ABSTRACT
Recent studies suggest that endocytosis of ROMK channels is important for regulation of K+ secretion in cortical collecting ducts. In this study the effect of V364D mutation is examined on the membrane turnover and stability of ROMK2 channel when expressing in Xenopus laevis oocytes. In this experimental study oocytes were isolated by standard protocols using collagenase [Type 1A]. Mutations of the cytoplasmic termini of ROMK2 were constructed using the quikchange approach for site-directed mutagensis. Xenopus oocytes were injected with cRNA encoding ROMK2 or V364D mutant three days prior to treatment with BFA solution [time 0]. Brefeldin A [BFA] was added to the OR3 medium [+BFA] at concentrations of 5-25 M [inhibit insertion of new proteins into the cell membrane] or ethanol as BFA vehicle [-BFA]. Two-electrode voltage clamp [TEVC] was used to measure oocyte ROMK-dependent currents and membrane potential. Data was analysed using Student's t-tests or ANOVA as appropriate. Incubation of oocytes expressing ROMK2 channels in both 5microM and 25 microM BFA caused a reduction in the normalized steady state currents. The effect of BFA was dose dependent. In oocytes expressing the V364D mutant, there was no decay in current at any time point during incubation with BFA at either 5 M or 25 M. The fractional current for ROMK2 at 48h following treatment of oocytes with BFA was 0.24 0.05 [n=16] which was significantly different to V364D mutant [1.17 0.09]. These results show that the V364D mutation increases the general stability of ROMK and renderes the protein resistant to endocytosis, consistent with the idea that there is an interaction between the C-terminal of ROMK2 and components of the endocytotic pathway. A functional PDZ domain [the S-E-V] plays a key role in determining stability of ROMK