Changes in NAD(P)H fluorescence and membrane current produced by glutamate uptake into salamander Müller cells.
1. Glutamate uptake into isolated, whole-cell patch-clamped glial cells was studied by monitoring the increase of cell fluorescence generated as glutamate and NAD(P) were converted into alpha-ketoglutarate and NAD(P)H by glutamate dehydrogenase. The current generated by the glutamate 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 fluorescence response and uptake current were abolished, showing that there is no sodium-independent glutamate uptake across the cell membrane. 4. Varying the glutamate concentration altered both the fluorescence response and the uptake current. The fluorescence response saturated at a lower glutamate concentration than the uptake current, and depended in a Michaelis-Menten fashion on the uptake current. 5. The fluorescence response 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 was altered or intracellular potassium was removed was the same as that seen when the external glutamate concentration was altered. 7. These fluorescence studies show that glutamate uptake is inhibited by depolarization 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 electrogenic sodium- and potassium-dependent glutamate uptake with fixed stoichiometry being the only significant influx route for glutamate. Other possible interpretations of the data are also discussed.
Barbour
B
B
Department of Physiology, University College London.
Magnus
C
C
Szatkowski
M
M
Gray
P T
PT
Attwell
D
D
D000200:Action Potentials / Q000187:drug effects; D000557:Ambystoma / Q000378:metabolism; D000818:Animals; D001693:Biological Transport, Active; D005453:Fluorescence; D005971:Glutamates / Q000493:pharmacokinetics*; D018698:Glutamic Acid; D066298:In Vitro Techniques; D007424:Intracellular Fluid / Q000378:metabolism; D008564:Membrane Potentials; D008954:Models, Biological; D009249:NADP / Q000378:metabolism*; D009457:Neuroglia / Q000378:metabolism*; D011188:Potassium / Q000378:metabolism; D012160:Retina / Q000166:cytology / Q000378:metabolism*; D012964:Sodium / Q000494:pharmacology
D005971:Glutamates; D018698:Glutamic Acid; D009249:NADP; D012964:Sodium; D011188:Potassium