The first-nucleotide binding domain of the cystic-fibrosis transmembrane conductance regulator is important for inhibition of the epithelial Na+ channel

Abstract

The cystic-fibrosis transmembrane conductance regulator (CFTR) functions as a cAMP-regulated Cl- channel and as a regulator of other membrane conductances. cAMP-dependent activation of CFTR inhibits epithelial Na+ channels (ENaC). The specificity of interaction between CFTR and ENaC was examined by coexpression of ENaC and ATP-binding cassette (ABC) proteins other than CFTR. In addition, we identified domains within CFTR that are of particular importance for the inhibition of ENaC. To that end, two-electrode voltage-clamp experiments were performed on Xenopus oocytes coexpressing ENaC together with CFTR, the multidrug resistance protein MDR1, the sulfonyl urea receptor SUR1, or the cadmium permease YCF1. Except for CFTR, none of the other ABC proteins were able to inhibit ENaC. Several truncated versions of CFTR were examined for their inhibitory effects on ENaC. In fact, it is shown that C-terminal truncated CFTR is able to inhibit ENaC on activation by intracellular cAMP. Moreover, the data also show that an intact first-nucleotide binding domain (NBF-1) is important for inhibition of ENaC. We conclude that NBF-1 of CFTR contains a CFTR-specific regulatory site that down-regulates ENaC. It is speculated that this regulatory site also is needed for CFTR-mediated interactions with other membrane proteins and that it is not present in NBF-1 of other ABC proteins.

Figure 1

Summary of the whole-cell conductances measured in Xenopus oocytes expressing human CFTR, mouse multidrug resistance protein MDR1, hamster sulfonyl urea receptor SUR1, and yeast cadmium factor YCF1. Water-injected oocytes served as controls. A marginal but significant increase in the nonspecific baseline conductance was observed in MDR1-, SUR1-, and YCF1-expressing oocytes. CFTR-expressing oocytes showed a slight but significant increase in baseline Cl⁻ conductance that was largely increased on stimulation with 1 mmol/liter IBMX and 10 μmol/liter forskolin (black bars). No additional currents were activated in MDR1-, SUR1-, and YCF1-expressing oocytes. The numbers of experiments are shown in parentheses. ∗, Significant difference from control (P < 0.0001). ^§, Significant difference from water-injected oocytes (P < 0.01).

Figure 2

Time course of the whole-cell conductance measured in oocytes coexpressing CFTR and α-, β-, and γ-ENaC (A) or MDR1 and α-, β-, and γ-ENaC (B). Amiloride (Amil; 10 μmol/liter) blocked most of the whole-cell conductance when applied under control conditions. In CFTR-expressing oocytes, the effect of amiloride was attenuated after stimulation with IBMX and forskolin. After washout of IBMX/forskolin (30 min), the initial effect of amiloride was recovered. In MDR1-expressing oocytes the effects of amiloride were independent of cAMP-dependent stimulation.

Figure 3

Summary of amiloride-sensitive whole-cell conductances measured in oocytes coexpressing α-, β-, and γ-ENaC and different ABC transporters. In oocytes expressing only ENaC (ENaC + water) or coexpressing ENaC with either MDR1, SUR1, or YCF1, no changes of the amiloride-sensitive ENaC conductance were observed on stimulation with IBMX and forskolin, whereas activation of CFTR significantly inhibited ENaC conductance. The numbers of experiments are shown in parentheses. ∗, Significant difference from control (P < 0.0001).

Figure 4

Summary of whole-cell conductances observed in oocytes expressing various truncated versions of CFTR. All truncations induced enhanced baseline Cl⁻ conductance. When stimulated with IBMX and forskolin, only oocytes expressing the N-terminal half of CFTR including the R domain (E831X) responded with an increase of the whole-cell conductance. The numbers of experiments are shown in parentheses. ∗, Significant difference from control (P < 0.002). ^§, Significant difference from water-injected oocytes (P < 0.01).

Figure 5

Cl⁻/I⁻ conductivity and permeability ratios for water-injected control oocytes and for oocytes expressing wild-type CFTR and truncated CFTR, respectively. When stimulated with IBMX and forskolin, only oocytes expressing either full-length CFTR or E831X had a change of the conductivity (G_Cl/G_I) and permeability (P_Cl/P_I) ratios toward Cl⁻ > I⁻, indicating activation of CFTR typical Cl⁻ conductance. The numbers of experiments are shown in parentheses. ∗, Significant difference from control (P < 0.01).

Figure 6

Time course of the whole-cell conductance measured in oocytes coexpressing C590X and α-, β-, and γ-ENaC (A) or M595-C and α-, β-, and γ-ENaC (B). Amiloride (Amil; 10 μmol/liter) blocked most of the whole-cell conductance when applied under control conditions. In C590X-expressing oocytes, whole-cell conductances were inhibited, and the effect of amiloride was attenuated after stimulation with IBMX and forskolin. In M595-C-expressing oocytes the effect of amiloride was independent of cAMP-dependent stimulation.

Figure 7

Summary of amiloride-sensitive whole-cell conductances (G_Amiloride) measured in oocytes coexpressing α-, β-, and γ-ENaC and different truncated versions of CFTR. G_Amiloride values measured under control conditions (white bars) were significantly smaller after stimulation of the oocytes with IBMX and forskolin (black bars), except for E402X and M959-C. The numbers of experiments are shown in parentheses. ∗, Significant difference from control (P < 0.01).