Functional characterization and visualization of a GABAA receptor-GFP chimera expressed in Xenopus oocytes.
The GABAA receptor is a ligand-gated chloride channel belonging to the superfamily of ligand-gated ion channels of which the nicotinic acetylcholine (nACh) receptor is prototypic. In the central nervous system the GABAA receptor mediates fast neuronal inhibition. To facilitate the study of this receptor, a GABAA receptor-green fluorescent protein (GABAAR-GFP) chimera was constructed by fusing green fluorescent protein (GFP) to the C-terminus region of the GABAA receptor alpha1 subunit. When expressed in Xenopus oocytes, this chimera responded in a manner indistinguishable from the wild-type GABAA receptor with respect to agonist potency, receptor desensitization, allosteric modulation, rectification, and ion selectivity of the channel. The addition of GFP to the GABAA receptor alpha1 subunit did not appear to alter the assembly or efficiency of expression of the GABAA receptor complex. The GABAAR-GFP chimera generated a strong fluorescent signal that was restricted to the animal pole of the oocyte plasma membrane. This signal was readily detectable using either epifluorescence or laser confocal microscopy. To confirm the extracellular location of the GFP portion of the chimera, non-permeabilized oocytes were immunolabeled with an anti-GFP antibody. Fluorescence microscopy showed that GFP was located extracellularly since it was accessible to the GFP antibody. These results confirm the predicted extracellular location of the C-terminus of the GABAA receptor alpha1 subunit and also demonstrate that GFP retains its fluorescent property when expressed extracellularly. The usefulness of the GABAAR-GFP chimera in receptor trafficking was investigated using non-hydrolyzable GTP analogues since GTP binding proteins participate in protein transport in oocytes. Microinjections of GTP-gamma-S but not GDP-beta-S reduced both GABA-gated chloride currents and cell surface GFP fluorescence in oocytes expressing the GABAAR-GFP chimera indicating that the chimera undergoes internalization upon stimulation of oocyte GTP-binding proteins. The results of the present study show that the GABAAR-GFP chimera is functionally similar to the wild-type GABAA receptor and can be used to study receptor trafficking in living cells. This is the first demonstration of a ligand-gated ion channel-GFP chimera for an ion channel belonging to this superfamily and also is the first example of the fusion of GFP to an extracellular domain of an integral membrane protein.
Bueno
O F
OF
Department of Pharmacology and Therapeutics, Louisiana State University Medical Center, Shreveport, 1501 Kings Highway, Shreveport, LA 71106, USA.
Robinson
L C
LC
Alvarez-Hernandez
X
X
Leidenheimer
N J
NJ
D000777:Anesthetics / Q000494:pharmacology; D000818:Animals; D001640:Bicuculline / Q000494:pharmacology; D002462:Cell Membrane / Q000737:chemistry / Q000502:physiology; D003900:Desoxycorticosterone / Q000031:analogs & derivatives / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D004594:Electrophysiology; D005260:Female; D018756:GABA Antagonists / Q000494:pharmacology; D018757:GABA Modulators / Q000494:pharmacology; D015870:Gene Expression; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000494:pharmacology; D007202:Indicators and Reagents; D015640:Ion Channel Gating / Q000187:drug effects / Q000502:physiology*; D008164:Luminescent Proteins / Q000235:genetics*; D008564:Membrane Potentials / Q000187:drug effects / Q000502:physiology; D018613:Microscopy, Confocal; D008874:Midazolam / Q000494:pharmacology; D009865:Oocytes / Q000502:physiology; D010424:Pentobarbital / Q000494:pharmacology; D010852:Picrotoxin / Q000494:pharmacology; D011963:Receptors, GABA-A / Q000235:genetics*; D011993:Recombinant Fusion Proteins / Q000235:genetics; D014982:Xenopus laevis; D005680:gamma-Aminobutyric Acid / Q000494:pharmacology
D000777:Anesthetics; D001640:Bicuculline; D003900:Desoxycorticosterone; D018756:GABA Antagonists; D018757:GABA Modulators; D049452:Green Fluorescent Proteins; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D007202:Indicators and Reagents; D008164:Luminescent Proteins; D008874:Midazolam; D010424:Pentobarbital; D010852:Picrotoxin; D011963:Receptors, GABA-A; D011993:Recombinant Fusion Proteins; D005680:gamma-Aminobutyric Acid; C009413:tetrahydrodeoxycorticosterone