Intrinsic voltage dependence of the epithelial Na+ channel is masked by a conserved transmembrane domain tryptophan

Abstract

Tryptophan residues critical to function are frequently located at the lipid-water interface of transmembrane domains. All members of the epithelial Na+ channel (ENaC)/Degenerin (Deg) channel superfamily contain an absolutely conserved Trp at the base of their first transmembrane domain. Here, we test the importance of this conserved Trp to ENaC/Deg function. Targeted substitution of this Trp in mouse ENaC and rat ASIC subunits decrease channel activity. Differential substitution with distinct amino acids in alpha-mENaC shows that it is loss of this critical Trp rather than introduction of residues having novel properties that changes channel activity. Surprisingly, Trp substitution unmasks voltage sensitivity. Mutant ENaC has increased steady-state activity at hyperpolarizing compared with depolarizing potentials associated with transient activation and deactivation times, respectively. The times of activation and deactivation change 1 ms/mV in a linear manner with rising and decreasing slopes, respectively. Increases in macroscopic currents at hyperpolarizing potentials results from a voltage-dependent increase in open probability. Voltage sensitivity is not influenced by divalent cations; however, it is Na+-dependent with a 63-mV decrease in voltage required to reach half-maximal activity per log increase in [Na+]. Mutant channels are particularly sensitive to intracellular [Na+] for removing this sodium abolishes voltage dependence. We conclude that the conserved Trp at the base of TM1 in ENaC/Deg channels protects against voltage by masking an inhibitory allosteric or pore block mechanism, which decreases activity in response to intracellular Na+.

FIGURE 1.

Substitution of a critical Trp at the base of TM1 decreases ENaC activity but not membrane expression. A, sequence alignment of the residues spanning the cytosolic-membrane interface for TM1 in ENaC/Deg channels. The subunit type is noted to the left. The position of the transmembrane domain is noted at the top. B, representative macroscopic currents before and after addition of amiloride (noted by arrow) for voltage-clamped CHO cells expressing wild-type and mutant ENaC containing one subunit with substituted Trp. Also shown are representative currents for channels containing α-subunits with the residues just before and after Trp substitution. Inward current is downwards. C, summary graph of activity reported as amiloride-sensitive current density for wild-type and mutant ENaC expressed in CHO cells. *, significant decrease compared with wild-type. D, summary graph of the relative membrane levels (to total expression levels) of wild-type and mutant ENaC. For each mutant, the subunit of interest was identified using an engineered Myc tag. Wild-type α-ENaC (having a Myc tag) was followed in the wild-type control group. The inset shows a typical experiment: the top Western blots were probed with anti-Myc antibody and contained the total cellular and membrane pool of proteins from control CHO cells expressing only GFP and cells expressing Myc-tagged α-mENaC with untagged β- and γ-mENaC. Membrane fractions isolated from 4× the amount of total cellular lysate loaded. As shown in the lower panel, these blots were stripped and counterprobed with anti-Fra-2, which is a cytosolic protein, to confirm good separation of the membrane fraction from total cellular pools. Molecular weights are noted to the left.

FIGURE 2.

Loss of the critical Trp at the base of TM1 causes decreases in activity. A, summary graph of activity of wild-type channels and mutant channels containing α-mENaC with differential substitution of the critical Trp at the base of TM1. Identical to experiments in Fig. 1, the activity of recombinant ENaC was quantified as the amiloride-sensitive current density in voltage-clamped CHO cells. *, significant decrease compared with wild-type. B, summary graph of the relative membrane levels of the α-subunits in wild-type and mutant ENaC containing α-subunits with differentially substituted Trp. The inset shows representative Western blots: the left and right blots contain total cellular proteins and membrane fractions (4× concentrated), respectively. As in Fig. 1, blots are probed with anti-Myc antibody to identify α-subunits in wild-type and mutant channels.

FIGURE 3.

ENaC containing α-subunits with substituted Trp sense voltage. A, representative families of macroscopic currents evoked by a voltage-step protocol (shown to the right) for wild-type (top) and mutant ENaC (containing W112C α-subunits; bottom) in voltage-clamped CHO cells in symmetrical solutions. B, normalized macroscopic I-V relation for instantaneous (t = 1) and steady-state (t = 2) currents for wild-type and mutant ENaC from voltage-clamped CHO cells as in A. Currents normalized to instantaneous current at −100 mV. C, summary G-V curve showing the voltage dependence of steady-state currents from mutant ENaC. The inset shows wild-type. Data are collected from experiments similar to those described in A.

FIGURE 4.

