Orai1–Orai2 complex is involved in store-operated calcium entry in chondrocyte cell lines

•Orai1 and/or Orai2 contribute to SOCE in OUMS-27 cells.•Orai1, Orai2, and STIM1 expressions were detected in mRNA and protein levels.•Orai1 and Orai2 were also expressed in human cartilage tissues in mRNA levels.•The dominant negative form of Orai1 and siOrai1 markedly reduced SOCE.•Orai2 attenuated SOCE by forming hetero-tetramer with Orai1.

Ca2+ influx via store-operated Ca2+ entry (SOCE) plays critical roles in many essential cellular functions. The Ca2+ release-activated Ca2+ (CRAC) channel complex, consisting of Orai and STIM, is one of the major components of store-operated Ca2+ (SOC) channels. Our previous study demonstrated that histamine can cause sustained Ca2+ entry through SOC channels in OUMS-27 cells derived from human chondrosarcoma. This SOCE was increased by low- and decreased by high-concentrations of 2-aminoethoxydiphenyl borate. Quantitative reverse transcription PCR and Western blot analyses revealed abundant expressions of Orai1, Orai2 and STIM1. Introduction of dominant negative mutant of Orai1, or siOrai1 knockdown significantly attenuated SOCE. Following histamine application, single molecule imaging using total internal reflection fluorescence (TIRF) microscopy demonstrated punctate Orai1–STIM1 complex formation in plasma membrane. In contrast, knockdown or over-expression of Orai2 resulted in an increase or a decrease in SOCE, respectively. Finally, TIRF imaging revealed direct coupling between Orai1 and Orai2, and suggested that Orai2 reduces Orai1 function by formation of a hetero-tetramer. These results provide substantial evidence that Orai1, Orai2 and STIM1 form functional CRAC channels in OUMS-27 cells and that these complexes are responsible for sustained Ca2+ entry in response to agonist stimulation.

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