Determinants of ion selectivity in ASIC1a- and ASIC2a-containing acid-sensing ion channels

Abstract

Trimeric acid-sensing ion channels (ASICs) contribute to neuronal signaling by converting extracellular acidification into excitatory sodium currents. Previous work with homomeric ASIC1a implicates conserved leucine (L7′) and consecutive glycine-alanine-serine (GAS belt) residues near the middle, and conserved negatively charged (E18′) residues at the bottom of the pore in ion permeation and/or selectivity. However, a conserved mechanism of ion selectivity throughout the ASIC family has not been established. We therefore explored the molecular determinants of ion selectivity in heteromeric ASIC1a/ASIC2a and homomeric ASIC2a channels using site-directed mutagenesis, electrophysiology, and molecular dynamics free energy simulations. Similar to ASIC1a, E18′ residues create an energetic preference for sodium ions at the lower end of the pore in ASIC2a-containing channels. However, and in contrast to ASIC1a homomers, ion permeation through ASIC2a-containing channels is not determined by L7′ side chains in the upper part of the channel. This may be, in part, due to ASIC2a-specific negatively charged residues (E59 and E62) that lower the energy of ions in the upper pore, thus making the GAS belt more important for selectivity. This is confirmed by experiments showing that the L7′A mutation has no effect in ASIC2a, in contrast to ASIC1a, where it eliminated selectivity. ASIC2a triple mutants eliminating both L7′ and upper charges did not lead to large changes in selectivity, suggesting a different role for L7′ in ASIC2a compared with ASIC1a channels. In contrast, we observed measurable changes in ion selectivity in ASIC2a-containing channels with GAS belt mutations. Our results suggest that ion conduction and selectivity in the upper part of the ASIC pore may differ between subtypes, whereas the essential role of E18′ in ion selectivity is conserved. Furthermore, we demonstrate that heteromeric channels containing mutations in only one of two ASIC subtypes provide a means of functionally testing mutations that render homomeric channels nonfunctional.

Coupling structure with function in acid‐sensing ion channels: challenges in pursuit of proton sensors

“…Interestingly, unlike pentameric ligand gated ion channels whose open state pore tends to collapse in molecular dynamics simulations (Damgen & Biggin, 2020), the open state of cASIC1 is reasonably stable, allowing more detailed examination of ion selectivity (Lynagh et al . 2017, 2020). However, these studies were not aimed at addressing how protonation drives transitions between functional states.…”

“…Indeed, past molecular dynamics work has explored interactions with psalmotoxin (Pietra, 2009), investigated a predicted calcium site at the extracellular mouth of the pore for ASIC3 (Zuo et al 2018), as well as studied the stability of a conserved salt bridge (Yang et al 2012) and the effects of mutations on dynamics (Roy et al 2013). Interestingly, unlike pentameric ligand gated ion channels whose open state pore tends to collapse in molecular dynamics simulations (Damgen & Biggin, 2020), the open state of cASIC1 is reasonably stable, allowing more detailed examination of ion selectivity (Lynagh et al 2017(Lynagh et al , 2020. However, these studies were not aimed at addressing how protonation drives transitions between functional states.…”

The current chemical biology tool box for studying ion channels

“…Furthermore, both approaches rely on ribosomal tolerance, which means certain backbone or side chain types cannot be incorporated (see section on Semi-synthetic approaches below). Lastly, and depending on the sequence context, incorporation efficiency can be highly variable, unwanted re-initiation from non-canonical start codons can occur or introduced stop codons can be read through, resulting in non-specific incorporation of (endogenous) amino acids at the site of interest (Kalstrup & Blunck, 2015;Lynagh et al 2018Lynagh et al , 2020Poulsen et al 2019;Khoo et al 2020). The latter issues are particularly problematic in the most N-and C-terminal parts of proteins.…”

“…Lastly, and depending on the sequence context, incorporation efficiency can be highly variable, unwanted re‐initiation from non‐canonical start codons can occur or introduced stop codons can be read through, resulting in non‐specific incorporation of (endogenous) amino acids at the site of interest (Kalstrup & Blunck, 2015; Lynagh et al . 2018, 2020; Poulsen et al . 2019; Khoo et al .…”

The M1 and pre-M1 segments contribute differently to ion selectivity in ASICs and ENaCs

“…Previous work indeed suggests that the selectivity filter of ENaCs is formed by the S of the G/SXS motif [19] and most ASIC structures available to date display a marked constriction at the level of this highly conserved M2 sequence feature [21, 22]. However, we have recently shown that the ASIC GAS sequence is unlikely to make major contribution to ion selectivity in ASIC1a and 2a, while acidic side chains in the lower M2 appear to be more important [17, 23]. Additionally, the latest ASIC structures show that the lower pore is lined by a reentrant loop formed by side chains from the N-terminus [22], and this pre-M1 region has previously been implicated in ion selectivity in ASICs [24, 25] and ENaCs [26, 27].…”