Dopamine and 5HT Transporter Amino And Carboxy Termini Contribute To Substrate Affinity And Psychostimulant Activity

Abstract

Determining how psychoactive drugs interact with their molecular targets is essential for understanding psychostimulant abuse and pharmacotherapies. The SLC6 carriers, DAT and SERT, are the primary targets for both addictive and therapeutic psychostimulants. Recent SLC6 structures have shed light on these molecular interactions. However, these structures lack much of the carriers' N‐ and C‐termini, raising questions regarding their potential contributions to the transport mechanism. To address this question, we generated DAT/SERT chimeras that exchange the N‐, C‐, or both termini, and tested their effect on substrate uptake, ligand binding and efflux kinetics. The SERT N‐terminus decreased DAT's affinity for DA and AMPH affinity two‐fold, whereas substituting the SERT C‐terminus had no effect on DAT substrate affinities. Moreover, replacing both DAT intracellular termini with SERT domains rescued DAT substrate affinity. In contrast, substituting the DAT N‐terminus had no effect on SERT substrate affinities, whereas the DAT C‐terminus decreased SERT substrate affinities and this was not rescued when both DAT termini were substituted onto SERT. Chimeric DAT and SERT protein affinities for cocaine were unaffected by interchanging domains, suggesting that DAT and SERT tail domains contribute in substrate translocation but not ligand binding. Ongoing studies test whether these intracellular domains contribute to AMPH‐stimulated substrate efflux and catalytic turnover rates and should shed considerable light onto the differential contribution of these domains to the DA vs. 5HT transport processes.