Function and Regulation of HCN Channels in Sinoatrial Myocytes

 描述(由申请人提供)：控制心率是一种治疗目标，已被证明对治疗各种疾病有好处。通过手术植入电子起搏器加速心率是治疗年龄相关性窦房结功能障碍(SND)的唯一有效方法，而通过超极化激活的环核苷酸敏感(HCN)通道的通用阻滞剂减慢心率正在成为心绞痛和心力衰竭的重要治疗方法。虽然这些治疗方法是有效的，但它们也有重要的局限性。因此，通过表达HCN通道来开发生物起搏器和开发阻断窦房型HCN通道的降心率药物已经投入了大量的精力。然而，由于缺乏关于窦房结特异性HCN4通道亚型的详细机制信息，这些目标的进展受到限制。如果在窦房结心肌细胞中，HCN4的突变与SND相关，并且在SAM中调节IF已经被证明是一种控制心率的手段，那么HCN4就会产生有趣的电流。上一次供资期间的结果和新的初步数据证实，存在两种新的、特定于HCN4的异构体激活剂。这项提案的中心目标是1)确定这些新型调节剂控制HCN4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和控制SAM中的起搏器活动。将使用一系列技术来实现这些目标，这些技术利用研究小组的互补专业知识。将使用蛋白质亲和层析、荧光共振能量转移、X射线结晶学和比较蛋白质组学来分析蛋白质相互作用和HCN4通道的翻译后修饰。新调控模式背后的生物物理机制将通过异源表达的野生型和突变型HCN4通道的膜片钳电生理学来定义。模拟和抑制新调节器的试剂正在开发中，并将评估它们对急性分离的小鼠SAM膜片钳记录中IF和起搏器活性的影响。如果成功，这些实验将提供关于HCN4异构体特异性调控的新颖和详细的机制信息，并将为小分子通过这些机制改变起搏器活动的能力提供概念验证数据。这些研究的结果可以指导控制心率的新治疗范例的开发。

 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.