Extracellular protons regulate human ENaC by modulating Na+ self-inhibition

Abstract

The epithelial Na(+) channel, ENaC, is exposed to a wide range of proton concentrations in the kidney, lung, and sweat duct. We, therefore, tested whether pH alters ENaC activity. In Xenopus oocytes expressing human alpha-, beta-, and gammaENaC, amiloride-sensitive current was altered by protons in the physiologically relevant range (pH 8.5-6.0). Compared with pH 7.4, acidic pH increased ENaC current, whereas alkaline pH decreased current (pH(50) = 7.2). Acidic pH also increased ENaC current in H441 epithelia and in human primary airway epithelia. In contrast to human ENaC, pH did not alter rat ENaC current, indicating that there are species differences in ENaC regulation by protons. This resulted predominantly from species differences in gammaENaC. Maneuvers that lock ENaC in a high open-probability state ("DEG" mutation, proteolytic cleavage) abolished the effect of pH on human ENaC, indicating that protons alter ENaC current by modulating channel gating. Previous work showed that ENaC gating is regulated in part by extracellular Na(+) ("Na(+) self-inhibition"). Based on several observations, we conclude that protons regulate ENaC by altering Na(+) self-inhibition. First, protons reduced Na(+) self-inhibition in a dose-dependent manner. Second, ENaC regulation by pH was abolished by removing Na(+) from the extracellular bathing solution. Third, mutations that alter Na(+) self-inhibition produced corresponding changes in ENaC regulation by pH. Together, the data support a model in which protons modulate ENaC gating by relieving Na(+) self-inhibition. We speculate that this may be an important mechanism to facilitate epithelial Na(+) transport under conditions of acidosis.

FIGURE 1.

Extracellular protons modulate human ENaC current. A, representative trace of current versus time recorded in Xenopus oocyte expressing human αβγ ENaC at holding potential of –60 mV. The extracellular bath was changed from pH 7.4 (open bars) to pH 8.5 and 6.5 (black bars) with or without 10 μm amiloride (black bars) as indicated. B, amiloride-sensitive current for human ENaC (relative to current at pH 7.4) with bathing solution pH varied from 8.5 to 5.5 (mean ± S.E., n = 37; error bars are hidden by the symbols). Data are fit to the Hill equation (R² = 0.9991).

FIGURE 2.

Extracellular protons modulate Na⁺ transport in epithelia. A and B, representative short-circuit current traces from H441 (A) and primary human airway (B) epithelia. pH changes and the addition of amiloride (Amil) to the apical bathing solution (10 μm) are indicated by the bars. 0.5-mV pulses were applied every 15 s to monitor resistance. C, percent increase in amiloride-sensitive current in response to pH 6.5 (compared with pH 7.4) for H441 epithelia (n = 6), primary airway epithelia (n = 6), and oocytes expressing human αβγ ENaC (n = 37) (mean ± S.E.). The asterisk indicates that the change in amiloride-sensitive current at pH 6.5 (compared with pH 7.4) is statistically significant (p < 0.001 for H441, p < 0.003 for airway, and p < 0.0001 for oocytes by Student's t test).

FIGURE 3.

Species specificity of pH regulation. A, representative trace of current versus time recorded in Xenopus oocyte expressing rat αβγ ENaC at holding potential of –60 mV. The extracellular bath was changed from pH 7.4 (open bars) to pH 8.5 and 6.5 (black bars). 10 μm amiloride (black bar) was added to the bathing solution as indicated. B, amiloride-sensitive current for human (H) or rat (R) ENaC or the indicated combinations of two human and one rat subunit (relative to current at pH 7.4) with bathing solution pH varied from 8.5 to 6 (mean ± S.E., n = 5–37; error bars are hidden by the symbols). The Hill equation was used to fit the data for human (R² = 0.9991), human βγ rat α (R² = 0.9962), and human αγ rat β (R² = 0.9995).

