Determinants of selective ion permeation in the epithelial Na⁺ channel

The epithelial Na⁺ channel (ENaC) is a key transporter mediating and controlling Na⁺ reabsorption in many tight epithelia. A very high selectivity for Na⁺ over other cations, including K⁺, is a hallmark of this channel. This selectivity greatly exceeds that of the closely related acid-sensing channels (ASICs). Here, we assess the roles of two regions of the ENaC transmembrane pore in the determination of cation selectivity. Mutations of conserved amino acids with acidic side chains near the cytoplasmic end of the pore diminish macroscopic currents but do not decrease the selectivity of the channel for Na⁺ versus K⁺ In the WT channel, voltage-dependent block of Na⁺ currents by K⁺ or guanidinium⁺, neither of which have detectable conductance, suggests that these ions permeate only ∼20% of the transmembrane electric field. According to markers of the electric field determined by Zn²⁺ block of cysteine residues, the site of K⁺ block appears to be nearer to the extracellular end of the pore, close to a putative selectivity filter identified using site-directed mutations. To test whether differences in this part of the channel account for selectivity differences between ENaC and ASIC, we substitute amino acids in the three ENaC subunits with those present in the ASIC homotrimer. In this construct, Li:Na selectivity is altered from that of WT ENaC, but the high Na:K selectivity is maintained. We conclude that a different part of the pore may constitute the selectivity filter in the highly selective ENaC than in the less-selective ASIC channel.