Dopamine transporter interaction with blockers and transportable substrates: insights from kinetics study/Dopamiini transporteri koostoime inhibiitorite ning transporditavate substraatidega: kineetika uuringutest jarelduv molekulaarne mehhanism

• Published in: Proceedings of the Estonian Academy of Sciences 2023-12, Vol.72 (4), p.418
• Main Author: Stepanov, Vladimir
• Format: Article
• Language: English
• Subjects: Dopamine; Medical research; Medicine, Experimental; Pharmacokinetics
• ISSN: 1736-6046
• DOI: 10.3176/proc.2023.4.07

Competition kinetic analysis was performed to examine the interaction mechanism of the dopamine transporter with dopamine, (S)-amphetamine, and cocaine, which play a central role in drug abuse phenomena connected with the dopaminergic system. Efficient dopamine transporter inhibitor [[.sup.3]H]PE2I was used as a reporter ligand for this analysis as this compound initiates slow isomerization of the transporter--ligand complex and thus ensures reliable results of the filtration radioligand assay. It was shown that the three investigated compounds do not initiate slow isomerization of their complexes with the transporter, but their presence inhibits the isomerization step of the radioactive reporter ligand. Secondly, it was shown that (S)-amphetamine and dopamine do not interfere with the fast step of the inhibitor ligand binding, pointing to the formation of the ternary complex, including transporter protein, reporter ligand, and an unlabeled compound. This is possible if the two molecules bind to non-overlapping sites on the transporter. Binding of cocaine results in slightly improved binding of the reporter ligand, pointing to a positive allosteric interaction between these binding processes. Keywords: dopamine transporter, ligand interaction mechanism, competition kinetic analysis, amphetamine, cocaine, dopamine. Dopamiini, (S)amfetamiini ning kokaiini toime molekulaarsete mehhanismide selgitamine on oluline moistmaks nende ainete moju aju dopamiinergilise susteemi komponentidele ning sellega seotud narkosoltuvuse ilmingutele, mis pohjustavad nii meditsiinilisi kui ka tosiseid sotsiaalseid probleeme. Selles toos uuriti nende ainete interaktsiooni hiire aju juttkeha dopamiini transportvalguga, kasutades selleks erinevalt eelnevatest uuringutest kineetilise analuusi meetodit radioaktiivse reporterligandi [[.sup.3]H]PE2I toimel, mis on tohus dopamiini transporteri inhibiitor. Too tulemused naitavad, et ukski kolmest uuritud uhendist ei algata nende ainete ja transportvalgu kompleksi aeglast isomerisatsiooni, mis on iseloomulik paljudele madala molekulmassiga uhendite sidumisprotessidele valkudega. Teiseks naidati, et (S)amfetamiin ning dopamiin ei mojuta reporterligandi ja transporteri vahelise kompleksi moodustumise kiiret tasakaalu. Seega on reporterligandi nende uhendite seostumine transportvalgu erinevatele ja omavahel mittekattuvatele sidumiskohtadele ilmne. Samal ajal aga koik kolm ainet aeglustavad [[.sup.3]H]PE2I seostumise kiirele staadiumile jargne