Molecular cloning and expression of a high affinity L-proline transporter expressed in putative glutamatergic pathways of rat brain

Abstract

We have used the polymerase chain reaction (PCR) with degenerate oligonucleotides derived from two conserved regions of the norepinephrine and gamma-aminobutyric acid transporters to identify novel Na(+)-dependent transporters in rat brain. One PCR product hybridized to a 4.0 kb RNA concentrated in subpopulations of putative glutamatergic neurons including mitral cells of the olfactory bulb, pyramidal cells of layer V of the cerebral cortex, pyramidal cells of the piriform cortex, and pyramidal cells of field CA3 of the hippocampus. Transient expression of the cognate cDNA conferred Na(+)-dependent L-proline uptake in HeLa cells that was saturable (Km = 9.7 microM) and exhibited a pharmacological profile similar to that for high affinity L-proline transport in rat brain slices. The cloned transporter cDNA predicts a 637 aa protein with 12 putative transmembrane domains and exhibits 44%-45% amino acid sequence identity with other members of the emerging family of neurotransmitter transporters. These findings support a synaptic role for L-proline in specific excitatory pathways in the CNS.