Inhibitory regulation of acid-sensing ion channel 3 by zinc

doi:10.1016/j.neuroscience.2010.05.043

Neuroscience

Volume 169, Issue 2, 25 August 2010, Pages 574-583

https://doi.org/10.1016/j.neuroscience.2010.05.043 Get rights and content

Abstract

Acid-sensing ion channel 3 (ASIC3) is a proton-gated, voltage-insensitive Na⁺ channel that is expressed primarily in peripheral sensory neurons and plays an important role in pain perception, particularly as a pH sensor following cardiac ischemia. We previously reported that ASIC3 currents are not affected by zinc at nanomolar concentrations. In this study, we examined the potential role of micromolar zinc in the regulation of ASIC3. In CHO cells expressing ASIC3, we found that ASIC3 currents triggered by dropping the pH from 7.4 to 6.0 were inhibited by pretreatment with zinc in a concentration-dependent manner; the half-maximum inhibitory concentration of zinc was 61 μM. ASIC currents activated by a relatively small drop in pH from 7.4 to 7.2 or 7.0 were also subject to inhibition by zinc. The inhibition was fast and pH independent, and occurred within a relatively narrow range of zinc concentrations between 30 and 300 μM. Further, increasing extracellular Ca²⁺ concentrations from 2 to 10 mM failed to affect inhibition of ASIC3 currents by zinc. Experimentally elevating intracellular zinc levels did not affect the inhibition of ASIC3 currents by equal concentrations of extracellular zinc, and modification of cysteine or histidine residues had no effect on the inhibition of ASIC3 currents by zinc. These collective results suggest that zinc is an important regulator of ASIC3 at physiological concentrations, that zinc inhibits ASIC3 in a pH- and Ca²⁺-independent manner, and that inhibition of ASIC3 currents is dependent upon the interaction of zinc with extracellular domain(s) of ASIC3.

Section snippets

Tissue culture and ASICs transient expression

Tissue culture and transfection of CHO cells with various ASIC subunits were described in detail previously (Chu et al., 2004, Chu et al., 2006). Briefly, CHO cells were maintained in standard F12 medium (American Type Culture Collection, Manassas, VA, USA) supplemented with 10% fetal bovine serum (Invitrogen, Grand Island, NY, USA) 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. At∼50%

Zinc does not affect ASIC3 channels in an open state

Our previous study demonstrated that TPEN, a high-affinity zinc chelator, enhances ASIC1a, but not ASIC1b, ASIC2a, or ASIC3 currents. This suggests that zinc inhibits ASIC1a, but not ASIC1b, ASIC2a or ASIC3, subunits with a high-affinity (Chu et al., 2004). To investigate whether zinc might affect ASIC3 currents at physiologically relevant (i.e. at micromolar) concentrations, CHO cells expressing ASIC3 were recorded using a whole-cell voltage clamp configuration. Rapid inward ASIC3 currents

Discussion

Previous studies have shown that zinc regulates ASICs (Baron et al., 2001, Chu et al., 2004, Wu et al., 2004, Poirot et al., 2006, Xu and Xiong, 2007). It inhibits ASIC1a and ASIC1a-containing channels with a high affinity (IC₅₀∼10 nM) (Chu et al., 2004) but potentiates ASIC2a and ASIC2a-containing channels with a low affinity (EC₅₀∼110 μM) (Baron et al., 2001). Inhibition of recombinant ASIC3 channels by zinc at a concentration of 300 μM has also been described (Poirot et al., 2006). In the

Acknowledgments

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).

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Acid-sensing ion channels (ASICs) are involved in numerous physiological and pathological processes in the central nervous system. Development of pharmacological tools capable to inhibit or potentiate these channels is important for our knowledge about roles of ASICs in the neuronal network and can be promising for treatment of some disorders. Recently we described four hydrophobic monoamines that potentiate and inhibit ASICs depending on subunit composition of the channel and peculiarities of the drug structure. In the present work we performed structure-activity relationship analysis using derivatives of adamantane, phenylcyclohexyl and 9-aminoacridine to reveal the main determinants of action of amine-containing compounds on recombinant ASIC1a and ASIC2a homomers expressed in CHO cells. We found that the most active compounds are monocations with protonatable aminogroup. In general, potentiators and inhibitors of ASIC1a we found, but only potentiators for ASIC2a. Flat aromatic structure of the headgroup determines inhibition of ASIC1a while “V-shape” structure of the hydrophobic moiety favors potentiation of ASIC2a. Moreover, for some series of monoamines there was a correlation between action on ASIC1a and ASIC2a, the weaker ASIC1a inhibition, the stronger ASIC2a potentiation. Decay of response was accelerated by ASIC1a inhibitors as well as by potentiators. All compounds potentiating ASIC2a slowed down desensitization. Our results suggest that hydrophobic amines cause complex action on ASICs.
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Acid-sensing ion channels (ASICs) are widely expressed in the body and represent good sensors for detecting protons. The pH drop in the nervous system is equivalent to ischemia and acidosis, and ASICs are very good detectors in discriminating slight changes in acidity. ASICs are important pharmacological targets being involved in a variety of pathophysiological processes affecting both the peripheral nervous system (e.g., peripheral pain, diabetic neuropathy) and the central nervous system (e.g., stroke, epilepsy, migraine, anxiety, fear, depression, neurodegenerative diseases, etc.). This review discusses the role played by ASICs in different pathologies and the pharmacological agents acting on ASICs that might represent promising drugs. As the majority of above-mentioned pathologies involve not only neuronal dysfunctions but also microvascular alterations, in the next future, ASICs may be also considered as potential pharmacological targets at the vasculature level. Perspectives and limitations in the use of ASICs antagonists and modulators as pharmaceutical agents are also discussed.
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Development of the pharmacology of Acid-Sensing Ion Channels (ASICs) has become a key challenge to study their structure, their molecular and cellular functions and their physiopathological roles. This review provides a summary of the different compounds that directly interact with these channels, either with inhibitory or stimulatory effect, and with high selectivity or poor specificity. They include drugs and endogenous regulators, natural compounds of vegetal origin, and peptides isolated from animal venoms. The in vivo use of some of these pharmacological modulators in animal models and a few small clinical studies in humans have provided substantial data on the physiological and physiopathological roles of ASIC channels. Modulation of these channels will certainly provide new therapeutic opportunities in neurological and psychiatric diseases including pain, stroke, epilepsy, anxiety, depression or traumatic injury, as well as in some non-neurological pathologies.
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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.

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Cellular and Molecular NeuroscienceResearch PaperInhibitory regulation of acid-sensing ion channel 3 by zinc

Abstract

Section snippets

Tissue culture and ASICs transient expression

Zinc does not affect ASIC3 channels in an open state

Discussion

Acknowledgments

J Biol Chem

Neuroscience

J Biol Chem

Neuron

J Neurosci Methods

Adv Protein Chem

J Biol Chem

J Pain

Neuron

Neuroscience

J Biol Chem

Trends Neurosci

Neuroscience

J Biol Chem

J Biol Chem

Pharmacol Ther

J Biol Chem

Neuroscience

Neuron

J Biol Chem

Pain

Prog Neurobiol

Biophys J

J Biol Chem

Curr Opin Neurobiol

Trends Neurosci

J Biol Chem

Curr Opin Pharmacol

Cellular and Molecular Neuroscience
Research Paper
Inhibitory regulation of acid-sensing ion channel 3 by zinc