Anionic phospholipids differentially regulate the epithelial sodium channel (ENaC) by interacting with alpha, beta, and gamma ENaC subunits

Abstract

Anionic phospholipids (APs) present a variety of lipids in the cytoplasmic leaflet of the plasma membrane, including phosphatidylinositol (PI), PI-4-phosphate (PI(4)P), phosphatidylserine (PS), PI-4,5-bisphosphate (PI(4,5)P(2)), PI-3,4,5-trisphosphate (PI(3,4,5)P(3)), and phosphatidic acid (PA). We previously showed that PI(4,5)P(2) and PI(3,4,5)P(3) upregulate the renal epithelial sodium channel (ENaC). Further studies from others suggested that PI(4,5)P(2) and PI(3,4,5)P(3) respectively target beta- and gamma-ENaC subunit. To determine whether PI(4,5)P(2) and PI(3,4,5)P(3) selectively bind to beta and gamma subunit, we performed lipid-protein overlay experiments. Surprisingly, the results reveal that most APs, including PI(4)P, PS, PI(4,5)P(2), PI(3,4,5)P(3), and PA, but not PI, non-selectively bind to not only beta and gamma but also alpha subunit. To determine how these APs regulate ENaC, we performed inside-out patch-clamp experiments and found that PS, but not PI or PI(4)P, maintained ENaC activity, that PI(4,5)P(2) and PI(3,4,5)P(3) stimulated ENaC, and that PA, however, inhibited ENaC. These data together suggest that APs differentially regulate ENaC by physically interacting with alpha-, beta-, and gamma-ENaC. Further, the data from cell-attached patch-clamp and confocal microscopy experiments indicate that PA, a product of phospholipase D, may provide one of the pathways for inhibition of ENaC by endothelin receptors.

Fig. 1

Each individual ENaC subunit expressed in HEK293 cells binds to most anionic phospholipids. a A diagram for lipid-protein overlay experiments, as described in “Materials and methods”. b HEK293 cells were transiently transfected with ECFP-tagged rat α-ENaC (α-rENaC-ECFP), EYFP-tagged rat β-ENaC (β-rENaC-EYFP), or DsRed-tagged rat γ-ENaC (γ-rENaC-DsRed). PIP Strips were respectively incubated with protein extracts from cells expressing each construct. α-rENaC-ECFP (left) and β-rENaC-EYFP (middle) were detected with anti-green-fluorescent protein antibody, while γ-rENaC-Red (right) was detected with anti-DsRed antibody. The data represent three experiments showing similar results

Fig. 2

The αβγ ENaC complex natively expressed in H441 cells also binds to most anionic phospholipids. PIP Strips™ were incubated with protein extract from H441 cells endogenously expressing ENaC subunits (α-, β-, and γ-hENaC) either in the absence (a) or in the presence of either the antigen peptide (b) or 10 μg/ml poly-L-lysine (c), and then detected with antibodies to human α-(left), β-(middle), and γ-ENaC (right) subunits, respectively. The data represent four experiments showing similar results

Fig. 3

Phosphatidylserine (PS) maintains ENaC activity in inside-out patches. a ENaC single-channel current before and after addition of saline (as a control) to the “cytoplasmic” bath. b–d ENaC single-channel current before and after addition of phosphatidylinositol (PI) (50 μM), PI(4)P (50 μM), or PS (50 μM) to the “cytoplasmic” bath, respectively. In this figure and the following figures, mean ENaC P_os before (−3 to 0 min) and after each experimental manipulation (0–3 min and 3–6 min) were listed under each representative single-channel trace. e Percent change in ENaC P_o (3–6 min after additions of saline (Con), PI, PI(4)P, or PS versus −3 to 0 min before each addition)

Fig. 4

Stimulation of ENaC by phosphatidylinositol (PI) (4,5)P₂ and PI(3,4,5)P₃ in inside-out patches is dependent on negative charges. a, b ENaC single-channel current before and after addition of PI(4,5)P₂ (50 μM) to the “cytoplasmic” bath either alone or in the presence of 10 μg/ml poly-L-lysine. c, d ENaC single-channel current before and after addition of PI(3,4,5)P₃ (50 μM) to the “cytoplasmic” bath either alone or in the presence of 10 μg/ml poly-L-lysine. e ENaC single-channel current before and after addition of poly-L-lysine alone. f Percent change in ENaC P_o (3–6 min after additions of PI(4,5)P₂ and PI(3,4,5)P₃ in the absence or presence of poly-L-lysine (poly-L-lysine alone serves as a control) versus −3 to 0 min before each addition)

Fig. 5

Phosphatidic acid (PA) inhibits ENaC in inside-out patches. a, b ENaC single-channel current recorded before and after addition of either saline (as a control) or PA (50 μM) to the “cytoplasmic” bath. c Percent reduction of ENaC P_o (3 to 6 min after addition of PA versus −3 to 0 min before the addition)

Fig. 6

Endothelin-1 (ET-1) induces phospholipase D (PLD)-dependent inhibition of ENaC in cell-attached patches. a, b ENaC single-channel current before and after addition of either saline (as a control) or ET-1 (20 nM) to the basolateral bath. c ENaC single-channel current before and after addition of ET-1 (20 nM) to the basolateral bath in the presence of 1% 1-butanol (1-But). d Percent reduction of ENaC P_o (3 to 6 min after additions of either saline or ET-1 in the absence or presence of 1% 1-But versus −3 to 0 min before each addition)

Fig. 7

Endothelin-1 (ET-1) induces translocation of phospholipase D (PLD) to the apical membrane. a Under control conditions, PLD (green fluorescence) was sporadically localized in the cytoplasm of A6 cells. b After the cells were basolaterally treated with 20 nM ET-1, PLD accumulated near the apical membrane. c In contrast, no green fluorescence was detected when the cells were incubated with the fluorescent secondary antibody alone. In a–c, nuclei were stained with Hoechst 33258 trihydrochloride, shown in blue. Confocal microscopy XY scanning was performed across A6 cell monolayer on the edge of folded filter (see “Materials and methods”). The figure represents three experiments showing similar results