Abstract
Epithelial Na+ channel (ENaC)-mediated Na+ transport has a key role in the regulation of extracellular fluid volume, blood pressure, and extracellular [K+]. Among the thousands of human ENaC variants, only a few exist whose functional consequences have been experimentally tested. Here, we used the Xenopus oocyte expression system to investigate the functional roles of four nonsynonymous human ENaC variants located within the β7-strand and its adjacent loop of the α-subunit extracellular β-ball domain. αR350Wβγ and αG355Rβγ channels exhibited 2.5- and 1.8-fold greater amiloride-sensitive currents than WT αβγ human ENaCs, respectively, whereas αV351Aβγ channels conducted significantly less current than WT. Currents in αH354Rβγ-expressing oocytes were similar to those expressing WT. Surface expression levels of three mutants (αR350Wβγ, αV351Aβγ, and αG355Rβγ) were similar to that of WT. However, three mutant channels (αR350Wβγ, αH354Rβγ, and αG355Rβγ) exhibited a reduced Na+ self-inhibition response. Open probability of αR350Wβγ was significantly greater than that of WT. Moreover, other Arg-350 variants, including αR350G, αR350L, and αR350Q, also had significantly increased channel activity. A direct comparison of αR350W and two previously reported gain-of-function variants revealed that αR350W increases ENaC activity similarly to αW493R, but to a much greater degree than does αC479R. Our results indicate that αR350W along with αR350G, αR350L, and αR350Q, and αG355R are novel gain-of-function variants that function as gating modifiers. The location of these multiple functional variants suggests that the αENaC β-ball domain portion that interfaces with the palm domain of βENaC critically regulates ENaC gating.
Highlights
Epithelial Na؉ channel (ENaC)-mediated Na؉ transport has a key role in the regulation of extracellular fluid volume, blood pressure, and extracellular [K؉]
When we examined human gene variant databases (NCBI dbSNP, 1000 Genome Project, TopMed [22] and ExAC (Exome Aggregation Consortium [23]), we noted that several nonsynonymous human SCNN1A variants (␣R350W, ␣V351A, ␣H354R, and ␣G355R) are present in the 7-strand and its following loop that form part of the -ball of the ␣-subunit (Fig. 1, A and B)
To investigate the effects of these 7-strand human ENaC variants, we generated the ␣R350W, ␣V351A, ␣H354R, and ␣G355R mutations and co-expressed wildtype (WT) or mutant human ␣-subunit with WT human - and ␥-subunits in Xenopus oocytes
Summary
ENaCs are expressed in the apical plasma membranes of specific epithelia and, in parallel with the basolateral Naϩ,Kϩ-ATPase, mediate the absorption of Naϩ from the lumen of the aldosterone-sensitive distal nephron (ASDN), the distal colon, and the airway and alveolae. Other than well-defined Liddle syndrome mutations that disrupt or result in a loss of a Pro-Tyr (PY) motif in the C terminus of the - or ␥-subunit, correlating nonsynonymous ENaC variants that alter channel activity with predicted changes in blood pressure in humans has been challenging. This may reflect that fact that only two of the common ENaC nonsynonymous variants alter ENaC function in heterologous expression systems, and these have not been clearly linked to changes in blood pressure in humans (9 –14). Among these variants, ␣R350W exhibited the most robust effect on enhancing channel activity and open probability (Po)
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