Gating mechanism of TRPCs and Orai1 by STIM1 & their role in cardiac hypertrophy

Ca^* 信号传导介导心脏和平滑肌发育和增殖。异常钙^* 信号传导与心血管系统的主要疾病密切相关;然而， 异常的Ca 2+信号传导尚未确定。我们建议研究的门控机制， 瞬时受体电位（TRPC）和Orail（CRAC）通道，以更好地了解异常的 心肌肥大和心力衰竭中的Ca^* 信号传导。在目标1中，我们将确定 STIM 1和Homer的TRPC以及STIM 1和Homer如何协同工作以调节TRPC。我们将 研究STIM 1上的最后2个正电荷（^'' KK <$^^）和两个保守的负电荷 TRPC C-末端的氨基酸通过STIM 1功能性地与门控TRPC相互作用。荷马结合位点 TRPC（PXXF）与带负电荷的残基仅相距4个残基，并且与ST 1 M1不同，Homer 通过将TRPC耦合到IP 3RS，使TRPC保持关闭状态。因此，我们将确定（a）如果H ia（短形式 Homer）和H1（W24 A）（均为显性阴性）增加STIM 1接近和与TRPC 1的结合;（B）如果 在2个负电荷和PXXF之间添加aa's分离了它们对TRPC 1的影响;以及（c）如果Homer 和STIM 1竞争结合TRPC 1。在目标2中，我们将通过以下方式检查Orail的门控机制： STMI和吸收我们的知识的Orail和TRPC门控的背景下，本地SOC。我们将（a） Rriap激活Orail所需的最小STMI区域和与之相互作用的Orail结构域 （B）确定该最小STMI区域的Orail活性的动力学性质是否类似于 以及该区域外的结构域是否调节该活性;以及（c）确定全长STIMI的结构域， 天然Orail和天然TRPC对天然SOC的贡献。在目标3中，我们将评估STIM 1的作用， Orail和TRPC在心脏肥大中的作用，通过（a）测量来自心肌细胞的电流和SOC活性 分离自用血管紧张素II、内皮素-1或内皮素-2处理的STIM 1、Orail和TRPC 1/3/6敲除（KO）小鼠， 苯肾上腺素;和（B）测量胸主动脉结扎（TAB）压力超负荷对这些KO的影响 通过测定小鼠的核NFAT量; RCAN 1、p-MHC和ANF mRNA水平; HW/BW比; 心肌细胞大小和形态;以及通过超声心动图测定的心脏功能。

Ca^* signaling mediates cardiac and smooth muscle development and proliferation. Aberrant Ca^* signaling has been firmly linked to major diseases of the cardiovascular system; however, the cause of the aberrant Ca^"" signaling has yet to be determined. We propose to study the gating mechanism of the Transient Receptor Potential (TRPC) and Orail (CRAC) channels towartJs better understanding the aberrant Ca^* signaling in cardiac hypertrophy and heart failure. In Aim 1, we will determine the gating mechanism of TRPCs by STIM1 and Homer and how STIM1 and Homer work in tandem to regulate the TRPCs. We will investigate how the last 2 positive (^¿''KK¿^^) charges on STIM1 and the two conserved, negatively charged amino acids in TRPC C-terminus functionally interact to gate TRPCs by STIM1. The Homer binding site on TRPCs (PXXF) is only 4 residues away from the negatively charged residues, and unlike ST1M1, Homer keeps TRPCs in a closed state by coupling them to IP3RS. Therefore, we will determine (a) if H ia (short form of Homer) and H1{W24A) (both dominant negative) increase STIM1 access and binding to TRPC1; (b) if adding aa's between the 2 negative charges and PXXF separates their effects on TRPC1; and (c) if Homer and STIM1 compete for binding to TRPC1. In Aim 2, we will examine the gating mechanism of Orail by STIMI and assimilate our knowledge of Orail and TRPC gating in the context of native SOCs. We will (a) rriap the minimal STIMI region required for activation of Orail and the Orail domain(s) that interact with STIMI; (b) determine if the kinetic properties of Orail activity by this minimal STIMI region are siniilar to that of full length STIMI and if domains outside this region modulate this activity; and (c) determine the contribution of native Orail and native TRPCs to native SOCs. In Aim 3, we will assess the roles of STIM1, Orail, and TRPCs in cardiac hypertrophy by (a) measuring current and SOCs activity from cardiomyocytes isolated from STIM1, Orail and TRPC1/3/6 knocikout (KO) mice treated with angiotensin II, endothelin-1, or phenylephrine; and (b) measuring the effects of thoracic aorta banding (TAB) pressure overload on these KO mice by assaying for nuclear NFAT amounts; RCAN1, p-MHC and ANF mRNA levels; HW/BW ratio; myocyte size and morphology; and cardiac function by echocardiography.