PROTEIN-KINASE-C ACTIVATION REGULATES HUMAN SEROTONIN TRANSPORTERS INHEK-293 CELLS VIA ALTERED CELL-SURFACE EXPRESSION

Antidepressant- and cocaine-sensitive serotonin (5-hydroxytryptamine, 5-HT) transporters (SERTs) dictate clearance of extracellular 5-HT aft er release. To explore protein kinase C-mediated SERT regulation, we g enerated a stable human SERT (hSERT)-expressing cell line (293-hSERT) and evaluated modulation of 5-HT activity via studies of 5-HT flux, hS ERT-mediated currents under voltage clamp, and surface distribution of SERT protein. 293-hSERT cells exhibit saturable, high-affinity, and a ntidepressant-sensitive 5-HT uptake as well as hSERT-dependent whole-c ell currents. In these cells, the protein kinase C activator beta-PMA caused a time-dependent reduction in 5-HT uptake capacity (V-max) afte r acute application and a reduction in SERT-mediated currents. Effects of beta-PMA were mimicked by the phorbol ester beta-PDBu, were not ob served with the inactive alpha-isomers, and could be blocked by treatm ent of cells with the protein kinase C inhibitor staurosporine. Biotin ylation/immunoblot analyses showed that activity reductions are parall eled by a staurosporine-sensitive loss of surface SERT protein. These data indicate that altered surface abundance, rather than reduced cata lytic transport efficiency, mediates acute PKC-dependent modulation of 5-HT uptake.