GABAC receptor binding of quantum-dot conjugates of variable ligand valency.
Highly fluorescent CdSe quantum dots (qdots) can serve as a platform for tethering multiple copies of a receptor-targeted ligand, affording study of how the level of multivalency affects receptor binding. We previously showed that qdots conjugated with long PEG chains terminated by muscimol, a known GABA(C) agonist, exhibit specific binding to the surface membrane of GABA(C) receptor-expressing Xenopus oocytes. The present report addresses the effect of varying the number, i.e., valency, of muscimol- (M-) terminated PEG chains attached to the qdot on binding of the resulting conjugate to GABA(C) receptors. M-PEG-qdots of differing muscimol valency were prepared by conjugating AMP-CdSe/ZnS qdots with muscimol-terminated and methylamine-terminated PEG chains in proportions designed to yield varying percentages of muscimol-terminated chains among the total approximately 150-200 chains bound to the qdot. The investigated valencies represented 0%, approximately 25%, approximately 50%, and 100% loading with muscimol (preparations termed M-PEG-qdot0, M-PEG-qdot25, M-PEG-qdot50, and M-PEG-qdot100, respectively. Binding of a given conjugate to surface membranes of GABA(C) receptor-expressing oocytes was analyzed by quantitative fluorescence microscopy following defined incubation with approximately 30 nM of the conjugate. With 5-20 min incubation, the fluorescence signal resulting from incubation with M-PEG-qdot25 exceeded, by approximately 6-fold, the fluorescence level obtained with M-PEG-qdot preparations that lacked muscimol-terminated chains (M-PEG-qdot0). M-PEG-qdot50 yielded a net signal roughly similar to that of M-PEG-qdot25, and that produced by M-PEG-qdot100 exceeded, by approximately 30-50%, those for M-PEG-qdot25 and M-PEG-qdot50. The time course of changes in oocyte surface membrane fluorescence resulting from the introduction of and removal of M-PEG-qdots in the medium bathing the oocyte indicated only a modest dependence of both binding and wash-out kinetics on muscimol valency. The results demonstrate a dependence of the binding activity of the M-PEG-qdot conjugates on muscimol valency, presumably reflecting higher GABA(C) avidity and/or affinity of the muscimol at high valency, and provide insight on the interactions of membrane receptor proteins with qdot conjugates containing multiple copies of a receptor-targeting ligand.
Gussin
Hélène A
HA
Departments of Ophthalmology and Visual Sciences, Bioengineering, Neurology and Rehabilitation, University of Illinois at Chicago, 1855 W. Taylor St., Chicago, IL 60612, USA.
Tomlinson
Ian D
ID
Muni
Niraj J
NJ
Little
Deborah M
DM
Qian
Haohua
H
Rosenthal
Sandra J
SJ
Pepperberg
David R
DR
D001665:Binding Sites; D019187:Cadmium Compounds / Q000737:chemistry*; D005453:Fluorescence; D008024:Ligands; D008744:Methylamines / Q000737:chemistry; D015394:Molecular Structure; D009118:Muscimol / Q000737:chemistry; D011092:Polyethylene Glycols / Q000737:chemistry; D045663:Quantum Dots; D018079:Receptors, GABA / Q000737:chemistry*; D018036:Selenium Compounds / Q000737:chemistry*; D013440:Sulfides / Q000737:chemistry; D017967:Zinc Compounds / Q000737:chemistry
D019187:Cadmium Compounds; C087200:GABA-C receptor; D008024:Ligands; D008744:Methylamines; D018079:Receptors, GABA; D018036:Selenium Compounds; D013440:Sulfides; D017967:Zinc Compounds; D009118:Muscimol; D011092:Polyethylene Glycols; C058667:cadmium selenide; C027451:methylamine; C031238:zinc sulfide