Publication
Orai1- and Orai2-, but not Orai3-mediated I
Publisher:
Wiley
Date:
25-12-2022
DOI:
10.1113/JP282502
Abstract: 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 2+ entry mechanism. When co‐expressed with STIM1, Orai1, Orai2 and Orai3 form highly selective Ca 2+ channels with properties of Ca 2+ release‐activated Ca 2+ (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 2+ ‐dependent inactivation, inhibition otentiation 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 cl ing 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 litude and the kinetics of Ca 2+ ‐dependent inactivation. In contrast, Orai2 litude, 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. image It has been shown previously that Orai1/stromal interaction molecule 1 (STIM1)‐mediated Ca 2+ release‐activated Ca 2+ 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 litude of Orai2 current is affected by the changes in pH i similarly to the litude of Orai1. However, unlike Orai1, fast Ca 2+ ‐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 .