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bioRxiv · 10.1101/2020.11.01.364299

Orai1- and Orai2-, but not Orai3-mediated ICRAC is regulated by intracellular pH

Rychkov, G.·Zhou, F. H.·Adams, M. K.·Brierley, S. M.·Ma, L.·Barritt, G. J.
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Abstract

Three Orai (Orai1, Orai2 and Orai3) and two STIM (STIM1 and STIM2; stromal interaction molecule) 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-APB 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. 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 intracellular pH (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.

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Rychkov, G., Zhou, F. H., Adams, M. K., Brierley, S. M., Ma, L., Barritt, G. J.. 2020-11-02. Orai1- and Orai2-, but not Orai3-mediated ICRAC is regulated by intracellular pH. https://doi.org/10.1101/2020.11.01.364299

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