title: Functional implications of mutations in the human renal outer medullary potassium channel (ROMK2) identified in Bartter syndrome.
abstract: Bartter syndrome is an autosomal recessive heterogeneous renal tubular disorder affecting NaCl reabsorption in the thick ascending limb of Henle's loop (TAL). The aim of this study was to elucidate the functional implications of mutations in the predominant human ROMK isoform in TAL, hROMK2, involved in Bartter syndrome type II. cRNA of flag-tagged hROMK2 and eight mutants identified in seven non-related patients was expressed in Xenopus laevis oocytes. hROMK2 activity was measured by two-electrode voltage-clamp analysis and defined as the Ba2+ -sensitive current at a holding potential of -75 mV. The subcellular localization of hROMK2 in oocytes was studied by immunocytochemistry. Injection of 25 pg hROMK2 cRNA resulted in an inwardly rectifying Ba2+ -sensitive current of 522+/-43 nA ( n=22). The mutants could be divided into three distinct groups. First, at 25 pg injection mutants W80C, V103E and T313/350X exhibited no significant currents and could only be detected intracellularly. Upon 8 ng injection, plasma membrane presence was observed as well as currents up to 60% of wild-type current. Second, mutants V53E and V296G exhibited no Ba2+ -sensitive current, but were present in the plasma membrane at 0.1 ng and 8 ng injection levels. Third, mutants P91L and A179T were detectable on the plasma membrane (0.1 ng) and yielded currents of 98% and 80% of wild-type, respectively, at 25 pg injection. S294C yielded currents that were 45% of wild-type and were detected both on and just below the plasma membrane at 0.1 ng injection. This study has unraveled three distinct mechanisms by which mutations in hROMK2 could impair channel function in Bartter syndrome. Future experiments on kidney epithelial cell lines will have to confirm this classification, after which specific pharmacological treatments could be considered for each group of mutations.
authors.affiliation: Department of Cell Physiology, University Medical Centre, PO Box 9101, 6500 HB, Nijmegen, The Netherlands.
authors.forename: Patrick G J F
authors.initials: PG
authors.lastname: Starremans
authors.forename: Annemiete W C M
authors.initials: AW
authors.lastname: van der Kemp
authors.forename: Nine V A M
authors.initials: NV
authors.lastname: Knoers
authors.forename: Lambertus P W J
authors.initials: LP
authors.lastname: van den Heuvel
authors.forename: René J M
authors.initials: RJ
authors.lastname: Bindels
mesh_terms: D000818:Animals; D001477:Bartter Syndrome / Q000235:genetics / Q000503:physiopathology*; D006801:Humans; D007150:Immunohistochemistry; D007679:Kidney Medulla / Q000503:physiopathology*; D008564:Membrane Potentials / Q000502:physiology; D016296:Mutagenesis / Q000502:physiology; D009865:Oocytes / Q000502:physiology; D018408:Patch-Clamp Techniques; D015221:Potassium Channels / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D024661:Potassium Channels, Inwardly Rectifying; D017434:Protein Structure, Tertiary; D014882:Water-Electrolyte Balance / Q000502:physiology; D014982:Xenopus laevis
chemical_list: C086989:KCNJ1 protein, human; D015221:Potassium Channels; D024661:Potassium Channels, Inwardly Rectifying; C571352:ROMK2 protein, human