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

P01 HL110873 NHLBI NIH HHS

             sodium ion transmembrane transport

	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.

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