L-proline has been postulated to be a neurotransmitter or neuromodulator in the mammalian central nervous system (CNS). Studies to define the role of L-proline in discrete pathways in the mammalian CNS have not been possible due to the inability to block its biosynthesis or high affinity transport in nervous tissue. Our recent cloning of a high affinity, Na+- dependent L-proline transporter expressed in subpopulations of putative glutamatergic neurons provides compelling support for a specific role for L-proline in excitatory transmission. Glutamatergic synapses have been implicated in diverse physiologic processes including the acquisition of associative learning, synapse formation, and neuroendocrine regulation. Abnormalities of glutamatergic transmission have been implicated in the pathophysiology of excitotoxic neurologic disorders. Thus an understanding of the functional role(s) of the L-proline transporter in specific glutamatergic neurons could provide insights into synaptic regulatory mechanisms involved in synaptic plasticity and memory acquisition or their disruption in diseases such as Alzheimer's disease. The long term goal of this research is to elucidate the role(s) of L-proline, and its transporter, in synaptic transmission.
Specific Aim l will undertake the first detailed studies of the regional, cellular, and subcellular distribution of the high affinity L-proline transporter protein in rat brain. The precise projection pathways of individual neurons that express L-proline transporter mRNA will be determined by combining retrograde dye tract-tracing with in situ hybridization histochemistry. Knowledge of these pathways will be crucial to the design of physiological studies of the role of L-proline in synaptic transmission. Studies will also examine the possible co-localization of high affinity L-proline and L-glutamate transporters in glutamatergic nerve terminals.
Specific Aim 2 will use the L-proline transporter as a model neurotransmitter transporter, to investigate the structure-function properties and regulation of this important class of synaptic proteins using chimeric transporter strategies and site-directed mutagenesis. The final Specific Aim will investigate potential synaptic roles of L-proline. A Xenopus oocyte expression assay will be used to investigate the hypothesis that L-proline modulates signal transduction at a glutamate receptor subtype, analogous to glycine modulation of the NMDA receptor. This assay will also be used to investigate the possibility that distinct L-proline receptors exist in mammalian CNS. Monoclonal antibodies that selectively block L-proline transport will be generated, for use as transport inhibitors, to examine the hypothesis that L-proline modulates the synthesis and/or release of glutamate from nerve terminals. The potential discovery of novel regulatory mechanisms in glutamatergic synapses is the incentive which drives the research described in this proposal.

National Institute of Health (NIH)
National Institute of Neurological Disorders and Stroke (NINDS)
Research Project (R01)
Project #
Application #
Study Section
Neurological Sciences Subcommittee 1 (NLS)
Project Start
Project End
Budget Start
Budget End
Support Year
Fiscal Year
Total Cost
Indirect Cost
Duke University
Schools of Medicine
United States
Zip Code
Crump, F T; Fremeau, R T; Craig, A M (1999) Localization of the brain-specific high-affinity l-proline transporter in cultured hippocampal neurons: molecular heterogeneity of synaptic terminals. Mol Cell Neurosci 13:25-39
Renick, S E; Kleven, D T; Chan, J et al. (1999) The mammalian brain high-affinity L-proline transporter is enriched preferentially in synaptic vesicles in a subpopulation of excitatory nerve terminals in rat forebrain. J Neurosci 19:21-33
Galli, A; Jayanthi, L D; Ramsey, I S et al. (1999) 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. J Neurosci 19:6290-7
Velaz-Faircloth, M; McGraw, T S; alandro, M S et al. (1996) Characterization and distribution of the neuronal glutamate transporter EAAC1 in rat brain. Am J Physiol 270:C67-75
Fremeau Jr, R T; Velaz-Faircloth, M; Miller, J W et al. (1996) A novel nonopioid action of enkephalins: competitive inhibition of the mammalian brain high affinity L-proline transporter. Mol Pharmacol 49:1033-41
Shafqat, S; Velaz-Faircloth, M; Henzi, V A et al. (1995) Human brain-specific L-proline transporter: molecular cloning, functional expression, and chromosomal localization of the gene in human and mouse genomes. Mol Pharmacol 48:219-29
Velaz-Faircloth, M; Guadano-Ferraz, A; Henzi, V A et al. (1995) Mammalian brain-specific L-proline transporter. Neuronal localization of mRNA and enrichment of transporter protein in synaptic plasma membranes. J Biol Chem 270:15755-61