The cystic fibrosis transmembrane conductance regulator (CFTR) inhibits ENaC through an increase in the intracellular Cl- concentration

Abstract

Activation of the CFTR Cl- channel inhibits epithelial Na+ channels (ENaC), according to studies on epithelial cells and overexpressing recombinant cells. Here we demonstrate that ENaC is inhibited during stimulation of the cystic fibrosis transmembrance conductance regulator (CFTR) in Xenopus oocytes, independent of the experimental set-up and the magnitude of the whole-cell current. Inhibition of ENaC is augmented at higher CFTR Cl- currents. Similar to CFTR, ClC-0 Cl- currents also inhibit ENaC, as well as high extracellular Na+ and Cl- in partially permeabilized oocytes. Thus, inhibition of ENaC is not specific to CFTR and seems to be mediated by Cl-.

Fig. 1. Continuous recording (A) and summary (B) of whole-cell conductances obtained from Xenopus oocytes coexpressing CFTR and ENaC. The effects of amiloride (10 µmol/l) and low bath Cl^– (5 mmol/l) were compared before and after stimulation with IBMX (1 mmol/l) and forskolin (10 µmol/l). No net change in the whole-cell conductance (G_m) was observed during activation (IBMX/forskolin; black bars) of the Cl^– conductance (G_Cl–) and inhibition of ENaC (G_Amiloride). (C) Stimulation of CFTR in the presence of high (105 mmol/l) but not low (5 mmol/l) extracellular Cl^– inhibited ENaC. Asterisks indicate statistical significance (paired t-test, P <0.05) (number of experiments shown in parentheses). The experiments were performed with OOC-1 and Warner OC725C amplifiers with two bath electrodes..

Fig. 2. (A) Summaries of Cl^– (G_Cl–) and ENaC (G_Amiloride) whole-cell conductance in Xenopus oocytes expressing low or high levels of CFTR and ENaC. Conductances were obtained before (white bars) and after (black bars) stimulation with IBMX and forskolin. (B) Summary of Cl^– (G_Cl–) and ENaC (G_Amiloride) from experiments showing two different ratios of expression for G_CFTR:G_ENaC of ∼2:1 and 10:1. Stronger inhibition of ENaC is observed at the higher ratio. Asterisks indicate statistical significance (paired t-test, P <0.05). # indicates significant difference of ENaC inhibition at high and low ratios (unpaired t-test, P <0.05) (number of experiments shown in parentheses). (C) Continuous recording of the whole-cell current generated by expression of ENaC and ClCl-0 in a Xenopus oocyte. Oocytes were voltage clamped from –60 to +40 mV in steps of 10 mV. Note that the effect of amiloride is significantly and reversible inhibited at high bath Cl^– concentration. (D) Summary of the whole-cell conductances obtained from experiments shown in (C). G_Amiloride is significantly (paired t-test, P <0.05) inhibited at high extracellular Cl^– concentration (number of experiments shown in parentheses). Experiments were performed with GeneClamp 500 and Warner OC725C amplifiers using two bath electrodes.

Fig. 3. (A) Amiloride-sensitive whole-cell currents in Xenopus oocytes after subtraction of leak currents and summary of the calculated amiloride sensitive whole-cell conductances (G_Amiloride). Oocytes were voltage clamped from –50 to +20 mV in steps of 10 mV. Currents are shown before and after permeabilization with amphotericin B (Ampho; 1 µmol/l). ENaC is inhibited by high Na⁺ after permeabilization. (B) I/V curves obtained from ENaC expressing oocytes before (dotted line) and after (solid line) application of amiloride, and before (–Ampho) and after (+Ampho) permeabilization. After permeabilization, ENaC was inhibited by an increase of extracellular Cl^– to 50 mmol/l. Inserts summarize total conductance and effect of amiloride before and after permeabilization. (C) Amiloride-sensitive Na⁺ conductances (G_Amiloride) in permeabilized oocytes at various bath Cl^– concentrations. (D) Amiloride-sensitive Na⁺ conductances (G_Amiloride) in permeabilized oocytes in the presence of 5 and 105 mmol/l Cl^–, Br^– or I^– in the bath solution. (E) Inhibition of ENaC by activation of CFTR at different extracellular Cl^– concentrations. Asterisks indicate statistical significance (paired t-test, P <0.05) (number of experiments shown in parentheses). Experiments were performed with GeneClamp 500 and Warner OC725C amplifiers using two bath electrodes.