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）提供两个关键功能：细胞兴奋性的调节和CA2+进入的调节。 CA2+功能障碍伴随成人疾病进展，例如心脏肥大。因此，因此，依赖电压依赖性Ca2+通道表达的慢性调节对于心脏和骨骼肌的功能至关重要。该提议的指导假设是，与RAS相关的GTPase REM通过与电压门控Ca2+通道的β-单位相互作用的新机制来调节心脏和骨骼肌中的Ca2+通道活性，以阻止其与Alpha1-Subunits的关联。我们假设这是通过阻止对质膜的功能性钙通道的运输来抑制通道活性的。该假设是由以下试点研究激励的：1）REM在心脏和骨骼肌中高度表达。 2）REM与β亚基结合。 3）野生型REM的过表达抑制异源表达系统中的离子电流表达，重要的是，原发性心肌细胞中的离子电流表达。 4）T型Ca 2+通道不需要附件亚基，并且REM不会通过此通道系列抑制电流的表达。这表明REM以beta-subunit依赖性方式调节Ca2+通道活动。 5）尚不清楚REM介导的CAV1通道的调节，但是初步研究定义了REM C末端结构域在此过程中的关键作用。我们提出了三个特定的目的，旨在阐明REM作为电压依赖性CA2+通道活性的调节剂的功能。特定的目标1将确定REM中的氨基酸序列，而Ca2+通道β亚基对于它们的相互作用很重要。具体的目标2将确定REM的形成：Beta-Subunit复合物在体内受到14-3-3蛋白结合和GTPase活性的调节。研究还将检查C末端结构域在REM介导的Ca2+通道调节中的作用。特定的目标3将通过检查REM对alpha1和beta-subunits和通道运输的效果来表征REM介导的Alpha1通道活性调节的机制。这些研究将通过阐明一种用于控制慢性Ca2+通道活性的新机制，以及在与RAS相关的GTPase和电信号通路之间实现交叉谈话的新机制。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。