Communication between N terminus and loop2 tunes Orai activation

Abstract

Ca2+ release-activated Ca2+ (CRAC) channels constitute the major Ca2+ entry pathway into the cell. They are fully reconstituted via intermembrane coupling of the Ca2+-selective Orai channel and the Ca2+-sensing protein STIM1. In addition to the Orai C terminus, the main coupling site for STIM1, the Orai N terminus is indispensable for Orai channel gating. Although the extended transmembrane Orai N-terminal region (Orai1 amino acids 73–91; Orai3 amino acids 48–65) is fully conserved in the Orai1 and Orai3 isoforms, Orai3 tolerates larger N-terminal truncations than Orai1 in retaining store-operated activation. In an attempt to uncover the reason for these isoform-specific structural requirements, we analyzed a series of Orai mutants and chimeras. We discovered that it was not the N termini, but the loop2 regions connecting TM2 and TM3 of Orai1 and Orai3 that featured distinct properties, which explained the different, isoform-specific behavior of Orai N-truncation mutants. Atomic force microscopy studies and MD simulations suggested that the remaining N-terminal portion in the non-functional Orai1 N-truncation mutants formed new, inhibitory interactions with the Orai1-loop2 regions, but not with Orai3-loop2. Such a loop2 swap restored activation of the N-truncation Orai1 mutants. To mimic interactions between the N terminus and loop2 in full-length Orai1 channels, we induced close proximity of the N terminus and loop2 via cysteine cross-linking, which actually caused significant inhibition of STIM1-mediated Orai currents. In aggregate, maintenance of Orai activation required not only the conserved N-terminal region but also permissive communication of the Orai N terminus and loop2 in an isoform-specific manner.

Mapping the functional anatomy of Orai1 transmembrane domains for CRAC channel gating

“…This conclusion is consistent with other recent studies that have examined the contribution of the membrane-proximal N terminus for Orai1 gating (16,31). We do not yet know how the Orai1 N terminus contributes to channel function, but possible explanations could include an interaction with another part of Orai1 in the open state (40) or a role in ion permeation (41).…”

Pore properties of Orai1 calcium channel dimers and their activation by the STIM1 ER calcium sensor

“…1C). Recent evidence indicates that the 2-3 loop mediates important communication with the M1-ext helix (43). Although the C-terminal M4-ext is the primary site for STIM1 binding to gate the channel, we would speculate that the entire cytosolic domain (M1-ext, 2-3 loop, and M4-ext) exists as a conformationally coupled entity, the alteration of which affects both STIM1 binding and gating of the channel.…”

Molecular basis of allosteric Orai1 channel activation by STIM1

“…observed that the loss of current in Orai1 N‐terminal truncation mutants (∆1–78) can be partially restored by replacing the native Orai1 TM2–3 loop with that of Orai3 (Fahrner et al . ). They postulated that the inhibition of current seen in the N‐terminally truncated Orai1 channels occurs because of an interaction between the truncated Orai1 N‐terminus and the TM2–3 loop (Fahrner et al .…”

“…They postulated that the inhibition of current seen in the N‐terminally truncated Orai1 channels occurs because of an interaction between the truncated Orai1 N‐terminus and the TM2–3 loop (Fahrner et al . ). Consistent with this idea, cysteine cross‐linking between the N‐terminus and the TM2–3 loop inhibited currents in STIM1‐gated and P245L constitutively active mutant channels (Fahrner et al .…”

“…Consistent with this idea, cysteine cross‐linking between the N‐terminus and the TM2–3 loop inhibited currents in STIM1‐gated and P245L constitutively active mutant channels (Fahrner et al . ). This interesting finding suggests that an Orai1 N‐terminus–TM2–3 loop interaction inhibits gating.…”