Regulation of Calcium Channel Function by the Rem GTPase

Rem GTPase 对钙通道功能的调节

基本信息

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
项目类别：
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

项目摘要

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。