Epithelial Na⁺channel mutants causing Liddle's syndrome retain ability to respond to aldosterone and vasopressin

Abstract

Liddle's syndrome is a monogenic form of hypertension caused by mutations in the PY motif of the COOH terminus of beta- and gamma-epithelial Na+ channel (ENaC) subunits. These mutations lead to retention of active channels at the cell surface. Because of the critical role of this PY motif in the stability of ENaCs at the cell surface, we have investigated its contribution to the ENaC response to aldosterone and vasopressin. Mutants of the PY motif in beta- and gamma-ENaC subunits (beta-Y618A, beta-P616L, beta-R564stop, and gamma-K570stop) were stably expressed by retroviral gene transfer in a renal cortical collecting duct cell line (mpkCCDcl4), and transepithelial Na+ transport was assessed by measurements of the benzamil-sensitive short-circuit current (Isc). Cells that express ENaC mutants of the PY motif showed a five- to sixfold higher basal Isc compared with control cells and responded to stimulation by aldosterone (10(-6) M) or vasopressin (10(-9) M) with a further increase in Isc. The rates of the initial increases in Isc after aldosterone or vasopressin stimulation were comparable in cells transduced with wild-type and mutant ENaCs, but reversal of the effects of aldosterone and vasopressin was slower in cells that expressed the ENaC mutants. The conserved sensitivity of ENaC mutants to stimulation by aldosterone and vasopressin together with the prolonged activity at the cell surface likely contribute to the increased Na+ absorption in the distal nephron of patients with Liddle's syndrome.

Early Aldosterone-Induced Gene Product Regulates the Epithelial Sodium Channel by Deubiquitylation

“…That the stimulatory action of Usp2-45 on ENaC depends on previous ENaC ubiquitylation indicates, however, that Usp2-45 induction cannot explain the effect of aldosterone on Liddletype mutant ENaC (see the beginning of this article) (23,24). Therefore, it is expected that still other aldosterone-induced gene products, possibly also identified in our screen, act via different pathways on ENaC expression and function.…”

“…This has been indicated by the results of experiments that were performed with mpkCCD cl4 cells that expressed Liddle-type ENaC channels or with mice that expressed an ENaC-␤ subunit with Liddle mutation, thus displaying impaired ENaC/Nedd4-2 interaction (23,24). In these systems, aldosterone was still able to stimulate transepithelial sodium transport.…”

Functional Domains of the Epithelial Sodium Channel

“…More recently, single-channel studies in mouse models of Liddle syndrome (pseudohypoaldosteronism) that lack the PY motif in the ␤-ENaC subunit show that the ENaC response to elevation in plasma aldosterone level is drastically increased in Liddle mice compared with wild-type mice (66). In a cortical CD cell line that expressed ENaC wild-type or mutants with mutations in the PY motif, the rate of increase in Na ϩ transport induced by aldosterone during the early response was similar (67). These observations suggest that the PY motif regulates ENaC activity at the cell surface independent of a stimulation of the aldosterone signaling pathway.…”

Dietary Sodium Intake Regulates the Ubiquitin-Protein Ligase Nedd4-2 in the Renal Collecting System

“…Cells were cultured on plastic tissue-culture flasks as described previously (35). For studying the effect of aldosterone on Nedd4-2 expression, cells were grown on collagencoated filters (Transwell 0.4-m pore, 4.7 cm 2 ).…”