title: ROMK expression remains unaltered in a mouse model of familial hyperkalemic hypertension caused by the CUL3Δ403-459 mutation.
abstract: Familial hyperkalemic hypertension (FHHt) is a rare inherited form of salt-dependent hypertension caused by mutations in proteins that regulate the renal Na(+)-Cl(-) cotransporter NCC Mutations in four genes have been reported to cause FHHt including CUL3 (Cullin3) that encodes a component of a RING E3 ligase. Cullin-3 binds to WNK kinase-bound KLHL3 (the substrate recognition subunit of the ubiquitin ligase complex) to promote ubiquitination and proteasomal degradation of WNK kinases. Deletion of exon 9 from CUL3 (affecting residues 403-459, CUL3(Δ403-459)) causes a severe form of FHHt (PHA2E) that is recapitulated closely in a knock-in mouse model. The loss of functionality of CUL3(Δ403-459) and secondary accumulation of WNK kinases causes substantial NCC activation. This accounts for the hypertension in FHHt but the origin of the hyperkalemia is less clear. Hence, we explored the impact of CUL3(Δ403-459) on expression of the distal secretory K channel, ROMK, both in vitro and in vivo. We found that expressing wild-type but not the CUL3(Δ403-459) mutant form of CUL3 prevented the suppression of ROMK currents by WNK4 expressed in Xenopus oocytes. The mutant CUL3 protein was also unable to affect ROMK-EGFP protein expression at the surface of mouse M-1 cortical collecting duct (CCD) cells. The effects of CUL3 on ROMK expression in both oocytes and M-1 CCD cells was reduced by addition of the neddylation inhibitor, MLN4924. This confirms that neddylation is important for CUL3 activity. Nevertheless, in our knock-in mouse model expressing CUL3(Δ403-459) we could not show any alteration in ROMK expression by either western blotting whole kidney lysates or confocal microscopy of kidney sections. This suggests that the hyperkalemia in our knock-in mouse and human PHA2E subjects with the CUL3(Δ403-459) mutation is not caused by reduced ROMK expression in the distal nephron.
authors.affiliation: Division of Experimental Medicine and Immunotherapeutics, Department of Medicine, University of Cambridge, Cambridge, CB2 2QQ, United Kingdom.
authors.forename: Meena
authors.initials: M
authors.lastname: Murthy
authors.affiliation: MRC Protein Phosphorylation and Ubiquitylation Unit, College of Life Sciences University of Dundee, Dow Street, Dundee DD15EH, Scotland, United Kingdom.
authors.forename: Thimo
authors.initials: T
authors.lastname: Kurz
authors.affiliation: Division of Experimental Medicine and Immunotherapeutics, Department of Medicine, University of Cambridge, Cambridge, CB2 2QQ, United Kingdom kmo22@medschl.cam.ac.uk.
authors.forename: Kevin M
authors.initials: KM
authors.lastname: O'Shaughnessy
mesh_terms: D048868:Adaptor Proteins, Signal Transducing; D000818:Animals; D015415:Biomarkers / Q000097:blood; D002460:Cell Line; D044842:Cullin Proteins / Q000235:genetics*; D055879:Gene Knock-In Techniques; D020022:Genetic Predisposition to Disease; D007668:Kidney / Q000201:enzymology*; D008564:Membrane Potentials; D008822:Mice, Transgenic; D008840:Microfilament Proteins / Q000235:genetics / Q000378:metabolism; D009154:Mutation; D009865:Oocytes; D010641:Phenotype; D011188:Potassium / Q000097:blood; D024661:Potassium Channels, Inwardly Rectifying / Q000235:genetics / Q000378:metabolism*; D011499:Protein Processing, Post-Translational; D017346:Protein Serine-Threonine Kinases / Q000235:genetics / Q000378:metabolism; D011546:Pseudohypoaldosteronism / Q000097:blood / Q000201:enzymology* / Q000235:genetics; D014162:Transfection; D014982:Xenopus laevis
chemical_list: D048868:Adaptor Proteins, Signal Transducing; D015415:Biomarkers; C503646:Cul3 protein, mouse; D044842:Cullin Proteins; C000598025:KLHL3 protein, mouse; C510693:Kcnj1 protein, mouse; D008840:Microfilament Proteins; D024661:Potassium Channels, Inwardly Rectifying; C499024:Prkwnk4 protein, mouse; D017346:Protein Serine-Threonine Kinases; D011188:Potassium
keywords: Cullin‐3; ROMK; WNK kinases; hyperkalemia; proteasome; ubiquitin‐ligase complex