Regulation of Calcium Channel Function by the Rem GTPase

Rem GTPase 对钙通道功能的调节

• 批准号: 6736938
• 负责人: Douglas Allen Andres
• 金额: $ 36.81万
• 依托单位: UNIVERSITY OF KENTUCKY
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-04-15 至 2007-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6736938
• 关键词: calcium channel; cardiac myocytes; cell membrane; computer simulation; genetic regulation; guanine nucleotide binding protein; intracellular transport; laboratory mouse; membrane activity; myocardium; protein protein interaction; protein sequence; protein structure function; site directed mutagenesis; striated muscles; voltage gated channel; yeast two hybrid system

DESCRIPTION (provided by applicant): Voltage-dependent calcium channels (VDCCs) serve two critical functions: the regulation of cellular excitability, and the regulation of Ca2+ entry. Ca2+ dysfunction accompanies adult disease progression such as in cardiac hypertrophy. Thus, it follows that the chronic regulation of voltage-dependent Ca2+ channel expression is critical to the function of the heart and skeletal muscle. The guiding hypothesis of this proposal is that the Ras-related GTPase Rem regulates Ca2+ channel activity in cardiac and skeletal muscle by the novel mechanism of interacting with the beta-subunits of voltage-gated Ca2+ channels to block their association with alpha1-subunits. We hypothesize that this inhibits channel activity by blocking trafficking of functional calcium channels to the plasma membrane. This hypothesis was motivated by the following pilot studies: 1) Rem is highly expressed in cardiac and skeletal muscle. 2) Rem binds to beta- subunits. 3) Over-expression of wild-type Rem inhibits ionic current expression in heterologous expression systems, and importantly, in primary ventricular myocytes. 4) T-type Ca 2+ channels do not require accessory subunits and Rem does not inhibit expression of currents through this family of channels. This suggests that Rem regulates Ca2+ channel activity in a beta-subunit-dependent fashion. 5) Rem-mediated regulation of CaV1 channels is not understood, but initial studies have defined a crucial role for the Rem C-terminal domain in this process. We propose three specific aims to elucidate the function of Rem as a regulator of voltage-dependent Ca2+ channel activity. Specific Aim 1 will identify the amino acid sequences in Rem and the Ca2+ channel beta- subunit important for their interaction. Specific Aim 2 will determine if formation of the Rem:beta-subunit complex is regulated in vivo by 14-3-3 protein binding and GTPase activity. Studies will also examine the role of the C-terminal domain in Rem-mediated Ca2+ channel regulation. Specific Aim 3 will characterize the mechanism of Rem-mediated regulation of alpha1 channel activity by examining Rem effects on the association of alpha1 and beta-subunits and channel trafficking. These studies will advance knowledge by elucidating a new mechanism for controlling chronic Ca2+ channel activity as well as a novel mechanism for achieving cross talk between Ras-related GTPases and electrical signaling pathways. This knowledge could aid understanding of the regulation of voltage-dependent Ca2+ channels in both normal and disease states.

描述（由申请人提供）：电压依赖性钙通道（VDCC）具有两个关键功能：细胞兴奋性的调节和 Ca2+ 进入的调节。 Ca2+ 功能障碍伴随着成人疾病的进展，例如心脏肥大。因此，电压依赖性 Ca2+ 通道表达的长期调节对于心脏和骨骼肌的功能至关重要。该提议的指导性假设是 Ras 相关 GTPase Rem 通过与电压门控 Ca2+ 通道的 β 亚基相互作用以阻断其与 α1 亚基的关联的新机制来调节心肌和骨骼肌中的 Ca2+ 通道活性。我们假设这通过阻止功能性钙通道向质膜的运输来抑制通道活性。这一假设是由以下初步研究推动的：1) Rem 在心肌和骨骼肌中高度表达。 2) Rem 与 β 亚基结合。 3) 野生型Rem的过度表达抑制异源表达系统中的离子电流表达，重要的是，抑制原代心室肌细胞中的离子电流表达。 4) T型Ca 2+ 通道不需要辅助亚基，并且Rem不会抑制通过该通道家族的电流表达。这表明 Rem 以依赖 β 亚基的方式调节 Ca2+ 通道活性。 5) Rem 介导的 CaV1 通道调节尚不清楚，但初步研究已经确定了 Rem C 端结构域在此过程中的关键作用。我们提出了三个具体目标来阐明 Rem 作为电压依赖性 Ca2+ 通道活动调节器的功能。具体目标 1 将鉴定 Rem 和 Ca2+ 通道 β 亚基中对其相互作用至关重要的氨基酸序列。具体目标 2 将确定 Rem:β 亚基复合物的形成是否在体内受到 14-3-3 蛋白结合和 GTP 酶活性的调节。研究还将检验 C 末端结构域在 Rem 介导的 Ca2+ 通道调节中的作用。具体目标 3 将通过检查 Rem 对 α1 和 β 亚基关联以及通道运输的影响来表征 Rem 介导的 α1 通道活性调节机制。这些研究将通过阐明控制慢性 Ca2+ 通道活性的新机制以及实现 Ras 相关 GTP 酶和电信号通路之间串扰的新机制来增进知识。这些知识可以帮助理解正常和疾病状态下电压依赖性 Ca2+ 通道的调节。