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

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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