Structure and function of potassium channels

描述(申请人提供)：钾通道(K通道)是电信号的关键组成部分，是神经和肌肉功能所必需的基本生物过程。通道本身是门控小孔；当小孔打开时(通过电压或信号分子)，K离子流过小孔，产生一种离子电流，使神经或肌肉细胞电“松弛”。电压和钙激活的“MAXI-K”通道是理解K通道调制和门控的原型。在生理学方面，Maxi-K通道在血管平滑肌松弛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。人们希望，对MAXI-K通道结构和功能的更好理解将最终导致神经和心血管疾病的治疗进展。 与其他电压依赖性K通道一样，MAXI-K通道在门控时会经历一系列构象变化。在这项提案中，我们的目标是利用膜片钳记录和时间分辨荧光光谱学的组合来测试和扩展最近关于这些门控运动的假说。我们的膜片钳和光谱实验将在完整的活细胞中的通道上进行，因此可以回答无法用结晶学解决的问题。通过检测通道中连接的荧光团和内源性侧链之间的特定荧光猝灭相互作用，我们可以估计分子间的距离，从而了解通道的结构。我们的具体目标是：1)通过使用独立但互补的电生理学和荧光光谱方法估计侧链之间的距离来确定Maxi-K通道的二级结构特征；2)通过测量在碘化物(一种水溶液猝灭剂)存在下荧光标记通道的激发态寿命来确定通道上特定氨基酸位置的溶剂可及性；以及3)通过对膜片钳全细胞中开放和关闭的通道进行荧光光谱来定位感受门控运动的氨基酸位置。这些实验的结合将有助于我们在K通道门控方面的知识的显著进步。

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.