The Effect of Phosphorylation and Dephosphorylation of the Serine Residue (S362) on ROMK2 (Kir1.1b) Endocytosis

In this study, the effects of S362A and S362D mutations on the membrane turnover and the stability of ROMK2 channel when expressing in Xenopus laevis oocytes are examined. Methods and Materials:In this experimental study, oocytes were isolated by standard protocols using collagens (Type 1A). Mutations of the cytoplasmic termini of ROMK2 were constructed using the quick-change approach for site-directed mutagenesis. Xenopus oocytes were injected with cRNA encoding ROMK2 or S362A or S362D 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. In oocytes was expressing ROMK2 and/or the S362A mutant, there was significant reduce in current and membrane voltage of K. The fractional currents for ROMK2 and S362D mutant demonstrated a slight difference 48h following treatment of oocytes with BFA, 0.160.05(n=18) and 0.110.05(n=18) respectively. This was; however, significantly different from the fractional current of S362A mutant which stood at 0.960.05(n=24). Mutant Serine residue S362A which causes phosphorylation in endocytosis and helps determine the number of ROMK2, plays an important part in PDZ domain.