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 β‐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 β‐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.