Charged residues in the M2 region of α-hENaC play a role in channel conductance

Abstract

The epithelial Na(+) channel (ENaC) is a low-conductance channel that is highly selective for Na(+) and Li(+) over K(+) and impermeable to anions. The molecular basis underlying these conduction properties is not well known. Previous studies with the ENaC subunits demonstrated that the M2 region of alpha-ENaC is critical to channel function. Here we examine the effects of reversing the negative charges of highly conserved amino acids in alpha-subunit human ENaC (alpha-hENaC) M1 and M2 domains. Whole cell and single-channel current measurements indicated that the M2 mutations E568R, E571R, and D575R significantly decreased channel conductance but did not affect Na(+):K(+) permeability. We observed no functional perturbations from the M1 mutation E108R. Whole cell amiloride-sensitive current recorded from oocytes injected with the M2 alpha-hENaC mutants along with wild-type (wt) beta- and gamma-hENaC was low (46-93 nA) compared with the wt channel (1-3 microA). To determine whether this reduced macroscopic current resulted from a decreased number of mutant channels at the plasma membrane, we coexpressed mutant alpha-hENaC subunits with green fluorescent protein-tagged beta- and gamma-subunits. Confocal laser scanning microscopy of oocytes demonstrated that plasma membrane localization of the mutant channels was the same as that of wt. These experiments demonstrate that acidic residues in the second transmembrane domain of alpha-hENaC affect ion permeation and are thus critical components of the conductive pore of ENaC.

Second Transmembrane Domains of ENaC Subunits Contribute to Ion Permeation and Selectivity

“…2A). Interestingly, all mutants with cysteine substitutions of negatively charged residues displayed smaller currents than wild type, consistent with the observations of Langloh et al (9). Because levels of Na ϩ current expression vary between different batches of oocytes, we compared current expression of wild type and mutant mENaCs in a paired manner.…”

“…Our results indicate that point mutations at any of the three negatively charged residues in the M2 domain of ␣mENaC subunit significantly reduced amiloride-sensitive Na ϩ currents, and expression of channels with either double (␣␤D544C ␥D562C) or triple (␣D602C ␤D544C ␥D562C) mutations of the negatively charged residues with ENaC M2 domains failed to produce measurable amiloride-sensitive Na ϩ current in agreement with observations of Langloh et al (9). The reduction in whole cell Na ϩ currents observed with point mutations of polar residues within mENaC M2 domains, as well as the reduction in single-channel Li ϩ conductance previously reported with ␣D575R␤␥ hENaC (human ␣Asp 575 and mouse ␣Asp 602 are at equivalent positions within ␣ENaC), are consistent with the notion that these acidic residues may line the conduction pore and their side chains may provide ion-binding sites (9). Alternatively, these charged residues may have a role in maintaining proper channel conformation, and mutations within M2 domains may disrupt or destabilize the conformation of the channel pore, leading to a reduction of ion conduction.…”

“…No detectable amiloridesensitive Na ϩ currents were observed in oocytes injected with double (␣␤D544C ␥D562C) or triple mutants (␣D602C ␤D544C ␥D562C). Although the exact role of the negatively charged residues within M2 domains of ENaC subunits is not clear, these results suggest that polar residues in M2 domains have an important role in the functional expression of ENaC, as suggested by Langloh et al (9).…”

“…The interdomain electrostatic interactions between transmembrane domains have also been suggested to mediate folding of voltage-gated K ϩ channels (20). Mutations that alter the channel conformation may interfere with its assembly or trafficking to the plasma membrane, although Langloh et al (9) have shown that reversal of the three negatively charged residues within human ␣ENaC does not significantly alter the surface expression of the mutant channels based on confocal images of oocytes expressing mutant ␣hENaC with green fluorescence proteintagged ␤ or ␥ hENaC (9). Reduction of whole cell Na ϩ currents observed with mutations within the M2 domain may also reflect changes in channel open probability, although Langloh et al (9) reported that channel gating is not significantly altered by mutations of negatively charged M2 residues.…”

“…Recent work suggests that M2 domains of ENaC also contribute to the formation of the pore. Langloh et al (9) reported that mutations of the negative-charged amino acids within the M2 domains of human ␣ENaC (E568R, E571R, and D575R) nearly eliminated channel activity, although these mutations did not alter the levels of protein expression at the plasma membrane. Furthermore, mutations of positively charged residues immediately following the ␣-subunit M2 domain altered cation selectivity of human ENaC (10).…”