Analyses of epithelial Na+ channel variants reveal that an extracellular β-ball domain critically regulates ENaC gating

Abstract

Edited by Mike Shipston 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 ␤7-strand and its adjacent loop of the ␣-subunit extracellular ␤-ball domain. ␣R350W␤␥ and ␣G355R␤␥ channels exhibited 2.5-and 1.8-fold greater amiloride-sensitive currents than WT ␣␤␥ human ENaCs, respectively, whereas ␣V351A␤␥ channels conducted significantly less current than WT. Currents in ␣H354R␤␥-expressing oocytes were similar to those expressing WT. Surface expression levels of three mutants (␣R350W␤␥, ␣V351A␤␥, and ␣G355R␤␥) were similar to that of WT. However, three mutant channels (␣R350W␤␥, ␣H354R␤␥, and ␣G355R␤␥) exhibited a reduced Na ؉ self-inhibition response. Open probability of ␣R350W␤␥ was significantly greater than that of WT. Moreover, other Arg-350 variants, including ␣R350G, ␣R350L, and ␣R350Q, also had significantly increased channel activity. A direct comparison of ␣R350W and two previously reported gain-of-function variants revealed that ␣R350W increases ENaC activity similarly to ␣W493R, but to a much greater degree than does ␣C479R. Our results indicate that ␣R350W along with ␣R350G, ␣R350L, and ␣R350Q, and ␣G355R are novel gain-of-function variants that function as gating modifiers. The location of these multiple functional variants suggests that the ␣ENaC ␤-ball domain portion that interfaces with the palm domain of ␤ENaC critically regulates ENaC gating.

Extracellular intersubunit interactions modulate epithelial Na+ channel gating

“…For αQ441CβE499Cγ channels, DTT not only activated the channel but also significantly increased the Na + self-inhibition magnitude of αQ441CβE499Cγ (36 ± 12%, n = 12, p < 0.0001 versus the first response prior to DTT (see aforementioned) from paired Student's t test. These results are in contrast to the inverse relationship between the magnitude of Na + self-inhibition and ENaC activity that has been frequently observed in mutagenesis studies (see Discussion section) ( 41 , 45 , 46 ).…”

“…3 , G and H ). This relationship between channel activity and the magnitude of the Na + self-inhibition response is strikingly similar to what has been observed in gain-of-function mutations and specific ENaC activators that affect channel gating ( 20 , 21 , 26 , 37 , 38 , 39 , 40 , 42 , 45 , 46 , 49 , 62 , 63 , 64 , 65 , 68 , 70 , 73 , 74 , 75 , 76 , 77 ).…”

“…Open probability of ENaC is, in general, proportional to the magnitude of Na + self-inhibition ( 3 , 14 , 42 ). Other extracellular regulators, including proteases, modulate ENaC activity in part by altering Na + self-inhibition ( 3 , 14 , 15 , 16 , 26 , 29 , 37 , 46 , 49 , 57 , 58 , 59 , 60 , 61 , 62 , 63 , 64 , 65 , 66 , 67 , 68 , 69 , 70 , 71 , 72 ). We examined the Na + self-inhibition response of the double Cys mutant channels before and after inducing or breaking a disulfide bond to determine if it was altered.…”

Rare Variants in Genes Encoding Subunits of the Epithelial Na + Channel Are Associated With Blood Pressure and Kidney Function

“…[43][44][45] Additionally, an ENaC gain-of-function variant in SCNN1A was associated with a modest Liddle syndrome-like phenotype and a blunting of the inhibitory effect of extracellular Na + . 2,46,47 We noted that variants in SCNN1B and SCNN1D are associated with eGFR. Although DBP is contributing to the variance of eGFR in our population, we controlled for DBP in our eGFR analyses and the associations of SCNN1B with eGFR are independent of DBP.…”

“…43-45 Additionally, an ENaC gain-of-function variant in SCNN1A was associated with a modest Liddle syndrome–like phenotype and a blunting of the inhibitory effect of extracellular Na + . 2,46,47…”