A Combined Computational and Functional Approach Identifies New Residues Involved in pH-dependent Gating of ASIC1a

Justyna Iwaszkiewicz,Simon Bernèche,Olivier Michielin,Stephan Kellenberger,Benoîte Bargeton,Sophie Roy,Luz Angélica Liechti

doi:10.1074/jbc.m109.092015

Abstract

Acid-sensing ion channels (ASICs) are key receptors for extracellular protons. These neuronal nonvoltage-gated Na(+) channels are involved in learning, the expression of fear, neurodegeneration after ischemia, and pain sensation. We have applied a systematic approach to identify potential pH sensors in ASIC1a and to elucidate the mechanisms by which pH variations govern ASIC gating. We first calculated the pK(a) value of all extracellular His, Glu, and Asp residues using a Poisson-Boltzmann continuum approach, based on the ASIC three-dimensional structure, to identify candidate pH-sensing residues. The role of these residues was then assessed by site-directed mutagenesis and chemical modification, combined with functional analysis. The localization of putative pH-sensing residues suggests that pH changes control ASIC gating by protonation/deprotonation of many residues per subunit in different channel domains. Analysis of the function of residues in the palm domain close to the central vertical axis of the channel allowed for prediction of conformational changes of this region during gating. Our study provides a basis for the intrinsic ASIC pH dependence and describes an approach that can also be applied to the investigation of the mechanisms of the pH dependence of other proteins.

Highlights

Residues Involved in ASIC Gating—Fig. 7B and the supplemental video show on a single ASIC1a subunit residues whose mutation has affected ASIC pH dependence in this and previous functional studies
Hypothesis on ASIC Gating—The pKa calculation and the functional data suggest that in the closed conformation the thumb is located farther away from the ␤-ball and the finger than it is in the crystal structure, due to the repulsion of negatively charged residues
The results presented in this work, together with previous studies, provide strong evidence that ASIC gating does not depend on the protonation and deprotonation of only a few pH-sensing residues but that many different residues in each subunit contribute to pH sensing for ASIC gating

Summary

Introduction

Residues Involved in ASIC Gating—Fig. 7B and the supplemental video show on a single ASIC1a subunit residues whose mutation has affected ASIC pH dependence in this and previous functional studies. Mutations of residues of the ␣5 helix of the thumb (Asp-347, Asp-351, and Glu-355), of the ␤-ball (␤4-␤5 loop, Arg-190; ␤7-␤8 loop, Asp-253 and Glu254), and on the interacting finger loop that originates in the strands ␤6 and ␤7 of the ␤-ball (Glu-235, Asp-237, and Glu238) affected ASIC activation

Results

Conclusion