Identification of SPLUNC1's ENaC-inhibitory domain yields novel strategies to treat sodium hyperabsorption in cystic fibrosis airways

Abstract

The epithelial sodium channel (ENaC) is responsible for Na+ and fluid absorption across colon, kidney, and airway epithelia. We have previously identified SPLUNC1 as an autocrine inhibitor of ENaC. We have now located the ENaC inhibitory domain of SPLUNC1 to SPLUNC1's N terminus, and a peptide corresponding to this domain, G22-A39, inhibited ENaC activity to a similar degree as full-length SPLUNC1 (∼2.5 fold). However, G22-A39 had no effect on the structurally related acid-sensing ion channels, indicating specificity for ENaC. G22-A39 preferentially bound to the β-ENaC subunit in a glycosylation-dependent manner. ENaC hyperactivity is contributory to cystic fibrosis (CF) lung disease. Addition of G22-A39 to CF human bronchial epithelial cultures (HBECs) resulted in an increase in airway surface liquid height from 4.2±0.6 to 7.9±0.6 μm, comparable to heights seen in normal HBECs, even in the presence of neutrophil elastase. Our data also indicate that the ENaC inhibitory domain of SPLUNC1 may be cleaved away from the main molecule by neutrophil elastase, which suggests that it may still be active during inflammation or neutrophilia. Furthermore, the robust inhibition of ENaC by the G22-A39 peptide suggests that this peptide may be suitable for treating CF lung disease.

Figure 1.

Identification of the ENaC inhibitory domain of SPLUNC1. A) Effect of C-terminal SPLUNC1 deletions on the amiloride-sensitive ENaC current. αβγ-ENaC subunits were coinjected into Xenopus oocytes with the corresponding SPLUNC1 truncants, and the relative ENaC current was measured by the 2-electrode voltage-clamp method (n=10–14). B–D) Oocytes were coinjected with α-, β^S518C-, γ-ENaC subunits and incubated for 1 h with G22-A39 or the control peptide, ADG (all n=11). B) Effect of 10 and 100 μM G22-A39 on the amiloride-sensitive ENaC current. C) Effect of MTSET on the amiloride-sensitive ENaC current in the presence or absence of 10 or 100 μM G22-A39. D) Effect of 10 and 100 μM ADG on the amiloride-sensitive ENaC current. *P < 0.05 vs. basal or nontransfected current.

Figure 2.

G22-A39 peptide does not affect the function of ASIC1a, ASIC2a, and ASIC3 channels. Whole-cell currents were measured from CHO cells stably expressing ASIC subunits, voltage clamped to −60 mV. Stimulations lasted 5 s and were performed every 45 s. A) Typical experiment with an ASIC1a-expressing cell. Cells were stimulated 3 times with pH 6.6 (ASIC1a and ASIC3) or pH 4 (ASIC2a). Between stimulations, cells were returned to a pH 7.4 conditioning solution for 40 s to allow recovery from inactivation. The conditioning solution was then switched to a pH 7.4 solution containing 10 μM G22-A39. Three stimulations in the presence of 10 μM G22-A39 were performed before washing off the peptide. B) Current amplitudes of the above described experiments were normalized to the first control value and plotted as a function of time. G22-A39 was added at t = 0 s. Squares, ASIC1a; circles, ASIC2a; triangles, ASIC3; all n = 3. C) Cells were incubated for 40 s in a pH 7.1 (ASIC1a and ASIC3) or 5.6 (ASIC2a) conditioning solution, then activated using an acidic stimulus (pH 5 for ASIC1a and ASIC3; pH 4 for ASIC2a). Experiments were performed with or without 10 μM G22-A39 in the conditioning solution. Current amplitudes measured during the acidic stimulus were normalized to the control amplitude obtained with a pH 7.4 conditioning solution. Open bars, control; solid bars, G22-A39; all n = 3–4. D) Cells expressing ASIC1a were stimulated 3 times with a pH 6.6 stimulus before 40 μg/ml trypsin was added to the pH 7.4 solution. Stimulations were performed every 45 s. Protocol was performed with or without 10 μM G22-A39 in all solutions. Average current is plotted as a function of time. Trypsin was added at t = 0. Open circles, control; solid squares, G22-A39; all n = 3–5.

