EAAT2 density at the astrocyte plasma membrane and Ca(2 + )-regulated exocytosis.
We studied whether regulated exocytosis affects the glutamate transporter density in cultured astrocytes, in which the expression of a fluorescently labeled excitatory amino acid transporter 2 (EAAT2-EGFP) predominantly labeled the plasma membrane. The addition of ionomycin that elevates cytosolic Ca(2+) strongly increased the fluorescence of FM 4-64 membrane area dye, confirming the presence of regulated exocytosis in transfected astrocytes. However, concomitant with Ca(2+)-dependent FM 4-64 fluorescence increase, ionomycin induced a significant steady-state decrease in EAAT2-EGFP fluorescence. This is likely due to a secondary inner filter effect since,(i) in the absence of FM 4-64, ionomycin stimulation was ineffective in changing the EAAT2-EGFP fluorescence, and (ii) fluorescence changes in FM 4-64 and EAAT2-EGFP were inversely correlated. To test whether subcellular EAAT2-EGFP structures are translocated from the cytoplasm to the plasma membrane during ionomycin stimulation, EAAT2-EGFP fluorescence was monitored locally at the plasma membrane and a few microns away in the adjacent cytoplasm. Measurements revealed sites with an increase in EAAT2-EGFP plasma membrane fluorescence correlated with a fluorescence decrease beneath the plasma membrane, and sites with plasma membrane fluorescence decrease correlated with fluorescence increase within the adjacent cytoplasm. The sites of rapid translocation/retrieval of EAAT2-EGFP structures to/from the plasma membrane appeared to be distributed in a punctuate pattern around the cell perimeter. The density of EAAT2-EGFP was regulated in a Ca(2+)-dependent manner, since in the absence of extracellular Ca(2+) local translocation/retrieval events were absent, revealing rapid surface density regulation of EAAT2 in astrocytes by regulated exo/endocytosis.
Stenovec
Matjaz
M
Celica Biomedical Center, Ljubljana, Slovenia.
Kreft
Marko
M
Grilc
Sonja
S
Pangrsic
Tina
T
Zorec
Robert
R
D000818:Animals; D001253:Astrocytes / Q000187:drug effects / Q000378:metabolism*; D001693:Biological Transport, Active; D002118:Calcium / Q000378:metabolism; D002462:Cell Membrane / Q000378:metabolism*; D002478:Cells, Cultured; D027342:Excitatory Amino Acid Transporter 2 / Q000235:genetics / Q000378:metabolism*; D005089:Exocytosis; D005456:Fluorescent Dyes; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D015759:Ionomycin / Q000494:pharmacology; D007476:Ionophores / Q000494:pharmacology; D018613:Microscopy, Confocal; D011726:Pyridinium Compounds; D000644:Quaternary Ammonium Compounds; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D014162:Transfection
D027342:Excitatory Amino Acid Transporter 2; C092350:FM 4-64; D005456:Fluorescent Dyes; D007476:Ionophores; D011726:Pyridinium Compounds; D000644:Quaternary Ammonium Compounds; D011993:Recombinant Fusion Proteins; C493395:Slc1a2 protein, rat; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D015759:Ionomycin; D002118:Calcium