Expression of GFP-tagged neuronal glutamate transporters in cerebellar Purkinje neurons.
Of the five excitatory amino acid transporters (EAATs) identified, two genes are expressed by neurons (EAAT3 and EAAT4) and give rise to transporters confined to neuronal cell bodies and dendrites. At an ultrastructural level, EAAT3 and EAAT4 proteins are clustered at the edges of postsynaptic densities of excitatory synapses. This pattern of localization suggests that postsynaptic EAATs may help to limit spillover of glutamate from excitatory synapses. In an effort to study transporter localization in living neurons and ultimately to manipulate uptake at intact synapses, we have developed viral reagents encoding neuronal EAATs tagged with GFP. We demonstrate that these fusion proteins are capable of Na(+)-dependent glutamate uptake, that they generate ionic conductances indistinguishable from their wild-type counterparts, and that GFP does not alter their glutamate dose-dependence. Two-photon microscopy was used to examine fusion protein expression in Purkinje neurons in acute cerebellar slices. Both EAAT3-GFP and EAAT4-GFP were observed at high levels in the dendritic spines of transfected Purkinje neurons. These findings indicate that functional EAAT fusion proteins can be synthesized and appropriately trafficked to postsynaptic compartments. Furthermore, they validate a powerful system for looking at EAAT function in situ.
Meera
Pratap
P
Department of Neurobiology and Brain Research Institute, David Geffen School of Medicine at UCLA, Los Angeles, CA 90095, USA.
Dodson
Paul D
PD
Karakossian
Movses H
MH
Otis
Thomas S
TS
D027322:Amino Acid Transport System X-AG / Q000145:classification / Q000378:metabolism*; D000818:Animals; D000831:Animals, Newborn; D001224:Aspartic Acid / Q000031:analogs & derivatives / Q000494:pharmacology; D002460:Cell Line; D002531:Cerebellum / Q000166:cytology*; D003001:Cloning, Molecular / Q000379:methods; D006224:Cricetinae; D004305:Dose-Response Relationship, Drug; D005786:Gene Expression Regulation / Q000502:physiology*; D018698:Glutamic Acid / Q000378:metabolism*; D049452:Green Fluorescent Proteins / Q000378:metabolism*; D006801:Humans; D066298:In Vitro Techniques; D008564:Membrane Potentials / Q000187:drug effects / Q000502:physiology / Q000528:radiation effects; D008845:Microinjections / Q000379:methods; D036641:Microscopy, Fluorescence, Multiphoton / Q000379:methods; D009865:Oocytes; D018408:Patch-Clamp Techniques / Q000379:methods; D011689:Purkinje Cells / Q000378:metabolism*; D051381:Rats; D012964:Sodium / Q000378:metabolism; D014162:Transfection / Q000379:methods; D014316:Tritium / Q000378:metabolism; D014981:Xenopus
D027322:Amino Acid Transport System X-AG; D014316:Tritium; D049452:Green Fluorescent Proteins; C011274:3-hydroxyaspartic acid; D001224:Aspartic Acid; D018698:Glutamic Acid; D012964:Sodium