Open probability of the epithelial sodium channel is regulated by intracellular sodium

Abstract

The regulation of epithelial Na(+) channel (ENaC) activity by Na(+) was studied in Xenopus oocytes using two-electrode voltage clamp and patch-clamp recording techniques. Here we show that amiloride-sensitive Na(+) current (I(Na)) is downregulated when ENaC-expressing cells are exposed to high extracellular [Na(+)]. The reduction in macroscopic Na(+) current is accompanied by an increase in the concentration of intracellular Na(+) ([Na(+)](i)) and is only slowly reversible. At the single-channel level, incubating oocytes in high-Na(+) solution reduces open probability (P(o)) approximately twofold compared to when [Na(+)] is kept low, by increasing mean channel closed times. However, increasing P(o) by introducing a mutation in the beta-subunit (S518C) which, in the presence of [2-(trimethylammonium) ethyl] methane thiosulfonate (MTSET), locks the channel in an open state, could not alone abolish the downregulation of macroscopic current measured with exposure to high external [Na(+)]. Inhibition of the insertion of new channels into the plasma membrane using Brefeldin A revealed that surface channel lifetime is also markedly reduced under these conditions. In channels harbouring a beta-subunit mutation, R564X, associated with Liddle's syndrome, open probability in both high- and low-Na(+) conditions is significantly higher than in wild-type channels. Increasing the P(o) of these channels with an activating mutation abrogated the difference in macroscopic current observed between groups of oocytes incubated in high- and low-Na(+) conditions. These findings demonstrate that reduction of ENaC P(o) is a physiological mechanism limiting Na(+) entry when [Na(+)](i) is high.

Figure 1. Increased [Na⁺]_i inhibits ENaC currents

Oocytes were injected with either wild-type (αβγ; wt) or mutant (αβR564Xγ) ENaC and incubated in low-Na⁺ (1 mm) MBS overnight. The following day, Na⁺ current was measured by TEVC following the voltage-step protocol described in the Methods. A, normalized change in I_Na over 8 experiments with assessment of 2 batches of oocytes (wt, I₀ = 14 ± 3 μA; mutant, I₀ = 18 ± 3 μA). Current measurements were taken at intervals shown while oocytes were maintained in 110 mm NaCl bath solution. Amiloride (10 μm) was bath applied to the oocyte at the end of every experiment to ensure that currents measured were amiloride sensitive. After 80 min, wt currents had fallen to ∼40% of their initial value, while currents in the mutant stayed the same or increased. B, Na⁺ current reversal potentials (E_rev) were noted, and used to calculate [Na⁺]_i by the Nernst Equation. C, [Na⁺]_i rises over the course of the experiment. Error bars represent the s.e.m.

Figure 2. Reversal of [Na⁺]_i does not reverse effects of high [Na⁺] on macroscopic ENaC current

Oocytes were injected with αβγ ENaC and incubated in low-Na⁺ MBS overnight. A, normalized change in macroscopic I_Na in response to changing extracellular Na⁺ solutions is monitored by TEVC. Filled and shaded bars indicate periods of high (110 mm) and low (5 mm) bath [Na⁺], respectively. At approximately 90 and 120 mins, bath solution was briefly switched from low to high [Na⁺], and I_Na measured. B, while intracellular Na⁺ levels return to, or go below, their pre-high-Na⁺ levels, currents do not significantly recover (see partA), or recover slowly (n = 8). C, oocytes expressing ENaC were incubated overnight in high-Na⁺ solution. The next day, after amiloride-sensitive Na⁺ currents were measured, oocytes were transferred to low-Na⁺ MBS. However, Na⁺ currents were still not as high in this group (17 ± 2 μA) as in those oocytes incubated continuously for 2 days in low [Na⁺] (28 ± 3 μA; n = 19–28). *P < 0.05 compared to high-Na⁺ condition; †P < 0.05 compared to high-Na⁺, then low- Na⁺ condition.

Figure 3. Feedback inhibition is specific for ENaC current

Rat ROMK2 cRNA was co-injected into oocytes with αβγ ENaC cRNA, and incubated in low-Na⁺ MBS. The next day, amiloride- and Ba²⁺-sensitive currents were assessed. After a further overnight incubation in high-Na⁺ MBS, currents were again measured and normalized to current measured the previous day. While ENaC current declined over this time, ROMK current was not affected (n = 10; means ± s.e.m.).

Figure 4. Effects of high- and low-Na⁺ MBS on ENaC kinetics

Oocytes injected with αβγ ENaC cRNA were incubated overnight in MBS solution containing high [Na⁺], low [Na⁺], or high [Na⁺] with 200 nm ouabain. A, macroscopic amiloride-sensitive Na⁺ current is inhibited by high [Na⁺] (high [Na⁺], n = 53; low [Na⁺], n = 52; ouabain, n = 30). B, exemplar cell-attached patch recordings taken from oocytes incubated in high- or low-Na⁺ MBS, with Li⁺ as the charge carrier. The number (N) of channels in each patch and estimated P_o are indicated to the left of each trace. Segments of recording are 1 min in length and correspond to a pipette voltage of +90 mV. Downward deflections show inward current from the pipette to the cell. C, single-channel i–V relationships were determined by measuring amplitude of transition levels at the voltages indicated (n = 19–146 for each point; means ± s.e.m.). Single-channel conductance (g) of ENaC was 7.4 ± 0.1 pS in low-Na⁺ solution and 7.5 ± 0.3 pS in high-Na⁺ solution. D, estimated P_o of ENaC in high-Na⁺ solution (0.30 ± 0.03; n = 13) was lower than P_o of ENaC in low-Na⁺ solution (0.57 ± 0.05; n = 11). *P < 0.05 compared to high-Na⁺ condition.

