Small G-protein Regulation of Calcium Channels

说明(申请人提供)：高电压激活的钙通道(CaV1/CaV2)是兴奋性细胞功能所必需的。抑制CaV1/CaV2通道的分子对生理有很强的调节作用，对高血压、神经性疼痛、心律失常和帕金森病等多种严重疾病具有重要或潜在的治疗作用。CaV1/CaV2通道被称为RGK(Rad，Rem，Rem2，Gem/Kir)蛋白的四成员单体G蛋白家族有效地抑制。RGKs在兴奋性组织中表达，其表达水平随疾病的变化而变化，提示其对CaV1/CaV2的强烈调控具有广泛的病理生理意义。工程RGK作为基因编码的CAV通道阻滞剂(CCB)具有潜在的治疗应用前景，可治疗多种疾病。对于特定的应用，基因编码的抑制剂可能比传统的小分子CCB提供更高的治疗指数，因为它们可以在局部表达，从而实现有效的CAV通道阻断，同时将非靶点效应降至最低。RGKs抑制CaV1/CaV2通道的确切分子机制尚不清楚。我们的初步数据提示，定制和复杂性的程度令人惊讶，其中不同的RGK蛋白不同地使用多种机制和结构决定因素来抑制单个CaV1/CaV2通道亚型。准确了解定制的RGK抑制CaV1/CaV2通道的机制对于深入了解这一通道调节的病理生理分支以及努力设计有用的新的遗传编码CCB至关重要。我们的长期目标是提供对RGK抑制CaV1/CaV2通道的不同分子机制和结构决定因素的基本理解，并将这些见解连接到：(I)对这一过程的病理生理意义的新认识 频道调制；以及(Ii)设计新的、有用的遗传编码的CCB作为潜在的疗法。我们结合了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、通道工程和生化方法来解决三个特定目标：(1)剖析RGK蛋白Rem抑制重组CaV1.2通道的机制。(2)确定和对比跨CaV1/CaV2通道家族的RGK抑制机制。(3)分析RGK抑制心肌细胞天然CaV1.2通道的作用机制。

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.