An epithelial sodium channel (ENaC) is composed of three homologous subunits: ␣, ␤, and ␥. To elucidate the function of the cytoplasmic, NH 2 terminus of rat ENaC (rENaC) subunits, a series of mutant cDNAs was constructed and the cRNAs for all three subunits were expressed in Xenopus oocytes. Amiloride-sensitive Na ؉ currents (I Na ) were measured by the two-electrode voltage clamp technique. Deletion of the cytoplasmic, NH 2 terminus of ␣ (⌬2-109), ␤ (⌬2-49), or ␥-rENaC (⌬2-53) dramatically reduced I Na . A series of progressive, NH 2 -terminal deletions of ␣-rENaC were constructed to identify motifs that regulate I Na . Deletion of amino acids 2-46 had no effect on I Na : however, deletion of amino acids 2-51, 2-55, 2-58, and 2-67 increased I Na by ϳ4-fold. By contrast, deletion of amino acids 2-79, 2-89, 2-100, and 2-109 eliminated I Na . To evaluate the mechanism whereby ⌬2-67-␣-rENaC increased I Na , single channels were evaluated by patch clamp. The single-channel conductance and open probability of ␣,␤,␥-rENaC and ⌬2-67-␣,␤,␥-rENaC were similar. However, the number of active channels in the membrane increased from 6 ؎ 1 channels per patch with ␣,␤,␥-rENaC to 11 ؎ 1 channels per patch with ⌬2-67-␣,␤,␥-rENaC. Laser scanning confocal microscopy confirmed that there were more ⌬2-67-␣,␤,␥-rENaC channels in the plasma membrane than ␣,␤,␥-rENaC channels. Deletion of amino acids 2-67 in ␣-rENaC reduced the endocytic retrieval of channels from the plasma membrane and increased the half-life of the channel in the membrane from 1.1 ؎ 0.2 to 3.5 ؎ 1.1 h. We conclude that the cytoplasmic, NH 2 terminus of ␣-, ␤-, and ␥-rENaC is required for channel activity. The cytoplasmic, NH 2 terminus of ␣-rENaC contains two key motifs. One motif regulates the endocytic retrieval of the channel from the plasma membrane. The second motif is required for channel activity.An amiloride-sensitive, epithelial sodium channel (ENaC) 1 mediates Na ϩ transport across the apical membrane of a variety of epithelia including the kidney, lung, and intestine and, thereby, plays a vital role in maintaining Na ϩ and fluid homeostasis (1-4). ENaC is composed of three subunits: ␣, ␤, and ␥ (5, 6). The expression of the ␣ subunit in Xenopus oocytes produces very small currents, and the expression of ␤ and/or ␥ subunits generates no current (5, 6). However, coexpression of ␣-, ␤-, and ␥-rENaC produces large Na ϩ currents in oocytes (5, 6). The ENaC subunits are members of a growing family of ion channels that include the FMRFamide-gated Na ϩ channel, Na ϩ channels in brain (BNC1 and BNC2), and the degenerins of Caenorhabditis elegans that encode mechanosensitive channels (e.g. DEG-1, MEC-4, and MEC-10) (4, 7).Amino acid sequence analysis and biochemical studies suggest that the ENaC subunits have cytoplasmic NH 2 and COOH termini, two hydrophobic transmembrane domains (M1 and M2) and a large extracellular domain (4 -8). Several lines of evidence suggest that the ␣-, ␤-, and ␥-subunits interact to form a heteromultimeric channel complex and that this c...

We tested the hypothesis that an arginine-rich region immediately following the second transmembrane domain may constitute part of the inner mouth of the epithelial Na+ channel (ENaC) pore and, hence, influence conduction and/or selectivity properties of the channel by expressing double point mutants in Xenopus oocytes. Double point mutations of arginines in this post-M2 region of the human alpha-ENaC (alpha-hENaC) led to a decrease and increase in the macroscopic conductance of alphaR586E,R587Ebetagamma- and alphaR589E,R591Ebetagamma-hENaC, respectively, but had no effect on the single-channel conductance of either double point mutant. However, the apparent equilibrium dissociation constant for Na+ was decreased for both alphaR586E,R587Ebetagamma- and alphaR589E,R591Ebetagamma-hENaC, and the maximum amiloride-sensitive Na+ current was decreased for alphaR586E,R587Ebetagamma-hENaC and increased for alphaR589E,R591Ebetagamma-hENaC. The relative permeabilities of Li+ and K+ vs. Na+ were increased 11.25- to 27.57-fold for alphaR586E,R587Ebetagamma-hENaC compared with wild type. The relative ion permeability of these double mutants and wild-type ENaC was inversely related to the crystal diameter of the permeant ions. Thus the region of positive charge is important for the ion permeation properties of the channel and may form part of the pore itself.

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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.