Regulation of CLC-Ka/barttin by the ubiquitin ligase Nedd4-2 and the serum- and glucocorticoid-dependent kinases.
BACKGROUND
ClC-Ka and ClC-Kb, chloride channels participating in renal tubular Cl- transport, require the coexpression of barttin to become functional. Mutations of the barttin gene lead to the Bartter's syndrome variant BSND, characterized by congenital deafness and severe renal salt wasting. Barttin bears a proline-tyrosine motif, a target structure for the ubiquitin ligase Nedd4-2, which mediates the clearance of channel proteins from the cell membrane. Nedd4-2 is, in turn, a target of the serum- and glucocorticoid-inducible kinase SGK1, which phosphorylates and, thus, inactivates the ubiquitin ligase. ClC-Ka also possesses a SGK1 consensus site in its sequence. We hypothesized that ClC-Ka/barttin is stimulated by SGK1, and down-regulated by Nedd4-2, an effect that may be reversed by SGK1 and its isoforms, SGK2 or SGK3.


METHODS
To test this hypothesis, ClC-Ka/barttin was heterologously expressed in Xenopus oocytes with or without the additional expression of Nedd4-2, SGK1, SGK2, SGK3, constitutively active S422DSGK1, or inactive K127NSGK1.


RESULTS
Expression of ClC-Ka/barttin induced a slightly inwardly rectifying current that was significantly decreased upon coexpression of Nedd4-2, but not the catalytically inactive mutant C938SNedd4-2. The coexpression of S422DSGK1, SGK1, or SGK3, but not SGK2 or K127NSGK1 significantly stimulated the current. Moreover, S422DSGK1, SGK1, and SGK3 also phosphorylated Nedd4-2 and thereby inhibited Nedd4-2 binding to its target. The down-regulation of ClC-Ka/barttin by Nedd4-2 was abolished by elimination of the PY motif in barttin.


CONCLUSIONS
ClC-Ka/barttin channels are regulated by SGK1 and SGK3, which may thus participate in the regulation of transport in kidney and inner ear.
Embark
Hamdy M
HM
Department of Physiology I, University of Tübingen, Germany.
Böhmer
Christoph
C
Palmada
Monica
M
Rajamanickam
Jeyaganesh
J
Wyatt
Amanda W
AW
Wallisch
Sabine
S
Capasso
Giovambattista
G
Waldegger
Petra
P
Seyberth
Hannsjörg W
HW
Waldegger
Siegfried
S
Lang
Florian
F
D020816:Amino Acid Motifs / Q000502:physiology; D000818:Animals; D018118:Chloride Channels / Q000378:metabolism* / Q000502:physiology; D004594:Electrophysiology; D056827:Endosomal Sorting Complexes Required for Transport; D005260:Female; D006801:Humans; D017874:Immediate-Early Proteins; D008565:Membrane Proteins / Q000737:chemistry / Q000378:metabolism* / Q000502:physiology; D000075702:Nedd4 Ubiquitin Protein Ligases; D009687:Nuclear Proteins / Q000378:metabolism / Q000502:physiology*; D009865:Oocytes; D018408:Patch-Clamp Techniques; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism / Q000502:physiology*; D044767:Ubiquitin-Protein Ligases / Q000378:metabolism / Q000502:physiology*; D029867:Xenopus Proteins; D014982:Xenopus laevis; D000099184:Serum-Glucocorticoid Regulated Kinases
D018118:Chloride Channels; D056827:Endosomal Sorting Complexes Required for Transport; D017874:Immediate-Early Proteins; D008565:Membrane Proteins; D000075702:Nedd4 Ubiquitin Protein Ligases; D009687:Nuclear Proteins; D017346:Protein Serine-Threonine Kinases; D044767:Ubiquitin-Protein Ligases; D029867:Xenopus Proteins; D000099184:Serum-Glucocorticoid Regulated Kinases; C440005:BSND protein, human; C489932:CLCNKA protein, human; C000618482:Nedd4 protein, Xenopus; C000618483:Nedd4 protein, human; C000618485:Nedd4L protein, human; C000618460:nedd4l protein, Xenopus