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

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 ?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.

SUBMITTER: Wang X

PROVIDER: S-EPMC6851303 | biostudies-literature | 2019 Nov

REPOSITORIES: biostudies-literature

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

Wang Xueqi X Chen Jingxin J Shi Shujie S Sheng Shaohu S Kleyman Thomas R TR

The Journal of biological chemistry 20190924 45

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 ...[more]

PMID: 31551351

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Project description:CLC-2 is a voltage-gated chloride channel that contributes to electrical excitability and ion homeostasis in many different mammalian tissues and cell types. Among the nine mammalian CLC homologs, CLC-2 is uniquely activated by hyperpolarization, rather than depolarization, of the plasma membrane. The molecular basis for the divergence in polarity of voltage gating mechanisms among closely related CLC homologs has been a long-standing mystery, in part because few CLC channel structures are available, and those that exist exhibit high conformational similarity. Here, we report cryoEM structures of human CLC-2 at 2.46 - 2.76 Å, in the presence and absence of the potent and selective inhibitor AK-42. AK-42 binds within the extracellular entryway of the Cl--permeation pathway, occupying a pocket previously proposed through computational docking studies. In the apo structure, we observed two distinct apo conformations of CLC-2 involving rotation of one of the cytoplasmic C-terminal domains (CTDs). In the absence of CTD rotation, an intracellular N-terminal 15-residue hairpin peptide nestles against the TM domain to physically occlude the Cl--permeation pathway from the intracellular side. This peptide is highly conserved among species variants of CLC-2 but is not present in any other CLC homologs. Previous studies suggested that the N-terminal domain of CLC-2 influences channel properties via a "ball-and-chain" gating mechanism, but conflicting data cast doubt on such a mechanism, and thus the structure of the N-terminal domain and its interaction with the channel has been uncertain. Through electrophysiological studies of an N-terminal deletion mutant lacking the 15-residue hairpin peptide, we show that loss of this short sequence increases the magnitude and decreases the rectification of CLC-2 currents expressed in mammalian cells. Furthermore, we show that with repetitive hyperpolarization WT CLC-2 currents increase in resemblance to the hairpin-deleted CLC-2 currents. These functional results combined with our structural data support a model in which the N-terminal hairpin of CLC-2 stabilizes a closed state of the channel by blocking the cytoplasmic Cl--permeation pathway.

Dataset Information

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

Publications

Analyses of epithelial Na<sup>+</sup> channel variants reveal that an extracellular β-ball domain critically regulates ENaC gating.

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