L-proline and L-pipecolate induce enkephalin-sensitive currents in human embryonic kidney 293 cells transfected with the high-affinity mammalian brain L-proline transporter

The high-affinity mammalian brain L-proline transporter (PROT) belongs to t he GAT1 gene family, which includes Na- and Cl-dependent plasma membrane ca rriers for neurotransmitters, osmolites, and metabolites. These transporter s couple substrate flux to transmembrane electrochemical gradients, particu larly the Na gradient. In the nervous system, transporters clear synapses a nd help to replenish transmitters in nerve terminals. The localization of P ROT to specific excitatory terminals in rat forebrain suggests a role for t his carrier in excitatory transmission (Renick et al., 1999). We investigat ed the voltage regulation and electrogenicity of this novel transporter, us ing human embryonic kidney (HEK) 293 cells stably transfected with rat PROT cDNA. In physiological solutions between -140 and -40 mV, L-proline (PRO) and its six-member ring congener (PIP) induced inward current. The current- voltage relationship of PROT or in an externally accessible pore. and the v ariance of current fluctuations were similar for PRO- and PIP-induced curre nt, and the ratio of induced variance to the mean current ranged from 20 to 60 fA, Des-Tyr-Leu-enkephalin (GGFL), a competitive peptide inhibitor of P ROT, reduced the rat PROT-associated current to control levels. GGFL alone did not elicit currents, and the GGFL-sensitive substrate-induced current w as absent in nontransfected cells. Finally, GGFL inhibited PROT-mediated tr ansport only when applied to the extracellular face of PROT. These data sug gest that (1) PROT uptake is electrogenic, (2) individual transporter curre nts are voltage-independent, and (3) GGFL is a nonsubstrate inhibitor that interacts either with an extracellular domain of PROT or in an externally a ccessible pore.