The mechanism of Orai channels dependence on intracellular pH

G.Y. Rychkov,¹ F.H. Zhou,¹ M. Adams,¹ L. Ma² and G.J. Barritt,² ¹School of Medicine, University of Adelaide, and South Australian Health and Medical Research Institute, Adelaide, SA 5005, Australia and ²College of Medicine and Public Health, Flinders University, Adelaide, SA 5001, Australia.

The activity of store-operated Ca²⁺ channels formed by Orai1 and STIM1 proteins has been shown to strongly depend on the changes of intracellular pH (pH_i) within the physiological ranges, however, the amino acids responsible for this dependence have not been yet identified (Beck et al., 2014, Gavriliouk et al., 2017, Tsujikawa et al., 2015). Furthermore, it is not known whether these amino acids are localized in Orai1 or STIM1 polypeptides, and whether Ca²⁺ channels made of Orai1 homologues, Orai2 and Orai3, exhibit pH_i dependence similar to that of Orai1.

In this study we investigated dependence of Orai2- and Orai3-mediated Ca²⁺ currents on pH_i using whole-cell patch clamping of HEK293T cells heterologously expressing STIM1 and either Orai2 or Orai3 proteins. Intracellular pH has been varied by application of different concentrations of sodium propionate (C₂H₅COONa) or ammonium chloride (NH₄Cl) to the bath solution.

It was found that intracellular acidification achieved by applying 60 mM propionate to the bath solution inhibited Orai2-mediated Ca²⁺ current by ∼80-90%, compared to the current recorded under control conditions (pH_i 7.3). Intracellular alkalinisation using 15 mM NH₄Cl in the bath strongly potentiated Orai2 current amplitude, but only when EGTA was used as intracellular Ca²⁺ buffer. This dependence of Orai2 amplitude on pH_i was very similar to that exhibited by Orai1-mediated current (Gavriliouk et al., 2017). However, unlike in Orai1, the kinetics of Orai2 fast Ca²⁺ dependent inactivation was not affected by intracellular acidification. In contrast, Orai3-mediated store-operated Ca²⁺ current exhibited no dependence on pH_i, suggesting that amino acids that mediate pH_i dependence of Orai1 and Orai2 are localized in intracellular C- or N-termini, or the intracellular loop of these proteins, and are absent in Orai3. Replacement of Orai1 C- and N-termini with those of Orai3 did not affect either Orai1 amplitude or kinetics dependence on pH_i. Replacement of Orai1 intracellular loop with that of Orai3 did, however, abolish Orai1 dependence on pH_i. Surprisingly, mutating the only potentially protonatable glutamates E162 and E164 in the intracellular loop of Orai1, which are absent in Orai3, to glutamine, had no effect on pH_i dependence of Orai1 current, suggesting that protonatable site is localized in STIM1.

Beck A, Fleig A, Penner R, Peinelt C. (2014). Cell Calcium 56, 235-243.

Gavriliouk D, Scrimgeour NR, Grigoryev S, Ma L, Zhou FH, Barritt GJ, Rychkov GY. (2017). Scientific Reports 7:9829 doi: 10.1038/s41598-017-06371-0

Tsujikawa H, Yu AS, Xie J, Yue Z, Yang W, He Y & Yue L. (2015). Scientific Reports 5:16747 doi: 10.1038/srep16747