Abstract
Purpose::
ρ1 GABAC receptors (GABACR) are activated by chain-derivatized forms of muscimol (M), a known GABACR agonist. This finding has potential application for the construction of molecular devices containing tethered M that could modulate retinal GABA R activity (1). We showed by quantitative imaging that M, covalently joined to highly fluorescent quantum dots (qdots) via a long PEG chain, binds to Xenopus oocyte-expressed ρ1 GABACR (2). To test whether the number of Ms tethered to a given qdot (i.e., M valency) affects binding, we analyzed the surface membrane fluorescence of GABAC-expressing oocytes treated with M-PEG-qdots of differing M valency.
Methods::
Preparations of M-PEGs and PEG chains in varying proportion were conjugated with AMP®-CdSe/ZnS qdots. Conjugates containing ~ (a) 150-200, (b) 100-150, (c) 75-100 and (d) 37-50 M-PEGs per qdot were studied. Oocytes expressing human ρ1 GABACR and nonexpressing control oocytes (1,2) were incubated for 5-20 min with 34-37 nM of either (a), (b), (c), or (d) M-PEG-qdots. Fluorescence images of the oocyte surface membrane and surrounding medium (background) (confocal microscopy; exc= 476 nm; em= 580-620 nm) were quantified (0-255 scale) using MetaMorph (2).
Results::
Incubation of GABAC-expressing oocytes with M-PEG-qdot preparations yielded fluorescent halos at the oocyte surface membrane. Surface fluorescence was absent following similar treatment of nonexpressing oocytes. Halo fluorescence intensities (mean ± SD) of GABAC-expressing oocytes following incubation with (a), (b), (c) and (d) were 56.21 ± 24.22, 35.22 ± 18.97, 34.61 ± 21.37 and 17.41 ± 14.37, respectively. Background intensities were 13.97 ± 11.80, 7.85 ± 8.98, 17.97 ± 13.98 and 9.76 ± 9.87, respectively. While intensities for (a) vs (d) differed significantly with respect to both halo (F=1037.46, p<10-18) and background (F=39.07, p=10-6), the intensity difference between halos exceeded that between backgrounds (F=236.82, p=10-12). Determinations of fluorescence quenching for (a)-(d) indicated a variability of ~5%, insufficient to account for the observed difference in halo fluorescence.
Conclusions::
Binding of qdot-conjugated M-PEG to ooctye-expressed ρ1 GABACR increases with M-valency, presumably reflecting higher GABAC affinity and/or avidity at high M valency. The results provide further information on the interactions of chain-derivatized M with ρ1 GABACR, a model system for developing tethered-M regulators of retinal GABA R. (1) Vu et al. (2005) Biomaterials 26:1895-1903. (2) Gussin et al. (2006) J Am Chem Soc 128:15701-15713.
Keywords: neurotransmitters/neurotransmitter systems • retina: neurochemistry