title: Orai1-STIM1 formed store-operated Ca2+ channels (SOCs) as the molecular components needed for Pb2+ entry in living cells.
abstract: Heavy metal lead (Pb2+) is a pollutant and causes severe toxicity when present in human tissues especially the nervous system. Recent reviews have suggested that Pb2+ can target Ca2+-related proteins within neurons and that Ca2+ channels might be a candidate for Pb2+ entry. This study's main aim was to identify the functional entry pathway of Pb2+ into living cells. We firstly characterized the endogenous expression of Orai1 and STIM1 mRNA together with the level of thapsigargin (TG) stimulated capacitative Ca2+ entry in PC12 and HeLa cells; this was done by RT-PCR and time-lapse Ca2+ imaging microscopy, respectively. Our data supported Orai1 and STIM1 as contributing to store-operated Ca2+ channel (SOC) basal activity. Secondly, using the indo-1 quenching method with the SOC blocker 2-APB, we observed that Pb2+ was able to enter cells directly through unactivated SOCs without TG pretreatment. Thirdly, we further demonstrated that co-expression of Orai1 and STIM1 differentially enhanced SOC functional activity (4-fold with PC12 and 5-fold with HeLa cells) and Pb2+ entry (5- to 7-fold with PC12 and 2-fold with HeLa cells). Furthermore, after a 1 h of Pb2+ exposure, the depolarization- and histamine-induced Ca2+ responses were significantly decreased in both PC12 and HeLa cells in a dose-dependent manner. This result indicated that the decreased Ca2+ responses were, in part, due to Pb2+ entry. In summary, our results suggest that SOCs are responsible for Pb2+ permeation and that the Orai1-STIM1 protein complex formed by functional SOCs is one of the molecular components involved in Pb2+ entry.
authors.affiliation: Department of Medical Research and Education, Taipei Veterans General Hospital, Taiwan, Republic of China.
authors.forename: Yu-Fen
authors.initials: YF
authors.lastname: Chang
authors.forename: Hsiao-Chuan
authors.initials: HC
authors.lastname: Teng
authors.forename: Sha-Yen
authors.initials: SY
authors.lastname: Cheng
authors.forename: Chin-Tien
authors.initials: CT
authors.lastname: Wang
authors.forename: Shi-Hwa
authors.initials: SH
authors.lastname: Chiou
authors.forename: Lung-Sen
authors.initials: LS
authors.lastname: Kao
authors.forename: Fu-Jen
authors.initials: FJ
authors.lastname: Kao
authors.forename: Arthur
authors.initials: A
authors.lastname: Chiou
authors.forename: De-Ming
authors.initials: DM
authors.lastname: Yang
mesh_terms: D000818:Animals; D001692:Biological Transport; D001896:Boron Compounds / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism; D002121:Calcium Channel Blockers / Q000494:pharmacology; D015220:Calcium Channels / Q000032:analysis / Q000187:drug effects / Q000378:metabolism*; D002413:Cations, Divalent / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D007854:Lead / Q000378:metabolism*; D008565:Membrane Proteins / Q000032:analysis / Q000378:metabolism*; D009363:Neoplasm Proteins / Q000032:analysis / Q000378:metabolism*; D000071740:ORAI1 Protein; D016716:PC12 Cells; D051381:Rats; D000071737:Stromal Interaction Molecule 1
chemical_list: D001896:Boron Compounds; D002121:Calcium Channel Blockers; D015220:Calcium Channels; D002413:Cations, Divalent; D008565:Membrane Proteins; D009363:Neoplasm Proteins; D000071740:ORAI1 Protein; C510746:ORAI1 protein, human; C103530:STIM1 protein, human; D000071737:Stromal Interaction Molecule 1; D007854:Lead; C109986:2-aminoethoxydiphenyl borate; D002118:Calcium