Figure 5. Comparison of P_n measured and P_n predicted by binomial distribution

The probability that a channel resides at a particular open level (n) was either determined directly by dividing level dwell time by the total time of recording, or calculated from the binomial distribution as described in the text. A, comparison of measured and predicted P_n values for a recording with 4 channels in the patch, taken from an oocyte incubated in high-Na⁺ solution. B, comparison of measured and predicted P_n values for recording with 5 channels in the patch, taken from an oocyte incubated in low-Na⁺ solution.

Figure 6. Effects of high and low [Na⁺] on single-channel kinetics of ENaC Liddle's mutant αβR564Xγ

Oocytes injected with αβR564Xγ ENaC cRNA were incubated overnight in MBS solution containing high [Na⁺], low [Na⁺], or high [Na⁺] with 200 nm ouabain. A, I_Na in wild-type (wt) and βR564X mutant channels in the same batches of oocytes. Mutant channels are still sensitive to high [Na⁺], but fractional decrease in macroscopic amiloride-sensitive Na⁺ current is less than in wild-type channels (high [Na⁺], n = 12 wt, n = 25 mutant; low [Na⁺], n = 16 wt, n = 26 mutant; ouabain, n = 11 wt, n = 15 mutant). B, exemplar cell-attached patch recordings taken from oocytes incubated in high- or low-Na⁺ MBS, with Li⁺ as the charge carrier. Segments of recording are 1 min in length and correspond a pipette voltage of +90 mV. Downward deflections show movement of inward current from the pipette to the cell. C, single-channel i–V relationships were determined by measuring amplitude of transition levels at the voltages indicated (n = 7–84 for each point; means ± s.e.m.). Single-channel conductance (g) of ENaC was 8.5 ± 0.6 pS in low-Na⁺ solution and 8.1 ± 0.1 pS in high-Na⁺ solution. D, estimated P_o of ENaC in high-Na⁺ solution (0.52 ± 0.05; n = 7) was lower than P_o of ENaC in low-Na⁺ solution (0.81 ± 0.04; n = 6); P < 0.05. *P < 0.05 compared to high-Na⁺ condition; †P < 0.05 compared to wt channels under the same condition.

Figure 7. Effects of increasing single-channel P_o on macroscopic currents

A, oocytes expressing wt ENaC were incubated in high- or low-Na⁺ solution, and I_Na measured by TEVC. MTSET was then dissolved in 110 mm NaCl to a final concentration of 1 mm immediately before use. Currents in the presence of MTSET were measured every 1–2 min until they stabilized; amiloride-sensitive currents were subsequently assessed by perfusing 10 μm amiloride into the bath (n = 3 high [Na⁺]; n = 3 low [Na⁺]). B, oocytes expressing βS518C ENaC were incubated in high- or low-Na⁺ solution, and I_Na measured by TEVC. MTSET (1 mm) was then bath applied to oocytes, and amiloride-sensitive currents assessed (n = 14 high [Na⁺]; n = 12 low [Na⁺]). C, representative recordings taken from oocytes expressing βS518C ENaC incubated in either high- or low-Na⁺ solution overnight, showing response to MTSET and amiloride application. *P < 0.05 compared to wt high-Na⁺ condition; †P < 0.05 compared to mutant high-Na⁺ condition.

Figure 8. Effect of brefeldin A on macroscopic Na⁺ current

Oocytes injected with αβγ ENaC cRNA were incubated overnight in MBS solution containing high or low [Na⁺]. Sixteen to 24 hours after injection, macroscopic amiloride-sensitive Na⁺ current was assessed by TEVC. Brefeldin A (BFA; 5 μm) was then added to the incubation media, and Na⁺ current assessed every 2 h by TEVC. Each point represents the mean of 4–11 oocytes. Data were fitted by an exponential decay function (I = Ae^−t/τ + B). In high-Na⁺ solution, A = 3.0 μA, τ = 2.1 h and B = 0.3 μA; in low-Na⁺ solution, A = 5.0 μA, τ = 2.1 h and B = 1.9 μA. Error bars represent the s.e.m.

Figure 9. Effects of increasing single-channel P_o of βR564X ENaC on macroscopic currents

A, oocytes were injected with αβS518C–R564Xγ ENaC and incubated in high- or low-Na⁺ solution. The following day, after I_Na was measured, MTSET was then dissolved in 110 mm NaCl to a final concentration of 1 mm immediately before use and applied to oocytes. Currents in the presence of MTSET were measured every 1–2 min until they stabilized; amiloride-sensitive currents were subsequently assessed by perfusing 10 μm amiloride into the bath (n = 11 high [Na⁺]; n = 15 low [Na⁺]). B, representative recordings taken from oocytes expressing βS518C–R564X ENaC incubated in either high- or low-Na⁺ solution overnight, showing response to MTSET and amiloride application. *P < 0.05 compared to high-Na⁺ condition.