Properties of GluR3 receptors tagged with GFP at the amino or carboxyl terminus.
Anatomical visualization of neurotransmitter receptor localization is facilitated by tagging receptors, but this process can alter their functional properties. We have evaluated the distribution and properties of WT glutamate receptor 3 (GluR3) alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptors (WT GluR3) and two receptors in which GFP was tagged to the amino terminus (GFP-GluR3) or to the carboxyl terminus (GluR3-GFP). Although the fluorescence in Xenopus oocytes was stronger in the vegetal hemisphere because of localization of internal structures (probable sites of production, storage or recycling of receptors), the insertion of receptors into the plasma membrane was polarized to the animal hemisphere. The fluorescence intensity of oocytes injected with GluR3-GFP RNA was approximately double that of oocytes injected with GFP-GluR3 RNA. Accordingly, GluR3-GFP oocytes generated larger kainate-induced currents than GFP-GluR3 oocytes, with similar EC(50) values. Currents elicited by glutamate, or AMPA coapplied with cyclothiazide, were also larger in GluR3-GFP oocytes. The glutamate- to kainate-current amplitude ratios differed, with GluR3-GFP being activated more efficiently by glutamate than the WT or GFP-GluR3 receptors. This pattern correlates with the slower decay of glutamate-induced currents generated by GluR3-GFP receptors. These changes were not observed when GFP was tagged to the amino terminus, and these receptors behaved like the WT. The antagonistic effects of 6-nitro-7-sulfamoylbenzo[f]quinoxaline-2,3-dione (NBQX) and 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) were not altered in any of the tagged receptors. We conclude that GFP is a useful and convenient tag for visualizing these proteins. However, the effects of different sites of tag insertion on receptor characteristics must be taken into account in assessing the roles played by these receptor proteins.
Limon
Agenor
A
Department of Neurobiology and Behavior, University of California, Irvine, CA 92697-4550, USA.
Reyes-Ruiz
Jorge Mauricio
JM
Eusebi
Fabrizio
F
Miledi
Ricardo
R
D000818:Animals; D002462:Cell Membrane / Q000378:metabolism; D004594:Electrophysiology; D018698:Glutamic Acid / Q000737:chemistry / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000378:metabolism*; D020128:Inhibitory Concentration 50; D007608:Kainic Acid / Q000378:metabolism; D018613:Microscopy, Confocal; D008954:Models, Biological; D009865:Oocytes / Q000378:metabolism; D010957:Plasmids / Q000378:metabolism; D017434:Protein Structure, Tertiary; D018091:Receptors, AMPA / Q000737:chemistry / Q000378:metabolism*; D014981:Xenopus
D018091:Receptors, AMPA; C104723:glutamate receptor ionotropic, AMPA 3; D049452:Green Fluorescent Proteins; D018698:Glutamic Acid; D007608:Kainic Acid