Identification of amino acid residues in the alpha, beta, and gamma subunits of the epithelial sodium channel (ENaC) involved in amiloride block and ion permeation

Abstract

The amiloride-sensitive epithelial Na channel (ENaC) is a heteromultimeric channel made of three alpha beta gamma subunits. The structures involved in the ion permeation pathway have only been partially identified, and the respective contributions of each subunit in the formation of the conduction pore has not yet been established. Using a site-directed mutagenesis approach, we have identified in a short segment preceding the second membrane-spanning domain (the pre-M2 segment) amino acid residues involved in ion permeation and critical for channel block by amiloride. Cys substitutions of Gly residues in beta and gamma subunits at position beta G525 and gamma G537 increased the apparent inhibitory constant (Ki) for amiloride by > 1,000-fold and decreased channel unitary current without affecting ion selectivity. The corresponding mutation S583 to C in the alpha subunit increased amiloride Ki by 20-fold, without changing channel conducting properties. Coexpression of these mutated alpha beta gamma subunits resulted in a non-conducting channel expressed at the cell surface. Finally, these Cys substitutions increased channel affinity for block by external Zn2+ ions, in particular the alpha S583C mutant showing a Ki for Zn2+ of 29 microM. Mutations of residues alpha W582L, or beta G522D also increased amiloride Ki, the later mutation generating a Ca2+ blocking site located 15% within the membrane electric field. These experiments provide strong evidence that alpha beta gamma ENaCs are pore-forming subunits involved in ion permeation through the channel. The pre-M2 segment of alpha beta gamma subunits may form a pore loop structure at the extracellular face of the channel, where amiloride binds within the channel lumen. We propose that amiloride interacts with Na+ ions at an external Na+ binding site preventing ion permeation through the channel pore.

Figure 1

Linear model of an ENaC subunit showing two transmembrane domains M1 and M2 with a pre-M2 segment (arrow) dipping into the membrane. The pre-M2 segment corresponds to the H2 segment in the linear model of ENaC subunits proposed by Canessa et al. (1994b). Sequence alignment of the pre-M2 segments of ENaC αβγ subunits is shown below. These sequences are identical in X. Laevis, rat, and human ENaC genes. Numbering refers to the ENaC rat sequence.

Figure 2

Representative tracings of macroscopic inward Li⁺current recorded in three different oocytes expressing wild-type ENaC αβγ subunits. Substitution of 20 mM KCl with 20 mM LiCl in the external sucrose buffer medium (•) induced an inward Li⁺current that was inhibited by addition of 10 nM, 100 nM, 330 nM, 660 nM, 5 μM indicated by the filled triangles (▴).

Figure 3

Cell surface expression and macroscopic I_Liof wt ENaC (n = 39), triple Cys mutant (α_SCβ_SCγ_SC) (n = 36) and βγ subunits (n = 41). Filled bars represents I_Li. Hatched bars represents specific binding per oocyte of M₂AB ([¹²⁵I]M₂IgG₁) antibodies (fmols/oocyte) directed against a FLAG epitope introduced in the ectodomain of β and γ wt and mutant subunits. *Denotes statistical significance <0.001.

Figure 4

Effects of introducing Cys residues in pre M2 segment at positions αS583, βG525, and γG537. (A) Single channel tracings of βG525C mutant recorded with Na⁺as permeating ion. (B) Left: I-V relationships of wt ENaC and αS583C mutant. Cord conductance for wt ENaC (wt) in the presence of Na⁺was 5.1 and 10 pS with Li⁺. Li⁺conductance of αS583C was 8.9 pS. Right: I-V relationships for βG525C and γG537C. In the presence of Na⁺and Li⁺ions, cord conductances were, respectively, 3.2 and 5.8 pS for βG525C, 4 and 6.23 pS for γG534C. Dotted lines represent fit of the data of the I-V relationships according to the constant field equation. Each point represents mean value of two to five channels.

