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

描述(申请人提供)：钙信号调节心肌和平滑肌的发育和增殖。钙信号的异常与心血管系统的主要疾病密切相关；然而，钙信号异常的原因尚未确定。我们建议研究瞬时受体电位(TRPC)和ORAIL(CRAG)通道的门控机制，以更好地了解心肌肥厚和心力衰竭中钙信号的异常。在目标1中，我们将确定STIM1和Hmer对TRPC的门控机制，以及STIM1和Hmer如何协同调节TRPC。我们将研究ST1M1上的最后两个正电荷(^‘*KK^)和TRPC C末端的两个保守的负电荷是如何通过STIM1与GATE TRPC相互作用的。TRPC上的荷马结合位点(PXXF)距离带负电荷的残基只有4个残基，与STIM1不同的是，荷马通过将TRPC与IP3RS偶联来保持TRPC处于关闭状态。因此，我们将确定(A)HI a(Hmer的缩写)和H1(W24A)(均为负)是否增加了STIM1的访问和与TRPC1的结合；(B)如果在两个负电荷和PXXF之间添加AA是否分离了它们对TRPC1的影响；以及(C)Hmer和STIM1是否竞争与TRPC1的结合。在目标2中，我们将通过STIM1研究ORAIL的选通机制，并在本地SOC的背景下吸收我们对ORAIL和TRPC选通的知识。我们将(A)定位激活ORAIL所需的最小STIM1区域以及与STIM1相互作用的ORAIL结构域(S)；(B)确定这个最小ST1M1区域的ORAIL活性是否与全长STIM1相似，以及该区域外的结构域是否调节这一活性；以及(C)确定本地ORAIL和本地TRPC对本地SOC的贡献。在目标3中，我们将通过以下方式评估STIM1、ORAIL和TRPC在心肌肥厚中的作用：(A)检测经血管紧张素II、内皮素-1或苯肾上腺素处理的STIM1、ORAIL和TRPC1/3/6基因敲除(KO)小鼠的心肌细胞电流和SOCs活性；(B)通过超声心动图检测胸主动脉缩窄(TAB)压力超负荷对这些KO小鼠的影响；RCAN1、p-MHC和ANF mRNA水平；HW/BW比率；心肌细胞大小和形态；以及心功能。相关性(参见说明)：钙信号的异常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。我们建议研究钙离子通道的门控机制，以更好地理解钙离子信号和病理性心脏重塑之间的联系。

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