Distinct Functional Roles of the N- and C-Terminal STIM1 Binding Sites in Orai1 for Trapping and Gating of CRAC Channels

Abstract

Store-operated Ca2+ release-activated Ca2+ (CRAC) channels are activated through a mechanism wherein depletion of intracellular calcium stores results in the aggregation of the ER Ca2+ sensor, STIM1, and the CRAC channel protein, Orai1, to overlapping sites in the ER- and plasma-membranes. Redistribution of CRAC channels is driven through direct STIM1-Orai1 binding, a critical step that not only controls CRAC channel gating, but also regulates its pore properties including calcium selectivity. Previous findings indicate that Orai1 harbors two STIM1 binding sites, located in the intracellular N- and C-termini. Here, we compared the functional contributions of these sites for puncta formation, gating, and modulation of CRAC channel ion selectivity. Consistent with past studies, C-terminal mutations that impaired STIM1-Orai1 association and puncta formation also abrogated channel gating. However, gating was fully restored in some of these mutants by directly tethering a STIM1 fragment containing the minimal CRAC activation domain (CAD) in duplicate to Orai1 (Orai1-SS channels). By contrast, mutants lacking the N-terminal CAD binding site displayed no channel activity despite retaining significant STIM1-Orai1 binding, and this defect could not be restored in Orai1-SS channels. Analysis of ion selectivity indicated that the N-terminal site is essential for conferring high calcium selectivity to Orai1 while the C-terminal site is dispensable if CAD is directly linked to Orai1. Thus, the structural requirements for gating and modulation of ion selectivity are similar but substantively different from those for Orai1 puncta formation. Collectively, these results are broadly consistent with a two-step model for Orai channel activation, in which the C-terminal binding site tethers and retains CAD to Orai1, thereby allowing functionally critical interactions at the Orai1 N-terminus to gate the channel and modulate its pore properties.