Function and Regulation of HCN Channels in Sinoatrial Myocytes

描述(申请人提供)：心脏的每一次跳动开始于窦房结中特殊起搏器细胞的自发电去极化。交感神经系统主要通过从支配窦房结的神经释放去甲肾上腺素来增加心率。在窦房肌细胞内，交感战斗或逃跑反应的这一方面需要对去甲肾上腺素作出反应的两个肾上腺素能受体(2AR)和共同控制动作电位的时间和形状的离子通道之间的通信。该项目的长期目标是了解在窦房肌细胞中产生和调节起搏器活动的分子机制。本提案中概述的实验主要集中在2AR和超极化激活的环核苷酸敏感(HCN，或起搏器)离子通道之间信号传递的某些方面。HCN通道由2AR激活，被认为对设定静息心率和调节两种激动剂的正性变时性作用都是至关重要的。然而，HCN通道参与起搏的生物物理机制，以及HCN通道与2种肾上腺素能受体之间的功能和物理关系尚不清楚。本项目将检验的工作假设是，HCN通道产生的泄漏电流对窦房肌细胞的起搏活动至关重要，而对起搏的交感调节需要包含2AR和HCN通道的大分子信号复合体。这些问题将通过表达的HCN通道、急性分离的小鼠窦房肌细胞和培养的窦房肌细胞来解决。可采用的主要技术有膜片钳电生理学、共聚焦免疫荧光显微镜和免疫亲和层析。具体目的有三：(1)了解舒张期HCN通道活动的生物物理机制；(2)描述2ARs和HCN通道之间控制窦房肌细胞放电频率的功能关系；(3)确定参与交感神经起搏控制的蛋白质的亚细胞定位和物理相互作用。

DESCRIPTION (provided by applicant): Each beat of the heart begins as a spontaneous electrical depolarization of specialized pacemaker cells in the sinoatrial node. The sympathetic nervous system increases heart rate primarily by releasing norepinephrine from nerves that innervate the sinoatrial node. Within the sinoatrial myocytes, this aspect of the sympathetic fight-or-flight response requires communication between the 2 adrenergic receptors (2ARs) that respond to the norepinephrine and the ion channels that collectively control the timing and shape of action potentials. The long-term goal of this project is to understand the molecular machinery that produces and regulates pacemaker activity in sinoatrial myocytes. Experiments outlined in this proposal focus on some aspects of signaling between 2ARs and hyperpolarization-activated, cyclic nucleotide sensitive (HCN, or pacemaker) ion channels. HCN channels are activated by 2ARs and are thought to be critical both for setting the resting heart rate and for mediating the positive chronotropic effect of 2 agonists. However, the biophysical mechanisms for HCN channel involvement in pacemaking, and the functional and physical relationships between HCN channels and 2 adrenergic receptors are poorly understood. The working hypotheses to be tested in this project are that a leak current produced by HCN channels is critical for pacemaker activity in sinoatrial myocytes, and that sympathetic regulation of pacemaking requires a macromolecular signaling complex that contains 2ARs and HCN channels. These questions will be addressed using expressed HCN channels, acutely isolated murine sinoatrial myocytes and cultured sinoatrial myocytes. The principle techniques to be employed are patch clamp electrophysiology, confocal immunofluorescent microscopy, and immunoaffinity chromatography. There are three specific aims: (1) To understand the biophysical mechanisms for HCN channel activity during diastole, (2) To describe the functional relationships between 2ARs and HCN channels that control firing rate in sinoatrial myocytes, and (3) To determine the subcellular localization and physical interactions of proteins that participate in sympathetic control of pacemaking.