Orai1-and Orai2-, but not Orai3-mediated I-CRAC regulated by intracellular pH

Three Orai (Orai1, Orai2, and Orai3) and two stromal interaction molecule (STIM1 and STIM2) mammalian protein homologues constitute major components of the store-operated a Ca2+ entry mechanism. When co-expressed with STIM1, Orai1, Orai2 and Orai3 form highly selective Ca2+ channels with properties of Ca2+ release-activated Ca2+ (CRAG) channels. Despite the high level of homology between Orai proteins, CRAG 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 Grail, Orai2- and Orai3-mediated CRAC current (I-CRAC) by intracellular pH (pH(i)). Using whole-cell patch clamping of HEK293T cells heterologously expressing Orai and STIM1, we show that I-CRAC formed by each Oral homologue has a unique sensitivity to changes in pH(i). Grail-mediated I-CRAC exhibits a strong dependence on pH(i), of both current amplitude and the kinetics of Ca2+-dependent inactivation. In contrast, Orai2 amplitude, but not kinetics, depends on pH(i), whereas Orai3 shows no dependence on pH(i) at all. Investigation of different Orai1-Orai3 chimeras suggests that pH(i) dependence of Orail resides in both the N-terminus and intracellular loop 2, and may also involve pH-dependent interactions with STIM1.

Automated summary

Three Orai proteins (Orai1, Orai2, and Orai3) and two STIM proteins (STIM1 and STIM2) are main components of the store-operated calcium entry mechanism. When co-expressed with STIM1, Orai1, Orai2, and Orai3 can form highly selective calcium channels with properties of calcium release-activated calcium (CRAC) channels. Different Orai isoforms in CRAC channels have unique properties, especially in terms of calcium-dependent inactivation, modulation by 2-aminoethyl diphenylborinate, and sensitivity to reactive oxygen species. This study shows that the sensitivity of the CRAC current (I-CRAC) mediated by Grail, Orai2, and Orai3 to intracellular pH (pH(i)) varies, with Grail being highly pH(i)-dependent, Orai2 amplitude being pH(i)-dependent, and Orai3 showing no pH(i) dependence. The study suggests that the pH(i) dependence of Orai1 resides in both the N-terminus and intracellular loop 2, and may involve pH-dependent interactions with STIM1.