Photoaffinity labeling of the monoamine transporter of bovine chromaffin granules and other monoamine storage vesicles using 7-azido-8-[125I]iodoketanserin.
An iodinated azido derivative of ketanserin, 7-azido-8-[125I]iodoketanserin ( [125I]AZIK), has been used to label the monoamine transporter of bovine chromaffin granule membranes by the technique of photoaffinity labeling. In the dark, this derivative was found to bind reversibly to the membranes, with an equilibrium dissociation constant estimated to be 6 nM at 0 degrees C. As for ketanserin, binding occurred at the tetrabenazine site: (i) [125I]AZIK was displaced efficiently from its binding site by tetrabenazine, ketanserin, and 7-azidoketanserin, whereas serotonin, which is a substrate for the transporter but has a low affinity for tetrabenazine binding site, was a poor displacer; pipamperone and pyrilamine, two antagonists of respectively serotonin S2 and histamine H1 receptors, were inactive. (ii) 7-Azidoketanserin was a competitive inhibitor of [3H]dihydrotetrabenazine binding, and it inhibited the ATP-dependent uptake of serotonin by chromaffin granule ghosts. Irradiation of [125I]AZIK with long-wavelength UV light, followed by electrophoresis on sodium dodecyl sulfate/polyacrylamide gels and autoradiography, revealed irreversible labeling of a membrane component with an apparent molecular weight of 73,000. Tetrabenazine inhibited the labeling of this 73-kDa band in a manner parallel to the binding of [125I]AZIK in the dark. Such a labeling is totally compatible with previous results obtained through photolabeling with a tetrabenazine derivative or by target size analysis. Moreover, preliminary experiments showed that [125I]AZIK can label the tetrabenazine binding sites of various sources including rat striatum, rabbit platelets, human pheochromocytoma, and human adrenal medulla. Therefore, this molecule appears to be an excellent probe to label the monoamine transporter of different amine storage vesicles even without purification.
Isambert
M F
MF
Institut de Biologie Physico-Chimique, Paris, France.
Gasnier
B
B
Laduron
P M
PM
Henry
J P
JP
D000042:Absorption; D000345:Affinity Labels; D000588:Amines / Q000378:metabolism*; D000818:Animals; D001386:Azides; D001665:Binding Sites; D001693:Biological Transport, Active; D002417:Cattle; D002837:Chromaffin Granules / Q000378:metabolism*; D002838:Chromaffin System / Q000378:metabolism*; D003624:Darkness; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D007425:Intracellular Membranes / Q000378:metabolism; D007457:Iodine Radioisotopes; D007650:Ketanserin / Q000031:analogs & derivatives*; D008970:Molecular Weight; D011817:Rabbits; D051381:Rats; D012701:Serotonin / Q000493:pharmacokinetics; D013045:Species Specificity; D013747:Tetrabenazine / Q000031:analogs & derivatives / Q000378:metabolism*
D000345:Affinity Labels; D000588:Amines; D001386:Azides; D007457:Iodine Radioisotopes; D012701:Serotonin; C044880:7-azidoketanserin; D007650:Ketanserin; D013747:Tetrabenazine