title: Regulation of endogenous and heterologous Ca²⁺ release-activated Ca²⁺ currents by pH.
abstract: Deviations from physiological pH (∼pH 7.2) as well as altered Ca(2+) signaling play important roles in immune disease and cancer. One of the most ubiquitous pathways for cellular Ca(2+) influx is the store-operated Ca(2+) entry (SOCE) or Ca(2+) release-activated Ca(2+) current (ICRAC), which is activated upon depletion of intracellular Ca(2+) stores. We here show that extracellular and intracellular changes in pH regulate both endogenous ICRAC in Jurkat T lymphocytes and RBL2H3 cells, and heterologous ICRAC 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 ICRAC. In the absence of IP3, external alkalization did not elicit endogenous ICRAC but was able to activate heterologous ICRAC in HEK293 cells expressing Orai1/2/3 and STIM1 or STIM2. Similarly, internal acidification reduced IP3-induced activation of endogenous and heterologous ICRAC, 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 ICRAC by external acidic pH as well as its facilitation by alkaline conditions. We conclude that intra- and extracellular pH differentially regulates ICRAC. While intracellular pH might affect aggregation and/or binding of STIM to Orai, external pH seems to modulate ICRAC through its channel pore, which in Orai1 is partially mediated by residues D110 and D112.
authors.affiliation: Queen's Center for Biomedical Research, Laboratory of Cell and Molecular Signaling, The Queen's Medical Center, Honolulu, HI 96813, United States; John A. Burns School of Medicine, University of Hawaii, Honolulu, HI 96813, United States; Department of Pharmacology and Toxicology, ZHMB, Saarland University, D-66421 Homburg, Germany.
authors.forename: Andreas
authors.initials: A
authors.lastname: Beck
authors.affiliation: Queen's Center for Biomedical Research, Laboratory of Cell and Molecular Signaling, The Queen's Medical Center, Honolulu, HI 96813, United States; John A. Burns School of Medicine, University of Hawaii, Honolulu, HI 96813, United States.
authors.forename: Andrea
authors.initials: A
authors.lastname: Fleig
authors.affiliation: Queen's Center for Biomedical Research, Laboratory of Cell and Molecular Signaling, The Queen's Medical Center, Honolulu, HI 96813, United States; John A. Burns School of Medicine, University of Hawaii, Honolulu, HI 96813, United States. Electronic address: rpenner@hawaii.edu.
authors.forename: Reinhold
authors.initials: R
authors.lastname: Penner
authors.affiliation: Queen's Center for Biomedical Research, Laboratory of Cell and Molecular Signaling, The Queen's Medical Center, Honolulu, HI 96813, United States; John A. Burns School of Medicine, University of Hawaii, Honolulu, HI 96813, United States; Department of Biophysics, Saarland University, ZHMB, 66421 Homburg, Germany. Electronic address: bpcpei@uks.eu.
authors.forename: Christine
authors.initials: C
authors.lastname: Peinelt
mesh_terms: D002118:Calcium / Q000378:metabolism*; D015220:Calcium Channels / Q000378:metabolism*; D020013:Calcium Signaling / Q000502:physiology*; D015815:Cell Adhesion Molecules / Q000378:metabolism*; D005453:Fluorescence; D057809:HEK293 Cells; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008565:Membrane Proteins / Q000378:metabolism*; D009363:Neoplasm Proteins / Q000378:metabolism*; D000071740:ORAI1 Protein; D061848:Optical Imaging; D018408:Patch-Clamp Techniques; D000071737:Stromal Interaction Molecule 1; D000071738:Stromal Interaction Molecule 2
chemical_list: D015220:Calcium Channels; D015815:Cell Adhesion Molecules; D008565:Membrane Proteins; D009363:Neoplasm Proteins; D000071740:ORAI1 Protein; C510746:ORAI1 protein, human; C103530:STIM1 protein, human; C513279:STIM2 protein, human; D000071737:Stromal Interaction Molecule 1; D000071738:Stromal Interaction Molecule 2; D002118:Calcium
keywords: HEK293; I(CRAC); Jurkat cells; Orai; RBL; STIM; Whole-cell patch clamp