Patch-clamp studies on epithelial sodium channels in salivary duct cells

Mouse mandibular salivary duct cells contain an amiloride-sensitive Na + current and express all three subunits of the epithelial Na + channel, ENaC. This amiloride-sensitive Na + current is subject to feedback regulation by intracellular Na + and we have previously demonstrated that this regulation is mediated by an ubiquitin-protein ligase, which we identified as Nedd4. The evidence supporting this identification is as follows: (1) antibodies raised against murine Nedd4 block Na + feedback inhibition; (2) a mutant of murine Nedd4 containing the WW domains but no HECT domain (ubiquitin-protein ligase) blocks Na + feedback inhibition; and (3) Nedd4 is expressed in mouse mandibular salivary duct cells. In the present studies, we have used whole-cell patch-clamp methods to further investigate the mechanisms by which ubiquitin-protein ligases regulate the amiloride-sensitive Na + conductance in mouse salivary duct cells. In particular, we have examined the possibility that the ubiquitin-protein which ubiquitin-protein ligases inhibit Na + channels. We have found that KIAA0439 is expressed in mouse mandibular ducts and interacts with the PY motifs of the α-, β-, and γ-subunits of ENaC in vitro. Furthermore, in whole-cell patch-clamp studies, a glutathione-S-transferase (GST)-fusion protein containing the WW motifs of human KIAA0439 was able to inhibit feedback regulation of the amiloride-sensitive Na + current by intracellular Na + . We also examined whether GST-fusion proteins containing the C-termini of the α-, β-, and γ-subunits of ENaC are able to interrupt Na + feedback regulation of the amiloride-sensitive Na + current. We found that the C-termini of the β- and γ-subunits were able to do so, whereas the C-terminus of the α-subunit was not. We conclude that KIAA0439 is, together with Nedd4, a potential mediator of the control of epithelial Na + channels in salivary duct cells by intracellular Na + . We further conclude that ubiquitin-protein ligases interact with the Na + channels through the C-termini of the β- and γ-subunits of the Na + channels.