WNK4 regulates activity of the epithelial Na+ channel in vitro and in vivo

Abstract

Homeostasis of intravascular volume, Na(+), Cl(-), and K(+) is interdependent and determined by the coordinated activities of structurally diverse mediators in the distal nephron and the distal colon. The behavior of these flux pathways is regulated by the renin-angiotensin-aldosterone system; however, the mechanisms that allow independent modulation of individual elements have been obscure. Previous work has shown that mutations in WNK4 cause pseudohypoaldosteronism type II (PHAII), a disease featuring hypertension with hyperkalemia, due to altered activity of specific Na-Cl cotransporters, K(+) channels, and paracellular Cl(-) flux mediators of the distal nephron. By coexpression studies in Xenopus oocytes, we now demonstrate that WNK4 also inhibits the epithelial Na(+) channel (ENaC), the major mediator of aldosterone-sensitive Na(+) (re)absorption, via a mechanism that is independent of WNK4's kinase activity. This inhibition requires intact C termini in ENaC beta- and gamma-subunits, which contain PY motifs used to target ENaC for clearance from the plasma membrane. Importantly, PHAII-causing mutations eliminate WNK4's inhibition of ENaC, thereby paralleling other effects of PHAII to increase sodium balance. The relevance of these findings in vivo was studied in mice harboring PHAII-mutant WNK4. The colonic epithelium of these mice demonstrates markedly increased amiloride-sensitive Na(+) flux compared with wild-type littermates. These studies identify ENaC as a previously unrecognized downstream target of WNK4 and demonstrate a functional role for WNK4 in the regulation of colonic Na(+) absorption. These findings support a key role for WNK4 in coordinating the activities of diverse flux pathways to achieve integrated fluid and electrolyte homeostasis.

Fig. 1.

Wild-type WNK4, but not PHAII-mutant WNK4, inhibits ENaC by way of a kinase-independent mechanism. cRNA encoding the indicated proteins was injected into Xenopus oocytes, and amiloride-sensitive, whole-cell Na⁺ currents were recorded as describe in Methods. (a) A diagram showing the clustering of PHAII-causing mutations in WNK4. (b) Representative current tracings from oocytes expressing ENaC alone, ENaC plus wild-type WNK4, or ENaC plus PHAII–WNK4. (c) Current–voltage (I–V) relationships of oocytes expressing indicated constructs from a representative experiment plotted as mean ± SE of the Na⁺ currents for each experimental group. (d Upper) Cumulative results of amiloride-sensitive currents measured at −100 mV are shown as a proportion of the ENaC control. Each group expresses ENaC plus the indicated construct; a minimum of 29 oocytes were studied in each group. The significance of differences between groups was calculated with two-tailed Student's t test. (d Lower) Results of Western blotting of oocyte lysates shows the expression of WNK4-HA in the corresponding experimental groups.

Fig. 2.

Inhibition of ENaC by WNK4 requires C-terminal domains in the channel. (a) A diagram of wild-type ENaC subunit and Liddle's syndrome mutations that truncate the C terminus of either β- or γ-subunits and delete the PY motif (in green) is shown (β and γ_LT represent Liddle's truncations in the respective ENaC subunits). (b and c) Activity of ENaC with Liddle's mutations in the presence or absence of WNK4. cRNA encoding ENaC harboring a Liddle's mutation in either the β-subunit (at least 11 oocytes per group) (b) or the γ-subunit (at least 22 oocytes per group) (c) with or without wild-type WNK4 was injected into Xenopus oocytes, and amiloride-sensitive, whole-cell Na⁺ currents were recorded and analyzed as in Fig. 1. Western blotting of oocyte lysates confirms the presence of WNK4-HA in the corresponding experimental groups. WNK4 does not inhibit ENaC activity in the presence of Liddle's mutations.

Fig. 3.

Immunolocalization of WNK4 in the mouse colon. Distal colons of mice were dissected, prepared, and stained with antibodies specific for WNK4 as described in Methods. A transverse section is shown. The results show strong staining of the surface epithelium and upper part of crypts. (Magnification: ×400.) (Inset) A higher magnification, demonstrating the strongest staining of the basolateral membrane. (Magnification: ×800.)

Fig. 4.

Increased amiloride-sensitive Na⁺ transport in the distal colon of PHAII mice. Segments of distal colonic epithelia were dissected and mounted in modified Ussing chambers, and amiloride-sensitive Na⁺ flux was measured as described in Methods. (a) A representative current tracing of amiloride-sensitive I_sc from wild-type and PHAII mice is shown, demonstrating a larger amiloride-sensitive current in PHAII versus wild-type mice. (b) Mean and SE of amiloride-sensitive Na⁺ flux across colonic epithelia from wild-type mice (n = 11) and PHAII mice (n = 11) is shown, demonstrating a significant increase in PHAII mice.