Cocaine abuse is a worldwide health problem. Cocaine inhibits monoamine reuptake proteins including: dopamine transporter (DAT), norepinephrine transporter (NET), and serotonin transporter (SERT). The location of a cocaine binding site on these proteins has not yet been identified.
The three monoamine transporters are integral membrane proteins that have multiple conformations; therefore, atomic resolution of a crystallized cocaine bound transporter protein is a very difficult task. To determine the cocaine binding site, alternative protein structure function analysis methods must be used to identify how cocaine inhibition occurs. Since cocaine inhibits these transporters at similar concentrations and these proteins share homology, it is presumed that they would share a similar cocaine binding site. This dissertation uses protein mutagenesis in three methods to locate the cocaine binding site: disulfide crosslinks, cocaine analog screening, and computer modeled inhibition.
Cysteine is a unique amino acid; within a protein, two neighboring cysteine residues can form a disulfide linkage identifying residue proximity. Cross-linking these residues can reveal the DAT protein tertiary structure and possible cocaine binding sites. A fully functional D. melanogaster DAT mutant with all non-critical cysteines removed (dDATx7) was mutated to include factor Xa protease recognition sequences (fXa-dDATx7). Using fXa-dDATx7, 54 single cysteine positional mutants were generated in six transmembrane domains (TMs) (1, 2, 3, 5, 7, and 12). Of these, 18 transported DA more than 50% of wild type dDAT. Functional mutants could be paired together in a single protein to determine if cross-linked residues affect cocaine inhibition or if cocaine inhibits cross-linking. However, crystal structures of a monoamine transporter homolog were solved revealing the overall tertiary structure of the transporter superfamily.
An alternative approach utilized cocaine analog compounds that contain different chemical modifications to the cocaine structure. Cocaine methiodide (CM), a charged cocaine analog, cannot pass the blood brain barrier. Therefore, the effects of systemic CM doses represent cocaine actions in peripheral tissues. However, the half maximal inhibition values (IC50) of CM have not been clearly determined for major cocaine targets: DAT, NET, SERT, and sodium channels. In cells transfected with individual monoamine transporters, mouse dorsal root ganglion neurons, and synaptosomes from adult male mice, the IC50 of CM were at least 31-fold to 184-fold higher than cocaine. These results indicate that an equal dose of this cocaine analog will not produce the equivalent inhibition effect that cocaine produces.
A more potent analog of cocaine is the DAT-selective uptake inhibitor, RTI-113. Using site-directed mutagenesis, putative aqueous-facing residues were switched between the mouse DAT and NET at the same position. Changing a specific tyrosine residue in NET to phenylalanine increased RTI-113 sensitivity. Conversely, switching a tyrosine into the phenylalanine expressing DAT decreased RTI-113 sensitivity. In contrast, neither mutation significantly altered the sensitivities to non-transporter-selective inhibitors or transporter function. Random mutagenesis at this residue of DAT or NET also does not significantly affect inhibitor sensitivity. This indicates a critical residue contributing to the potent uptake inhibitions of a cocaine analog and a possible residue at the cocaine binding site.