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. 2007 Sep 25;104(39):15526-30.
doi: 10.1073/pnas.0706773104. Epub 2007 Sep 19.

Properties of GluR3 receptors tagged with GFP at the amino or carboxyl terminus

Affiliations

Properties of GluR3 receptors tagged with GFP at the amino or carboxyl terminus

Agenor Limon et al. Proc Natl Acad Sci U S A. .

Abstract

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.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Visualization of GFP-tagged GluR3. (A–D) Confocal sections of oocytes injected in the equator with the indicated cRNAs, taken at 300 μm from the bottom of the oocyte. The animal hemisphere is at the right and the vegetal at the left. Note that the fluorescence is more intense in the vegetal hemisphere. Diagrams in A and B represent the GluR3 chimeras showing the extracellular or intracellular localization of the GFP. (C and D) Immunolocalization of GluR3 receptors in the plasma membrane of nonpermeabilized oocytes. (C) Oocyte expressing WT GluR3 incubated with an antibody against the amino terminus of GluR3, which is localized extracellularly and a secondary antibody (Alexa 568, red label). (D) Oocyte expressing GluR3-GFP incubated with the same primary and secondary antibodies. Note that the insertion of GluR3 is mainly polarized to the plasma membrane in the animal hemisphere. (Da and Db) Magnification of the membrane near the animal and vegetal poles of the oocyte shown in D. (Scale bar: 50 μm.) (E and F) Z-axis sequential scans in the vegetal hemisphere of an oocyte injected with GluR3-GFP. (Insets) Magnifications of the areas within the smaller squares. Images taken at 2.8 and 5.7 μm from the oocyte's surface. Below the surface, notice high fluorescence in elongated structures, resembling annulate lamellae. (G) Electron microscope image of an annulate lamellae (AL). (Scale bar: 1 μm.) Adapted from R.M. and C. Tate (unpublished work 1978).
Fig. 2.
Fig. 2.
Differences in antibody selectivity against GluR3 chimeras. Western blots against GluR3 receptors in the membranes of Xenopus oocytes in 10% (Upper) and 8% (Lower) gels. Notice that the anti-GluR2/3 fails to recognize properly the GluR3-GFP.
Fig. 3.
Fig. 3.
GluR3 GFP-constructs have different expressional potencies. (A) Fluorescence intensity (arbitrary units) of injected oocytes. The fluorescence of oocytes expressing WT GluR3 is the same as the native fluorescence observed in noninjected oocytes (n = 9 each). The fluorescence of oocytes expressing GluR3-GFP was double of that of oocytes expressing GFP-GluR3 (P < 0.05). (B) GluR3-GFP injected oocytes generated nearly three times more 100 μM Kai-current than oocytes injected with WT GluR3 or GFP-GluR3 cRNA (P < 0.05; n = 64). (C) Sample membrane currents evoked by 100 μM kainate in injected oocytes. The 2 μA calibration bar applies only to the GluR3-GFP current. (D) The currents elicited by 10 μM AMPA plus 10 μM CTZ were also larger in oocytes expressing the GluR3-GFP.
Fig. 4.
Fig. 4.
Glutamate-induced currents are larger in GluR3-GFP oocytes. (A) Normalized sample Kai- and Glu-currents generated by the different receptors. Kai was 100 μM and glutamate was 1 mM. (B) Glutamate is about three times more efficient in eliciting currents in oocytes injected with GluR3-GFP than in oocytes injected with WT GluR3 or GFP-GluR3 (n = 106).
Fig. 5.
Fig. 5.
Agonist and antagonist dose/current response relationships of oocytes expressing GluR3 receptors. (A) There was no statistical difference between kainate EC50 of WT GluR3, GFP-GluR3 and GluR3-GFP receptors. (B and C) GluR3-GFP has less sensitivity to glutamate alone, and to AMPA coapplied with 10 μM CTZ than the other GluR3 receptors. (D) Neither NBQX (left) nor CNQX (right) concentration/current relationships were altered by GFP tagging to WT GluR3. The action of CNQX and NBQX was evaluated on 30 μM Kai-currents. Currents were normalized to the maximum drug-elicited current for each oocyte (n = 3–9).
Fig. 6.
Fig. 6.
Carboxyl-terminal GFP tagging alters the decay of GluR3 receptors. (A) Glutamate (1 mM) plus CTZ (10 μM)-currents in oocytes injected with GluR3 or the GFP constructs. For better comparison, only the temporal course of activation and decay of the currents are shown. The inactivation was not complete after 20 s of agonist perfusion. After washing out the Glu plus CTZ, the currents returned to their basal level. (B) GluR3-GFP-currents decayed more slowly: τd = 1,102 ± 276 ms; n = 10; *, P < 0.05; vs. WT GluR3 (447 ± 44 ms; n = 10) and GFP-GluR3 (374 ± 20 ms; n = 12). Thicker traces are the exponential fits to the Glu-current traces. Currents were normalized for comparative purposes. Glu-current amplitudes were as follows: 600 nA for WT GluR3, 980 nA for GFP-GluR3, and 3,180 nA for GluR3-GFP.

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