Structure and function of potassium channels

描述（由申请人提供）：钾通道（K通道）是电信号的关键组成部分，是神经和肌肉功能必不可少的基本生物过程。通道本身为门控孔；当孔洞打开时（通过电压或信号分子），钾离子流过孔洞，产生一种离子电流，通过电“放松”神经或肌肉细胞。电压和钙激活的“最大K”通道已作为理解K通道调制和门控的原型。在生理上，大k通道在血管平滑肌舒张调节血压方面尤为重要。尽管我们知道这些通道的功能，但对它们的结构或功能的分子基础知之甚少。希望更好地了解max - k通道的结构和功能将最终导致神经和心血管疾病的治疗取得进展。

DESCRIPTION (provided by applicant): Potassium channels (K channels) are critical components of electrical signaling, a basic biological process that is essential to the function of nerve and muscle. The channels themselves are gated pores; K ions flow through the pore when it is opened (by voltage or signaling molecules), producing an ionic current that electrically "relaxes" the nerve or muscle cell. The voltage- and calcium-activated "maxi-K" channel has served as a prototype for understanding K channel modulation and gating. In terms of physiology, maxi-K channels are especially important in the relaxation of vascular smooth muscle to regulate blood pressure. Despite our knowledge of the function of these channels, little is known of their structure or of the molecular basis of their function. It is hoped that a better understanding of maxi-K channel structure and function will ultimately lead to advances in the treatment of neurological and cardiovascular disease. The maxi-K channel, like other voltage-dependent K channels, undergoes a series of conformational changes when it gates. In this proposal, we aim to test and expand upon recent hypotheses of these gating movements, using a combination of patch-clamp recording and time-resolved fluorescence spectroscopy. Our patch-clamp and spectroscopy experiments will be performed on channels in intact, living cells, and can thus answer questions that cannot be addressed with crystallography. By detecting specific fluorescence quenching interactions between attached fluorophores and endogenous sidechains in the channel, we can obtain estimates of intermolecular distances, and thus gain insight toward channel structure. Our specific aims are: 1) to determine secondary structural features of the maxi-K channel by estimating distances between sidechains, using independent but complementary approaches of electrophysiology and fluorescence spectroscopy; 2) to determine the solvent accessibility of specific amino acid positions on the channel, by measuring excited-state lifetimes of fluorescently-labeled channels in the presence of iodide (an aqueous quenching agent); and 3) to locate amino acid positions that sense gating movements, by performing fluorescence spectroscopy on open and closed channels in patch-clamped whole cells. This combination of experiments will contribute to significant advances in our knowledge of K channel gating.