Cell-specific regulation of L-WNK1 by dietary K

Abstract

Flow-induced K(+) secretion in the aldosterone-sensitive distal nephron is mediated by high-conductance Ca(2+)-activated K(+) (BK) channels. Familial hyperkalemic hypertension (pseudohypoaldosteronism type II) is an inherited form of hypertension with decreased K(+) secretion and increased Na(+) reabsorption. This disorder is linked to mutations in genes encoding with-no-lysine kinase 1 (WNK1), WNK4, and Kelch-like 3/Cullin 3, two components of an E3 ubiquitin ligase complex that degrades WNKs. We examined whether the full-length (or "long") form of WNK1 (L-WNK1) affected the expression of BK α-subunits in HEK cells. Overexpression of L-WNK1 promoted a significant increase in BK α-subunit whole cell abundance and functional channel expression. BK α-subunit abundance also increased with coexpression of a kinase dead L-WNK1 mutant (K233M) and with kidney-specific WNK1 (KS-WNK1), suggesting that the catalytic activity of L-WNK1 was not required to increase BK expression. We examined whether dietary K(+) intake affected L-WNK1 expression in the aldosterone-sensitive distal nephron. We found a paucity of L-WNK1 labeling in cortical collecting ducts (CCDs) from rabbits on a low-K(+) diet but observed robust staining for L-WNK1 primarily in intercalated cells when rabbits were fed a high-K(+) diet. Our results and previous findings suggest that L-WNK1 exerts different effects on renal K(+) secretory channels, inhibiting renal outer medullary K(+) channels and activating BK channels. A high-K(+) diet induced an increase in L-WNK1 expression selectively in intercalated cells and may contribute to enhanced BK channel expression and K(+) secretion in CCDs.

Keywords: high-conductance calcium-activated potassium channels; kidney-specific with-no-lysine kinase 1; long with-no-lysine kinase 1; potassium adaptation; pseudohypoaldosteronism; with-no-lysine kinase 1.

Fig. 1.

Long (L) with-no-lysine kinase 1 (WNK1) expression increases high-conductance Ca²⁺-activated K⁺ (BK) α-subunit abundance. HEK293 cells were transfected with green fluorescent protein (GFP, control), BK α-subunit, or BK α-subunit and L-WNK1. A: L-WNK1 expression increases whole cell BK α-subunit abundance. Whole cell lysates were subjected to immunoblot with an anti-myc antibody to detect BK α-subunit, an anti-hemagglutinin (HA) antibody to detect L-WNK1, or an anti-actin antibody. B: summary of analyses of BK α-subunit whole cell expression. Immunoblots were quantified as described in materials and methods (n = 11, *P < 0.001) (Student's t-test). Results were normalized to BK α-subunit expression in the absence of exogenous L-WNK1.

Fig. 2.

L-WNK1 expression increases whole cell BK channel activity. HEK293 cells were transfected with BK α-subunit alone or with L-WNK1. Patch-clamp studies were performed with the amphotericin B perforated whole cell technique (A–C) or with conventional whole cell patch-clamp (D), as described in materials and methods. BK currents were evoked by voltage steps from −80 to 100 mV in 10-mV increments from a holding potential of −60 mV (A–C) or by voltage steps from −80 to 200 mV in 20-mV increments from a holding potential of −80 mV (D). A and B: representative tracings obtained in the absence or presence of 100 nM iberiotoxin (IBTX) from cells transfected with myc-tagged BK α-subunit alone (A) or with L-WNK1 (B). C: summary of the effect of L-WNK1 on IBTX-sensitive whole cell currents measured at +80 mV (n = 6–7, *P < 0.01) (Student's t-test). D: normalized steady-state conductance-voltage (G–V) relationships for cells transfected with BK α-subunit alone (open circles) or with L-WNK1 (closed circles) (n = 9–11). Data were fitted to a Boltzmann function as described in materials and methods.

Fig. 3.

L-WNK1 increases whole cell BK expression in HEK293 L-WNK1 knockout (KO) cells. A: lack of endogenous L-WNK1 expression in HEK293 L-WNK1 KO cells. Lysates obtained from HEK293 and HEK293 L-WNK1 KO cells were subjected to immunoblot with an anti-L-WNK1 antibody as described in materials and methods. Each lane was loaded with 12 μg of protein. B: L-WNK1 increases whole cell BK expression in HEK293 L-WNK1 KO cells. Whole cell lysates from HEK293 L-WNK1 KO cells transfected with GFP (control), L-WNK1, BK α-subunit, or BK α-subunit with L-WNK1 were subjected to immunoblot with an anti-HA antibody to detect L-WNK1, an anti-myc antibody to detect BK α-subunit, or an anti-actin antibody. Lanes were loaded with 1–6 μg of protein. Representative of 4 independent experiments. C: linearity of the Western blot detection system for BK α-subunit abundance. The relative intensity of the bands in the immunoblot shown in B was calculated with ImageJ. Data from cells transfected with BK α-subunit alone are shown as open symbols while those from cells transfected with the BK α-subunit and L-WNK1 are shown as gray symbols. Data were analyzed by linear regression.

