Orai1- and Orai2-, but not Orai3-mediated I_CRAC 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 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 (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 Orai homologue has a unique sensitivity to changes in pH_i . Orai1-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 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 Ca²⁺ release-activated Ca²⁺ current (I_CRAC ) 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_i ). The amplitude of Orai2 current is affected by the changes in pH_i  similarly to the amplitude of Orai1. However, unlike Orai1, fast Ca²⁺ -dependent inactivation of Orai2 is unaffected by acidic pH_i . In contrast to both Orai1 and Orai2, Orai3 is not sensitive to pH_i  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_i . Reduction of I_CRAC dependence on pH_i seen in a STIM1-independent Orai1 mutant suggested that some parts of STIM1 are also involved in I_CRAC modulation by pH_i .