Genetically modified mice lacking connexin 30 (Cx30) have been reported to develop salt-sensitive hypertension (1) because of an increased activity of the epithelial sodium channel (ENaC) in the distal nephron (2). This suggests that ENaC is inhibited by Cx30. In the present study co-expression experiments were used to investigate a functional interaction of ENaC and Cx30. Human ENaC and human Cx30 were heterologously expressed in Xenopus laevis oocytes. In two-electrode-voltage clamp experiments ENaC-mediated whole-cell currents were determined by measuring amiloride (2 µM) sensitive currents. ENaC expression at the cell surface was estimated using a FLAG-tagged βENaC subunit and a chemiluminescence assay. Channel open probability (Po) was assessed in outside-out patch-clamp recordings. Site-directed mutagenesis was used to identify channel regions relevant for the functional interaction of ENaC and Cx30. Values are presented as mean ± SEM. Unpaired Student’s t-test was used for statistical analysis. Co-expression of Cx30 significantly reduced ENaC-currents by 56.3±3.0% (n=79, p<0.001) compared to those in control oocytes expressing ENaC alone. The inhibitory effect of Cx30 on ENaC was associated with a significant ~60% reduction of channel surface expression (p<0.001, n=72) without an apparent decrease in Po. Each of the three ENaC subunits (αβγ) contains a PY-motif within its cytosolic C-terminus. The PY-motifs are thought to be critically involved in Nedd4-2-dependent channel retrieval from the cell surface. Simultaneous truncation of the C-termini of all three ENaC subunits essentially abolished the Cx30-mediated channel inhibition. Interestingly, truncation of the γENaC C-terminus alone was sufficient to prevent ENaC inhibition by Cx30, whereas mutation of the prolines in the PY-motif of γENaC (623PPPxY627) did not significantly reduce the inhibitory effect of Cx30. This finding indicates that the inhibitory effect of Cx30 is not mediated by increased Nedd4-2-dependent channel retrieval. A putative clathrin adaptor protein 2 (AP-2) recognition motif (YxxΦ) is also present in the C-terminus of the γ-subunit and shares the tyrosine with the PY-motif. Importantly, mutating the leucine residue in this 627YxxL630-motif of γENaC significantly reduced the inhibitory effect of Cx30 on ENaC which averaged 23.5±4.0% (p<0.001, n=50). We conclude that Cx30 inhibits ENaC by reducing channel expression at the cell surface. Our finding that the inhibitory effect of Cx30 involves a putative AP-2 recognition motif in the C-terminus of the channel’s γ-subunit suggests that the effect may be mediated by stimulating channel retrieval via clathrin-dependent endocytosis. Reduced channel retrieval in the absence of Cx30 may explain an increased ENaC activity in mice lacking Cx30.