Fig. 4.

L-WNK1 increases whole cell BK expression in the absence of kinase activity. A: kinase dead L-WNK1 (K233M) increases whole cell BK α-subunit abundance. Whole cell lysates from HEK293 L-WNK1 KO cells transfected with GFP (control), BK α-subunit, or BK α-subunit with either wild-type L-WNK1 or L-WNK1 K233M were subjected to immunoblot with an anti-HA antibody to detect L-WNK1, an anti-myc antibody to detect BK α-subunit, or an anti-actin antibody. B: summary of analyses of BK α-subunit whole cell expression. Immunoblots were quantified as described in materials and methods (n = 8, *P < 0.01) (Student's t-test). Results were normalized to BK α-subunit expression in the absence of exogenous L-WNK1 or L-WNK1 K233M. Levels of expression of BK α-subunit were significantly increased by coexpression of L-WNK1 (P < 0.001) or by coexpression of the K233M mutant (P < 0.05) (Kruskal-Wallis test followed by Dunn's multiple-comparisons test).

Fig. 5.

kidney-specific (KS) WNK1 increases whole cell BK expression. A: both L-WNK1 and KS-WNK1 increase whole cell BK α-subunit expression. Lysates from HEK293 L-WNK1 KO cells transfected with GFP (control), BK α-subunit, or BK α-subunit with either wild-type L-WNK1, KS-WNK1, or both L-WNK1 and KS-WNK1 were subjected to immunoblot with an anti-myc antibody to detect BK α-subunit, an anti-HA antibody to detect L-WNK1 or KS-WNK1, or an anti-actin antibody. B: reduced levels of KS-WNK1 compared with levels of L-WNK1 in HEK293 L-WNK1 knockout cells. Whole cell lysates from cells transfected as indicated above were subjected to immunoblot with an anti-HA antibody to detect L-WNK1 or KS-WNK1 or an anti-GAPDH antibody. Note that the detection of KS-WNK1 required significantly longer exposure times than were required to detect L-WNK1. For this experiment, cells were harvested 24 h posttransfection. C: summary of analyses of BK α-subunit whole cell expression. Immunoblots were quantified as described in materials and methods (n = 9). Results were normalized to BK α-subunit expression in the absence of exogenous L-WNK1 or KS-WNK1. Levels of expression of BK α-subunit were significantly increased by coexpression of L-WNK1 (P < 0.001), KS-WNK1 (P < 0.01), or both L-WNK1 and KS-WNK1 (P < 0.001) (Kruskal-Wallis test followed by Dunn's multiple-comparisons test).

Fig. 6.

High-K⁺ (HK) diet increases L-WNK1 expression in rabbit cortical collecting ducts (CCDs). Single CCDs isolated from New Zealand White (NZW) rabbits fed a HK or a low-K⁺ (LK) diet for 10 days were microperfused and fixed for immunofluorescence microscopy. Immunolabeling was performed as indicated in materials and methods. A: L-WNK1 expression in CCDs from LK-fed animals. Immunostaining for L-WNK1 was difficult to visualize in CCDs from rabbits fed a LK diet. B: expression of L-WNK1 in rabbit CCD intercalated cells after 10 days on a HK diet. Intercalated cells (arrows) were identified by their apical labeling with rhodamine-conjugated peanut agglutinin (PNA). C: negative control incubated with Alexa488-conjugated goat anti-rabbit IgG antibody but not with primary antibody. D: antigen competition. Only modest basal membrane labeling was seen when the anti-L-WNK1 antibody was preincubated with a WNK1 peptide fragment (arrows indicate PNA⁺ cells).

Fig. 7.

Quantitation of L-WNK1 expression in the apical and subapical regions of PNA-positive (PNA⁺) and PNA-negative (PNA⁻) cells. A: representative confocal photomicrographs of L-WNK1 immunolabeling (green) in immunoperfused CCD isolated from a HK-fed rabbit. Type B intercalated cells are labeled with apical PNA (red). Rectangles identify PNA-positive β-intercalated cells (green), PNA-negative cells (purple), and lumen (background, yellow). B: relative fluorescence intensity of PNA⁺ and PNA⁻ cells. The L-WNK1 immunofluorescence intensity of PNA⁺ cells (1.00 ± 0.06) was 2.3-fold greater than that of PNA⁻ cells (0.44 ± 0.03). The relative fluorescence intensity of the apical and subapical region of PNA⁺ cells (1.00 ± 0.07) was 7.8-fold greater than that of PNA⁻ cells (0.13 ± 0.02). Fluorescence intensity was normalized to the average signal in PNA⁺ cells. Median comparisons between cell types made using a Mann-Whitney test. *P ≤ 0.001; n = 20 (whole cell) and n = 29 (apical-subapical region) (PNA⁺ and PNA⁻, each) in 4 tubules.