Dominant role of Orai1 with STIM1 on the cytosolic entry and cytotoxicity of lead ions.
Pb(2+) ions cause severe damages to living cells. In particular, our previous study showed that the Orai-STIM1 (stromal interacting protein 1)-formed store-operated Ca(2+) channels (SOCs) allow Pb(2+) entry. In relation to this, the present study investigates the molecular gating mechanism of Pb(2+) entry by Orai1 with STIM1, as well as the resulting cytotoxicity on human embryonic kidney HEK293 cells. The store-operated Ca(2+) entry (SOCE, activity of SOCs) and Pb(2+) entry were measured using the fura-2 imaging method and indo-1 quenching strategy, as well as through an atomic absorption spectrophotometer. The results of RT-PCR, Western blot, fast confocal, and fluorescent lifetime imaging microscopy indicated the endogenous expression of Orai1 and STIM1 in HEK cells and the functional interaction between these two proteins during SOCE. Both SOCE and Pb(2+) entry largely increased when Orai1 and STIM1 were overexpressed (3- and 1.64-folds, respectively) compared with nonfluorescent cells, and they were significantly attenuated when the E106Q mutation of Orail with STIM1 was cotransfected (6- and 2.25-folds decrease, respectively) compared with Orai1-STIM1 coexpressed cells. The ion gating for Pb(2+) could be governed by the E106 region of Orai1. After sorting and subsequent cultures, the Orai1-STIM1 positive expressed cells behaved more sensitively to Pb(2+) than the Orai1-STIM1 negative cells. In summary, the data suggest that Orai1, together with STIM1, plays a critical role in Pb(2+) entry and the toxicity of Pb(2+).
Department of Medical Research and Education, Taipei Veterans General Hospital, Taiwan, Republic of China.
Tai-Yu
TY
Chiu
Hsiao-Chuan
HC
Teng
Ping-Chun
PC
Huang
Fu-Jen
FJ
Kao
De-Ming
DM
Yang
D000818:Animals; D001692:Biological Transport; D015153:Blotting, Western; D002118:Calcium / Q000378:metabolism; D015220:Calcium Channels / Q000235:genetics / Q000378:metabolism*; D049109:Cell Proliferation / Q000187:drug effects; D002469:Cell Separation; D002470:Cell Survival / Q000187:drug effects; D003600:Cytosol / Q000378:metabolism*; D004721:Endoplasmic Reticulum / Q000187:drug effects / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005434:Flow Cytometry; D006801:Humans; D007854:Lead / Q000378:metabolism* / Q000633:toxicity*; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence; D009154:Mutation; D009363:Neoplasm Proteins / Q000235:genetics / Q000378:metabolism*; D000071740:ORAI1 Protein; D016716:PC12 Cells; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases / Q000037:antagonists & inhibitors / Q000378:metabolism; D013054:Spectrophotometry, Atomic; D000071737:Stromal Interaction Molecule 1; D019284:Thapsigargin / Q000494:pharmacology; D013997:Time Factors; D014162:Transfection
D015220:Calcium Channels; D004791:Enzyme Inhibitors; D008565:Membrane Proteins; D009363:Neoplasm Proteins; D000071740:ORAI1 Protein; C510746:ORAI1 protein, human; D011993:Recombinant Fusion Proteins; C103530:STIM1 protein, human; D000071737:Stromal Interaction Molecule 1; D007854:Lead; D019284:Thapsigargin; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases; D002118:Calcium