Rychkov, G.Y.Zhou, F.H.Adams, M.K.Brierley, S.M.Ma, L.Barritt, G.J.2022-01-202022-01-202021Journal of Physiology, 2021; 600(3):1-120022-37511469-7793https://hdl.handle.net/2440/134132Three Orai (Orai1, Orai2, and Orai3) and two stromal interaction molecule (STIM1 and STIM2) 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 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.en© 2021 The Authors. The Journal of Physiology © 2021 The Physiological SocietyCa2+ dependent inactivationICRACOrai3STIM1gatingpHJournal article10.1113/jp2825022022-01-19596646Rychkov, G.Y. [0000-0002-2788-2977]Zhou, F.H. [0000-0003-3113-1671]Brierley, S.M. [0000-0002-2527-2905]