Orai1- and Orai2-, but not Orai3-mediated I is 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 Ca entry mechanism. When co-expressed with STIM1, Orai1, Orai2 and Orai3 form highly selective Ca channels with properties of Ca release-activated Ca (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 Ca -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 (I ) by intracellular pH (pH ). Using whole-cell patch clamping of HEK293T cells heterologously expressing Orai and STIM1, we show that I formed by each Orai homologue has a unique sensitivity to changes in pH . Orai1-mediated I exhibits a strong dependence on pH of both current amplitude and the kinetics of Ca -dependent inactivation. In contrast, Orai2 amplitude, but not kinetics, depends on pH , whereas Orai3 shows no dependence on pH at all. Investigation of different Orai1-Orai3 chimeras suggests that pH dependence of Orai1 resides in both the N-terminus and intracellular loop 2, and may also involve pH-dependent interactions with STIM1. KEY POINTS: It has been shown previously that Orai1/stromal interaction molecule 1 (STIM1)-mediated Ca release-activated Ca current (I ) is inhibited by intracellular acidification and potentiated by intracellular alkalinisation. The present study reveals that CRAC channels formed by each of the Orai homologues Orai1, Orai2 and Orai3 has a unique sensitivity to changes in intracellular pH (pH ). The amplitude of Orai2 current is affected by the changes in pH  similarly to the amplitude of Orai1. However, unlike Orai1, fast Ca -dependent inactivation of Orai2 is unaffected by acidic pH . In contrast to both Orai1 and Orai2, Orai3 is not sensitive to pH  changes. Domain swapping between Orai1 and Orai3 identified the N-terminus and intracellular loop 2 as the molecular structures responsible for Orai1 regulation by pH . Reduction of I dependence on pH seen in a STIM1-independent Orai1 mutant suggested that some parts of STIM1 are also involved in I modulation by pH .