Regulation of the epithelial Na⁺ channel by intracellular Na⁺

The hypothesis that the intracellular Na⁺ concentration ([Na⁺](i)) is a regulator of the epithelial Na⁺ channel (ENaC) was tested with the Xenopus oocyte expression system by utilizing a dual-electrode voltage clamp. [Na⁺](i) averaged 48.1 ± 2.2 meq (n = 27) and was estimated from the amiloride-sensitive reversal potential. [Na⁺](i) was increased by direct injection of 27.6 nl of 0.25 or 0.5 M Na₂SO₄. Within minutes of injection, [Na⁺](i) stabilized and remained elevated at 97.8 ± 6.5 meq (n = 9) and 64.9 ± 4.4 (n = 5) meq 30 min after the initial injection of 0.5 and 0.25 M Na₂SO₄, respectively. This increase of [Na⁺](i) caused a biphasic inhibition of ENaC currents. In oocytes injected with 0.5 M Na₂SO₄ (n = 9), a rapid decrease of inward amiloride-sensitive slope conductance (g(Na)) to 0.681 ± 0.030 of control within the first 3 min and a secondary, slower decrease to 0.304 ± 0.043 of control at 30 min were observed. Similar but smaller inhibitions were also observed with the injection of 0.25 M Na₂SO₄. Injection of isotonic K₂SO₄ (70 mM) or isotonic K₂SO₄ made hypertonic with sucrose (70 mM K₂SO₄-1.2 M sucrose) was without effect. Injection of a 0.5 M concentration of either K₂SO₄, N-methyl-D-glucamine (NMDG) sulfate, or 0.75 M NMDG gluconate resulted in a much smaller initial inhibition (<14%) and little or no secondary decrease. Thus increases of [Na⁺](i) have multiple specific inhibitory effects on ENaC that can be temporally separated into a rapid phase that was complete within 2-3 min and a delayed slow phase that was observed between 5 and 30 min.