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Expression of γ-Aminobutyric Acid ρ1 and ρ1Δ450 as Gene Fusions with the Green Fluorescent Protein
Ataúlfo Martínez-Torres and Ricardo Miledi
Proceedings of the National Academy of Sciences of the United States of America
Vol. 98, No. 4 (Feb. 13, 2001), pp. 1947-1951
Published by: National Academy of Sciences
Stable URL: http://www.jstor.org/stable/3054981
Page Count: 5
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The functional characteristics and cellular localization of the γ-aminobutyric acid (GABA) ρ1 receptor and its nonfunctional isoform ρ1Δ450 were investigated by expressing them as gene fusions with the enhanced version of the green fluorescent protein (GFP). Oocytes injected with ρ1-GFP had receptors that gated chloride channels when activated by GABA. The functional characteristics of these receptors were the same as for those of wild-type ρ1 receptors. Fluorescence, because of the chimeric receptors expressed, was over the whole oocyte but was more intense near the cell surface and more abundant in the animal hemisphere. Similar to the wild type, ρ1Δ450-GFP did not lead to the expression of functional GABA receptors, and injected oocytes failed to generate currents even after exposure to high concentrations of GABA. Nonetheless, the fluorescence displayed by oocytes expressing ρ1Δ450-GFP was distributed similarly to that of ρ1-GFP. Mammalian cells transfected with the ρ1-GFP or ρ1Δ450-GFP constructs showed mostly intracellularly distributed fluorescence in confocal microscope images. A sparse localization of fluorescence was observed in the plasma membrane regardless of the cell line used. We conclude that ρ1Δ450 is expressed and transported close to, and perhaps incorporated into, the plasma membrane. Thus, ρ1- and ρ1Δ450-GFP fusions provide a powerful tool to visualize the traffic of GABA type C receptors.
Proceedings of the National Academy of Sciences of the United States of America © 2001 National Academy of Sciences