Communication between N terminus and loop2 tunes Orai activation

Marc Fahrner,Saurabh K Pandey,Martin Muik,Lukas Traxler,Carmen Butorac,Michael Stadlbauer,Vasilina Zayats,Adéla Krizova,Peter Plenk,Irene Frischauf,Rainer Schindl,Hermann J Gruber,Peter Hinterdorfer,Rüdiger Ettrich,Christoph Romanin,Isabella Derler

doi:10.1074/jbc.m117.812693

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.

Highlights

Introduction of the double point mutantL74E/W76E in Orai1 P245L did not completely abolish, but significantly reduced, constitutive currents, which were enhanced by the Orai3-loop2 in Orai1 (Orai1 L74E/W76E Orai3-loop2 P245L) (Fig. 4b)
STIM1-dependent function of this Orai1 N-truncation mutant was restored upon the swap with Orai3-loop2
We suggest that the function of Orai channels is ensured by a fine-tuned, permissive interplay between the N terminus and loop2

Summary

Introduction

Introduction of the double point mutantL74E/W76E in Orai P245L did not completely abolish, but significantly reduced, constitutive currents, which were enhanced by the Orai3-loop in Orai (Orai L74E/W76E Orai3-loop P245L) (Fig. 4b). The single point mutation K85E leaves Orai P245L and Orai Orai3-loop P245L inactive in the absence of STIM1, highlighting Lys as essential for general Orai channel function in support of Ref. 39 (see Ref. 71). Constitutive Orai mutants lost function in the N-terminal truncated Orai1 ⌬N1–78 form; their activity was restored upon the swap of Orai3-loop. Constitutive Orai mutants lost function in the N-terminal truncated Orai1 ⌬N1–78 form; their activity was restored upon the swap of Orai3-loop2 These results pointed to an inhibitory effect of Orai1-loop in the N-truncated mutants, which occurred independent of STIM1. We supposed that Orai1-loop and Orai3-loop induced distinct, isoform-specific Orai conformations, permissive or nonpermissive, that accounted for the functional differences and probably affected STIM1 binding in an indirect manner.

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Conclusion