Photoinactivation of serotonin uptake by an arylazido derivative of 5-hydroxytryptamine

A potential photoaffinity probe for the substrate-binding polypeptide of the neuronal serotonin uptake system has been synthesized. Under dark conditions, 3-(beta-(4-azidobenzamidino)ethyl-5-hydroxyindole (serotonin azidobenzamidine (SABA) was found to inhibit competitively [3H]5-hydroxytryptamine uptake by rat cortical synaptosomes with a K1 of 130 nM. The selectivity of this action was indicated by SABA's much lower potency as an inhibitor of synaptosomal [3H]norepinephrine uptake (K1 = 7 microM). When synaptosomes were irradiated in the presence of SABA, serotonin uptake was irreversibly inhibited in a concentration-dependent fashion with the maximum effect occurring at 1 microM SABA. At this concentration, approximately 40% of the serotonin uptake activity could not be recovered upon repeated washing of the synaptosomes. This inhibition was determined not to result from the production of a potent inhibitory photolysis product of SABA. The photoinactivation of serotonin transport by SABA was found to depend on the time of irradiation and could be prevented by the presence of agents that interact with the uptake system. Serotonin, p-chloroamphetamine, fenfluramine, and alaproclate protected the serotonin carrier against SABA's irreversible effects in a concentration-dependent manner. The presence of high concentrations of Tris or p-aminobenzoic acid, two nitrene-scavenging agents, did not reduce the level of photoinactivation of serotonin uptake by SABA, indicating that the irreversible inhibition is a result of true photoaffinity labeling of the carrier.