Kota P et al. (2018), The N terminus of α-ENaC mediates ENaC cleavage...

XB-ART-55082

J Gen Physiol 2018 Aug 06;1508:1179-1187. doi: 10.1085/jgp.201711860.

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The N terminus of α-ENaC mediates ENaC cleavage and activation by furin.

Kota P , Gentzsch M , Dang YL , Boucher RC , Stutts MJ .

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Epithelial Na+ channels comprise three homologous subunits (α, β, and γ) that are regulated by alternative splicing and proteolytic cleavage. Here, we determine the basis of the reduced Na+ current (INa) that results from expression of a previously identified, naturally occurring splice variant of the α subunit (α-ENaC), in which residues 34-82 are deleted (αΔ34-82). αΔ34-82-ENaC expression with WT β and γ subunits in Xenopus oocytes produces reduced basal INa, which can largely be recovered by exogenous trypsin. With this αΔ34-82-containing ENaC, both α and γ subunits display decreased cleavage fragments, consistent with reduced processing by furin or furin-like convertases. Data using MTSET modification of a cysteine, introduced into the degenerin locus in β-ENaC, suggest that the reduced INa of αΔ34-82-ENaC arises from an increased population of uncleaved, near-silent ENaC, rather than from a reduced open probability spread uniformly across all channels. After treatment with brefeldin A to disrupt anterograde trafficking of channel subunits, INa in oocytes expressing αΔ34-82-ENaC is reestablished more slowly than that in oocytes expressing WT ENaC. Overnight or acute incubation of oocytes expressing WT ENaC in the pore blocker amiloride increases basal ENaC proteolytic stimulation, consistent with relief of Na+ feedback inhibition. These responses are reduced in oocytes expressing αΔ34-82-ENaC. We conclude that the α-ENaC N terminus mediates interactions that govern the delivery of cleaved and uncleaved ENaC populations to the oocyte membrane.

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Species referenced: Xenopus
Genes referenced: furin prss1
GO keywords: sodium channel activity [+]

