| Proline is actively taken up by nervous tissue through a high-affinity Na{dollar}sp+{dollar}-dependent plasma membrane carrier that is structurally related to the uptake carriers of recognized neurotransmitters such as glutamate, glycine, GABA and the monoamines. Although circumstantial evidence suggests that proline may have a specific modulatory role at mammalian synapses, no proline receptors have been identified. Accordingly, in the absence of a specific postsynaptic 'prolinergic' pharmacology, a molecular and pharmacological characterization of neural proline transport was undertaken to help elucidate further the neurobiology of proline.; In the first part of the study, the human brain proline transporter (hProT) was cloned, sequenced and functionally expressed. Human ProT has {dollar}>{dollar}98% amino acid identity with its rat counterpart, the highest interspecies conservation in the major neurotransmitter transporter gene family. Northern hybridization analysis showed that expression of hProT is unique to brain tissue and heterogeneous within brain. Transient expression of the hProT cDNA in HeLa cells established that this transporter has high affinity for L-proline {dollar}(rm Ksb{lcub}m{rcub} = 6.2 mu{dollar}M) and exhibits remarkable structural specificity in substrate recognition.; ProT was also found to be potently inhibited by a group of peptides, characterized in the second part of the study. Leu-enkephalin (Tyr-Gly-Gly-Phe-Leu) and its subset peptides Gly-Gly-Phe-Leu and Gly-Phe-Leu inhibited the proline transporter with high potency (K{dollar}sb{lcub}rm i{rcub}{dollar}'s of 3-6 {dollar}mu{dollar}M). Lineweaver-Burke transformation of uptake data showed that this inhibition was competitive. ({dollar}sp3{dollar}H) Leu-enkephalin was not accumulated above control levels by hProT-transfected HeLa cells. Enkephalin-mediated inhibition was essentially specific for the proline transporter: the norepinephrine transporter was inhibited with a K{dollar}sb{lcub}rm i{rcub} sim 100 mu{dollar}M while other neurotransmitter transporters were much less sensitive (K{dollar}sb{lcub}rm i{rcub}{dollar}'s {dollar}>{dollar} 100 uM).; This work establishes that the high affinity proline transporter, a member of the Na{dollar}sp+{dollar}- and Cl{dollar}sp-{dollar}-dependent neurotransmitter transporter family, is brain-specific, heterogeneously distributed within brain, highly conserved during evolution from rat to human, and potently, competitively and selectively inhibited by enkephalin peptides. When considered with the available literature on proline, these findings strongly argue for a specific neural role for proline in brain function, perhaps involving an interaction with glutamate and/or enkephalins. |
