متوقف شدن روند آندوسيتوز كانال پتاسيمي ROMK2 (Kir1.1b در اثر موتاسيون S362A در غشاء اووسيت هاي Xenopus Laevis

The S362A mutation block ROMK2 (Kirl.lb)endocytosis in Xenopus laevis oocyte membrane

Introduction: ROMK channel is localized on the apical membrane of nephrons. 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 expressed in Xenopus laevis oocytes. Methods: oocytes were isolated by standard protocols using collagenase (Type 1A). Mutations of the cytoplasmic termini of ROMK2 were performed using the quik-change approach for site-directed mutagenesis. Xenopus oocytes were injected with cRNA encoding ROMK2 or S362A mutant 3 days prior to treatment with Brefeldin A added to the OR3 medium (+BFA) at the concentration of 25 jiM or ethanol as BFA vehicle (-BFA). BFA inhibits the insertion of new proteins into the cell membrane. Two-electrode voltage clamp (TEVC) was used to measure oocyte ROMK-dependent currents and membrane potential. Data was analyzed using Studentʹs t-tests or ANOVA as appropriate. Results: Incubation of oocytes expressing ROMK2 channels in 25 fiM 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 fiM. The fractional current for ROMK2 at 48h following treatment of oocytes with BFA was 0.24 ± 0.05 (n=24), which was significantly different from S362A mutant (0.96 ±0.05, n=24). Conclusion: These results show that the S362A mutation increases the general stability of ROMK and renders the protein resistance to endocytosis. This is 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 the stability of ROMK.