Structural and Functional Analysis of Gly212 Mutants Reveals the Importance of Intersubunit Interactions in ASIC1a Channel Function

Abstract

Acid-sensing ion channels (ASICs) act as pH sensors in neurons. ASICs contribute to pain sensation, learning, fear behavior and to neuronal death after ischemic stroke. Extracellular acidification induces a transient activation and subsequent desensitization of these Na +-selective channels. ASICs are trimeric channels made of identical or homologous subunits. We have previously shown that mutation of the highly conserved Gly212 residue of human ASIC1a to Asp affects the channel function. Gly212 is located in the proximity of a predicted Cl − binding site at a subunit interface. Here, we have measured the function of a series of Gly212 mutants. We show that substitution of Gly212 affects the ASIC1a pH dependence and current decay kinetics. Intriguingly, the mutations to the acidic residues Asp and Glu have opposing effects on the pH dependence and the current decay kinetics. Analysis of molecular dynamics simulation trajectories started with the coordinates of the closed conformation indicates that the immediate environment of residue 212 in G212E, which shifts the pH dependence to more alkaline values, adopts a conformation closer to the open state. The G212D and G212E mutants have a different pattern of intersubunit salt bridges, that, in the case of G212E, leads to an approaching of neighboring subunits. Based on the comparison of crystal structures, the conformational changes in this zone appear to be smaller during the open-desensitized transition. Nevertheless, MD simulations highlight differences between mutants, suggesting that the changed function upon substitution of residue 212 is due to differences in intra-and intersubunit interactions in its proximity.

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

“…The pH dependence of the F signal was in most mutants, even mutants associated with activation, shifted to more alkaline values than the pH dependence of current activation, and closer to the pH dependence of SSD. Such a shift has been observed in previous studies (Vullo et al, 2017, Bonifacio et al, 2014. It was observed here even with the non-desensitizing mutant A81C/Y417V/T209W, thus it does not indicate a link to desensitization.…”

“…The β5-β6 linker of the β-ball runs almost parallel to the membrane surface, above the β1-β2 linker. In the direction from Pro205 to Met210 it approaches a subunit interface (Figure 5A) (Lynagh et al, 2018, Bignucolo et al, 2020). Nine mutants were identified in which the placement of a Trp in the β5-β6 palm loop generated fluorescence signals (Figure 5A-B).…”

“…In previous studies we had observed rapid F signals with mutations located distantly from the channel gate (Bonifacio et al, 2014, Gwiazda et al, 2015, Vullo et al, 2017. For an accurate comparison of the kinetics of these mutants ( Figure 1B), they were measured here in a measuring chamber in which the current and F signal are measured from approximately the same oocyte surface (( Figure 1C, (Vullo et al, 2017)).…”

“…Homology models of human ASIC1a were constructed from chicken ASIC1a which shares 90% identity with its human homolog, using structures representing the closed (PDB code 5WKU), open (4NTW) and desensitized (4NYK) structures (Gonzales et al, 2009, Baconguis et al, 2014, Yoder and Gouaux, 2018, Yoder et al, 2018 with SWISS-MODEL (Biasini et al, 2014). The molecular systems were constructed as done previously (Bignucolo et al, 2020). They contained ~250 or ~220 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) molecules in each leaflet for the closed or open state, respectively, and the total number of atoms was 281'000 and 244'000.…”

“…The parameters for the steered molecular dynamics (SMD) were set as done previously (Bignucolo and Berneche, 2020), with the difference that here the constant force was replaced by a harmonic potential without initial velocity and with an initial force of 30 kJ/mol/Å. For each pair of residues, a target distance was defined as a function of the VCF-predicted distance change.…”