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Background: 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. Results: 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). Conclusion: Mutant Serine residue S362A which causes phosphorylation in endocytosis and helps determine the number of ROMK2, plays an important part in PDZ domain.

Keywords: : Potassium Channel, ROMK2, S362A Mutant, S362D Mutant, BFA, PDZ domain, Phosphorylation.

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