to have one type of Ca2+ sensor and one voltage sensor per subunit will be described by two-tiered 50 state allosteric gating mechanisms. Aim 2 will use the information obtained in Aim 1 together with additional experimental information to develop a comprehensive kinetic gating mechanism for wild type BK channels with their full complement of one voltage sensor and three different Ca2+ sensors per subunit. The hypothesis to be tested is that the Ca2+ and voltage dependent gating of BK channels is consistent with large two-tiered, recursive, 1250 state allosteric gating mechanisms. The kinetic gating mechanisms to be developed will specify the number of states the channel enters during gating, the transition pathways among the states, the rate constants for the transitions, the voltage and Ca2+ dependence of the rate constants, the allosteric changes in the opening and closings rates for each activated sensor, and the interactions among the various sensors. The ability of the model to describe gating will be tested for single-channel currents and also for published macroscopic ionic and gating currents. Defective BK channels are associated with hypertension, bladder disorder, epilepsy, paroxysmal movement disorder, autism, and mental retardation. The information to be obtained about gating mechanism should be useful towards identifying and understanding disease processes associated with BK channels and designing therapeutic interventions to restore disrupted physiological function. PUBLIC HEALTH RELEVANCE: Large conductance calcium and voltage activated potassium (BK) channels are involved in many key physiological processes including controlling skeletal and smooth muscle contraction, regulating the excitability of nerve cells, and modulating hormone and transmitter release. Defective or missing BK channels have been implicated in hypertension, bladder disorders, epilepsy, paroxysmal movement disorder, autism, and mental retardation. The proposed studies will provide insight into how BK channels function, which will be useful towards understanding disease processes associated with BK channels and in the development of therapeutic interventions to restore disrupted physiological function.