Protein kinase C activation regulates human serotonin transporters in HEK-293 cells via altered cell surface expression.
Antidepressant- and cocaine-sensitive serotonin (5-hydroxytryptamine, 5-HT) transporters (SERTs) dictate clearance of extracellular 5-HT after release. To explore protein kinase C-mediated SERT regulation, we generated a stable human SERT (hSERT)-expressing cell line (293-hSERT) and evaluated modulation of 5-HT activity via studies of 5-HT flux, hSERT-mediated currents under voltage clamp, and surface distribution of SERT protein. 293-hSERT cells exhibit saturable, high-affinity, and antidepressant-sensitive 5-HT uptake as well as hSERT-dependent whole-cell currents. In these cells, the protein kinase C activator beta-PMA caused a time-dependent reduction in 5-HT uptake capacity (Vmax) after acute application and a reduction in SERT-mediated currents. Effects of beta-PMA were mimicked by the phorbol ester beta-PDBu, were not observed with the inactive alpha-isomers, and could be blocked by treatment of cells with the protein kinase C inhibitor staurosporine. Biotinylation/immunoblot analyses showed that activity reductions are paralleled by a staurosporine-sensitive loss of surface SERT protein. These data indicate that altered surface abundance, rather than reduced catalytic transport efficiency, mediates acute PKC-dependent modulation of 5-HT uptake.
Qian
Y
Y
Department of Pharmacology and Center for Molecular Neuroscience, Vanderbilt School of Medicine, Nashville, Tennessee 37232-6600, USA.
Galli
A
A
Ramamoorthy
S
S
Risso
S
S
DeFelice
L J
LJ
Blakely
R D
RD
D000928:Antidepressive Agents / Q000494:pharmacology; D002352:Carrier Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism* / Q000502:physiology; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D004553:Electric Conductivity; D004789:Enzyme Activation / Q000502:physiology; D006801:Humans; D008562:Membrane Glycoproteins / Q000037:antagonists & inhibitors / Q000378:metabolism* / Q000502:physiology; D026901:Membrane Transport Proteins; D009419:Nerve Tissue Proteins; D011493:Protein Kinase C / Q000378:metabolism* / Q000502:physiology; D012701:Serotonin / Q000494:pharmacology; D050486:Serotonin Plasma Membrane Transport Proteins
D000928:Antidepressive Agents; D002352:Carrier Proteins; D008562:Membrane Glycoproteins; D026901:Membrane Transport Proteins; D009419:Nerve Tissue Proteins; C493230:SLC6A4 protein, human; D050486:Serotonin Plasma Membrane Transport Proteins; D012701:Serotonin; D011493:Protein Kinase C