Function and Regulation of HCN Channels in Sinoatrial Myocytes

 描述（由申请人提供）：控制心率是一个治疗目标，已证明对治疗各种疾病具有益处。通过手术植入电子起搏器的心率加速是年龄相关性窦房结功能障碍（SND）的唯一有效治疗方法，而通过超极化激活的环核苷酸敏感（HCN）通道的一般阻断剂降低心率正在成为心绞痛和心力衰竭的重要治疗方法。虽然这些治疗是有效的，但它们也有重要的局限性。因此，已经投入了大量的努力来开发通过表达HCN通道的生物起搏器和阻断窦房HCN通道的降低心率的药物。然而，由于缺乏关于窦房结特异性HCN 4通道亚型的详细机制信息，这些目标的进展受到限制。如果在窦房结肌细胞（SAM）中，HCN 4的突变与SND相关，则HCN 4产生有趣电流，并且SAM中If的调节已经被证明是控制心率的手段。上一个资助期的结果和新的初步数据确定了存在两种新型的HCN 4亚型特异性激活剂。该建议的中心目标是1）定义这些新调节剂控制HCN 4通道的分子和生物物理机制，以及2）确定这些调节剂如何影响并可用于控制SAM中的起搏器活性。这些目标将使用一系列技术来实现，这些技术将利用研究团队的互补专业知识。将使用蛋白亲和色谱、荧光共振能量转移、X射线晶体学和比较蛋白质组学分析来分析HCN 4通道的蛋白质相互作用和翻译后修饰。生物物理机制的新的监管模式将被定义为异源表达的野生型和突变型HCN 4通道的膜片钳电生理学。正在开发模拟和抑制新调节剂的试剂，并将评估其对急性分离小鼠SAM膜片钳记录中If和起搏器活性的影响。如果成功，这些实验将提供有关HCN 4亚型特异性调节的新颖和详细的机制信息，并将提供小分子通过这些机制改变起搏器活性的能力的概念验证数据。这些研究的结果可以指导控制心率的新治疗模式的开发。

 DESCRIPTION (provided by applicant): Control of heart rate is a therapeutic goal with proven benefit for treatment of a wide range of conditions. Heart rate acceleration via surgically implanted electronic pacemakers is the only effective treatment for age-related sinoatrial node dysfunction (SND), whereas heart rate reduction via a general blocker of hyperpolarization- activated, cyclic nucleotide-sensitive (HCN) channels is emerging as an important treatment for angina and heart failure. While these treatments are effective, they also have important limitations. Hence, substantial effort has been invested towards development of biological pacemakers via expression of HCN channels and towards heart rate lowering drugs that block sinoatrial HCN channels. However, progress towards these goals is limited by a lack of detailed mechanistic information about the sinoatrial node-specific HCN4 channel isoform. HCN4 produces the funny current, if, in sinoatrial node myocytes (SAMs), mutations in HCN4 are associated with SND, and modulation of If in SAMs is already a proven means to control heart rate. Results from the previous funding period and new preliminary data establish the existence of two novel, isoform- specific activators of HCN4. The central goals of this proposal are 1) to define the molecular and biophysical mechanisms by which these novel regulators control HCN4 channels, and 2) to determine how these regulators impact, and can be used to control, pacemaker activity in SAMs. These goals will be pursued using an array of techniques that leverage the complementary expertise of the research team. Protein interactions and post-translational modification of HCN4 channels will be assayed using protein affinity chromatography, fluorescence resonance energy transfer, X-ray crystallography, and comparative proteomic profiling. Biophysical mechanisms underlying the new regulatory modes will be defined by patch clamp electrophysiology of heterologous-expressed wild type and mutant HCN4 channels. Reagents that mimic and inhibit the new regulators are being developed and will be assessed for their effects on If and pacemaker activity in patch clamp recordings from acutely-isolated mouse SAMs. If successful, these experiments will provide novel and detailed mechanistic information about isoform-specific regulation of HCN4 and will deliver proof-of-concept data for the ability of small molecules to alter pacemaker activity via these mechanisms. Results of these studies could guide development of new treatment paradigms for control of heart rate.