FLUORESCENCE STUDIES OF THE SHAKER POTASSIUM CHANNEL

Shaker是电压依赖性钾离子通道的一个亚家族模型，它催化钾离子在膜去极化时的跨膜流动。 KV通道参与了包括心脏细胞复极化和神经元频率编码在内的无数生理过程。 虽然这种膜蛋白已被广泛的生物物理特性，有关通道的激活机制的细节是很少的。 为了阐明跨膜电场影响电压传感器运动的方式，将利用光学和电生理技术的组合。 定点诱变和巯基特异性荧光团的使用允许通过荧光技术测定特定的结构修饰，并与门控电荷置换相关。因此，本研究建议将集中在两个方面的振荡器通道电压传感器动力学：i）。使用基于镧系元素的荧光共振能量转移（LRET）确定S4残基的平衡位置，和ii.）使用位点特异性荧光表征局部构象变化的动力学从本研究中获得的见解将增加离子通道生物物理学的当代观点，并且除了许多通道病的病理生理学之外，还可能导致对正常生理学的更好理解。

Shaker, a model for the subfamily of voltage-dependent potassium (Kv) channels, catalyzes the transmembranous flow of potassium ions upon membrane depolarization. Kv channels are involved in a myriad of physiological processes including cardiac cell repolarization and frequency-encoding in neurons. Although this integral membrane protein has been subject to extensive biophysical characterization, details concerning the channel's activation mechanism are scant. In order to elucidate the manner in which the transmembrane electric field affects voltage sensor movement, a combination of optical and electrophysiological techniques will be utilized. The use of site-directed mutagenesis and thiol-specific fluorophores, allows specific structural modifications to be assayed via fluorescence techniques and correlated to gating charge displacement. Therefore, this research proposal will focus on two aspects of Shaker channel voltage-sensor dynamics: i.) determining equilibrium positions of S4 residues with the use of Lanthanide- Based Fluorescence Resonance Energy Transfer (LRET), and ii.) characterizing the kinetics of local conformational changes with the use of site-specific fluorescence The insight gained from this study will augment the contemporary view of ion channel biophysics and may result in a better understanding of normal physiology, in addition to the pathophysiology of numerous channelopathies.