Dataset Information

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 19kDa 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 Zn(2+), 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 2.9Å 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.

SUBMITTER: Gaffaney JD

PROVIDER: S-EPMC4394652 | biostudies-literature | 2014 Jul

REPOSITORIES: biostudies-literature

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

Gaffaney Jon D JD Shetty Madhur M Felts Bruce B Pramod Akula-Bala AB Foster James D JD Henry L Keith LK Vaughan Roxanne A RA

Neurochemistry international 20131120

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 19kDa 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 ...[more]

PMID: 24269640

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Project description:The dopamine transporter (DAT), a member of the neurotransmitter:sodium symporter family, mediates the reuptake of dopamine at the synaptic cleft. DAT is the primary target for psychostimulants such as cocaine and amphetamine. We previously demonstrated that cocaine binding and dopamine transport alter the accessibility of Cys342 in the third intracellular loop (IL3). To study the conformational changes associated with the functional mechanism of the transporter, we made cysteine substitution mutants, one at a time, from Phe332 to Ser351 in IL3 of the background DAT construct, X7C, in which 7 endogenous cysteines were mutated. The accessibility of the 20 engineered cysteines to polar charged sulfhydryl reagents was studied in the absence and presence of cocaine or dopamine. Of the 11 positions that reacted with methanethiosulfonate ethyl ammonium, as evidenced by inhibition of ligand binding, 5 were protected against this inhibition by cocaine and dopamine (S333C, S334C, N336C, M342C and T349C), indicating that reagent accessibility is affected by conformational changes associated with inhibitor and substrate binding. In some of the cysteine mutants, transport activity is disrupted, but can be rescued by the presence of zinc, most likely because the distribution between inward- and outward-facing conformations is restored by zinc binding. The experimental data were interpreted in the context of molecular models of DAT in both the inward- and outward-facing conformations. Differences in the solvent accessible surface area for individual IL3 residues calculated for these states correlate well with the experimental accessibility data, and suggest that protection by ligand binding results from the stabilization of the outward-facing configuration. Changes in the residue interaction networks observed from the molecular dynamics simulations also revealed the critical roles of several positions during the conformational transitions. We conclude that the IL3 region of DAT undergoes significant conformational changes in transitions necessary for both cocaine binding and substrate transport.

Dataset Information

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

Publications

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

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