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

描述（由申请人提供）：Ca^"^信号介导心脏和平滑肌的发育和增殖。异常的Ca^^信号传导与心血管系统的主要疾病密切相关；然而，异常Ca^ ^信号的原因尚未确定。我们建议研究瞬时受体电位（TRPC）和Orail （CRAG）通道的门控机制，以更好地理解心脏肥厚和心力衰竭中Ca^"^信号的异常。在Aim 1中，我们将确定STIM1和Homer对trpc的门控机制，以及STIM1和Homer如何协同调节trpc。我们将研究ST1M1上的最后2个正电荷（^^'*KK^^^）和TRPC c端上的两个保守的带负电荷的氨基酸如何通过STIM1功能性地相互作用来控制TRPC。trpc上的Homer结合位点（PXXF）距离带负电荷的残基只有4个残基，与STIM1不同，Homer通过与IP3RS偶联使trpc保持封闭状态。因此，我们将确定(a) HI a （Homer的简称）和H1(W24A)（均为显性阴性）是否会增加STIM1的接入和与TRPC1的结合；(b)在两个负电荷和PXXF之间添加aa's是否会分离它们对TRPC1的影响；(c) Homer和STIM1是否竞争与TRPC1的结合。在Aim 2中，我们将通过STIM1检查Orail的门控机制，并在原生soc的背景下吸收我们对Orail和TRPC门控的了解。我们将(a)绘制Orail激活所需的最小STIM1区域和与STIM1相互作用的Orail结构域；(b)确定该最小ST1M1区域的Orail活性的动力学性质是否与全长STIM1相似，以及该区域外的结构域是否调节该活性；(c)确定本地Orail和本地trpc对本地soc的贡献。在Aim 3中，我们将通过以下方法评估STIM1、Orail和TRPCs在心肌肥厚中的作用：(a)测量从STIM1、Orail和TRPC1/3/6敲除（KO）小鼠中分离的心肌细胞的电流和soc活性：血管紧张素II、内皮素-1或phenylephrine；(b)通过测定核NFAT量，测量胸主动脉束带（TAB）压力过载对KO小鼠的影响；RCAN1、p-MHC、ANF mRNA水平；HW / BW比率;心肌细胞大小和形态；通过超声心动图检测心脏功能。相关性：异常Ca^*信号与心血管系统的主要疾病密切相关，包括心脏肥厚和心力衰竭。我们建议研究储存操作的Ca^*通道的门控机制，以更好地理解Ca^*信号传导与病理性心脏重构之间的联系

DESCRIPTION (provided by applicant): 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 (CRAG) channels towards 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 ST1M1 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 STIM1, Homer keeps TRPCs in a closed state by coupling them to IP3RS. Therefore, we will determine (a) if HI a (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 STIM1 and assimilate our knowledge of Orail and TRPC gating in the context of native SOCs. We will (a) map the minimal STIM1 region required for activation of Orail and the Orail domain(s) that interact with STIM1; (b) determine if the kinetic properties of Orail activity by this minimal ST1M1 region are similar to that of full length STIM1 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 knockout (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. RELEVANCE (See Instructions): Aberrant Ca^* signaling has been firmly linked to major diseases of the cardiovascular system, including cardiac hypertrophy and heart failure. We propose to study the gating mechanism of store-operated Ca^* channels towards better understanding the link between Ca^* signaling and pathological cardiac remodeling