May 2007
Volume 48, Issue 13
Free
ARVO Annual Meeting Abstract  |   May 2007
Quantum Dot Conjugates With Variable Agonist Valency: Interaction With GABAC Receptors
Author Affiliations & Notes
  • H. A. Gussin
    Univ of Illinois - Chicago, Chicago, Illinois
    Ophthalmology & Visual Sciences,
  • I. D. Tomlinson
    Chemistry, Vanderbilt Univ, Nashville, Tennessee
  • D. M. Little
    Univ of Illinois - Chicago, Chicago, Illinois
    Neurology and Rehabilitation,
  • H. Qian
    Univ of Illinois - Chicago, Chicago, Illinois
    Ophthalmology & Visual Sciences,
  • S. J. Rosenthal
    Chemistry, Vanderbilt Univ, Nashville, Tennessee
  • D. R. Pepperberg
    Univ of Illinois - Chicago, Chicago, Illinois
    Ophthalmology & Visual Sciences,
  • Footnotes
    Commercial Relationships H.A. Gussin, None; I.D. Tomlinson, None; D.M. Little, None; H. Qian, None; S.J. Rosenthal, None; D.R. Pepperberg, None.
  • Footnotes
    Support NIH grants EY13693, EY16094, EY05494, EY01792, EB003728, EM72048, MH075791; Research to Prevent Blindness; Macular Degeneration Research grant from the American Health Assistance Foundation
Investigative Ophthalmology & Visual Science May 2007, Vol.48, 4590. doi:
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      H. A. Gussin, I. D. Tomlinson, D. M. Little, H. Qian, S. J. Rosenthal, D. R. Pepperberg; Quantum Dot Conjugates With Variable Agonist Valency: Interaction With GABAC Receptors. Invest. Ophthalmol. Vis. Sci. 2007;48(13):4590.

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      © ARVO (1962-2015); The Authors (2016-present)

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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 
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