Small G-protein Regulation of Calcium Channels

小 G 蛋白对钙通道的调节

• 批准号: 8695923
• 负责人: Henry M. Colecraft
• 金额: $ 30.4万
• 依托单位: COLUMBIA UNIVERSITY HEALTH SCIENCES
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-04-01 至 2018-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8695923
• 关键词: Address; Adult; Alzheimer' s Disease; Arrhythmia; Binding; Biochemical; Breathing; Calcium; Calcium Channel; Cardiac Myocytes; Cell membrane; Cells; Charge; Data; Disease; Dynamin; Electrophysiology (science); Endocytosis; Engineering; Family; Family member; Fluorescence Resonance Energy Transfer; Heart; Homeostasis; Hypertension; Individual; Light; Molecular; Molecular Biology; Monomeric GTP-Binding Proteins; Pain; Parkinson Disease; Physiological; Physiology; Probability; Protein Inhibition; Protein Isoforms; Proteins; Rattus; Reagent; Recombinants; Regulation; Relative (related person); Surface; Therapeutic; Therapeutic Index; Tissues; Work; channel blockers; density; design; inhibitor/antagonist; insight; nervous system disorder; novel; painful neuropathy; public health relevance; research study; sensor; small molecule; trafficking; voltage

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.

描述（由申请人提供）：高压激活的钙（CAV1/CAV2）通道对于可激发细胞的功能是必需的。抑制CAV1/CAV2通道的分子强大地调节生理，并且对于许多严重疾病，包括：高血压，神经性疼痛，心律不齐和帕金森氏病。 CAV1/CAV2通道被一个四成员的单体G-蛋白家族有效抑制，称为RGK（RAD，REM，REM，REM2，GEM/KIR）蛋白。 RGK在令人兴奋的组织中表达，其表达水平通常与疾病相关，这表明它们对CAV1/CAV2的强烈调节具有广泛的病理生理意义。工程的RGK具有潜在的治疗应用，作为遗传编码的CAV通道阻滞剂（CCB），可用于广泛的疾病。对于特定应用，遗传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通道块，同时最大程度地减少非目标效应。 RGK抑制CAV1/CAV2通道的精确分子机制尚不清楚。我们的初步数据暗示了一种令人惊讶的自定义和复杂性程度，其中不同的RGK蛋白差异化使用了多种机制和结构决定因素来抑制单个CAV1/CAV2通道同工型。对CAV1/CAV2通道的定制RGK抑制的基本机制的精确理解对于洞悉该通道调节的致病性分析以及为设计有用的新遗传编码CCB的努力而言至关重要。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，并将这些见解桥接到：（i）对此的病情生理学意义的新欣赏 通道调制； （ii）新型，有用的遗传编码CCB作为潜在疗法的设计。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，以解决三个特定目的：（1）RGK蛋白REM，REM，用于抑制重组的Cav1.2 Cav1.2 cav1.2频道。 （2）确定CAV1/CAV2通道家族中RGK抑制的对比度和对比机制。 （3）解剖心肌细胞中RGK抑制天然CAV1.2通道的机制。