Kinetic analysis of ASIC1a delineates conformational signaling from proton-sensing domains to the channel gate

Sabrina Vullo,Olivier Bignucolo,Nicolas Ambrosio,Jan P Kucera,Stephan Kellenberger

doi:10.7554/elife.66488

Sabrina Vullo, Olivier Bignucolo + Show 3 more

Open Access

https://doi.org/10.7554/elife.66488

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Journal: eLife	Publication Date: Mar 17, 2021
Citations: 6	License type: CC BY 4.0

Affiliation: University of Lausanne, University of Bern

Abstract

Acid-sensing ion channels (ASICs) are neuronal Na+ channels that are activated by a drop in pH. Their established physiological and pathological roles, involving fear behaviors, learning, pain sensation, and neurodegeneration after stroke, make them promising targets for future drugs. Currently, the ASIC activation mechanism is not understood. Here, we used voltage-clamp fluorometry (VCF) combined with fluorophore-quencher pairing to determine the kinetics and direction of movements. We show that conformational changes with the speed of channel activation occur close to the gate and in more distant extracellular sites, where they may be driven by local protonation events. Further, we provide evidence for fast conformational changes in a pathway linking protonation sites to the channel pore, in which an extracellular interdomain loop interacts via aromatic residue interactions with the upper end of a transmembrane helix and would thereby open the gate.

Highlights

This study investigates the activation mechanism of acid-sensing ion channels (ASICs), a family of H+gated Na+ channels of the nervous system (Waldmann et al, 1997; Wemmie et al, 2013; Kellenberger and Schild, 2015; Yang and Palmer, 2014)
Fast conformational changes in ASIC domains that are distant from the pore The aim of a first set of experiments was to compare the kinetics of conformational changes in different parts of ASIC1a
ASIC1a constructs containing engineered Cys residues for the docking of maleimide derivatives of fluorophores were expressed in Xenopus laevis oocytes, and oocytes were exposed to maleimide derivatives of AlexaFluor488 or CF488A prior to the measurement

Summary

Introduction

This study investigates the activation mechanism of acid-sensing ion channels (ASICs), a family of H+gated Na+ channels of the nervous system (Waldmann et al, 1997; Wemmie et al, 2013; Kellenberger and Schild, 2015; Yang and Palmer, 2014). ASICs respond to extracellular acidification with a transient current, because after opening, they enter a non-conducting desensitized state (Waldmann et al, 1997; Grunder and Pusch, 2015). Each ASIC subunit consists of short intracellular N- and C-terminal ends, two transmembrane domains TM1 and TM2, and a large extracellular region, with the shape of a hand, organized in defined domains that have been named palm, knuckle, b-ball, thumb, and finger (Figure 1A)

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