Allosteric Modulation of HCN Channels

项目摘要 超极化激活的环核苷酸敏感（HCN）通道是细胞凋亡的关键决定因素。 膜电位和兴奋性在许多类型的细胞在整个身体，包括心脏起搏器 细胞、中枢和外周神经元、多种类型的感觉细胞和结肠中的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 用于治疗一长串病症的靶点，包括心绞痛、心力衰竭、癫痫、神经性疼痛， 抑郁症、胃肠动力障碍和神经源性膀胱。然而，FDA批准的单一HCN 通道药物（伊伐布雷定）是有限的，部分原因是其非选择性阻断所有四种哺乳动物HCN 通道亚型及其对Kv11.1（hERG）、Nav1.5和Cav1.2通道的脱靶阻断。需要新的、 异构体特异性HCN通道激活剂和抑制剂已被广泛认可，但缺乏信息 关于HCN通道的变构和异构体特异性调节是开发新的 治疗学 该项目的长期目标是确定HCN通道的天然变构调节剂 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，并有助于新的HCN通道的发展 毒品目前的建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- HCN 4通道的特异性蛋白质相互作用伴侣。LRMP和IRAG是同源的ER跨膜蛋白 具有大的胞质结构域的蛋白质。重要的是，LRMP和IRAG仅调节HCN 4同种型。 此外，这两种蛋白质对HCN 4具有相反的作用：LRMP通过以下方式导致功能丧失（LOF） 降低cAMP诱导的电压依赖性的典型去极化位移，而IRAG引起增益， 通过将HCN 4的基础电压依赖性转移到更正的电位来实现功能（GOF）。初步 数据证实IRAG与HCN 4在心脏起搏细胞中共表达。拟议目标将确定 三种蛋白质上的相互作用位点，决定了不同亚型的分子机制- 特异性作用，并评估其在起搏细胞中的作用。

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.