Indirect Determinants of Ion Selectivity in Acid-Sensing Ion Channels and Epithelial Sodium Channels

Abstract

Members of the ENaC/DEG superfamily of ion channels are Na+-selective ion channels with a common trimeric architecture. Each of the three subunits has two transmembrane helices (M1 and M2), of which M2 lines the pore. Members of this family include the acid-sensing ion channels (ASICs), formed by identical or homologous subunits that mediate excitatory Na+ currents in the nervous system (relative Na+/K+ permeability of approx. 10/1). The epithelial sodium channels (ENaCs) are obligate heterotrimers and display a 10-fold higher Na+ selectivity than ASICs. The most recent findings indicate that this discrepancy might be due to different selectivity filter (SF) locations; while the ENaC SF is likely formed by the conserved G/S-X-S motif in the center of the pore, we have recently shown the mouse ASIC1a SF to be composed of two acidic side chains in the lower part of M2, namely E18’ and D21’. In order to elucidate if other parts of the channel contribute to the stark differences in Na+ selectivity between ASICs and ENaCs, we used conventional and non-canonical amino acid substitutions to probe the contribution of M1 residues to ion selectivity. Our results show that aromatic residues in ASIC M1 are important for ion selectivity. ENaC contains additional aromatic residues in M1. We hypothesize that these aromatics are similarly important for ion selectivity and that pore diameter plays an important role in both channels. Furthermore, the intracellular domains have previously been suggested to contribute to ion selectivity. Using a novel split intein-based approach we fuse partial ASIC1a constructs with recombinant or synthetic peptides corresponding to the N- or C-terminus of the full length protein. This enables us to introduce non-canonical amino acid substitutions, including post-translational modifications, into these less-studied regions of the channel.