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	Figure 1. Deletion of residues 34–82 of α-ENaC reduces ENaC current. (a) Topologic representations of rat α-, β-, and γ-ENaC subunits. The α-ENaC splice variant with the missing residues is shown as dashed lines. (b and c) WT or splice variant α-ENaC cRNA was injected into Xenopus oocytes with WT β- and γ-ENaC. Amiloride-inhibited current (INa) was recorded 20–24 h after injection. Trypsin (2.0 µg/ml) was added during continuous recording. n = 15 separate oocytes collected from three donor frogs. Mean basal INa (b) decreased by 73% and trypsin-stimulated INa (c) decreased by 24%, compared by Student’s t test. Error bars represent mean ± SEM.
	Figure 2. WCPO is unaffected by α-ENaC N-terminal splice deletion. (a) Oocytes injected with ENaC subunits as in Fig. 1, except that β-ENaCS518C replaced the WT β-ENaC RNA. After recording of basal INa, oocytes were exposed to 1 mM MTSET and two-electrode voltage clamp current recording continued for 3–5 min. (b) WCPO was calculated using the data from panel a (see Materials and methods). n = 20 (WT) or n = 21 (splice variant) oocytes from four frogs, two-way ANOVA. Error bars represent mean ± SEM.
	Figure 3. Trypsin overcomes αΔ34–82-ENaC resistance to MTSET. Oocytes injected with ENaC subunits, as in Fig. 2, with β-ENaCS518C replacing the WT β-ENaC RNA. (a) Representative two-electrode voltage clamp current traces illustrating WT (black trace) and αΔ34–82-ENaC (red trace) response to MTSET, followed by trypsin. (b) Summary data from the previous panel (a). n = 14 oocytes/group from three frogs. (c) After trypsin, αΔ34–82-, β-, and γ-ENaC was not resistant to MTSET. Similar experiment as in the other two panels (a and b), but trypsin exposure preceded MTSET. n = 6/group. Error bars represent mean ± SEM.
	Figure 4. The splice variant αΔNT reduces γ-ENaC furin site cleavage. (a) Crude membrane samples of uninjected oocytes or oocytes injected with WT or ΔNT α-ENaC plus WT β-ENaC and HA/V5 epitope-tagged γ-ENaC were prepared as described in Materials and methods and subjected to PAGE and blotted for the V5 epitope tag. In WT α-, β-, and γ-ENaC, samples appeared as a band consistent with full-length (FL) γ-ENaC and a more rapidly migrating band predictive of cleavage at the γ-ENaC furin site (FF). Deletion of N-terminal residues of α-ENaC sharply diminished FF staining in total and as a fraction of FL. All lysates generated a doublet unrelated to ENaC (NS). This result is representative of multiple similar experiments. (b) WT or ΔNT α-ENaC was injected, along with WT β-ENaC and γ-ENaC, to comprise two experimental groups of 60 oocytes each. One-half of each group was also injected with furin RNA (1.0 ng/oocyte). α-ENaCs were V5 epitope tagged at the C terminus. Samples were processed and analyzed as in the previous panel (a). Full-length ΔNT α-ENaC (labeled ΔNT FL) migrated faster than WT FL, as expected. Fragments consistent with furin cleavage—furin frag (FF)—migrated identically, as expected for C-terminal V5-tagged fragments. The fraction of FF/FL in each condition was obtained from densitometry and is indicated beneath each lane. Similar results were obtained in other trials. (c) We mutated residue 133E of γ-ENaCHA/V5 to 133C and coexpressed the mutant with WT β-ENaC and WT or ΔNT α-ENaC. After 24 h, oocytes were labeled with 40 µM Alexa Fluor 680 C2 maleimide in MBS++ buffer. Labeling was quenched with 10 mM cysteine, and oocytes were washed extensively. Western blots of total lysates, stained for the V5 epitope tag (Total) or for γ-ENaC labeled by fluorescent maleimide (Surface) are shown. Representative of multiple similar experiments.
	Figure 5. Residues 34–82 of α-NT are not required for γ-ENaC cleavage by matriptase. (a) Oocyte membrane samples prepared as in Fig. 4 were blotted for γ-ENaC (C-terminal V5 epitope tag) expressed with WT α- and β-subunits (three leftmost lanes) or αΔNT- and WT β-subunits (next three lanes to the right). ENaC groups were coinjected with furin or matriptase (Mtrp.). Labeled bands identify γ-ENaC as full length (FL), furin fragment (FF), and matriptase fragment (M). (b–e) Stimulation of INa by coexpressed furin (WT ENaC; b), (αΔNT-ENaC; c), or by coexpressed matriptase (WT ENaC; d), (αΔNT-ENaC; e). After recording basal INa (black bars), recording continued in the presence of 2 µg/ml. Current in each condition was normalized to stimulated INa in the control group. Error bars are mean ± SEM (10–15 recordings/group from two or three oocyte batches/condition).
	Figure 6. Amiloride stimulation of INa is impaired in αΔ34–82-ENaC. (a) Oocytes injected and incubated in Barth medium for 20 h and recorded as in Fig. 1 (−Amil). Oocytes from each injection group were then incubated in Barth medium with amiloride (10 µM) for 6 h and recorded (+Amil). (b) Oocytes were injected as in Fig. 1 and incubated for 24 h in Barth medium with or without 10 mM amiloride. Basal and trypsin-stimulated INa was recorded as above. Data collected from two to three oocyte batches, n = 6–18 recordings/condition. *, P < 0.05 for difference from −Amil by two-way ANOVA. Error bars represent mean ± SEM.
	Figure 7. Slower recovery from BFA exposure by αΔNT-ENaC. (a and b) Oocytes injected with WT β- and γ-ENaC subunits with WT or αΔNT-ENaC were incubated overnight in Barth medium containing 10 µM amiloride and 1 µM BFA. The next day, BFA was removed and basal (a) and simulated (b) INa was recorded after 2 and 6 h. Basal INa for WT increased (slope = 287 ± 44 nA/h) more rapidly than for αΔNT-ENaC (slope = 44 ± 17 nA/h). WT stimulated INa also increased (slope = 782 ± 91 nA/h) more rapidly than stimulated INa of αΔNT-ENaC (slope = 425 ± 84 nA/h). There were 10 oocytes in each injection condition, isolated from two frogs. The slopes and associated errors were generated by linear regression analyses and differ significantly (P < 0.01). (c) After overnight incubation in Barth medium with 10 µM amiloride, oocytes were exposed to 3 µM BFA or vehicle (DMSO). INa recorded after 5 h is expressed as fraction of the vehicle control at 5 h (n = 5/injection condition). Error bars represent mean ± SEM.

References [+] :

Abriel, Feedback inhibition of rat amiloride-sensitive epithelial sodium channels expressed in Xenopus laevis oocytes. 1999, Pubmed, Xenbase