Liddle’s syndrome (a hereditary form of hypertension) is associated with increased apical density of renal Na⁺ channels (ENaC). Proline-rich areas present in wild-type ENaC β- and γ-subunits normally interact with Nedd4 to tag membrane proteins prior to endocytosis, but these areas are disrupted in Liddle’s mutations (Hansson et al. 1995). Nedd4 downregulates wild-type ENaC (Goulet et al. 1998) and raising intracellular Ca²⁺ mobilizes Nedd4 to the apical membrane (Staub et al. 2000). Here, we report the effects of raising intracellular Ca²⁺ on amiloride-sensitive whole-cell current (I_amil) in Xenopus oocytes (donor killed by a Home Office approved method) expressing either wild-type or Liddle’s mutated human renal ENaC.

Oocyte nuclei were co-injected with either 3.5 ng cDNA for α_W, β_W and γ_W (wild-type channels), α_W, β_L and γ_W (β-Liddle’s channels), or α_W, β_W and γ_L (γ-Liddle’s channels). After incubating for 24-48 h, I_amil (10 µM amiloride) was evaluated between -140 and 40 mV by dual electrode voltage clamping before and after 30 min exposure to 10 µM A23187 (Ca²⁺ ionophore). Results were expressed as means ± S.E.M. and evaluated using Student’s paired t test, one-way ANOVA and Fisher’s LSD post hoc analyses. P values of < 0.05 were considered significant.

Basal I_amil at -100 mV was greater with β-Liddle’s (15.49 ± 2.30 µA, n = 47, P < 0.0001) and γ-Liddle’s (11.72 ± 1.97 µA, n = 40, P < 0.0001) channels compared with wild-type channels (2.59 ± 0.33 µA, n = 171). Within-group analysis showed that A23187 decreased normalised I_amil by 55 ± 10 % in oocytes expressing wild-type channels (P < 0.0005, n = 10), but the decreases in the oocytes expressing β- or γ-Liddle’s channels were insignificant (P = 0.4, n = 7 and P = 0.94, n = 6, respectively). Between-group analysis confirmed that the magnitude of the effect of A23187 was significantly smaller in oocytes expressing β- or γ-Liddle’s channels (P < 0.035 and P < 0.01, respectively) compared with those expressing wild-type channels. These data suggest that intracellular Ca²⁺ downregulates wild-type ENaC via the Nedd4/ubiquitination pathway, which is ineffective in Liddle’s syndrome.

G.G.R. was supported by the British Heart Foundation.