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ToxSci Advance Access published online on May 8, 2009
Toxicological Sciences, doi:10.1093/toxsci/kfp099
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© The Author 2009. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved. For permissions, please email: journals.permissions@oxfordjournals.org

Dominant Role of Orai1 with STIM1 on the Cytosolic Entry and Cytotoxicity of Lead Ions

Tai-Yu Chiua,b,+, Hsiao-Chuan Tenga,+, Ping-Chun Huanga,b, Fu-Jen Kaob and De-Ming Yanga,b,*

a Department of Medical Research and Education, Taipei Veterans General Hospital b Institute of Biophotonics, School of Medical Technology and Engineering, National Yang-Ming University, Taipei, Taiwan, Republic of China

* Correspondence to: Dr. De-Ming Yang, Department of Medical Research and Education, Taipei Veterans General Hospital; Institute of Biophotonics, National Yang-Ming University, Taipei 11217, Taiwan, Republic of China. Phone: 886-2-2875-7747 ; Fax: 886-2-2875-7435, E-mail: yang.deming{at}gmail.com

Received February 6, 2009; revision received April 30, 2009; accepted May 1, 2009


  Abstract

Pb2+ ions cause severe damages to living cells. In particular, our previous study showed that the Orai-STIM1-formed store-operated Ca2+ channels (SOCs) allow Pb2+ entry. In relation to this, the present study investigates the molecular gating mechanism of Pb2+ entry by Orai1 with STIM1, as well as the resulting cytotoxicity on human embryonic kidney HEK293 cells. The store-operated Ca2+ entry (SOCE, activity of SOCs) and Pb2+ 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 Pb2+ entry largely increased when Orai1 and STIM1 were overexpressed (3- and 1.64-folds, respectively) compared with non-fluorescent cells, and they were significantly attenuated when the E106Q mutation of Orail with STIM1 was co-transfected (6- and 2.25-folds decrease, respectively) compared with Orai1-STIM1 co-expressed cells. The ion gating for Pb2+ could be governed by the E106 region of Orai1. After sorting and subsequent cultures, the Orai1-STIM1 positive expressed cells behaved more sensitively to Pb2+ than the Orai1-STIM1 negative cells. In summary, the data suggest that Orai1, together with STIM1, plays a critical role in Pb2+ entry and the toxicity of Pb2+.

Key Words: Pb2+; HEK293 cells; Indo-1; store-operated Ca2+ channels; Orai1; STIM1; fluorescence lifetime imaging.

+ Equal contribution


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