ASICs are potential drug targets of interest. Their activation mechanism has however remained elusive. ASICs are neuronal, proton-gated, sodium-permeable channels that are expressed in the central and peripheral nervous system of vertebrates. They form a subfamily of the Epithelial Na channel / degenerin channel family, and contribute to pain sensation, fear, learning, and neurodegeneration after ischemic stroke. Depending on the extracellular pH, they exist in either one of three functional states: closed (resting), open and desensitized. While ASICs are at physiological pH 7.4 in the closed state, they open briefly upon extracellular acidification, before entering the non-conducting desensitized state. Crystal structures of the chicken ASIC1 channel in the desensitized and the open state were published several years ago. This structural information allowed, together with observations from functional studies, an understanding of the transitions between the open and the desensitized state. In contrast, the absence of structural information on the closed conformation of ASICs precluded so far a molecular understanding of their activation mechanism.
The Gouaux laboratory has now published structures of the homotrimeric chicken ASIC1 obtained at high pH by X-ray crystallography (2.95 Å resolution) and by single particle cryo-electron microscopy (3.7 Å) (1). These structures show a channel with a closed pore, representing likely the closed state. The overall structural organization is the same in all ASIC 3D structures published so far: each subunit consists of a large, complex ectodomain, two transmembrane domains, and short N- and C-termini (whose structure has not been resolved yet). The channel is formed by three identical subunits that are arranged around the central ion pore. A vestibule containing many acidic residues, the “acidic pocket”, is located on the outward-facing side of the ectodomain of each subunit, at 40-50 Å from the membrane. The main difference in the ectodomain between the closed ASIC structures and previously published open and desensitized structures is a wide opening of the acidic pocket in the structure of the closed channel.
Based on the comparison of closed, open and desensitized structures, the authors suggest the following activation mechanism: At physiological pH 7.4 the channel pore is closed and the acidic pocket has adapted an extended conformation. Extracellular acidification protonates acidic residues of the acidic pocket, thereby reducing repulsion between such residues and leading to a collapse of the acidic pocket. This movement is transmitted via central channel domains to the transmembrane helices, and leads to opening of the channel pore. A short time later, an additional movement in the central domains uncouples the ion pore from the acidic pocket and allows the transmembrane domains to relax to the non-conducting desensitized conformation. The acidic pocket will adapt its extended conformation only once the extracellular pH has returned to higher values.
This new 3D structure is undoubtedly a breakthrough in the understanding of the molecular mechanisms of ASIC activity. Some open questions remain however:
Several studies have shown that protonation events in domains other than the acidic pocket contribute to activation and desensitization, and it has also been shown that a channel in which most of the acidic residues in the acidic pocket have been neutralized can still be opened by extracellular acidification. These studies suggest that an important part of the drive for the conformational changes comes from protonation events outside the acidic pocket. This is different from the activation mechanism proposed by Yoder and colleagues, which relies on protonation events in the acidic pocket.
The cytoplasmic N- and C-termini of ASIC subunits contain sites important for ASIC function and ion selectivity. So far there is no structural information on these intracellular parts available. Future cryo-electron microscopy approaches will hopefully have the power to resolve the conformation of these domains.
Comments by Stephan Kellenberger, Université de Lausanne, Switzerland
1. Yoder, N., Yoshioka, C., and Gouaux, E. (2018) Gating mechanisms of acid-sensing ion channels. Nature, 555: 397-401. doi: 10.1038/nature25782. [PMID:29513651]