Characterization of Rb uptake into Sf9 cells using cation chromatography: evidence for a K-Cl cotransporter.

ABSTRACT

To assess cation-chloride cotransporter activity in Sf9 cells, cation chromatography was used to measure initial uptake rates of Rb. Rb exchanged with cellular K, with 30% of cellular K replaced after a 40 min exposure to Rb. Rb uptake into Sf9 cells was not inhibited by 50 µmol l(-1) ouabain. Rb uptake was approximately 65% inhibited by 250 µmol l(-1) bumetanide added to the assay solution, and was more than 95% inhibited when cells were pre-incubated for 20 min with bumetanide (100 and 1000 µmol l(-1)). Uptake of Rb and Cl followed simple Michaelis-Menten kinetics, with a K(m) for Rb of 17.1+/-2.2 mmol l(-1) and a K(m) for Cl of 93.7+/-5.6 mmol l(-1). Rb uptake was not dependent upon extracellular Na. Two min exposures to solutions with reduced [Na] or [Cl] produced small but significant changes in cellular Na content. We conclude that the primary Rb uptake pathway in Sf9 cells is a K-Cl cotransporter and that cation chromatography can be used to effectively study kinetic parameters of cotransporter function in tissue culture cells. Characterization of baseline cation-chloride cotransporter activity in Sf9 cells strengthens their utility as a tool for expression and characterization of exogenous proteins.