Antagonist-induced conformational changes in dopamine transporter extracellular loop two involve residues in a potential salt bridge

Abstract

Ligand-induced changes in the conformation of extracellular loop (EL) 2 in the rat (r) dopamine transporter (DAT) were examined using limited proteolysis with endoproteinase Asp-N and detection of cleavage products by epitope-specific immunoblotting. The principle N-terminal fragment produced by Asp-N was a 19 kDa peptide likely derived by proteolysis of EL2 residue D174, which is present just past the extracellular end of TM3. Production of this fragment was significantly decreased by binding of cocaine and other uptake blockers, but was not affected by substrates or Zn2+, indicating the presence of a conformational change at D174 that may be related to the mechanism of transport inhibition. DA transport activity and cocaine analog binding were decreased by Asp-N treatment, suggesting a requirement for EL2 integrity in these DAT functions. In a previous study we demonstrated that ligand-induced protease resistance also occurred at R218 on the C-terminal side of rDAT EL2. Here using substituted cysteine accessibility analysis of human (h) DAT we confirm cocaine-induced alterations in reactivity of the homologous R219 and identify conformational sensitivity of V221. Focused molecular modeling of D174 and R218 based on currently available Aquifex aeolicus leucine transporter crystal structures places these residues within 2.9 Å of one another, suggesting their proximity as a structural basis for their similar conformational sensitivities and indicating their potential to form a salt bridge. These findings extend our understanding of DAT EL2 and its role in transport and binding functions.

Insights into the Modulation of Dopamine Transporter Function by Amphetamine, Orphenadrine, and Cocaine Binding

“…Previous structure-based studies of DAT-substrate/drug interactions used LeuT, which shares 22% sequence identity with hDAT, as template. Despite the low sequence similarity, the conservation of the fold and local packing geometry near the substrate-binding site permitted to gain insights into DAT conformational changes involved in cocaine binding and DA translocation ( 13 , 18 – 20 , 65 – 69 ). Here, for the first time, we used as template, the recently resolved dDAT structure ( 33 ), which shares >50% sequence identity with hDAT, and we modeled the EL2 loop unresolved in the fruit fly orthologs using as constraint data from previous cross-linking studies ( 16 , 47 ) (Figure 1 ).…”

Substrate and Inhibitor–Specific Conformational Changes in the Human Serotonin Transporter Revealed by Voltage-Clamp Fluorometry

“…In the absence of the substrate (apo state), Na 1 -coupled transporters of the SLC6 fold are stabilized in the outwardfacing open conformation Gaffaney et al, 2014;Kazmier et al, 2014;Malinauskaite et al, 2014;Sandtner et al, 2014). Thus, prior to 5-HT application, the cell-surface population of hSERT is dominated by this apo state, whereas during 5-HT application, the transporter population becomes engaged in transport, adopting all potential conformational states of the alternating access cycle.…”

“…In contrast, it is poorly understood how drugs affect the overall conformational behavior of SERT. Current insight into mammalian SLC6 transporter conformations stabilized by drugs is mainly based on biochemical approaches, such as cysteine accessibility or protease and alkylation protection analyses (Zhang and Rudnick, 2006;Jacobs et al, 2007;Tavoulari et al, 2009;Koldso et al, 2013;Gaffaney et al, 2014) that do not offer direct experimental information on specific intraprotein motions occurring during drug binding. Studies of protein dynamics in purified bacterial SLC6 homologs using Förster resonance energy transfer and electron paramagnetic resonance spectroscopy techniques have identified specific motions associated with alternating access (Kazmier et al, 2014;Kohut et al, 2014); however, these techniques have not been available to study mammalian transporters.…”

Dopamine Transporter Amino and Carboxyl Termini Synergistically Contribute to Substrate and Inhibitor Affinities

“…Another factor that may contribute to role of the DAT N terminus in substrate/inhibitor affinities is the interaction of the DAT N terminus with PIP 2 and intracellular loop 4 (34, 47). A recent molecular dynamics study examining the hDAT N terminus on the dDAT background reported a PIP 2 -mediated interaction with hDAT N-terminal residues (Lys 3 , Lys 5 , and Arg 51 ) with intracellular loop 4 (34). This interaction appears to break salt bridges necessary for the conserved intracellular gate.…”

“…Determining how these drugs interact with their molecular targets is essential for understanding psychostimulant addiction, as well as psychiatric treatment. The monoamine transporters DAT 2 and SERT are the primary targets for a variety of psychostimulants, such as cocaine and amphetamines (AMPH, methamphetamine, and 3,4-methylenedioxymethamphetamine), as well as the SSRI and norepinephrine-dopamine reuptake inhibitor classes of antidepressants (1)(2)(3)5). Cocaine and SSRI/norepinephrine-dopamine reuptake inhibitors inhibit these transporters by directly blocking DA and 5HT reuptake, whereas amphetamine and its congeners are competitive DAT and SERT substrates.…”