FUNCTIONAL-CHARACTERIZATION AND VISUALIZATION OF A GABA(A) RECEPTOR-GFP CHIMERA EXPRESSED IN XENOPUS OOCYTES

The GABA(A) receptor is a ligand-gated chloride channel belonging to t he superfamily of ligand-gated ion channels of which the nicotinic ace tylcholine (nACh) receptor is prototypic. In the central nervous syste m the GABA(A) receptor mediates fast neuronal inhibition. To facilitat e the study of this receptor, a GABA(A) receptor-green fluorescent pro tein (GABA(A)R-GFP) chimera was constructed by fusing green fluorescen t protein (GFP) to the C-terminus region of the GABA(A) receptor alpha 1 subunit. When expressed in Xenopus oocytes, this chimera responded in a manner indistinguishable from the wild-type GABA(A) receptor with respect to agonist potency, receptor desensitization, allosteric modu lation, rectification, and ion selectivity of the channel. The additio n of GFP to the GABA(A) receptor alpha 1 subunit did not appear to alt er the assembly or efficiency of expression of the GABA(A) receptor co mplex. The GABA(A)R-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 las er confocal microscopy. To confirm the extracellular location of the G FP portion of the chimera, non-permeabilized oocytes were immunolabele d with an anti-GFP antibody. Fluorescence microscopy showed that GFP w as located extracellularly since it was accessible to the GFP antibody . These results confirm the predicted extracellular location of the C- terminus of the GABA(A) receptor alpha 1 subunit and also demonstrate that GFP retains its fluorescent property when expressed extracellular ly. The usefulness of the GABA(A)R-GFP chimera in receptor trafficking was investigated using non-hydrolyzable GTP analogues since GTP bindi ng proteins participate in protein transport in oocytes. Microinjectio ns of GTP-gamma-S but not GDP-beta-S reduced both GABA-gated chloride currents and cell surface GFP fluorescence in oocytes expressing the G ABA(A)R-GFP chimera indicating that the chimera undergoes internalizat ion upon stimulation of oocyte GTP-binding proteins. The results of th e present study show that the GABA(A)R-GFP chimera is functionally sim ilar to the wild-type GABA(A) receptor and can be used to study recept or trafficking in living cells. This is the first demonstration of a l igand-gated ion channel-GFP chimera for an ion channel belonging to th is superfamily and also is the first example of the fusion of GFP to a n extracellular domain of an integral membrane protein. (C) 1998 Elsev ier Science B.V. All rights reserved.