The transport of NaCl by the thick ascending limb of Henle’s loop couples a Na+-2Cl–-K+ cotransporter (NKCC2) with a secretory K+ channel (ROMK). In Bartter’s syndrome, mutations of the genes encoding these proteins disrupt NaCl reabsorption leading to salt wasting. M357T, a known Bartter’s mutation, has no effect on ROMK1 channel function (Schwalbe et al. 1998). In the current study we have evaluated the effect of this mutation on ROMK2 (M338T).
The M338T mutation in rat ROMK2 was produced by recombinant PCR. DNA encoding WT-ROMK2 and the M338T mutant were sub-cloned into the Xenopus expression vector PTLN and the EGFP-fusion expression vector, pEGFP-C2. Female Xenopus laevis were killed humanely and stage V-VI oocytes isolated. Oocytes were injected with either 50 nl H2O or 5 ng (0.1 ng nl-1) of cRNA encoding for WT-ROMK2 or the M357T mutant. Two-electrode voltage clamp was performed at room temperature 3-4 days after injection. The bath solution contained (mM): NaCl 96; KCl 2; MgCl2 1; CaCl2 1.8; Hepes 5. In Ba2+-containing solutions, BaCl2 replaced NaCl in equimolar quantities. The pH of solutions was adjusted to 7.5 using HCl or NaOH. HEK 293 cells were transfected with either pEGFP-C2 or EGFP-tagged constructs of WT-ROMK2 or the M338T mutant using Lipofectamine 2000TM reagent and cultured under standard conditions. The cells were viewed by fluorescence microscopy 24-48 h following transfection. Data are presented as means ± S.E.M. and statistical significance was assumed at the 5 % level by using an independent Student’s paired t test.
Oocytes expressing WT-ROMK2 displayed an outward current of 4.13 ± 0.13 µA (n = 7) at a holding potential of 0 mV, which was reduced by 58.3 ± 2.3 % following addition of 5 mM BaCl2. In oocytes expressing the M338T mutant, currents at 0 mV (4.17 ± 0.13 µA, n = 7) were not different from those in WT-ROMK2-expressing oocytes. In H2O-injected oocytes the current at 0 mV was 0.14 ± 0.01 µA (n = 7).
HEK cells expressing EGFP exhibited uniform fluorescence distributed throughout the cell interior and nucleus. In contrast, the distribution of fluorescence in HEK cells transfected with WT-ROMK2 or the M338T mutant was consistent with expression of the fusion protein in intracellular compartments and the plasma membrane.
These results indicate that the M338T mutation of ROMK2 does not affect trafficking of the protein to the cell membrane, or macroscopic channel function.
We thank Dr P. Fong (Baltimore) for a gift of the PTLN vector, Professor A. Surprenant (Sheffield) for advice regarding the use and maintenance of HEK293 cells, and the NKRF for financial support.