A C-Terminal Switch Gates the Orai1 Channel

Abstract

The mechanism by which Orai1 channels are activated by STIM1 remains unresolved. The closed Orai1 channel solved by crystallography reveals the fourth transmembrane domain (M4) has a cytosolic hinged extension. The M4 hinge (SHK; 263-265 in human) lies adjacent to the membrane surface. Immediately N-terminal of the hinge are two conserved residues (L261, V262) that lie close enough to L174/A175 in M3 for hydrophobic interactions to occur. We reveal the 5-amino acid extended hinge region (EHR) of the Orai1 M4 helix (261-265; LVSHK), is a crucial component of the STIM1-induced Orai1-gating site. Expression in HEK cells of EHR-mutated Orai1 (LVSHK to ANSGA), results in a constitutively open Orai1 with authentic ICRAC properties. Remarkably, we can remove the C-terminal STIM1-binding site or mutate it to prevent STIM1 binding (L273D), and the Orai1-ANSGA channel remains fully, constitutively open. The interaction between the M4-EHR and M3 appears crucial to channel activation. Diamide-induced disulfide interactions using the L174C/L261C or A175C/L261C paired cysteine substitutions, caused rapid increases in ICRAC. Mutations (L174D, A174K, L261D) aimed to disrupt M3-M4 interactions resulted in Orai1 remaining closed after store-depletion. Importantly, modification of the N-terminus of Orai1 (the site purported to bind STIM1 and gate Orai1), has precisely the same channel-blocking effect on the constitutively active (STIM1-independent) Orai1-ANSGA channel as on the STIM1-activated wildtype Orai1 channel, indicating STIM1-binding to the Orai1 N-terminus is not required for channel opening. Thus, the N-terminus of Orai1 (74-90) is merely part of the channel pore and not a gating site. Our data provide crucial new insights into the mechanism of Orai1 gating. EHR is adjacent to the STIM1 binding site, connected to M3, and entirely remote from the pore. Yet, its modification exactly mimics the action of STIM1 indicating its fundamental role in the gating of Orai1.