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

Ca^*信号介导心肌和平滑肌的发育和增殖。反常的钙^* 信号与心血管系统的主要疾病密切相关；然而， 异常的Ca^“”信号尚未确定。我们建议研究该系统的选通机制 瞬时受体电位(TRPC)和卵巢(CRAC)通道更好地理解异常 心肌肥厚和心力衰竭中的钙信号转导。在目标1中，我们将确定 STIM1和荷马的TRPC，以及STIM1和荷马如何协同工作来监管TRPC。我们会 研究STIM1上的最后两个正电荷和两个守恒的负电荷是如何 TRPC C末端的氨基酸通过STIM1与TRPC门相互作用。上的荷马结合位点 TRPC(PXXF)距离带负电荷的残基只有4个残基，与ST1M1不同，Hmer 通过将TRPC耦合到IP3R，使TRPC保持关闭状态。因此，我们将确定(A)是否Hia(缩写 和H1(W24A)(均为负显性)增加STIM1对TRPC1的访问和结合；(B)如果 在2个负电荷和PXXF之间加上AA可以分离它们对TRPC1的影响；以及(C)如果荷马 和STIM1竞争结合TRPC1。在目标2中，我们将通过以下方式研究ORAIL的门控机制 在本地SOC的背景下，STIMI和吸收我们在ORAIL和TRPC门控方面的知识。我们将(A) RRAP激活ORAIL所需的最小STIMI区域以及与其相互作用的ORAIL结构域(S (B)确定该最小STIMI区域的ORAIL活动的动力学特性是否与该区域相似 全长STIMI和该区域外的结构域调控这一活动；和(C)确定 本地ORAIL和本地TRPC对本地SOC的贡献。在目标3中，我们将评估STIM1、 ORAIL和TRPC在心肌肥厚中的作用(A)测量心肌细胞的电流和SOCs活性 分离自STIM1、ORAIL和TRPC1/3/6基因敲除(KO)小鼠，经血管紧张素II、内皮素-1或 苯肾上腺素；和(B)测量胸主动脉捆扎(TAB)压力超负荷对这些KO的影响。 检测小鼠核NFAT含量、RCAN1、p-MHC和ANF基因表达水平、HW/BW比值； 超声心动图显示心肌细胞的大小和形态；心功能。

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.