The S362A mutation block ROMK2 (Kir1.1b) endocytosis in Xenopus laevis oocyte membrane.

The S362A mutation block ROMK2 (Kir1.1b) endocytosis in Xenopus laevis oocyte membrane. Saeed Hajihashemi1, 1-Assistant professor, PhD in Physiology, Department of Physiology, School of Medical science, Arak University of Medical Sciences. ROMK channel is localized on the apical membrane of the nephron. 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 S362A 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 quik-change approach for site-directed mutagensis. Xenopus oocytes were injected with cRNA encoding ROMK2 or S362A mutant three days prior to treatment with BFA solution (time 0). Brefeldin A (BFA) was added to the OR3 medium (+BFA) at concentrations of 25µM (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 analyzed using Student’s t-tests or ANOVA as appropriate. Incubation of oocytes expressing ROMK2 channels in 25 μM BFA caused a reduction in the currents and membrane voltage. In oocytes expressing the S362A mutant, there was no decay in current and membrane voltage after 48 hours incubation with BFA at 25 µM. The fractional current for ROMK2 at 48h following treatment of oocytes with BFA was 0.24 0.05 (n=24) which was significantly different to S362A mutant (0.96 0.05 n=24). These results show that the S362A 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.