Expression of gamma-aminobutyric acid rho 1 and rho 1 Delta 450 as gene fusions with the green fluorescent protein.
The functional characteristics and cellular localization of the gamma aminobutyric acid (GABA) rho 1 receptor and its nonfunctional isoform rho 1 Delta 450 were investigated by expressing them as gene fusions with the enhanced version of the green fluorescent protein (GFP). Oocytes injected with rho 1-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 rho 1 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, rho 1 Delta 450-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 rho 1 Delta 450-GFP was distributed similarly to that of rho 1-GFP. Mammalian cells transfected with the rho 1-GFP or rho 1 Delta 450-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 rho 1 Delta 450 is expressed and transported close to, and perhaps incorporated into, the plasma membrane. Thus, rho 1- and rho 1 Delta 450-GFP fusions provide a powerful tool to visualize the traffic of GABA type C receptors.
Martinez-Torres
A
A
Laboratory of Cellular and Molecular Neurobiology, Department of Neurobiology and Behavior, University of California, Irvine, CA 92697, USA.
Miledi
R
R
D016475:3T3 Cells; D000818:Animals; D019677:Artificial Gene Fusion; D002460:Cell Line; D005453:Fluorescence; D015870:Gene Expression; D049452:Green Fluorescent Proteins; D006801:Humans; D008164:Luminescent Proteins / Q000096:biosynthesis / Q000235:genetics / Q000502:physiology; D051379:Mice; D020033:Protein Isoforms / Q000096:biosynthesis / Q000235:genetics / Q000502:physiology; D018079:Receptors, GABA / Q000096:biosynthesis* / Q000235:genetics / Q000502:physiology; D011963:Receptors, GABA-A; D018080:Receptors, GABA-B; D014982:Xenopus laevis; D005680:gamma-Aminobutyric Acid / Q000235:genetics / Q000378:metabolism
C106350:GABRR2 protein, human; C491398:Gabrr2 protein, mouse; D008164:Luminescent Proteins; D020033:Protein Isoforms; D018079:Receptors, GABA; D011963:Receptors, GABA-A; D018080:Receptors, GABA-B; D049452:Green Fluorescent Proteins; D005680:gamma-Aminobutyric Acid