Photoaffinity labeling of the acetylcholine transporter.
The acetylcholine (AcCh) binding site in the AcCh transporter-vesamicol receptor (AcChT-VR) present in synaptic vesicles isolated from the electric organ of Torpedo was characterized. A high-affinity analogue of AcCh containing an aryl azido group, namely, cyclohexylmethyl cis-N-(4-azidophenacyl)-N-methylisonipecotate bromide (AzidoAcCh), was synthesized in nonradioactive and highly tritiated forms. AzidoAcCh was shown to be a competitive inhibitor of [3H]AcCh active transport and binding of [3H]-vesamicol to the allosteric site. The [3H]AzidoAcCh saturation curve was determined. In all cases the AcChT.AzidoAcCh complex exhibited an inhibition or dissociation constant of about 0.3 microM. Binding of [3H]AzidoAcCh was inhibited by vesamicol and AcCh. AzidoAcCh irreversibly blocked greater than 90% of the [3H]vesamicol binding sites after multiple rounds of photolysis and reequilibration with fresh ligand. Autofluorographs of synaptic vesicles photoaffinity-labeled with [3H]AzidoAcCh showed specific labeling of material exhibiting a continuous distribution from 50 to 250 kDa after sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The result demonstrates that the AcChT has an unexpected structure highly suggestive of the synaptic vesicle proteoglycan.
Rogers
G A
GA
Department of Chemistry, University of California, Santa Barbara 93106.
Parsons
S M
SM
D000109:Acetylcholine / Q000031:analogs & derivatives / Q000138:chemical synthesis / Q000378:metabolism / Q000494:pharmacology; D000345:Affinity Labels; D000818:Animals; D001386:Azides / Q000138:chemical synthesis / Q000494:pharmacology; D001665:Binding Sites; D001667:Binding, Competitive; D001693:Biological Transport, Active / Q000187:drug effects; D002352:Carrier Proteins / Q000378:metabolism*; D004557:Electric Organ / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D007700:Kinetics; D026901:Membrane Transport Proteins; D010777:Photochemistry; D010880:Piperidines / Q000378:metabolism / Q000494:pharmacology; D017981:Receptors, Neurotransmitter / Q000378:metabolism*; D017478:Receptors, Phencyclidine; D013056:Spectrophotometry, Ultraviolet; D013572:Synaptic Vesicles / Q000737:chemistry; D014101:Torpedo; D014316:Tritium; D050494:Vesicular Acetylcholine Transport Proteins; D033921:Vesicular Transport Proteins
D000345:Affinity Labels; D001386:Azides; D002352:Carrier Proteins; D026901:Membrane Transport Proteins; D010880:Piperidines; D017981:Receptors, Neurotransmitter; D017478:Receptors, Phencyclidine; D050494:Vesicular Acetylcholine Transport Proteins; D033921:Vesicular Transport Proteins; D014316:Tritium; C075303:cyclohexylmethyl-N-(4-azidophenacyl)-N-methylisonipecotate; C006189:vesamicol; D000109:Acetylcholine