Abstract

L-Proline fulfills several of the classic criteria used to identify amino acid neurotransmitters, including the presence of a high affinity, Na(+)- (and Cl-)-dependent synaptosomal transport process and the Ca(2+)-dependent release of exogenously loaded radiolabeled L-proline from brain slices and synaptosomes after K(+)-induced depolarization. However, studies to define the role of L-proline in discrete pathways in the mammalian brain have been precluded by the inability to block its biosynthesis or high affinity transport in nervous tissue. We report the molecular cloning, functional expression, and chromosomal localization of a human brain-specific high affinity L-proline transporter (hPROT). The pharmacological specificity, kinetic properties, and ionic requirements of hPROT clearly distinguish this carrier from the other Na(+)-dependent plasma membrane carriers that transport L-proline. Multiple tissue Northern blot analysis revealed a prominent approximately 4-kb mRNA transcript in human brain tissue, whereas no specific hybridizing species were detected in peripheral tissue. An antipeptide antiserum directed against the carboxy-terminus of the predicted hPROT protein identified a single, broad immunoreactive protein of 68 kDa on immunoblots of synaptosomal membranes from various human brain regions. In contrast, no specific labeling was detected on immunoblots of membranes from human liver, kidney, or heart. A differential distribution of hPROT mRNA and protein was observed in the human corpus striatum, consistent with the hypothesis that the hPROT protein is synthesized in neuronal cell bodies in an extrastriatal location and axonally transported to the corpus striatum. These findings warrant the consideration of a synaptic regulatory role for this transporter and its presumed natural substrate, L-proline, in the mammalian central nervous system.