Figure 3.

G22-A39 interacts specifically with the β-ENaC subunit. A) Typical Western blot of the triple-transfected αβγ-ENaC peptide pulldown assay. The pulldown assay was performed with 1 V5-tagged subunit and 2 untagged subunits as designated. B) Typical Western blot of the G22-A39 peptide pulldown assay of individually expressed ENaC subunits. IN, input; PD, pulldown elution. C) Typical Western blot showing the pulldown assay performed with G22-A39 or ADG. No β-ENaC was observed in the elution with the ADG peptide, confirming that the observed β-ENaC is from specific interaction with the G22-A39; n = 3.

Figure 4.

β-ENaC/G22-A39 interaction is glycosylation dependent. A) Typical Western blot of the αβγ-ENaC peptide pulldown assay with the β-ENaC subunit V5-tagged and untagged α- and γ-ENaC subunits. Pulldown assay was performed, and the elution was treated with EndoH. B) Typical Western blot of the β-ENaC-only peptide pulldown assay with the β-ENaC subunit V5 tagged. Pulldown assay was performed, and the elution was treated with EndoH. PD, pulldown elution; PD+E, pulldown elution with EndoH treatment. C) Typical Western blot of the tunicamycin-treated β-ENaC-only pulldown assay. IN(T), input of tunicamycin-treated sample; PD (T), pulldown elution of tunicamycin-treated sample; n = 3.

Figure 5.

G22-A39 inhibits CF ASL hyperabsorption. A) Confocal micrographs of NL and CF ASL height 24 h after exposure to G22-A39 or vehicle (control). Scale bar = 7 μm. B) Mean ASL height over time in NL and CF HBECs with or without addition of G22-A39; dashed lines indicate NL HBECs (triangles, control; circles, G22-A39), nondashed lines indicate CF HBECs (diamonds, control; squares, G22-A39), n = 6. *P < 0.05 for NL vs. basal; ^†P < 0.05 for CF vs. basal. Inset: typical slot blot for SPLUNC1 lavaged from the mucosal surfaces of NL and CF HBECs at timed intervals. Mucosal surfaces were left undisturbed for 24 h, washed immediately prior to initiating ASL height experiments, and in parallel cultures, subsequent SPLUNC1 recovery was determined. C) Change in ASL height with increasing concentration of G22-A39 in NL (solid squares) and CF (open circles) HBECs. D) Thin-film transepithelial PD for NL HBECs. Open bars, control; solid bars, G22-A39; n = 10. *P < 0.05 vs. corresponding control. E) Thin-film transepithelial PD for CF HBECs. Open bars, control; solid bars, G22-A39; n = 6. ^†P < 0.05 vs. corresponding control. F) ASL height over time in NL HBECs in the presence of 100 μM bumetanide with (squares) or without G22-A39 (diamonds), n = 6. ^†P < 0.05 vs. no G22-A39.

Figure 6.

G22-A39 maintains ASL height of CF HBECs with no intrinsic structure. A) Far-UV CD spectra of G22-A39 at 25°C. B) ASL height of CF HBECs at 2 h. Open bar, control; shaded bar, G22-A39 at 21°C; solid bar, G22-A39 heated to 67°C then added to culture. *P < 0.05 vs. control.

Figure 7.

G22-A39 prevents ASL hyperabsorption in the presence of NE. A, B) Bar graphs of ASL height at 8 h in CF HBECs. A) Open bars, control; solid bars, 100 μM G22-A39; dark shaded bars, 10 μM aprotinin. APROT, aprotinin. NE was added at 100 nM. ANS was diluted 1:1 with PBS. All n = 6. *P < 0.05 vs. control, ^†P < 0.05 vs. aprotinin. B) Open bar, control; solid bars, 100 μM G22-A39 with 100 nM NE with or without 10 μM ONO; shaded bar, 100 nM NE with 10 μM ONO. All n = 6. *P < 0.05 vs. control. C) Sequence of SPLUNC1 obtained by mass spectrometry, with the observed residues after 5 min exposure to NE in solid black. G22-A39 peptide is underscored.