Orai1- and Orai2-, but not Orai3-mediated ICRAC is regulated by intracellular pH.

Three Orai (Orai1, Orai2 and Orai3) and two STIM (STIM1 and STIM2; stromal interaction molecule) mammalian protein homologues constitute major components of the store-operated Ca2+ entry mechanism. When co-expressed with STIM1, Orai1, Orai2 and Orai3 form highly selective Ca2+ channels with properties of Ca2+ release activated Ca2+ (CRAC) channels. Despite the high level of homology between Orai proteins, CRAC channels formed by different Orai isoforms have distinctive properties, particularly with regards to Ca2+ dependent inactivation, inhibition/potentiation by 2-Aminoethyl diphenylborinate (2-APB) and sensitivity to reactive oxygen species. This study characterises and compares the regulation of Orai1, Orai2- and Orai3-mediated CRAC current (ICRAC ) by intracellular pH (pHi ). Using whole-cell patch clamping of HEK293T cells heterologously expressing Orai and STIM1 we show that ICRAC formed by each Orai homologue has a unique sensitivity to changes in pHi . Orai1-mediated ICRAC exhibits a strong dependence on pHi of both current amplitude and the kinetics of Ca2+ dependent inactivation. In contrast, Orai2 amplitude, but not kinetics, depends on pHi , whereas Orai3 shows no dependence on pHi at all. Investigation of different Orai1-Orai3 chimeras suggests that pHi dependence of Orai1 resides in both, the N-terminus and intracellular loop 2, and may also involve pH- dependent interactions with STIM1. Abstract Figure. Ca2+ -release activated Ca2+ current (ICRAC ) mediated by each of Orai homologues, Orai1, Orai2 or Orai3, has a unique sensitivity to changes in the intracellular pH (pHi ). Domain swapping between Orai1 and Orai3 identified N-terminus and intracellular loop (loop2) as the molecular structures responsible for Orai1 regulation by pHi . This article is protected by copyright. All rights reserved.