COCAINE RECEPTOR--BIOCHEMICAL AND MOLECULAR STUDIES

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Because the dopamine transporter is a key target for psychostimulant drugs, a goal of this project is to characterize the dopamine transporter and other transporters. The characterization of this protein both as a drug receptor and as an important entity of the dopaminergic neuron is essential in understanding its function. We have previously shown that the dopamine transporter exists in multiple post-translationally modified forms. For example, the molecular weight of the transporter in the nucleus accumbens is somewhat higher than that in the striatum. We have found that there is evidence for post-translational variations during development and aging since the molecular weight of the dopamine transporter changes during this time. It appears that a significant part of this change is due to differences in glycosylation. Other proteins have also shown developmentally related changes in glycosylation. As there is evidence that the glycosylation is an important part of the functioning protein, these findings bear on the functional aspect of the transporter over aging. We previously found, for the first time, that dopamine reuptake is inhibited by nitric oxide. This appears to be an important issue because nitric oxide may be a transsynaptic regulator and may be able to diffuse from postsynaptic elements to presynaptic sites to alter function. Because it is known that glutamatergic synapses from cortical neurons are side by side with dopaminergic synapses from the midbrain, we investigated whether or not glutamate uptake would be affected by nitric oxide. In fact, we did find that it was inhibited by nitric oxide. This has important implications. For example, if nitric oxide is part of the excitatory amino acid receptor mediated toxic cycle, then inhibition of reuptake would enhance the toxicity of excitatory amino acids. Whether or not this turns out to be a relevant mechanism that occurs in vivo remains to be seen. We are currently investigating as to whether or not these mechanisms operate in vivo. Another project is the elucidation of the structure of the dopamine transporter protein by peptide mapping. In this procedure, the transporter is labeled with a photoaffinity analog of cocaine, treated with enzymes that break the protein and the fragments are then measured for molecular weight, content of the photoaffinity label, and glycosylation content. These results suggest that a photoaffinity label called DEEP which is based on GBR 12909 appears to bind within the first two transmembrane helices while RTI-82, a photoaffinity label based on cocaine, labels the protein between transmembrane domains 4 and 12. These results will ultimately help us understand the precise molecular sites where the different transport inhibitors bind. Overall, we continue to make substantial progress in understanding the nature of the dopamine transporter protein. We are currently focusing on the phosphorylation of the transporter protein.