Regulation of endogenous and heterologous Ca release-activated Ca currents by pH

Deviations from physiological pH (∼pH 7.2) as well as altered Ca²⁺ signaling play important roles in immune disease and cancer. One of the most ubiquitous pathways for cellular Ca²⁺ influx is the store-operated Ca²⁺ entry (SOCE) or Ca²⁺ release-activated Ca²⁺ current (I_CRAC), which is activated upon depletion of intracellular Ca²⁺ stores. We here show that extracellular and intracellular changes in pH regulate both endogenous I_CRAC in Jurkat T lymphocytes and RBL2H3 cells, and heterologous I_CRAC in HEK293 cells expressing the molecular components STIM1/2 and Orai1/2/3 (CRACM1/2/3). We find that external acidification suppresses, and alkalization facilitates IP₃-induced I_CRAC. In the absence of IP₃, external alkalization did not elicit endogenous I_CRAC but was able to activate heterologous I_CRAC in HEK293 cells expressing Orai1/2/3 and STIM1 or STIM2. Similarly, internal acidification reduced IP₃-induced activation of endogenous and heterologous I_CRAC, while alkalization accelerated its activation kinetics without affecting overall current amplitudes. Mutation of two aspartate residues to uncharged alanine amino acids (D110/112A) in the first extracellular loop of Orai1 significantly attenuated both the inhibition of I_CRAC by external acidic pH as well as its facilitation by alkaline conditions. We conclude that intra- and extracellular pH differentially regulates I_CRAC. While intracellular pH might affect aggregation and/or binding of STIM to Orai, external pH seems to modulate I_CRAC through its channel pore, which in Orai1 is partially mediated by residues D110 and D112.