FIGURE 4.

pH regulates ENaC gating. A and B, representative current traces at holding potential of –60 mV from the same Xenopus oocyte expressing human αβ_S520Cγ ENaC before (A) and after (B) covalent modification with 1 mm [2-(trimethylammonium)ethyl]methanethiosulfonate bromide (MTSET) for 2 min. C, representative current trace at holding potential of –60 mV from a Xenopus oocyte expressing wild-type human αβγENaC after proteolytic cleavage by trypsin (2 μg/ml for 2 min). In A–C, pH changes are indicated by open (pH 7.4) or black (pH 8.5 or 6.5) bars. 10 μm amiloride (Amil) was present in bathing solution, as indicated by the black bars. D, -fold change in amiloride-sensitive current in response to pH 8.5 or 6.5 (relative to current at pH 7.4) before and after application of MTSET (n = 5) or trypsin (n = 7) to oocytes expressing human αβ_S520Cγ or αβγ ENaC, respectively (mean ± S.E.). The asterisk indicates that the difference between the current at pH 6.5, and the current at pH 8.5 (I_6.5 – I_8.5) is statistically different between the indicated groups (p < 0.001).

FIGURE 5.

Extracellular pH modulates Na⁺ self-inhibition. A, representative current trace from Xenopus oocyte expressing humanαβγ ENaC (–60 mV). The extracellular bath pH was 7.4 unless otherwise indicated by black bars. The bath was rapidly changed from 1 to 116 mm NaCl (at pH 8.5 or 6.5) to observe the degree of Na⁺ self-inhibition. Peak current (I_P) and steady-state current (I_SS) are indicated. 10 μm amiloride (Amil) was added to quantitate ENaC current. B, plot of Na⁺ self-inhibition of amiloride-sensitive current ((I_P – I_SS)/I_P) at pH 8.5–6.5 (mean ± S.E., n = 14). C, plot of peak amiloride-sensitive current after a shift in bathing solution from 1 to 116 mm NaCl measured at pH 8.5–6.5 (relative to peak current at pH 7.4) (mean ± S.E., n = 14). D,Na⁺ self-inhibition induced by shift from 0 mm Na⁺ to 1–116 mm Na⁺ (plotted on the x axis) at pH 8.5, 7.4, and 6.5 (mean ± S.E., n = 3–5). Data are fit to the Hill equation; R² = 0.9984 (pH 8.5), 0.9998 (pH 7.4), and 0.9991 (pH 6.5).

FIGURE 6.

Regulation by extracellular pH requires Na⁺. A and B, representative current traces from Xenopus oocytes expressing human αβγ ENaC voltage-clamped at +30 mV. Extracellular bathing solution contained 1 mm Na⁺ (A) or 116 mm Na⁺ (B). Extracellular pH was changed from pH 7.4 (open bars) to pH 8.5 and 6.5 (black bars) as indicated. In panel B, the gray line indicates time-dependent drift in outward amiloride (Amil)-insensitive current. C, plot of -fold change in amiloride-sensitive current at pH 8.5 and 6.5 (relative to pH 7.4 (I_{pH x} – I_{pH 7.4})/I_{pH 7.4}). Inward currents were studied in 116 mm Na⁺ at –60 mV (n = 37), and outward currents were studied in 116 or 1 mm Na⁺ at +30 mV (n = 9 and 14, respectively) (mean ± S.E., *, p < 0.0001 versus 116 mm Na⁺ at –60 or +30 mV by Student's t test).

FIGURE 7.

Mutations that alter Na⁺ self-inhibition affect pH regulation. A and B, representative current traces at holding potential of –60 mV from Xenopus oocytes expressing human αβγ_H233R ENaC (A) or α_H255Rβγ ENaC (B). The extracellular bath was changed from pH 7.4 (open bars) to pH 8.5 and 6.5 (black bars) with or without 10 μm amiloride (Amil, black bars) as indicated. C, amiloride-sensitive current for wild-type or mutant ENaC (relative to current at pH 7.4) with bathing solution pH varied from 8.5 to 5.5 (mean ± S.E., n = 21–37; some error bars are hidden by the symbols). Data are fit to the Hill equation; R² = 0.9992 (α_H255Rβγ), 0.8767 (αβγ_H233R), and 0.9991 (wild type).