The mammalian, brain-specific, high affinity L-proline transporter (PROT) is a member of a gene family of Na and Cl dependent plasma membrane transport proteins that includes transporters for several neurotransmitters, osmolytes, and metabolites. This novel transporter is expressed in subpopulations of putative glutamatergic neurons, where the PROT protein is enriched in synaptic vesicles. These findings warrant the consideration of a presynaptic regulatory role for PROT in excitatory transmission at specific glutamatergic nerve terminals. The long term objective of the research is to elucidate the physiological role of mammalian brain PROT in excitatory synaptic transmission.
Specific aim 1 will investigate the functional role for PROT in synaptic vesicles. PROT is the only protein characterized at the molecular level that is specifically localized to synaptic vesicles in excitatory nerve terminals. The applicant will immunoisolate the PROT-containing synaptic vesicles and examine the neurotransmitter phenotype and biochemical properties of this unique subpopulation of synaptic vesicles. Are the PROT-containing vesicles prolinergic or glutamatergic, or do they represent a discrete set of excitatory nerve terminal vesicles with an unprecedented function? Little is known about how proteins are targeted to synaptic vesicles.
Specific aim 2 will use recombinant DNA techniques to identify putative synaptic vesicle targeting signals in PROT. Preliminary studies indicate that the primary amino acid sequence of PROT contains information necessary for targeting this protein to intracellular vesicles. Finally, the precise substrate binding site has not been identified or any member of this transporter family. Several recent advances indicate that PROT is a good model system to elucidate this critical region of the transporter protein.
Specific aim 3 will delineate the substrate binding domain and/or the translocation pore of PROT. The findings may provide new insights into presynaptic regulatory mechanisms involved in synaptic plasticity and excitotoxic nerve cell damage.

National Institute of Health (NIH)
National Institute of Neurological Disorders and Stroke (NINDS)
Research Project (R01)
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Neurological Sciences Subcommittee 1 (NLS)
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Kitt, Cheryl A
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Duke University
Schools of Medicine
United States
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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