Increases in P_o upon hyperpolarization underlie voltage sensitivity in mutant ENaC. Representative single channel current traces at test potentials ranging from 80 to −80 mV in excised, outside-out patches for wild-type (A) and mutant ENaC (B, containing W112C α-subunits) expressed in CHO cells are shown. Experiments were performed in symmetrical LiCl solutions. Inward current is down. C, summary plot showing open probability as a function of test potential for wild-type and mutant ENaC. Data are from experiments similar to those described in A and B. D, summary i-V plots for wild-type and mutant ENaC from experiments similar to those described in A and B.

FIGURE 5.

The tau of activation and deactivation for mutant ENaC change in a linear manner as a function of voltage. A, overlays of two representative families of macroscopic currents from mutant ENaC in a voltage-clamped CHO cell evoked by voltage protocols (shown to the left) built to quantify the tau of activation. Activation times were measured by stepping from the holding potential of 80 mV down to −100 mV (in 10-mV decrements) and from hold to −200 mV starting at 0 mV (in 10-mV decrements) with 1-s intervals at hold between each test potential. B, an overlay of a representative family of macroscopic currents from mutant ENaC in a voltage-clamped CHO cell evoked by a voltage protocol (shown to the left) built to quantify tau of deactivation. Deactivation times were measured by stepping to 100 mV (in 10-mV increments) from a holding potential of −80 mV with 1-s intervals at hold between each test potential. C, summary graph plotting τ_activation and τ_deactivation as a function of voltage. Data are from experiments similar to those described in A and B.

FIGURE 6.

I-V relation for mutant ENaC in symmetrical solutions with decreasing amounts of NaCl. Representative families of macroscopic currents and summary I-V graphs for instantaneous and steady-state currents from voltage-clamped CHO cells expressing wild-type (top) and mutant (bottom) ENaC. Data were collected in symmetrical 50 (A and B), 10 (C and D), and 2 mm [Na⁺] (E and F).

FIGURE 7.

Voltage sensitivity of mutant ENaC is [Na⁺]-dependent. A, summary G-V curves for steady-state currents from mutant ENaC acquired in symmetrical 150, 50, 10, and 2 mm [Na⁺]. B, summary graph showing the voltage resulting in half-maximal activity at steady state, as established from G-V curves, for mutant ENaC as a function of Na⁺ concentration.

FIGURE 8.

Mutant ENaC is sensitive to intracellular [Na⁺]. Current-voltage relations for instantaneous (t = 1) and steady-state (t = 2) macroscopic currents from wild-type and mutant (W112C) ENaC in voltage-clamped CHO cells in asymetrical (bath/pipette) 150/5 (A) and 5/150 (B) NaCl solutions. I-V relations for instantaneous (C) and steady-state (D) currents for wild-type and mutant ENaC corrected for reversal potential by subtracting E_rev from V_m and replotting the data with reversal through 0 mV. Inward currents were plotted from results with 150/5 gradients and outward currents from 5/150 gradients. E, G-V relation for steady-state currents from mutant ENaC in 150/5 and 5/150 solutions. F, summary graph showing the voltage resulting in half-maximal activity at steady state, as established from G-V curves, for mutant ENaC in asymetrical solutions as a function of intracellular [Na⁺]. The curve for V_1/2 versus [Na⁺] for mutant ENaC in symmetrical solutions from Fig. 7B is re-shown for comparison.

FIGURE 9.

Removal of intracellular Na⁺ abolishes the voltage dependence of mutant ENaC. A, representative single channel current traces at test potentials ranging from 0 to −100 mV in excised, outside-out patches for mutant ENaC containing W112C α-subunits expressed in CHO cells. Experiments performed in asymmetrical 150/0 NaCl solutions with no sodium in the solution bathing the intracellular face of the channel. Inward current is down. B, summary graph showing open probability as a function of test potential for mutant ENaC (assayed in outside-out patches) in asymetrical 150/0 NaCl. Data are from experiments similar to those described in A.

FIGURE 10.

Mutation of the critical Trp at the base of TM1 in β-ENaC also results in voltage sensitivity. A, representative family of macroscopic currents from mutant ENaC (containing wild-type α- and γ-subunits and W52I-substituted β-subunits) in asymetrical 150/5 Na⁺ solutions evoked by a voltage-step protocol in a voltage-clamped CHO cell. B, summary I-V curves for instantaneous and steady-state macroscopic currents from mutant ENaC containing W52I-substituted β-subunits. C, summary G-V curve for mutant ENaC containing the W52I β-subunit.