Elsevier

Neuroscience

Volume 193, 13 October 2011, Pages 89-99
Neuroscience

Cellular and Molecular Neuroscience
Research Paper
Cysteine 149 in the extracellular finger domain of acid-sensing ion channel 1b subunit is critical for zinc-mediated inhibition

https://doi.org/10.1016/j.neuroscience.2011.07.021Get rights and content

Abstract

Acid-sensing ion channel 1b (ASIC1b) is a proton-gated Na+ channel mostly expressed in peripheral sensory neurons. To date, the functional significance of ASIC1b in these cells is unclear due to the lack of a specific inhibitor/blocker. A better understanding of the regulation of ASIC1b may provide a clue for future investigation of its functional importance. One important regulator of acid-sensing ion channels (ASICs) is zinc. In this study, we examined the detailed zinc inhibition of ASIC1b currents and specific amino acid(s) involved in the inhibition. In Chinese hamster ovary (CHO) cells expressing rat ASIC1b subunit, pretreatment with zinc concentration-dependently inhibited the ASIC1b currents triggered by pH dropping from 7.4 to 6.0 with a half-maximum inhibitory concentration of 26 μM. The inhibition of ASIC1b currents by pre-applied zinc was independent of pH, voltage, or extracellular Ca2+. Further, we showed that the effect of zinc is dependent on the extracellular cysteine, but not histidine residue. Mutating cysteine 149, but not cysteine 58 or cysteine 162, located in the extracellular domain of the ASIC1b subunit abolished the zinc inhibition. These findings suggest that cysteine 149 in the extracellular finger domain of ASIC1b subunit is critical for zinc-mediated inhibition and provide the basis for future mechanistic studies addressing the functional significance of zinc inhibition of ASIC1b.

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).

References (61)

  • D.C. Immke et al.

    Protons open acid-sensing ion channels by catalyzing relief of Ca2+ blockade

    Neuron

    (2003)
  • Q. Jiang et al.

    Characterization of acid-sensing ion channels in medium spiny neurons of mouse striatum

    Neuroscience

    (2009)
  • Q. Jiang et al.

    Inhibitory regulation of acid-sensing ion channel 3 by zinc

    Neuroscience

    (2010)
  • O. Krishtal

    The ASICs: signaling molecules? Modulators?

    Trends Neurosci

    (2003)
  • E. Lingueglia

    Acid-sensing ion channels in sensory perception

    J Biol Chem

    (2007)
  • A. Mathie et al.

    Zinc and copper: pharmacological probes and endogenous modulators of neuronal excitability

    Pharmacol Ther

    (2006)
  • S.T. Nevin et al.

    Insights into the structural basis for zinc inhibition of the glycine receptor

    J Biol Chem

    (2003)
  • P. Paoletti et al.

    Zinc at glutamatergic synapses

    Neuroscience

    (2009)
  • Y. Pfister et al.

    A gating mutation in the internal pore of ASIC1a

    J Biol Chem

    (2006)
  • K.A. Sluka et al.

    Chronic hyperalgesia induced by repeated acid injections in muscle is abolished by the loss of ASIC3, but not ASIC1

    Pain

    (2003)
  • K.A. Sluka et al.

    ASIC3 in muscle mediates mechanical, but not heat, hyperalgesia associated with muscle inflammation

    Pain

    (2007)
  • T.G. Smart et al.

    Modulation of inhibitory and excitatory amino acid receptor ion channels by zinc

    Prog Neurobiol

    (1994)
  • S. Ugawa et al.

    Hypotonic stimuli enhance proton-gated currents of acid-sensing ion channel-1b

    Biochem Biophys Res Commun

    (2008)
  • R.Y. Walder et al.

    ASIC1 and ASIC3 play different roles in the development of Hyperalgesia after inflammatory muscle injury

    J Pain

    (2010)
  • R. Waldmann et al.

    Molecular cloning of a non-inactivating proton-gated Na+ channel specific for sensory neurons

    J Biol Chem

    (1997)
  • R. Waldmann et al.

    H+-gated cation channels: neuronal acid sensors in the ENaC/DEG family of ion channels

    Curr Opin Neurobiol

    (1998)
  • J.A. Wemmie et al.

    Acid-sensing ion channels: advances, questions and therapeutic opportunities

    Trends Neurosci

    (2006)
  • L.J. Wu et al.

    Characterization of acid-sensing ion channels in dorsal horn neurons of rat spinal cord

    J Biol Chem

    (2004)
  • Z.G. Xiong et al.

    Acid-sensing ion channels (ASICs) as pharmacological targets for neurodegenerative diseases

    Curr Opin Pharmacol

    (2008)
  • Z.G. Xiong et al.

    Neuroprotection in ischemia: blocking calcium-permeable acid-sensing ion channels

    Cell

    (2004)
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