Regulation of Calcium Channel Function by the Rem GTPase

描述（由申请人提供）：电压依赖性钙通道（VDCC）具有两种关键功能：调节细胞兴奋性和调节Ca2+内流。Ca2+功能障碍伴随成人疾病进展，如心脏肥大。因此，它遵循的电压依赖性Ca2+通道的表达的慢性调节是至关重要的心脏和骨骼肌的功能。该建议的指导性假设是Ras相关的GTdR Rem通过与电压门控Ca 2+通道的β亚基相互作用以阻断其与α 1亚基的结合的新机制来调节心脏和骨骼肌中的Ca 2+通道活性。我们推测这通过阻断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激发的：1）Rem在心肌和骨骼肌中高度表达。2)rem与β亚单位结合。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，抑制原代心室肌细胞中的离子电流表达。4)T型钙通道不需要辅助亚基，Rem不抑制通过该通道家族的电流表达。这表明Rem以β亚基依赖的方式调节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-末端结构域在此过程中的关键作用。我们提出了三个具体的目标来阐明雷姆作为电压依赖性钙通道活性的调节器的功能。具体目标1将确定Rem和Ca2+通道β亚基中对其相互作用重要的氨基酸序列。特异性目的2将确定Rem：β-亚基复合物的形成是否在体内受14 - 3 - 3蛋白结合和GTdR活性调节。研究还将检查C-末端结构域在Rem-mediated Ca2+通道调节中的作用。具体目标3将通过检查Rem对α 1和β亚基和通道运输的关联的影响来表征Rem介导的α 1通道活性调节的机制。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推进知识。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。

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.