Cellular and Molecular NeuroscienceResearch PaperCysteine 149 in the extracellular finger domain of acid-sensing ion channel 1b subunit is critical for zinc-mediated inhibition
Highlights
▶Pretreatment with zinc concentration-dependently inhibits ASIC1b currents. ▶Zinc inhibition of ASIC1b is independent of pH, membrane potential and extracellular Ca2+. ▶Extracellular domain of ASIC1b subunit is involved in the zinc-mediated inhibition. ▶Cysteine 149 residue in the ASIC1b is essential for zinc-mediated inhibition.
Section snippets
ASICs transient expression in Chinese hamster ovary cells
Tissue culture and transfection of Chinese hamster ovary (CHO) cells with various ASIC subunits were described in detail previously (Chu et al., 2004, Chu et al., 2006, Jiang et al., 2010). Briefly, CHO cells were maintained in standard F12 medium (American Type Culture Collection, Manassas, VA, USA) supplemented with 10% fetal bovine serum at 37 °C in a CO2 incubator. Cells were split with trypsin-EDTA, plated on a 35-mm culture dish at 10% confluence, and allowed to recover for 24 h at 37 °C.
Pretreatment with zinc, but not co-application, concentration-dependently inhibits ASIC1b currents
In contrast to ASIC1a currents, ASIC1b currents are not affected by zinc at nanomolar concentrations (Chu et al., 2004). However, it has been demonstrated that, at micromolar concentrations, zinc significantly inhibited the ASIC1b currents (Poirot et al., 2006). To determine the detailed concentration–response relationship for zinc effect on ASIC1b current, CHO cells expressing ASIC1b were pretreated with varying zinc concentrations for 2 min before decreasing the pH, under a whole-cell voltage
Discussion
In the present study, we examined the effect of zinc on homomeric ASIC1b channels expressed in CHO cells. We showed that: (1) homomeric ASIC1b channels are suppressed by pre-applied zinc with an IC50 of 26 μM, but co-application of zinc with acidic solution does not have an inhibitory effect on ASIC1b currents; (2) zinc-mediated inhibition of ASIC1b channels is independent of pH activation, steady-state desensitization and membrane potential; (3) Zn2+ and Ca2+ inhibit ASIC1b by binding to
Acknowledgments
We thank X.M. Zha for critical comments on the manuscript. This work was supported in part by American Heart Association Scientist Development Grant 0735092N, University of Missouri Research Board and University of Missouri-Kansas City School of Medicine Start-up Funding (X.P.C), and NIH grant R01NS047506 (Z.G.X).
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