(A) Referring to molecular determinants of function in the ENaC/DEG family with amino acid numbers of various channels makes comparisons confusing. E.g. it is not immediately clear that ENaC α subunit (species not reported) S589 (Waldmann et al., 1995) and rat ENaC (rENaC) α subunit S589 (Kellenberger et al., 1999a) mutations, both of which alter ion selectivity in those studies, actually refer to adjacent positions (GSS and GSS, respectively). Similarly, it is not immediately clear that A443 in mouse ASIC1a (mASIC1a) (Carattino and Della Vecchia, 2012), A444 in human ASIC1a (hASIC1a) (Yang et al., 2009) and G530 in rENaC β, (Kellenberger et al., 1999b) each of which determine ion permeability to some extent, actually occupy the equivalent position. To avoid such confusion, we have adopted a new numbering system, based on various functional data, recent structural data and the comparison of a diverse range of subunits from the family (similar to the numbering systems for pore-lining residues in Cys-loop receptors or S4 charges in voltage-gated ion channels). According to this analysis, the equivalent residues, D434 that contributes to ion permeation in hASIC1a (Yang and Palmer, 2014), D433 whose side chain is oriented into the top of the channel pore of chick ASIC1 (Baconguis et al., 2014), and D431 that contributes to Ca2+ permeability in HyNaC2 (Dürrnagel et al., 2012), are simply referred to as D0’. mASIC1a A443, hASIC1a A444 and rENaC β G530 (described above), are equivalent and now referred to as A11’, A11’ and G11’, respectively. The amino acid sequence alignment (Edgar, 2004) in (A) details the prime numbering system. Yellow highlighting indicates residues whose mutation is reported to alter channel function in at least two family members, as follows. 0’: lamprey ASIC1 D433N alters gating (Li et al., 2011a); hASIC1a D434N decreases ion conduction (Yang and Palmer, 2014); hydra HyNaC D431C decreases Ca2+ permeability (Dürrnagel et al., 2012). 4’: lamprey ASIC1 Q437C is water-accessible (Li et al., 2011b); chick ASIC1 Q437A slows desensitization (Baconguis et al., 2014). 7’: hASIC1a L440A decreases Na+/K+ selectivity (Yang et al., 2009); lamprey ASIC1 L440C is water-accessible (Li et al., 2011b); mASIC1a L439A decreases Na+/K+ selectivity (present study). 10’: rENaC β G529S decreases Na+/K+ selectivity (Kellenberger et al., 1999b); mENaC α G587C decreases Na+/K+ selectivity (Sheng et al., 2000). 11’: ENaC α S589I alters gating and Na+ conduction (species not reported [Waldmann et al., 1995]); hASIC1a A444G decreases Na+/K+ selectivity (Yang et al., 2009). 12’: C. elegans MEC-4 S455F decreases function (Hong and Driscoll, 1994); rENaC α S589C/D decreases Na+/K+ selectivity (Kellenberger et al., 1999a). 15’: C. elegans MEC-4 T458I decreases function (Waldmann et al., 1995); ENaC α S593I alters gating (species not reported (Waldmann et al., 1995); mASIC1a T447V prevents functional expression (Figure 4—figure supplement 1). 18’: C. elegans MEC-4 E461K decreases function (Hong and Driscoll, 1994); mENaC α E595C decreases Na+/K+ selectivity (Sheng et al., 2001); mASIC1a E450D/Q decreases Na+/K+ selectivity (present study). (B) Left, voltage ramps at rest and during activation of currents by pH 6.0 (dashed line) in different extracellular cations (indicated by color) at oocytes injected with WT mRNA, A11’UAG mRNA + tRNA-α, or A11’UAG mRNA + tRNA (the latter as a measure of non-specific incorporation). Right, current-voltage relationships of proton-gated current, where ramp during rest has been subtracted.