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

Abstract

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+).

Potential effect of resistin on the ET‐1‐increased reactions of blood pressure in rats and Ca2+ signaling in vascular smooth muscle cells

“…RNA samples for RT‐PCR were incubated for 30 min at 37°C with RNase‐free DNase I, and then for 10 min at 100°C to inactivate the DNase I. With Finnzymes PCR reagents (Finnzymes OY, Finland), RT‐PCR of Orai and STIM isoforms from the A10 cells was performed according to previously described procedures, including primer design, the general protocol for RNA extraction, and cDNA synthesis (Chang et al, 2008; Chiu et al, 2009).…”

“…Whole cell lysates for the Western blot analysis of Orais and STIMs (Chiu et al, 2009) were prepared by sonication of the cells in lysis buffer (1% Triton X‐100, 50 mM KCl, 25 mM HEPES, pH 7.8, 10 µg/ml leupeptin, 20 µg/ml aprotinin, 125 µM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride, and 1 mM sodium orthovanadate). Samples (100 µg of total protein) in 50 µl reducing sample buffer were then boiled for 10 min and resolved on 10% SDS gels.…”

“…The essential players of SOCs consist of at least two or three kinds of proteins: Transient receptor potentials (TRPs) (Parekh and Putney, 2005; Saleh et al, 2008; Birnbaumer, 2009; Saleh et al, 2009) and Ca 2+ release‐activated Ca 2+ modulators (CRACMs/Orais) (Feske et al, 2006; Vig et al, 2006), together with ER membrane protein stromal interaction molecules (STIMs) (Liou et al, 2005; Takahashi et al, 2007). On the functional aspect of SOCs, specifically, the driving of store‐operated Ca 2+ entry (SOCE), the translocation of STIM1 from the ER to the periplasmic region, and the interaction of STIM1 with Orai1 (Chiu et al, 2009; Potier et al, 2009), TRPC1, (Yuan et al, 2007; Li et al, 2008), or both (Birnbaumer, 2009), have been proven to be critical steps during the activation of SOCs. These SOC components play important pathophysiological roles in both the proliferation and migration of SMCs and the coupling of Ca 2+ ‐contraction/regulation of blood pressure (Potier et al, 2009; Bisaillon et al, 2010).…”

Chronic Kidney Disease and Exposure to Nephrotoxic Metals

“…In addition, exposure to Pb has been shown to alter the subcellular distribution of calcium in renal cells [262]. Specifically, exposure to Pb resulted in elevated levels of calcium in the cytoplasm and mitochondria while calcium levels in the endoplasmic reticulum were depleted.…”

“…Specifically, exposure to Pb resulted in elevated levels of calcium in the cytoplasm and mitochondria while calcium levels in the endoplasmic reticulum were depleted. These changes in intracellular calcium ratios may lead to significant alterations in intracellular signaling pathways and eventual apoptosis [262]. Interestingly, administration of the calcium channel blockers, verapamil and nimodipine, have been shown to decrease lipid oxidation and increase the activities of superoxide dismutase and glutathione peroxidase [263].…”