2-Nitroimipramine: a photoaffinity probe for the serotonin uptake/tricyclic binding site complex

[3H]2-Nitroimipramine ([3H]2-NI), a compound with high affinity for the serotonin uptake system, is shown to be an effective photoaffinity probe which incorporates covalently into membrane homogenates prepared from human platelets, as well as rat brain and liver. In all cases, [3H]2-NI preferentially incorporated into a minor membrane component of 30 kd protein, as determined by SDS-polyacrylamide gel electrophoresis and subsequent fluorography. A number of selective and general serotonin uptake inhibitors quantitatively chased labeling of the 30-kd band at nanomolar concentrations. Pharmacological characterizing agents unrelated to the serotonin uptake system generally had little effect on labeling. In platelet membranes, a broad band of approximately 35-kd protein was also labeled by [3H]2-NI, but this labeling was not inhibited by any of the selective serotonin uptake blockers. Interestingly, serotonin itself increased incorporation into the 30-kd band and selectively decreased labeling of the 35-kd band. Photolytic incorporation into the 30-kd band was of high affinity, saturable, and Scatchard analyses of irreversible labeling were linear. In contrast, Scatchard transformations of [3H]2-NI equilibrium binding saturation isotherms were markedly curvilinear. Cross-linking unlabeled 2-NI to intact platelets, followed by extensive dialysis, decreased the maximal velocity (Vmax) of platelet serotonin uptake, but did not alter the affinity (Km) of serotonin for its transport site. These results are noteworthy since current theories implicate prejunctional allosteric interactions between serotonin and imipramine at serotonergic synapses.(ABSTRACT TRUNCATED AT 250 WORDS)