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.
