title: Orai1-Orai2 complex is involved in store-operated calcium entry in chondrocyte cell lines.
abstract: Ca(2+) influx via store-operated Ca(2+) entry (SOCE) plays critical roles in many essential cellular functions. The Ca(2+) release-activated Ca(2+) (CRAC) channel complex, consisting of Orai and STIM, is one of the major components of store-operated Ca(2+) (SOC) channels. Our previous study demonstrated that histamine can cause sustained Ca(2+) 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 Ca(2+) entry in response to agonist stimulation.
authors.affiliation: Department of Molecular & Cellular Pharmacology, Graduate School of Pharmaceutical Sciences, Nagoya City University, Nagoya 467-8603, Japan.
authors.forename: Munenori
authors.initials: M
authors.lastname: Inayama
authors.affiliation: Department of Molecular & Cellular Pharmacology, Graduate School of Pharmaceutical Sciences, Nagoya City University, Nagoya 467-8603, Japan.
authors.forename: Yoshiaki
authors.initials: Y
authors.lastname: Suzuki
authors.affiliation: Department of Molecular & Cellular Pharmacology, Graduate School of Pharmaceutical Sciences, Nagoya City University, Nagoya 467-8603, Japan.
authors.forename: Satoshi
authors.initials: S
authors.lastname: Yamada
authors.affiliation: Department of Molecular & Cellular Pharmacology, Graduate School of Pharmaceutical Sciences, Nagoya City University, Nagoya 467-8603, Japan.
authors.forename: Takashi
authors.initials: T
authors.lastname: Kurita
authors.affiliation: Department of Molecular & Cellular Pharmacology, Graduate School of Pharmaceutical Sciences, Nagoya City University, Nagoya 467-8603, Japan.
authors.forename: Hisao
authors.initials: H
authors.lastname: Yamamura
authors.affiliation: Department of Molecular & Cellular Pharmacology, Graduate School of Pharmaceutical Sciences, Nagoya City University, Nagoya 467-8603, Japan; Department of Pharmacology, Division of Pathological Sciences, Kyoto Pharmaceutical University, Kyoto 607-8414, Japan.
authors.forename: Susumu
authors.initials: S
authors.lastname: Ohya
authors.affiliation: Faculty of Kinesiology, University of Calgary, Calgary, Alberta T2N 1N4, Canada.
authors.forename: Wayne R
authors.initials: WR
authors.lastname: Giles
authors.affiliation: Department of Molecular & Cellular Pharmacology, Graduate School of Pharmaceutical Sciences, Nagoya City University, Nagoya 467-8603, Japan. Electronic address: yimaizum@phar.nagoya-cu.ac.jp.
authors.forename: Yuji
authors.initials: Y
authors.lastname: Imaizumi
mesh_terms: D002118:Calcium / Q000378:metabolism*; D015220:Calcium Channels / Q000187:drug effects / Q000235:genetics / Q000502:physiology*; D002460:Cell Line; D002478:Cells, Cultured; D019902:Chondrocytes / Q000166:cytology / Q000378:metabolism*; D055785:Gene Knockdown Techniques; D006801:Humans; D008565:Membrane Proteins / Q000187:drug effects / Q000235:genetics / Q000502:physiology*; D009363:Neoplasm Proteins / Q000502:physiology; D000071740:ORAI1 Protein; D000071741:ORAI2 Protein; D024681:Potassium Channels, Calcium-Activated / Q000502:physiology; D034741:RNA, Small Interfering / Q000494:pharmacology; D000071737:Stromal Interaction Molecule 1
chemical_list: D015220:Calcium Channels; D008565:Membrane Proteins; D009363:Neoplasm Proteins; D000071740:ORAI1 Protein; C510746:ORAI1 protein, human; D000071741:ORAI2 Protein; C514506:ORAI2 protein, human; D024681:Potassium Channels, Calcium-Activated; D034741:RNA, Small Interfering; C103530:STIM1 protein, human; D000071737:Stromal Interaction Molecule 1; D002118:Calcium
keywords: Chondrocyte; Orai1; Orai2; Store-operated calcium entry (SOCE); Stromal interaction molecule 1 (STIM1); Total internal reflection fluorescence (TIRF) microscopy