Small G-protein Regulation of Calcium Channels

描述（由申请人提供）：高电压激活的钙（CaV 1/CaV 2）通道对于可兴奋细胞的功能是必需的。抑制CaV 1/CaV 2通道的分子有力地调节生理学，并且是许多严重疾病的重要或潜在的治疗剂，包括：高血压、神经性疼痛、心律失常和帕金森病。CaV 1/CaV 2通道被称为RGK（Rad、Rem、Rem 2、Gem/Kir）蛋白的单体G蛋白的四成员家族强力抑制。RGKs在可兴奋组织中表达，其表达水平经常与疾病相关地变化，表明其对CaV 1/CaV 2的强烈调节具有广泛的病理生理意义。工程RGKs作为基因编码的CaV通道阻滞剂（CCB）具有广泛的疾病的潜在治疗应用。对于特定应用，遗传编码的抑制剂可以提供比传统小分子CCB更高的治疗指数，因为它们可以局部表达，从而实现有效的CaV通道阻断，同时使脱靶效应最小化。RGKs抑制CaV 1/CaV 2通道的确切分子机制尚未完全了解。我们的初步数据暗示了令人惊讶的定制程度和复杂性，其中不同的RGK蛋白差异性地使用多种机制和结构决定因素来抑制单个CaV 1/CaV 2通道亚型。精确理解定制RGK抑制CaV 1/CaV 2通道的机制对于深入了解这种通道调节的病理生理学后果以及努力设计有用的新遗传编码的CCBs至关重要。我们的长期目标是提供对RGK抑制CaV 1/CaV 2通道的不同分子机制和结构决定因素的基本理解，并将这些见解与以下方面联系起来：（i）对这一过程的病理生理意义的新认识。 通道调节;和（ii）设计新的，有用的遗传编码的CCBs作为潜在的治疗剂。我们结合联合收割机全细胞和单通道电生理学、荧光共振能量转移（FRET）、分子生物学、通道工程和生物化学方法来解决三个特定的目标：（1）剖析RGK蛋白Rem用于抑制重组CaV 1.2通道的机制。(2)确定和对比RGK抑制CaV 1/CaV 2通道家族的机制。(3)分析RGK抑制心肌细胞中天然CaV1.2通道的机制。

DESCRIPTION (provided by applicant): High-voltage-activated calcium (CaV1/CaV2) channels are necessary for the function of excitable cells. Molecules that inhibit CaV1/CaV2 channels powerfully regulate physiology, and are important or potential therapeutics for many serious diseases including: hypertension, neuropathic pain, cardiac arrhythmias, and Parkinson's disease. CaV1/CaV2 channels are potently inhibited by a four-member family of monomeric G- proteins known as RGK (Rad, Rem, Rem2, Gem/Kir) proteins. RGKs are expressed in excitable tissues, and their expression level often changes correlatively with disease, suggesting their strong regulation of CaV1/CaV2 has broad patho-physiological implications. Engineered RGKs have potential therapeutic applications as genetically-encoded CaV channel blockers (CCBs) for a broad range of diseases. For specific applications, genetically encoded inhibitors may provide a higher therapeutic index than traditional small molecule CCBs because they can be locally expressed, thereby achieving effective CaV channel block while minimizing off- target effects. The precise molecular mechanisms by which RGKs inhibit CaV1/CaV2 channels are not well- understood. Our preliminary data hint at a surprising degree of customization and complexity where distinct RGK proteins differentially use multiple mechanisms and structural determinants to inhibit individual CaV1/CaV2 channel isoforms. Precise understanding of the mechanisms underlying customized RGK inhibition of CaV1/CaV2 channels is critical for insights into the patho-physiological ramifications of this channel regulation, as well as efforts to engineer useful new genetically-encoded CCBs. Our long-term objective is to furnish fundamental understanding of the diverse molecular mechanisms and structural determinants underlying RGK inhibition of CaV1/CaV2 channels and to bridge these insights to: (i) a new appreciation of the patho-physiological implications of this channel modulation; and (ii) the design of novel, useful genetically-encoded CCBs as potential therapeutics. We combine whole-cell and single-channel electrophysiology, fluorescence resonance energy transfer (FRET), molecular biology, channel engineering, and biochemical approaches to address three specific Aims: (1) Dissect mechanisms the RGK protein, Rem, uses to inhibit recombinant CaV1.2 channels. (2) Determine and contrast mechanisms of RGK inhibition across the CaV1/CaV2 channel family. (3) Dissect mechanisms of RGK inhibition of native CaV1.2 channels in cardiomyocytes.