CHANGES IN NAD(P)H FLUORESCENCE AND MEMBRANE CURRENT PRODUCED BY GLUTAMATE UPTAKE INTO SALAMANDER MULLER CELLS

1. Glutamate uptake into isolated, whole-cell patch-clamped glial cell s was studied by monitoring the increase of cell fluorescence generate d as glutamate and NAD(P) were converted into alpha-ketoglutarate and NAD(P)H by glutamate dehydrogenase. The current generated by the gluta mate uptake carrier was recorded simultaneously. 2. L-Glutamate evoked an increase of cell fluorescence and an inward uptake current. L- and D-aspartate generated an uptake current but no fluorescence response, consistent with the amino acid specificity of glutamate dehydrogenase . 3. In the absence of external sodium the glutamate-evoked fluorescen ce response and uptake current were abolished, showing that there is n o sodium-independent glutamate uptake across the cell membrane. 4. Var ying the glutamate concentration altered both the fluorescence respons e and the uptake current. The fluorescence response saturated at a low er glutamate concentration than the uptake current, and depended in a Michaelis-Menten fashion on the uptake current. 5. The fluorescence re sponse and the uptake current were reduced by membrane depolarization, and also by removal of intracellular potassium. 6. The dependence of the fluorescence response on uptake current when membrane potential wa s altered or intracellular potassium was removed was the same as that seen when the external glutamate concentration was altered. 7. These f luorescence studies show that glutamate uptake is inhibited by depolar ization and by removal of intracellular potassium, consistent with the conclusion of earlier work in which uptake was monitored solely as a membrane current. The data are consistent with high-affinity electroge nic sodium- and potassium-dependent glutamate uptake with fixed stoich iometry being the only significant influx route for glutamate. Other p ossible interpretations of the data are also discussed.