Response of the GFP‐tagged rat Na,K‐ATPase α1 isoform to phorbol esters

The membrane‐bound Na,K‐ATPase establishes and maintains the high internal K + and low internal Na + concentrations in mammalian cells. The functional molecule consists of 2 subunits, an α (110 kDa) and a β subunit (55 kDa), each one existing as several isoforms. The aim of this study was to investigate if the introduction of a GFP tag at the N‐terminus of the rat α1 Na,K‐ATPase alters the response to phorbol esters. Opossum Kidney (OK) cells transfected with rat GFP‐tagged Na,K‐ATPase α1 isoform were used and changes in Na,K‐ATPase‐mediated transport were monitored in cells treated with or without phorbol myristate acetate (PMA). Flame photometry was performed to evaluate the intracellular Na + content and modifications in Na,K‐ATPase cellular distribution induced by phorbol esters were assessed by confocal microscopy. We observed a 25% transport increase in OK cells transfected with wild‐type rat α1 isoform treated with PMA (p<0.01). A similar increase was found in cells expressing the GFP‐tagged isoform treated with PMA (20%, p<0.01). Differences in surface staining were observed in GFP transfected cells after PMA treatment and blocked by specific cytoskeletal inhibitors. In conclusion, the introduction of the GFP tag at the N‐terminus of the Na,K‐ATPase α1 isoform does not seem to alter the response to PMA. [This work was supported by NIH Grant RR‐19799 and the TTUHSC Summer Accelerated Biomedical Research program]