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 ligase, KIAA0439, which is closely related to Nedd4, may mediate Na+ feedback control of amiloride-sensitive Na+ channels. Furthermore, we have attempted to define the mechanism by 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 alpha-, beta-, and gamma-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 alpha-, beta-, and gamma-subunits of ENaC are able to interrupt Na+ feedback regulation of the amiloride-sensitive Na+ current. We found that the C-termini of the beta- and gamma-subunits were able to do so, whereas the C-terminus of the alpha-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 beta- and gamma-subunits of the Na+ channels.
Cook
David I
DI
Department of Physiology, University of Sydney, NSW, Australia. davidc@physiol.usyd.edu.au
Dinudom
Anuwat
A
Komwatana
Permsak
P
Kumar
Sharad
S
Young
John A
JA
D000584:Amiloride / Q000494:pharmacology; D000818:Animals; D002135:Calcium-Binding Proteins; D002712:Chlorides / Q000378:metabolism; D003600:Cytosol / Q000737:chemistry / Q000378:metabolism; D056827:Endosomal Sorting Complexes Required for Transport; D004847:Epithelial Cells / Q000378:metabolism; D053503:Epithelial Sodium Channels; D005246:Feedback; D008025:Ligases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D000075702:Nedd4 Ubiquitin Protein Ligases; D018408:Patch-Clamp Techniques; D018987:Salivary Ducts / Q000378:metabolism*; D012964:Sodium / Q000378:metabolism; D015222:Sodium Channels / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism*; D013363:Submandibular Gland / Q000033:anatomy & histology; D044767:Ubiquitin-Protein Ligases
D002135:Calcium-Binding Proteins; D002712:Chlorides; D056827:Endosomal Sorting Complexes Required for Transport; D053503:Epithelial Sodium Channels; D015222:Sodium Channels; D000584:Amiloride; D012964:Sodium; D000075702:Nedd4 Ubiquitin Protein Ligases; C000618483:Nedd4 protein, human; C000618485:Nedd4L protein, human; C000618461:Nedd4l protein, mouse; D044767:Ubiquitin-Protein Ligases; D008025:Ligases