Cell surface expression of the epithelial Na channel and a mutant causing Liddle syndrome: a quantitative approach

Abstract

The epithelial amiloride-sensitive sodium channel (ENaC) controls transepithelial Na+ movement in Na(+)-transporting epithelia and is associated with Liddle syndrome, an autosomal dominant form of salt-sensitive hypertension. Detailed analysis of ENaC channel properties and the functional consequences of mutations causing Liddle syndrome has been, so far, limited by lack of a method allowing specific and quantitative detection of cell-surface-expressed ENaC. We have developed a quantitative assay based on the binding of 125I-labeled M2 anti-FLAG monoclonal antibody (M2Ab*) directed against a FLAG reporter epitope introduced in the extracellular loop of each of the alpha, beta, and gamma ENaC subunits. Insertion of the FLAG epitope into ENaC sequences did not change its functional and pharmacological properties. The binding specificity and affinity (Kd = 3 nM) allowed us to correlate in individual Xenopus oocytes the macroscopic amiloride-sensitive sodium current (INa) with the number of ENaC wild-type and mutant subunits expressed at the cell surface. These experiments demonstrate that: (i) only heteromultimeric channels made of alpha, beta, and gamma ENaC subunits are maximally and efficiently expressed at the cell surface; (ii) the overall ENaC open probability is one order of magnitude lower than previously observed in single-channel recordings; (iii) the mutation causing Liddle syndrome (beta R564stop) enhances channel activity by two mechanisms, i.e., by increasing ENaC cell surface expression and by changing channel open probability. This quantitative approach provides new insights on the molecular mechanisms underlying one form of salt-sensitive hypertension.

Figure 1

(A) FLAG-epitope placement into the rat ENaC sequence. The alignment of the extracellular loop sequences for the rat, human, and Xenopus ENaC subunits demonstrated the most important divergence in the region near the first transmembrane domain M1. This region was chosen to insert (α and γ subunits) or to replace (β subunit) the FLAG sequence (DYKDDDDK) at the indicated positions. (B) Immunoprecipitation of the FLAG-tagged ENaC subunits, using M₂Ab*. Oocytes were injected with an α, β, or γ nontagged subunit alone (lane 1), or with an α, β, or γ FLAG-tagged subunit alone (lanes 2 and 3). Oocytes were metabolically labeled with [³⁵S]methionine for a 4-h pulse followed by a 4-h chase period. Membranes were prepared, and proteins were immunoprecipitated and analyzed on 5–8% gradient SDS/PAGE. Immunoprecipitations of the samples corresponding to lane 3 were performed in the presence of 1000-fold molar FLAG peptide excess over the antibody concentration.

Figure 2

Equilibrium binding of M₂Ab* and M₂Fab* to the FLAG-tagged ENaC subunits. Stage V oocytes were injected, either with 10 ng of FLAG-tagged or with nontagged ENaC cRNAs. Twenty-four hours later, these oocytes were tested for M₂Ab* (A) or M₂Fab (C) binding. Twelve oocytes were transferred into 100 μl (M₂Ab) or 50 μl (M₂Fab) binding buffer and then incubated with increasing concentrations of M₂Ab* or M₂Fab* for 4 h on ice. The concentration of M₂Ab* and M₂Fab varied from 0.03–20 nM and 5–50 nM, respectively. Specific binding was calculated as the difference of the binding between the oocytes injected with FLAG-tagged and the oocytes injected with nontagged ENaC cRNAs and transformed into Scatchard plots for M₂Ab* (B) or M₂Fab* (D). (A and C) The means ± SEM of three independent experiments, each performed on 12 oocytes.

Figure 3

Specific binding of the M₂Fab* and M₂Ab* to Xenopus oocytes expressing FLAG-tagged ENaC subunits. Binding of the 50 nM M₂Fab* (dotted columns) or 12 nM M₂Ab* (shaded columns) was measured in parallel on the oocytes expressing either FLAG-tagged (Fl) or nontagged ENaC subunits. Specificity of the binding was tested, using either 1000-molar excess of the FLAG peptide (M₂Fab* and M₂Ab*) or 200-fold excess of unlabeled M₂Ab (M₂Ab*). FLAG peptide and unlabeled M₂Ab were added in the binding solution simultaneously with the ¹²⁵I-labeled agents. Shown are means ± SEM from four independent experiments, each performed on 12 oocytes.

Figure 4

Single-channel properties of FLAG-tagged ENaC channels. (Upper) A single-channel recording performed in cell attached configuration with Na⁺ ions as major cation in the pipette. Pipette voltage (V_pip) was 80 mV. Downward deflections represent channel openings. The four distinct current levels indicate the presence of at least four conducting channels in the patch. (Lower) Current-voltage relationship in the presence of Na⁺ or Li⁺ ions in the pipette. Single-channel conductance estimated from regression analysis between −50 mV and −150 mV was 5.5 pS for Na⁺ and 8.8 pS for Li⁺.

Figure 5

Specific M₂Ab* binding to the oocytes injected with different combinations of ENaC subunits. X. laevis oocytes were injected with FLAG-tagged cRNAs (3 ng) of α, β, and γ ENaC, or combinations of αβ, αγ, βγ, αβγ, or nontagged αβγ as control. Binding analysis was performed with 12 nM M₂Ab*, and the specific binding has been determined as difference between the oocytes injected with the FLAG-tagged ENaC subunits and the oocytes injected with nontagged α, β, and γ ENaC subunits. The data are means ± SEM of three independent experiments, each performed on 12 oocytes. ∗, P < 0.001.

Figure 6

Relationships between macroscopic amiloride-sensitive Na⁺ current (I_Na) and surface expression of ENaC channel subunits. Specific M₂Ab* binding is expressed as a function of I_Na measured at −100 mV. Each symbol represent one oocyte, the type of symbol refers to different batches of oocytes. (A) Experiments (n = 4) performed with oocytes incubated in the low sodium solution. Regression analysis on all the data points gave a linear correlation (r²= 0.85) with a slope (solid line; dotted lines are 95% confidence limit) of 4.82 μA/fmol crossing the origin at 0.09 μA. (B) Oocytes incubated in the high sodium medium. Regression analysis of the correlation between I_Na and the bound M₂Ab* molecules gave a slope of 1.13 μA/fmol (r² = 0.83) and a y value at the origin of −0.13.

Figure 7

Effects of βR564stop mutation on I_Na and channel expression at the cell surface. Five independent experiments were performed in which 10 to 12 oocytes were injected with either β subunit or βR564stop mutant, together with α and γ ENaC wild type. (A) Mean I_Na and M₂Ab* binding was, respectively, 0.94 ± 0.27 μA and 0.29 ± 0.06 fmol for wt and 5.31 ± 1.01 μA and 0.56 ± 0.1 fmol for the β564R stop. ∗, A statistical significance P < 0.05. (B) Correlation between INa measured at −100 mV and specific M₂Ab* binding in individual oocytes. Regression analysis (regression line, straight line; dotted lines are 95% confidence limit) gave a linear correlation with a slope of 1.8 μA/fmol M₂Ab* (r² = 0.6) for the wt and 9.1 μA/fmol M₂Ab* (r² = 0.77) for βR564stop.