Proton binding sites involved in the activation of acid-sensing ion channel ASIC2a

doi:10.1016/j.neulet.2007.07.047

Neuroscience Letters

Volume 426, Issue 1, 9 October 2007, Pages 12-17

https://doi.org/10.1016/j.neulet.2007.07.047 Get rights and content

Abstract

Most acid-sensing ion channel (ASIC) subunits are activated by protons, but ASIC2b (a splice variant of ASIC2a) is acid-insensitive. Differences in protonatable residues between the extracellular loop regions of ASIC2a and ASIC2b may explain this difference. Site-directed mutagenesis, combined with immunocytochemistry and whole-cell patch clamp, demonstrated that mutating any one of five ASIC2a sites produces channels that traffic normally to the cell surface membrane but are insensitive to protons. One of the mutants forms functional heteromers with ASIC1a and ASIC2a, demonstrating that ion transport is intact in this mutant. These five sites may be involved in the activation of ASIC2a by protons.

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Acknowledgements

This work was supported by a Medical Research Council PhD studentship (E.St.J.S.) and by the Biotechnology and Biological Sciences Research Council (X.Z., H.C. and P.A.M.). We thank Prof. M. Lazdunski for the gift of rat ASIC1a, ASIC2a and ASIC2b plasmids and Prof. G. Lewin for the ASIC2 antibody. The advice and assistance of Dr. Jiehong Huang was greatly appreciated.

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Citation Excerpt :
All constructs were expressed in Chinese hamster ovary (CHO) cells that lack endogenous ASICs (17), and whole-cell patch clamp electrophysiology was used to measure responses to a pH 4.0 stimulus from a starting pH of pH 7.4. Although mASIC3 and rASIC3 robustly responded to protons with a stereotypical transient ASIC-like current (mASIC3: 69 ± 13 pA/pF, n = 26, Fig. 1A, and rASIC3: 627 ± 92 pA/pF, n = 18, Fig. 1B), nmrASIC3 failed to respond with an ASIC-like response to protons, even using a pH 3.0 stimulus, but rather produced a very small, non-inactivating response, similar to that which we have observed previously in non-transfected CHO cells (17) and in CHO cells transfected with the proton-insensitive ASIC2b (22) (pH 4.0, n = 24 and pH 3.0, n = 13, 7 separate transfections, Fig. 1C); a summary of all data are given in Table 1. We also investigated whether the non-proton ASIC3 agonist GMQ could activate nmrASIC3, but whereas we observed GMQ-mediated inward currents in cells expressing mASIC3 (Fig. 1D, 14.5 ± 3.0 pA/pF, 6 of 18 pH-sensitive cells responded) and rASIC3 (Fig. 1E, 180.6 ± 55.1 pA/pF, n = 8, all pH-sensitive cells responded), nmrASIC3 failed to respond (Fig. 1F, n = 11 cells from 4 transfections).
Acid-sensing ion channels (ASICs) form both homotrimeric and heterotrimeric ion channels that are activated by extracellular protons and are involved in a wide range of physiological and pathophysiological processes, including pain and anxiety. ASIC proteins can form both homotrimeric and heterotrimeric ion channels. The ASIC3 subunit has been shown to be of particular importance in the peripheral nervous system with pharmacological and genetic manipulations demonstrating a role in pain. Naked mole-rats, despite having functional ASICs, are insensitive to acid as a noxious stimulus and show diminished avoidance of acidic fumes, ammonia, and carbon dioxide. Here we cloned naked mole-rat ASIC3 (nmrASIC3) and used a cell-surface biotinylation assay to demonstrate that it traffics to the plasma membrane, but using whole-cell patch clamp electrophysiology we observed that nmrASIC3 is insensitive to both protons and the non-proton ASIC3 agonist 2-guanidine-4-methylquinazoline. However, in line with previous reports of ASIC3 mRNA expression in dorsal root ganglia neurons, we found that the ASIC3 antagonist APETx2 reversibly inhibits ASIC-like currents in naked mole-rat dorsal root ganglia neurons. We further show that like the proton-insensitive ASIC2b and ASIC4, nmrASIC3 forms functional, proton-sensitive heteromers with other ASIC subunits. An amino acid alignment of ASIC3s between 9 relevant rodent species and human identified unique sequence differences that might underlie the proton insensitivity of nmrASIC3. However, introducing nmrASIC3 differences into rat ASIC3 (rASIC3) produced only minor differences in channel function, and replacing the nmrASIC3 sequence with that of rASIC3 did not produce a proton-sensitive ion channel. Our observation that nmrASIC3 forms nonfunctional homomers may reflect a further adaptation of the naked mole-rat to living in an environment with high-carbon dioxide levels.

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¹: Present address: Max-Delbruck Centre for Molecular Medicine, Robert-Rossle Str. 10, 13125 Berlin-Buch, Germany.

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Proton binding sites involved in the activation of acid-sensing ion channel ASIC2a

Abstract

Section snippets

Acknowledgements

J. Biol. Chem.

J. Biol. Chem.

Neuron

Trends Neurosci.

Neuroscience

J. Biol. Chem.

Neuroscience

Neuroscience

Neuroscience

Neuron

Trends Neurosci.

A new member of the acid-sensing ion channel family

Neuroreport

Endogenous proton-gated cation channels in cell lines and Xenopus oocytes

J. Physiol.