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

Abstract

The ability to discriminate between different ionic species, termed ion selectivity, is a key feature of ion channels and forms the basis for their physiological function. Members of the degenerin/epithelial sodium channel (DEG/ENaC) superfamily of trimeric ion channels are typically sodium selective, but to a surprisingly variable degree. While acid-sensing ion channels (ASICs) are weakly sodium selective (sodium:potassium around 10:1), ENaCs show a remarkably high preference for sodium over potassium (>500:1). The most obvious explanation for this discrepancy may be expected to originate from differences in the pore-lining second transmembrane segment (M2). However, these show a relatively high degree of sequence conservation between ASICs and ENaCs and previous functional and structural studies could not unequivocally establish that differences in M2 alone can account for the disparate degrees of ion selectivity. By contrast, surprisingly little is known about the contributions of the first transmembrane segment (M1) and the preceding pre-M1 region. In this study, we use conventional and non-canonical amino acid-based mutagenesis in combination with a variety of electrophysiological approaches to show that the pre-M1 and M1 regions of mammalian ASIC1a channels are major determinants of ion selectivity. Mutational investigations of the corresponding regions in human ENaC show that they contribute less to ion selectivity, despite affecting ion conductance. In conclusion, our work supports the notion that the remarkably different degrees of sodium selectivity in ASICs and ENaCs are achieved through different mechanisms. This further highlights how subtle sequence variations in related channels can translate into notably different functional outcomes.

A flexible GAS belt responds to pore mutations changing the ion selectivity of proton-gated channels

“…Mutations of H28 have been shown to alter ion selectivity ( Sheikh et al, 2021 ). Among the numerous substitutions tested here ( Fig.…”

“…ASICs are made of three pore-forming subunits, and each subunit has a large extracellular domain (ECD), two transmembrane (TM) helices, and a short intracellular N-terminus and C-terminus. Although it has been known that mutations in a conserved segment of the N-terminus proximal to TM1 alter ion selectivity and kinetics ( Coscoy et al, 1999 ; Pfister et al, 2006 ; Sheikh et al, 2021 ), the lack of structural information of this domain has made it difficult to explain the mechanism(s) underlying the functional changes induced by those mutations. A genetic form of human pseudohypoaldosteronism produced by a mutation of the glycine residue in the His-Gly (HG) motif of the β-subunit of ENaC reduces activity of the channel, most likely by decreasing the open probability ( Chang et al, 1996 ; Gründer et al, 1997 , 1999 ).…”

Single ion free energy calculation in ASIC1: the importance of the HG loop

“…2 The role of pre-TM1 and TM1 residues in ion selectivity have recently been shown, supporting the structural observations. 13 Here, in order to study the impact of the re-entrant 'HG' loop on ion permeation, we calculated single ion free energy profiles of two ions, Na + and chloride (Cl À ), using two different ASIC1 structures. The first structure used is the open structure of ASIC1 stabilised with a snake toxin (PDB entry 4NTW, method X-ray diffraction, resolution 2.07 10 ).…”