Open AccessROMK expression remains unaltered in a mouse model of familial hyperkalemic hypertension caused by the CUL 3 Δ403‐459 mutation
Author(s) -
Murthy Meena,
Kurz Thimo,
O'Shaughnessy Kevin M.
Publication year - 2016
Publication title -
physiological reports
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.918
H-Index - 39
ISSN - 2051-817X
DOI - 10.14814/phy2.12850
Subject(s) - mutation , hyperkalemia , medicine , endocrinology , genetics , bioinformatics , biology , gene
Familial hyperkalemic hypertension ( FHH t) 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 FHH t including CUL 3 (Cullin3) that encodes a component of a RING E3 ligase. Cullin‐3 binds to WNK kinase‐bound KLHL 3 (the substrate recognition subunit of the ubiquitin ligase complex) to promote ubiquitination and proteasomal degradation of WNK kinases. Deletion of exon 9 from CUL 3 (affecting residues 403‐459, CUL 3 Δ403‐459 ) causes a severe form of FHH t ( PHA 2E) that is recapitulated closely in a knock‐in mouse model. The loss of functionality of CUL 3 Δ403‐459 and secondary accumulation of WNK kinases causes substantial NCC activation. This accounts for the hypertension in FHH t but the origin of the hyperkalemia is less clear. Hence, we explored the impact of CUL 3 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 CUL 3 Δ403‐459 mutant form of CUL 3 prevented the suppression of ROMK currents by WNK 4 expressed in Xenopus oocytes. The mutant CUL 3 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 CUL 3 on ROMK expression in both oocytes and M‐1 CCD cells was reduced by addition of the neddylation inhibitor, MLN 4924. This confirms that neddylation is important for CUL 3 activity. Nevertheless, in our knock‐in mouse model expressing CUL 3 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 PHA 2E subjects with the CUL 3 Δ403‐459 mutation is not caused by reduced ROMK expression in the distal nephron.