Monitoring EAAT2 density in astrocyte plasma membrane optically

Matjaž Stenovec, Marko Kreft, Tina Pangršič, Sonja Grilc, and Robert Zorec

Celica Biomedical Sciences Center, Stegne 21, 1000 Ljubljana, Slovenia, Laboratory of Neuroendocrinology - Molecular Cell Physiology, Institute of Pathophysiology, Medical School, University of Ljubljana, 1000 Ljubljana, Slovenia.

Astrocytes are non-neuronal cells in the central nervous system, which like neurons are capable of releasing neuroactive molecules, such as glutamate, possibly via regulated exocytosis. Astrocytes also remove glutamate from synaptic cleft, mainly through glutamate transporters, such as EAAT2. The successful removal of glutamate depends among other factors also on the density of glutamate transporters. We investigated the putative involvement of regulated exocytosis in glutamate transporter density control in astrocyte plasma membrane by transfecting cells with a construct to express excitatory amino acid transporter 2-tagged with enhanced green fluorescent protein (EAAT2-EGFP). This construct predominantly labeled the astrocyte plasma membrane. To monitor exocytosis we labeled the cell membrane with the FM 4-64. Addition of ionomycin to increase cytosolic Ca2+ strongly increased the FM 4-64 staining, indicating the presence of regulated exocytosis in astrocytes. However, concomitant with Ca2+-dependent FM 4-64 intensity increase, a similar ionomycin-induced increase in EAAT2-EGFP intensity could not be detected. Interestingly, while the steady state fluorescence intensity of EAAT2-EGFP decreased and the surface area of the plasma membrane increased as monitored by FM 4-64, these data suggest that the density of EAAT2-EGFP molecules in the plasma membrane diminishes due to Ca2+-dependent exocytosis. A reduction of glutamate density in astrocyte membrane may affect the time course of synaptic transmission.

Email: tina.pangrsic@mf.uni-lj.si

Address: Inštitut za patološko fiziologijo, Medicinska fakulteta, Zaloška 4, 1000 Ljubljana