Figure 5

Amiloride sensitivity of αβγ Cys mutants. (A) Original tracings of amiloride inhibition of inward Li⁺current in oocytes expressing βG525C mutant together with α and γ subunits. Triangles (▴) represent addition of external amiloride at increasing concentrations of 0.1, 10, 70, and 100 μM, and • indicates return to the starting K solution. (B) Titration curve of Li⁺current by amiloride. I/I₀ is the ratio of blocked over unblocked Li⁺current. Li⁺ current was measured in individual oocytes in the presence of four different amiloride concentrations (wt-ENaC, n = 82; αS583C, n = 27; βG525C, n = 59; γG537C, n = 14) Dotted lines represent fit of data to a Langmuir inhibition isotherm (see materials and methods).

Figure 6

Block of wt ENaC and mutants by external Zn²⁺. Concentration dependence of Zn²⁺ inhibition of inward Li⁺current. I/I₀represents the current ratio measured in the presence/absence of external Zn²⁺. Inhibitory constants (K _i) were obtained from best fit of the data to a Langmuir inhibition isotherm (dotted lines). Zn²⁺ K _i were >10 mM for wt ENaC (n = 34), 29 μM for αS583C (n = 42), 1.1 mM for βG525C (n = 10), and 1.9 mM for γG537C (n = 14).

Figure 7

Single channel recording of αS583C mutant in absence or with 0.2 mM Zn²⁺in the external solution. In the presence of Zn²⁺ channel activity (n · P _o) was 0.64 at −80 mV, 0.46 at −100 mV, 0.44 at −120 mV, and 0.35 at −150 mV.

Figure 8

Effects on single channel conductance of introducing negatively charged residues Asp in the pre-M2 segment of αβ subunits. (A) Upper tracing shows single channel recording in oocytes expressing α, βG522D, γ mutants. The lower tracings were recorded from an oocyte expressing both βG522D and β wild type together with α and γ subunits. Two channels with different conductances can be observed. (B) Left: I-V relationship of αS580D, βG522D, and βG522S in the presence of with 1.8 mM Ca²⁺ in the patch pipet. Cord Li⁺conductance was 9.6 pS for βG522S. Right: I-V relationship of S580D, G522D in the absence of Ca²⁺ in the pipet. Cord conductance were 6.7 and 11 pS for S580D in the presence of Na⁺and Li⁺respectively and 11 pS for βG522D with Li⁺ions. Each point represents a mean value of three to five channels.

Figure 9

Block of αS580D, βG522D, γG534E mutants by external Ca²⁺. Mutant subunits were coexpressed with α, γ wild-type subunits. (A) Concentration dependence of inhibition of Li⁺current by external Ca²⁺. The ratio I/I₀ represents the measured I_Liin the presence/absence of external Ca²⁺ions. Ca²⁺ inhibitory constants (K _i) obtained from fit to inhibition Langmuir isotherm (dotted lines) were 2.1 mM for the αS580D (n = 8), 0.4 mM for βG522D (n = 10), and 2.2 mM for γG534E (n = 8). (B) Voltage dependence of βG522D mutant block by external Ca²⁺ at concentrations of 0.5, 1, and 2 mM (n = 4). Dotted lines represent fit of the data to Eq. 2 (see materials and methods) gave K _i(0) values of 0.67, 0.97, and 0.49, z′ values of 0.26, 0.28, and 0.35 for the experiments done with respectively 0.5, 1, and 2mM Ca²⁺.

Figure 10

Decreased sensitivity of βG522D mutant to amiloride. Dose-dependent inhibition of Li⁺current by amiloride. Inhibitory constants (K _i) obtained from fit of the data to inhibition Langmuir isotherm were 0.14 μM for αS580D (n = 12), 0.06 μM for γG534E (n = 8), 3.4 μM for βG522D (n = 16), and 0.6 μM for βG522S (n = 10).