pubmed.ncbi.nlm.nih.gov

Incorporation of the δ-subunit into the epithelial sodium channel (ENaC) generates protease-resistant ENaCs in Xenopus laevis - PubMed

  • ️Mon Jan 01 2018

Incorporation of the δ-subunit into the epithelial sodium channel (ENaC) generates protease-resistant ENaCs in Xenopus laevis

Lukas Wichmann et al. J Biol Chem. 2018.

Abstract

The epithelial sodium channel (ENaC) is a critical regulator of vertebrate electrolyte homeostasis. ENaC is the only constitutively open ion channel in the degenerin/ENaC protein family, and its expression, membrane abundance, and open probability therefore are tightly controlled. The canonical ENaC is composed of three subunits (α, β, and γ), but a fourth δ-subunit may replace α and form atypical δβγ-ENaCs. Using Xenopus laevis as a model, here we found that mRNAs of the α- and δ-subunits are differentially expressed in different tissues and that δ-ENaC predominantly is present in the urogenital tract. Using whole-cell and single-channel electrophysiology of oocytes expressing Xenopus αβγ- or δβγ-ENaC, we demonstrate that the presence of the δ-subunit enhances the amount of current generated by ENaC due to an increased open probability, but also changes current into a transient form. Activity of canonical ENaCs is critically dependent on proteolytic processing of the α- and γ-subunits, and immunoblotting with epitope-tagged ENaC subunits indicated that, unlike α-ENaC, the δ-subunit does not undergo proteolytic maturation by the endogenous protease furin. Furthermore, currents generated by δβγ-ENaC were insensitive to activation by extracellular chymotrypsin, and presence of the δ-subunit prevented cleavage of γ-ENaC at the cell surface. Our findings suggest that subunit composition constitutes an additional level of ENaC regulation, and we propose that the Xenopus δ-ENaC subunit represents a functional example that demonstrates the importance of proteolytic maturation during ENaC evolution.

Keywords: Xenopus laevis; chymotrypsin; delta-subunit; electrophysiology; epithelial sodium channel (ENaC); furin; patch clamp; protease.

© 2018 Wichmann et al.

PubMed Disclaimer

Conflict of interest statement

The authors declare that they have no conflicts of interest with the contents of this article.

Figures

Figure 1.
Figure 1.

The δ-ENaC subunit is predominantly expressed in urogenital tissues in X. laevis. Tissues from adult X. laevis were screened for expression of δ- and α-ENaC subunit mRNA via RT-PCR. Amplicons for δ-ENaC (320 bp) were found in tissue samples from kidney, urinary bladder, testes, ovary, oocytes, and the cloaca. With the exception of the ovary, these tissues also expressed α-ENaC (291 bp). Amplification of β-actin mRNA (610 bp) served as a control. Control reactions have been performed without reverse transcriptase but are omitted from the figure for clarity. These reactions did not show any DNA signals in the MidoriGreen fluorescence of agarose gels after PCR. Numbers in parentheses indicate the quantity of positively tested samples with respect to the total amount of samples from different animals.

Figure 2.
Figure 2.

The δ-subunit alters ENaC characteristics. a, current (IM)/voltage (VM) plots of oocytes expressing αβγ- or δβγ-ENaC in the absence (left) or presence (right) of amiloride. b, representative IM recordings of oocytes expressing αβγ- or δβγ-ENaC at −60 mV. Black bars, application of amiloride (a). c, amiloride-sensitive current fractions (ΔIami) in both ENaC isoforms derived from the second application of amiloride (Student's unpaired t test with Welch's correction). d and e, representative current traces (d) and corresponding mean values of IM (e) of αβγ- or δβγ-ENaC–expressing oocytes, depicting the enhanced current rundown of δ-ENaC–containing channels over an extended time period. At any given time point, IM mediated by δ-containing channels was significantly increased compared with αβγ-ENaC–mediated currents (*, p < 0.0003, Mann–Whitney U test). Lines and error bars, mean and S.E.

Figure 3.
Figure 3.

Xenopus αβγ- and δβγ-ENaC single-channel characteristics. a, representative current traces from cell-attached patch-clamp recordings of αβγ- or δβγ-ENaC–expressing oocytes (VM = −100 mV). Amplitude histograms depict Gaussian distribution of channel amplitudes. Dotted squares, areas of trace magnification (c, closed; 1, 2, number of open channels). b, number of visible channels in cell-attached recordings as presented in a (Student's unpaired t test). c, slope conductance (Gslope) of αβγ- and δβγ-ENaC derived from linear regression of unitary channel conductance at −40 to −100 mV. d, apparent open probability of both ENaC isoforms (Student's unpaired t test). e, estimation of single-channel kinetics revealed an increased open time (topen) of δβγ-ENaC, but no significant change in closed time (tclosed) between ENaC isoforms (Dunn's multiple-comparison test). Evaluation of all single-channel characteristics was performed using current recordings with a duration of 120–180 s with a maximum of eight channels per patch. f, cell-attached recording of a δβγ-ENaC–expressing oocyte indicating a rundown of ENaC activity that impedes a precise determination of channel open probability. Lines and error bars, mean and S.E.

Figure 4.
Figure 4.

