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H.A. Gussin, I.D. Tomlinson, D.M. Little, H. Qian, S.J. Rosenthal, D.R. Pepperberg; Quantitative Image Analysis of the GABAC Receptor Interaction With Muscimol–Conjugated Quantum Dots . Invest. Ophthalmol. Vis. Sci. 2006;47(13):5384.
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© ARVO (1962-2015); The Authors (2016-present)
Chain–derivatized analogs of neurotransmitters are of interest for the design of molecular devices that could modulate the activity of retinal postsynaptic receptors (1). We recently showed by visualization that the GABAC receptor agonist muscimol, linked to highly fluorescent quantum dots (qdots) via a PEG chain, exhibits specific binding to Xenopus oocytes expressing ρ1 GABAC receptors (2). To quantify the binding of muscimol–qdots (m–q), we have analyzed the fluorescence of m–q treated GABAC–expressing oocytes and controls.
Oocytes expressing human ρ1 GABAC receptors (1) were incubated for 3–10 min with 34 nM m–q. Control, non–expressing oocytes were similarly incubated for 10–20 min. A fluorescence image (2) was then obtained. For each image, a multi–segment line was visually traced over the imaged portion of the oocyte surface membrane ("border"); another line of similar overall length was drawn through the imaged surrounding medium ("background"). Pixelated fluorescence intensities (full scale: 0–255) were obtained (MetaMorph) and analyzed by 2–way ANOVA and chi–square. To test for GABA/m–q competition, the GABAC–expressing oocyte was incubated sequentially with (a) 100 or 500 µM GABA (20 min), (b) 34 nM m–q plus 100 or 500 µM GABA (15 min); and, following washing, (c) 34 nM m–q (15 min). Images were obtained at the end of (b) and (c).
Incubation of GABAC–expressing oocytes with m–q yielded a border fluorescence intensity of 88+65 (mean + SD; n=11); this significantly (p<0.0001) exceeded background (31+35). Similarly treated control oocytes exhibited a border value of 15+22 (n=14), which did not differ significantly from background (16+22). A competition experiment performed with 100 µM GABA yielded border and background values of 22+32 and 7+12, respectively, after (b); and 120+84 and 12+15, respectively, after (c). A second competition experiment with 500 µM GABA yielded border and background values of 44+48 and 11+17, respectively, after (b); and border and background values of 124+84 and 8+13, respectively, after (c). For both experiments there was a significant shift in the distribution of border intensities between (b) and (c) (p<0.0001) but not in the background distribution.
The results quantify the interaction of ρ1 GABAC receptors with m–q. They support the feasibility of determining localization/binding properties of m–q and related qdot–conjugated neurotransmitter analogs at native retinal GABA postsynaptic receptors. (1) Vu et al. (2005) Biomaterials 26:1895–1903. (2) Gussin et al., ARVO 2005.
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