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

Abstract

Epithelial sodium channels (ENaC) are composed of three structurally related subunits (␣, ␤, and ␥). Each subunit has two transmembrane domains termed M1 and M2, and residues conferring cation selectivity have been shown to reside in a pore region immediately preceding the M2 domains of the three subunits. Negatively charged residues are interspersed within the M2 domains, and substitution of individual acidic residues within human ␣-ENaC with arginine essentially eliminated channel activity in oocytes, suggesting that these residues have a role in ion permeation. We examined the roles of M2 residues in contributing to the permeation pore by individually mutating residues within the M2 domain of mouse ␣ENaC to cysteine and systematically characterizing functional properties of mutant channels expressed in Xenopus oocytes by two-electrode voltage clamp. The introduction of cysteine residues at selected sites, including negatively charged residues (␣Glu 595 , ␣Glu 598 , and ␣Asp 602 ) led to a significant reduction of expressed amiloride-sensitive Na ؉ currents. Two mutations (␣E595C and ␣D602C) resulted in K ؉ -permeable channels whereas multiple mutations altered Li ؉ / Na ؉ current ratios. Channels containing ␣D602K or ␣D602A also conducted K ؉ whereas more conservative mutations (␣D602E and ␣D602N) retained wild type selectivity. Cysteine substitution at the site equivalent to ␣Asp 602 within ␤ mENaC (␤D544C) did not alter either Li ؉ /Na ؉ or K ؉ /Na ؉ current ratios, although mutation of the equivalent site within ␥ mENaC (␥D562C) significantly increased the Li ؉ /Na ؉ current ratio. Mutants containing introduced cysteine residues at ␣Glu 595 , ␣Glu 598 , ␣Asp 602 , or ␣Thr 607 did not respond to externally applied sulfhydryl reagent with significant changes in macroscopic currents. Our results suggest that some residues within the M2 domain of ␣ENaC contribute to the channel's conduction pore and that, in addition to the pore region, selected sites within M2 (␣Glu 595 and ␣Asp 602 ) may have a role in conferring ion selectivity.The epithelial sodium channel (ENaC 1 ) mediates Na ϩ transport across high resistance epithelia and has a key role in Na ϩ homeostasis and blood pressure control. This channel is a member of the ENaC/degenerin gene superfamily and is composed of three structurally related subunits, termed ␣, ␤, and ␥ (1). Members of the ENaC/degenerin family are homo-or heterooligomeric proteins whose subunits share a common topology of two membrane spanning domains (M1 and M2) and intracellular amino and carboxyl termini. All three ENaC subunits contribute to the formation of the ion-conducting pore (2-7). Hydropathy analyses of ENaC subunits have identified two large hydrophobic domains consisting of about 45 residues. The second hydrophobic domain contains two regions that are distinct in structure and function. The amino-terminal region is functionally similar to the pore region of many cation channels and is a key element determining pore properties of ENaC, including selectivity, gating, condu...

Maturation of the Epithelial Na+ Channel Involves Proteolytic Processing of the α- and γ-Subunits

“…Amiloridesensitive sodium currents measured in oocytes by two-electrode voltage clamp the following day indicated that all combinations of epitope-tagged subunits produce functional sodium channels with amiloride-sensitive whole cell currents in the range of 7-27 A/oocyte, similar to whole cell currents we have observed with wild type ENaC (27,29). Because these data indicate that the epitope-tagged ␣␤␥ENaC subunits produced functional amiloride-sensitive sodium channels, they were used to study ENaC assembly and processing.…”

“…The mENaC subunit cRNAs (1 ng per subunit) were injected into oocytes, and electrophysiological measurements were performed after 24 h as described previously (27). The difference in measured current in the presence and absence of amiloride (100 M) was used to identify ENaC currents produced by different subunit combinations.…”

Asymmetric Organization of the Pore Region of the Epithelial Sodium Channel

“…The residues that form the putative amiloride-binding site (␣Ser 583 , ␤Gly 525 , and ␥Gly 542 ) are at the site where the pore ␣-helix transitions to an extended selectivity filter (17). Our earlier work indicated that the second membrane-spanning domains of ENaC have a secondary selectivity site, and we suggested that M2 may have an "inverted teepee" structure similar to that of KcsA (18). The apparent lack of accessibility of ␣␤G525C␥ to MTSET is consistent with a pore region structure similar to that of the KcsA K ϩ channel.…”

In vitro selection of a sodium-specific DNAzyme and its application in intracellular sensing

“…To our knowledge, no Na + -specific nucleic acid, whether naturally occurring or in vitro selected has been previously reported. In the protein world, although K + channels are quite selective for K + over other metal ions, the Na + channels are much less selective (75,76). Being able to obtain the NaA43 DNAzyme with such a high selectivity for Na + will not only provide a highly selective sensor for Na + , as demonstrated here, it will also give us an opportunity to elucidate the origin of such a high selectivity.…”