Imaging GABAc receptors with ligand-conjugated quantum dots.
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.
PubMed ID: 18437227
PMC ID: PMC2323551
Article link: J Biomed Biotechnol.
Genes referenced: boc dcc nhs slc6a4
Article Images: [+] show captions
|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. .|