Allosteric Modulation of HCN Channels

项目总结 超极化激活的环核苷酸敏感(HCN)通道是 全身多种细胞的膜电位和兴奋性，包括心脏起搏器 细胞、中枢和外周神经元、多种感觉细胞和Cajal间质细胞。 膀胱。与这种广泛的分布一致，hcn通道已被确定为潜在的药物。 治疗一系列疾病的目标，包括心绞痛、心力衰竭、癫痫、神经性疼痛、 抑郁、胃肠动力障碍和神经性膀胱。然而，FDA批准的单一HCN 通道药物(伊夫拉定)是有限的，部分原因是它对所有四种哺乳动物的HCN都没有选择性阻断 Kv11.1(HERG)、NaV1.5和Cav1.2通道的通道异构体及其脱靶阻断。对新的、 异构体特异性HCN通道激活剂和抑制剂已被广泛认识，但缺乏信息 关于HCN通道的变构和异构体特异性调节是新药开发的障碍 治疗学。 该项目的长期目标是确定HCN通道的自然发生的变构调节因子 并了解它们的作用机制。实现这些目标将促进对 HCN通道的生理和分子功能及其对新的HCN通道的作用 毒品。目前的提议集中在我们令人兴奋的发现LRMP和IRAG作为两个新的，异构体- HCN4通道的特异性蛋白质相互作用伙伴。LRMP和IRAG是同源的内质网跨膜 具有大的细胞质结构域的蛋白质。重要的是，LRMP和IRAG只调节HCN4亚型。 此外，这两种蛋白质对HCN4有相反的作用：LRMP通过以下途径导致功能丧失(LOF) 减少cAMP引起的电压依赖的典型去极化漂移，而IRAG引起增益- 通过将HCN4的基础电压依赖转移到更正的电位来实现功能(GOF)。初步 数据表明，在心脏起搏细胞中，IRAG与HCN4共表达。建议的目标将确定 这三种蛋白质上的相互作用部位，决定了不同和异构体的分子机制。 并评估它们在起搏器细胞中的作用。

PROJECT SUMMARY Hyperpolarization-activated, cyclic nucleotide-sensitive (HCN) channels are critical determinants of membrane potential and excitability in many types of cells throughout the body, including cardiac pacemaker cells, central and peripheral neurons, many types of sensory cells, and interstitial cells of Cajal in the colon and bladder. Consistent with this widespread distribution, HCN channels have been identified as potential drug targets for treatment of a long list of conditions including angina, heart failure, epilepsy, neuropathic pain, depression, gastrointestinal dysmotility, and neurogenic bladder. However, the single FDA-approved HCN channel drug (ivabradine) is limited, owing in part to its non-selective block of all four mammalian HCN channels isoforms and its off-target block of Kv11.1 (hERG), Nav1.5, and Cav1.2 channels. The need for new, isoform-specific HCN channel activators and inhibitors has been widely recognized but the lack of information about allosteric and isoform-specific regulation of HCN channels is a roadblock to the development of novel therapeutics. The long-term goals of this project are to identify naturally-occurring, allosteric regulators of HCN channels and to understand their mechanisms of action. Achieving these goals will advance understanding of the physiological and molecular functions of HCN channels and aid in the development of new HCN channel drugs. The current proposal focuses on our exciting discovery of LRMP and IRAG as two novel, isoform- specific protein interaction partners of HCN4 channels. LRMP and IRAG are homologous ER transmembrane proteins that have large cytoplasmic domains. Importantly, LRMP and IRAG only modulate the HCN4 isoform. Moreover, the two proteins have opposing effects on HCN4: LRMP causes a loss-of-function (LOF) by decreasing the canonical depolarizing shift in voltage-dependence induced by cAMP while IRAG causes gain- of-function (GOF) by shifting the basal voltage dependence of HCN4 to more positive potentials. Preliminary data establish that IRAG is co-expressed with HCN4 in cardiac pacemaker cells. Proposed aims will identify interaction sites on the three proteins, determine the molecular mechanisms for the distinct and isoform- specific effects, and evaluate their role in pacemaker cells.