Regulation of Calcium Channel Function by the Rem GTPase

Rem GTPase 对钙通道功能的调节

• 批准号: 6877990
• 负责人: Douglas Allen Andres
• 金额: $ 36.83万
• 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/6877990
• 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(申请人提供)：电压依赖性钙通道(VDCC)具有两个重要功能：调节细胞兴奋性和调节钙离子内流。Ca~(2+)功能障碍伴随着成人疾病的进展，如心肌肥厚。因此，电压依赖性钙通道表达的慢性调节对心脏和骨骼肌的功能至关重要。Ras相关的GTPase Rem通过与电压门控钙通道的β亚基相互作用来阻断其与α1亚基的联系，从而调节心肌和骨骼肌的钙通道活性。我们假设，这是通过阻止功能性钙通道向质膜的运输来抑制通道活性的。这一假说源于以下先导研究：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的过表达抑制了异源表达系统中离子电流的表达，更重要的是抑制了原代培养的心室肌细胞中离子电流的表达。4)T型钙通道不需要辅助亚基，Rem不抑制通过该家族通道的电流表达。这表明，Rem以一种β亚基依赖的方式调节钙通道的活动。5)Rem介导的CaV1通道的调节尚不清楚，但初步研究确定了Rem C-末端结构域在这一过程中的关键作用。我们提出了三个特定的目标来阐明Rem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中的氨基酸序列和对它们相互作用至关重要的钙通道β亚基。特异性目标2将确定Rem：β-亚基复合体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。研究还将探讨C-末端结构域在Rem介导的钙通道调节中的作用。具体目标3将通过检测Rem对Alpha1和β-亚基的关联以及通道贩运的影响来表征Rem介导的Alpha1通道活动的调节机制。这些研究将通过阐明控制慢性钙通道活性的新机制以及实现RAS相关GTP酶与电信号通路之间的串扰的新机制来促进知识的进步。这一知识有助于理解电压依赖性钙通道在正常和疾病状态下的调节。