The MTSET-sensitive βS540C-ENaC mutant reveals an increased open probability of ENaCs containing the δ-subunit. a, amiloride-sensitive current fractions (ΔIami) of oocytes expressing αβγ- or δβγ-ENaC containing either WT β-ENaC or the βS540C-ENaC mutation (Student's unpaired t test). b and c, representative IM traces of oocytes expressing αβS540Cγ- or δβS540Cγ-ENaC. The open probability of both channel isoforms was determined by assessing ΔIami before and after the application of MTSET (1 m

m

). d, statistical comparison of ΔIami of oocytes expressing αβS540Cγ- or δβS540Cγ-ENaC before (initial) and after application of MTSET (initial, Student's unpaired t test; MTSET, Student's unpaired t test with Welch's correction). e, open probability of αβS540Cγ- and δβS540Cγ-ENaC determined by the ratio of ΔIami initial/ΔIami MTSET (Student's unpaired t test). Lines and error bars, mean and S.E.

Figure 5.
Figure 5.

The δ-subunit is not subject to proteolytic maturation, and δβγ-ENaC membrane abundance is not changed compared with α-containing channels. a, schematic depiction of epitope-tagged ENaC subunits. The numbers indicate approximate molecular mass of peptides with/without furin cleavage, detected by immunoblots, as presented in b. b, immunoblots of whole-cell lysates from oocytes expressing αHA/V5βγ- or δHA/V5βγ-ENaC using anti-HA and anti-V5 antibodies. c, a migration shift of the δHA/V5-subunit due to treatment with PNGase F indicates glycosylation of the protein. d, representative IM recordings of oocytes expressing αHA/V5βγ- or δHA/V5βγ-ENaC. e, amiloride-sensitive current fractions (ΔIami) of αHA/V5βγ- and δHA/V5βγ-ENaC (Student's unpaired t test). f, immunoblot using an anti-V5-antibody of membrane (mem.) and cytosolic fractions from oocytes expressing αHA/V5βγ- or δHA/V5βγ-ENaC. The bottom blot shows the results from reprobing of the same membrane with an anti-pan-actin antibody to verify proper fractionation. g, membrane abundance of αHA/V5βγ- and δHA/V5βγ-ENaC derived from densitometric analysis of immunoblots as shown in f (Student's unpaired t test). Lines and error bars, mean and S.E.

Figure 6.
Figure 6.

ENaCs containing the δ-subunit are insensitive to activation by extracellular proteases. a, representative IM traces of oocytes expressing αβγ-ENaC. Proteolytic channel activation was determined by assessing amiloride-sensitive current fractions (ΔIami) before and after application of chymotrypsin (CT; 2 μg/ml) together with amiloride (a). Current traces of oocytes previously incubated with furin inhibitor I (40 μ

m

) are depicted as dotted gray lines. b, comparison of ΔIami before and after application of chymotrypsin in oocytes incubated with (+) or without (−) furin inhibitor I (one-way ANOVA; F = 15.31, p < 0.0001; Tukey's multiple comparison test). c, comparison of -fold activation in ΔIami between experimental groups (chymotrypsin +) and control groups (chymotrypsin −) (one-way ANOVA; F = 44.4, p < 0.0001; Tukey's multiple comparison test). d–f, IM traces (d) and statistical evaluation of changes in ΔIami in oocytes expressing δβγ-ENaC. Data presentation is equivalent to a–c. e, Kruskal–Wallis test, p = 0.376; Dunn's multiple-comparison test. f, one-way ANOVA; F = 1.393, p = 0.2598; Tukey's multiple-comparison test. Lines and error bars, mean and S.E.

Figure 7.
Figure 7.

The presence of the δ-ENaC subunit prevents cleavage of the γ-subunit by extracellular proteases. a, comparison of ΔIami from oocytes expressing epitope-tagged αHA/V5βγ- or δHA/V5βγ-ENaC with (+) or without (−) prior incubation with chymotrypsin (2 μg/ml, 5 min). b, V5-targeted immunoblots of cytosolic and membrane fractions from oocytes expressing αHA/V5βγ- or δHA/V5βγ-ENaC that were treated with (+) or without (−) chymotrypsin. Blots were reprobed for pan-actin to verify proper isolation of membrane fractions. c, ΔIami of oocytes expressing epitope-tagged αβγHA/V5- or δβγHA/V5-ENaC with (+) or without (−) prior incubation with chymotrypsin. d, V5-targeted immunoblots of cytosolic and membrane fractions from oocytes expressing αβγHA/V5- or δβγHA/V5-ENaC, which were treated with (+) or without (−) chymotrypsin. Lines and error bars, mean and S.E.

Similar articles

Cited by

References

    1. Rossier B. C., Baker M. E., and Studer R. A. (2015) Epithelial sodium transport and its control by aldosterone: The story of our internal environment revisited. Physiol. Rev. 95, 297–340 10.1152/physrev.00011.2014 - DOI - PubMed
    1. Kleyman T. R., Kashlan O. B., and Hughey R. P. (2018) Epithelial Na+ channel regulation by extracellular and intracellular factors. Annu. Rev. Physiol. 80, 263–281 10.1146/annurev-physiol-021317-121143 - DOI - PMC - PubMed
    1. Fronius M. (2013) Treatment of pulmonary edema by ENaC activators/stimulators. Curr. Mol. Pharmacol. 6, 13–27 10.2174/1874467211306010003 - DOI - PubMed
    1. Baines D. (2013) Kinases as targets for ENaC regulation. Curr. Mol. Pharmacol. 6, 50–64 10.2174/18744672112059990028 - DOI - PubMed
    1. Chraïbi A., and Horisberger J. D. (2002) Na self inhibition of human epithelial Na channel: temperature dependence and effect of extracellular proteases. J. Gen. Physiol. 120, 133–145 10.1085/jgp.20028612 - DOI - PMC - PubMed

MeSH terms

Substances

LinkOut - more resources