Ca2+dependent K+Channels: Allosteric Gating

描述(申请人提供)：本研究的长期目标 了解电压、钙离子和镁离子的分子机制 大电导K+通道(BK通道)的依赖激活。BK 经络具有道路生理功能，包括调制 神经递质释放与血管管径控制。作为一名 这些生理功能的结果是BK通道具有重要意义 临床重要性。例如，BK通道的异常活动已被 在动物模型中与高血压有关；他们活动增加可能 降低缺血再灌注性心律失常的发生率。在……里面 BK通道电压的激活引起电压传感器的运动 在通道中，Ca~(2+)或Mg~(2+)与通道结合导致构象 通道蛋白的变化打开了激活门。现在，它的结构 K+通道孔已被解决；潜在的He激活的蛋白质序列 栅极、电压传感器和钙离子结合部位已经确定。 然而，电压传感器运动、钙离子或镁离子结合的方式 耦合到激活门的打开仍然未知。直到 阐明了这些耦合的结构和能量基础，如何电压， 在不同的BK通道中调制Ca2+和Mg2+敏感度以辅助 它们的生理功能无法被理解。基于之前的研究， 我们假设通道蛋白的一个结构域，即 物理上接近激活门(RCK结构域用于调节 K+通道的电导)是这些耦合的中心。最近，X光片 解决了RCK结构域的晶体结构问题。在结构的指导下 数据我们将使用分子生物学和 确定其对钙离子、镁离子或电压对能量贡献的影响 使用我们最近开发的电生理方法打开通道。 我们还将使用蛋白质生物化学和核磁学的方法 利用核磁共振技术定位特定的分子内蛋白质 在通道激活期间不能改变的相互作用，从而控制 通道功能。这些实验将为理解 不同的BK通道如何在生理过程中发挥作用并定义 BK通道上的靶点用于治疗目的。他们还将为 我们对离子通道门控的一般理解。

DESCRIPTION (provided by applicant): The long-term objective of this research is to understand the molecular mechanism of the voltage, Ca2+, and Mg2+ dependent activation of large-conductance K+ channels (BK channels). BK channels have road physiological functions, including the modulation of neurotransmitter release and the control of blood vessel diameters. As a consequence of these physiological functions BK channels are of significant clinical importance. For example, abnormal activity of BK channels has been associated with hypertension in animal models; their increased activity may reduce the incidence of ischemia- reperfusion-induced cardiac arrhythmia. In the activation of BK channels voltage induces movements of the voltage sensor in the channel, Ca2+ or Mg2+ binds to the channel to cause conformational changes in the channel protein to open he activation gate. Now the structure of the K+ channel pore has been solved; protein sequences underlying he activation gate, the voltage sensor, and the Ca2+ binding site have been identified. However, the manner n which voltage sensor movements, Ca2+ or Mg2+ binding are coupled to the opening of the activation gate remains unknown. Until the structural and energetic basis of these couplings is elucidated, how voltage, Ca2+ and Mg2+ sensitivities are modulated in various BK channels to subserve their physiological functions cannot be understood. Based on previous studies, we hypothesize that a structural domain of the channel protein that is physically close to the activation gate (the RCK domain for Regulating the conductance of K+ channels) is central in these couplings. Recently, the X-ray crystal structure of the RCK domain has been solved. Guided by the structural data we will perturb the channel structure using molecular biology and determine its impact on the energetic contribution of Ca2+, Mg2+, or voltage to channel opening using our recently developed electrophysiological approaches. We will also use approaches of protein biochemistry and nuclear magnetic resonance spectroscopy (NMR) to map specific intramolecular protein interactions that nay be altered during channel activation and hence control channel function. These experiments will provide a foundation for understanding how various BK channels play their role in physiological processes and define targets on BK channels for therapeutic purposes. They will also contribute to our understanding of ion channel gating in general.