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

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 Km for Rb of 17.1±2.2 mmol l−1 and a Km 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.