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（VDCCs）具有两个关键功能：细胞兴奋性调节和Ca2+进入调节。Ca2+功能障碍伴随着成人疾病的进展，如心脏肥厚。因此，由此可见，电压依赖性Ca2+通道表达的慢性调节对心脏和骨骼肌的功能至关重要。该建议的指导假设是ras相关的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α - 1亚基的关联的新机制调节心脏和骨骼肌中的Ca2+通道活性。我们假设这通过阻断功能性钙通道到质膜的运输来抑制通道活性。这一假设是基于以下初步研究：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的过表达抑制了异源表达系统中离子电流的表达，重要的是，在原代心室肌细胞中。4) t型ca2 +通道不需要辅助亚基，Rem不抑制通过该通道的电流表达。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通道调控尚不清楚，但初步研究已经确定了Rem c端结构域在这一过程中的关键作用。我们提出了三个具体的目标来阐明Rem作为电压依赖性Ca2+通道活性的调节剂的功能。特异性目标1将确定Rem和Ca2+通道β -亚基对它们相互作用的重要氨基酸序列。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到14-3-3蛋白结合和GTPase活性的调节。研究还将检查c端结构域在rem介导的Ca2+通道调节中的作用。特异性Aim 3将通过研究Rem对alpha1和β亚基的关联以及通道运输的影响，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控制慢性Ca2+通道活性的新机制以及实现ras相关GTPases和电信号通路之间串扰的新机制来推进知识。这一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.