Binding of muscimol-conjugated quantum dots to GABAc receptors

被引:65
作者
Gussin, Helene A.
Tomlinson, Ian D.
Little, Deborah M.
Warnement, Michael R.
Qian, Haohua
Rosenthal, Sandra J.
Pepperberg, David R. [1 ]
机构
[1] Univ Illinois, Lions Illinois Eye Res Inst, Dept Ophthalmol & Visual Sci, Dept Neurol & Rehabil, Chicago, IL 60612 USA
[2] Univ Illinois, Ctr Cognit Med, Chicago, IL 60612 USA
[3] Vanderbilt Univ, Dept Chem, Nashville, TN 37232 USA
关键词
D O I
10.1021/ja064324k
中图分类号
O6 [化学];
学科分类号
0703 ;
摘要
Functionalization of highly fluorescent CdSe/ZnS core-shell nanocrystals ( quantum dots, qdots) is an emerging technology for labeling cell surface proteins. We have synthesized a conjugate consisting of similar to 150-200 muscimols (a GABA receptor agonist) covalently joined to the qdot via a poly(ethylene glycol) (PEG) linker (similar to 78 ethylene glycol units) and investigated the binding of this muscimol-PEG-qdot conjugate to homomeric rho 1 GABA(C) receptors expressed in Xenopus oocytes. GABAC receptors mediate inhibitory synaptic signaling at multiple locations in the central nervous system (CNS). Binding of the conjugate was analyzed quantitatively by determining the fluorescence intensity of the oocyte surface membrane in relation to that of the surrounding incubation medium. Upon 5- to 10-min incubation with muscimol-PEG-qdots (34 nM in qdot concentration), GABA(C)-expressing oocytes exhibited a fluorescent halo at the surface membrane that significantly exceeded the fluorescence of the incubation medium. This halo was absent following muscimol-PEG-qdot treatment of oocytes lacking GABAC receptors. Incubation of the oocyte with free muscimol (100 mu M-5 mM), PEG-muscimol (500 mu M), or GABA (100 mu M - 5 mM) substantially reduced or eliminated the fluorescence halo produced by muscimol-PEG-qdots, and the removal of GABA or free muscimol led to a recovery of muscimol-PEG-qdot binding. Unconjugated qdots and PEG-qdots that lacked conjugated muscimol neither exhibited significant binding activity nor diminished the subsequent binding of muscimol-PEG-qdots. The results indicate that muscimol joined to qdots via a long-chain PEG linker exhibits specific binding activity at the ligand-binding pocket of expressed GABAC receptors, despite the presence of both the long PEG linker and the sterically bulky qdot.
引用
收藏
页码:15701 / 15713
页数:13
相关论文
共 66 条
[1]   Nanocrystal targeting in vivo [J].
Åkerman, ME ;
Chan, WCW ;
Laakkonen, P ;
Bhatia, SN ;
Ruoslahti, E .
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 2002, 99 (20) :12617-12621
[2]   Perspectives on the physical chemistry of semiconductor nanocrystals [J].
Alivisatos, AP .
JOURNAL OF PHYSICAL CHEMISTRY, 1996, 100 (31) :13226-13239
[3]   The use of nanocrystals in biological detection [J].
Alivisatos, P .
NATURE BIOTECHNOLOGY, 2004, 22 (01) :47-52
[4]   Highly fluorescent streptavidin-coated CdSe nanoparticles:: Preparation in water, characterization, and micropatterning [J].
Bäumle, M ;
Stamou, D ;
Segura, JM ;
Hovius, R ;
Vogel, H .
LANGMUIR, 2004, 20 (10) :3828-3831
[5]   Surface modification to reduce nonspecific binding of quantum dots in live cell assays [J].
Bentzen, EL ;
Tomlinson, ID ;
Mason, J ;
Gresch, P ;
Warnement, MR ;
Wright, D ;
Sanders-Bush, E ;
Blakely, R ;
Rosenthal, SJ .
BIOCONJUGATE CHEMISTRY, 2005, 16 (06) :1488-1494
[6]   Semiconductor nanocrystals as fluorescent biological labels [J].
Bruchez, M ;
Moronne, M ;
Gin, P ;
Weiss, S ;
Alivisatos, AP .
SCIENCE, 1998, 281 (5385) :2013-2016
[7]   Quantum dot bioconjugates for ultrasensitive nonisotopic detection [J].
Chan, WCW ;
Nie, SM .
SCIENCE, 1998, 281 (5385) :2016-2018
[8]   Channel opening locks agonist onto the GABAC receptor [J].
Chang, YC ;
Weiss, DS .
NATURE NEUROSCIENCE, 1999, 2 (03) :219-225
[9]   GABAC receptor ion channels [J].
Chebib, M .
CLINICAL AND EXPERIMENTAL PHARMACOLOGY AND PHYSIOLOGY, 2004, 31 (11) :800-804
[10]   Trichosanthin induced calcium-dependent generation of reactive oxygen species in human choriocarcinoma cells [J].
Chun-Yang, Z ;
Yi-Xuan, G ;
Hui, M ;
Cheng-Cai, A ;
Die-Yan, C .
ANALYST, 2000, 125 (09) :1539-1542