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J Biomed Biotechnol
2007 Jan 01;2007:76514. doi: 10.1155/2007/76514.
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Imaging GABAc receptors with ligand-conjugated quantum dots.
Tomlinson ID, Gussin HA, Little DM, Warnement MR, Qian H, Pepperberg DR, Rosenthal SJ.
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We report a methodology for labeling the GABAc receptor on the surface membrane of intact cells. This work builds upon our earlier work with serotonin-conjugated quantum dots and our studies with PEGylated quantum dots to reduce nonspecific binding. In the current approach, a PEGylated derivative of muscimol was synthesized and attached via an amide linkage to quantum dots coated in an amphiphilic polymer derivative of a modified polyacrylamide. These conjugates were used to image GABA(C) receptors heterologously expressed in Xenopus laevis oocytes.
Figure 1. A comparison of nonspecific adsorption of AMP-coated qdots to the surfaces
of 6 different cell types. These
experiments employed AMP-coated qdots that were either unconjugated (upper row)
or conjugated to PEG2000 (lower row).
Figure 2. Serotonin-coated qdots used to label SERT-expressing cells.
Figure 3. Muscimol, a GABAC and GABAA receptor agonist.
Scheme 1. Synthesis of Muscimol: (i) DBU, (ii) NH3, and (iii) BH3.
Scheme 2. Synthesis of muscimol ligand: (i) BOC anhydride, (ii) NHS, DCC, (iii)
Muscimol, (iv) TFA,(v) BOC-PEG3400-NHS, and (vi) TFA.
Figure 4. Agarose gel electrophoresis of qdots conjugates (1% agarose gel;
Tris-acetate-EDTA buffer containing ethidium bromide for DNA visualization; 80 V potential difference). A 1-kb DNA
ladder (Lane 1; New England Biolabs, Ipswich, Mass, USA), with DNA fragments ranging
from 0.5–10 kilobases (kb) as indicated, was utilized to illustrate relative
electrophoretic mobility of the qdot conjugates. Unconjugated AMP-coated
qdots (Lane 2) have an increased mobility by comparison with both
muscimol-conjugated qdots (Lane 3) and qdots conjugated with methoxy terminated
PEG2000 (Lane 4), indicating successful functionalization of the qdot surface.
Figure 5. Fluorescence images (top row) and
bright-field images (bottom row) of oocytes incubated with qdot-containing
compounds for 10 minutes. The
bright-field images illustrate the plane of focus of the opaque oocyte. Panels
A and B show results from a human ρ1 GABAC-expressing oocyte incubated with 34 nM muscimol-conjugated AMP-coated qdots. Panels C and D show a human ρ1 GABAC-expressing oocyte incubated with a 34 nM solution of unconjugated AMP-coated qdots. Panels
E and F show a nonexpressing oocyte incubated with 34 nM muscimol-conjugated
AMP-coated qdots. Adapted from Gussin et al. [65].
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