Photoaffinity labeling of the vesamicol receptor of cholinergic synaptic vesicles

On the basis of the high-affinity vesamicol analog 4-aminobenzovesamicol (ABV), a tritiated, arylazido ligand (azidoABV) of the vesamicol receptor (VR) in cholinergic synaptic vesicles was synthesized. azidoABV is an inhibitor of acetylcholine (AcCh) active transport, and it binds to the VR with higher affinity than vesamicol. The rate of dissociation of azidoABV from synaptic vesicles is 0.058 +/- 0.003 min-1 at 20 degrees C (about 3-fold slower than that of vesamicol), and the equilibrium dissociation constant is 2 nM (about 4-fold lower than that of vesamicol). Photolysis of [3H]azidoABV in the presence of a stoichiometric excess of the VR led to incorporation of 28% of the radiolabel, of which 57% was blocked by 50 microM vesamicol. Sodium dodecyl sulfate polyacrylamide gel electrophoretic analysis of the labeled vesicles revealed, after autofluorography, specific labeling over a broad molecular weight range that extended from about 50 to 200 kDa. This labeling pattern was essentially the same as that obtained with an azido analog of AcCh that was used to label the AcCh transporter (Rogers, G.A., & Parsons, S. M. (1992) Biochemistry 31, 5770-5777). In addition, about 6% of the radioligand that was specifically incorporated into proteins with M(r) greater than 12 kDa labeled four polypeptides that corresponded to bands in the Coomassie image at M(r) = 23, 33, 35, and 38 kDa. The results suggest that the VR exists as part of a complex system of subunits.