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The Dopamine Transporter (DAT) is responsible for re-uptake of dopamine from the synaptic cleft

Oligomerization of the Dopamine Transporter: cocaine-analog-induced conformational changes at a homodimer interface Jonathan A. Javitch, MD,PhD Columbia University. The Dopamine Transporter (DAT) is responsible for re-uptake of dopamine from the synaptic cleft. Amph.

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The Dopamine Transporter (DAT) is responsible for re-uptake of dopamine from the synaptic cleft

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  1. Oligomerization of the Dopamine Transporter: cocaine-analog-induced conformational changes at a homodimer interfaceJonathan A. Javitch, MD,PhDColumbia University

  2. The Dopamine Transporter (DAT) is responsible for re-uptake of dopamine from the synaptic cleft Amph

  3. Although the inhibition of DAT by cocaine and amphetamine is widely documented, the structural basis of DA transport and its inhibition by cocaine and other psychostimulants such as amphetamine is poorly understood. • Accessibility of endogenous cysteines and DAT topology • Conformational changes associated with substrate transport and inhibitor binding to DAT • Oligomerization of DAT • Trafficking of DAT induced by amphetamine, cocaine, and manipulation of signal transduction pathways • Mechanism of amphetamine-mediated DA efflux • Bacterial and archaeal sodium-dependent transporters as model systems for structural studies

  4. Oligomerization of neurotransmitter transporters?? • (The horror! The horror!) • Serotonin transporter: • Co-IP • effects of cysteine modification • FRET • GABA transporter • FRET • Glycine transporter:dimer intracellular and not on surface • DAT: • Radiation inactivation consistent with dimer and/or tetramer • FRET

  5. Flag-HA-synDAT

  6. Cross-linking of Flag-HA-DAT

  7. DAT runs as a broad band ~85k Da DAT is cross-linked by copper phenanthroline to a broad band ~195k Da DAT is cross-linked by bis-MTSEA to a broad band ~195k Da Cross-linking is reversed by reduction with DTT Are we cross-linking a DAT homodimer or a heterodimer between DAT and another protein?

  8. Myc-His-DAT

  9. Coimmunoprecipitation of Flag-HA-DAT and Myc-His-DAT

  10. DAT is cross-linked in mouse striatal membranes

  11. Which cysteine is responsible? MTSET blocks cross-linking so…

  12. Cys306 is necessary for DAT cross-linking

  13. Flag-HA-CD-DAT

  14. Cys306 is sufficient for DAT cross-linking

  15. The G(V/I)XXG(V/I)XX(A/T) motif is conserved in TM6 of neurotransmitter transporters

  16. TM6 Deviation from helical periodicity GVXXGVXXA/T Dimerization motif

  17. Mutation of Gly323 and Gly327 abolished uptake

  18. Summary of Dimerization • DAT in the plasma membrane can be cross-linked into a dimer by bis-EA or CuP. • Cys-306 at the extracellular end of TM6 is necessary and sufficient for cross-linking. • DAT is a symmetrical dimer (at least). • Cross-linking does not alter uptake or binding. • The GVXXGCXXA motif is presumably involved in dimerization of TM6. • “Only” 24 TMs to sort out…. • But wait!

  19. DAT is a tetramer in the membrane: a second symmetrical interface in TM4 is crosslinked by Cu++ or HgCl2

  20. Cocaine analogs block crosslinking of the TM4 interface but not the TM6 interface – conformational change

  21. Targeting and trafficking of the dopamine transporter Activation of PKC leads to acute ’downregulation’ of DAT

  22. DAT internalization upon direct PKC activation and upon activation of a co-expressed SP receptor Control 200 nM SP 1 mM PMA HEK-293 cells co-expressing EGFP-hDAT and the substance P receptor (NK-1)

  23. The hDAT contains multiple putative Ser/Thr phosphorylation sites PKC consensus sites Non-consensus sites Other putative internalization motifs

  24. No effect of mutating multiple serines and threonines in different combinations Surface biotinylation Uptake

  25. Direct protein kinase C activation and activation of a co-expressed SP receptor increase phosphorylation of DAT Phosphorylation 0 SP PMA 0 SP PMA 0 SP PMA 105 kD Control FLAG-hDAT FLAG-HA-hDAT- D1-22 Western blot

  26. Role of phosphorylation? Phosphorylation of a non-DAT substrate mediates internalization

  27. Properties of N-terminal truncation mutant • Internalization is normal in response to PMA • Internalization is normal in response to SP • Surface expression is normal/enhanced • Km of dopamine and tyramine are normal • Vmax/surface DAT is normal • Oligomerization is normal

  28. Acknowledgements Shonit Das – bacterial transporters Yvette Dehnes – DAT IL3 Jasmine Ferrer – DAT - the beginning Naomi Goldberg - bacterial transporters Wen Guo – receptor Hanne Hastrup – DAT X-linking George Liapakis – receptor Matthias Quick - bacterial transporters Namita Sen – DAT regulation Lei Shi - receptor Merrill Simpson - receptor Mark Sonders - DAT Arthur Karlin NIDA Charlotta Grånäs Claus Juul Loland Ulrik Gether Habibeh Khoshbouei Aurelio Galli Bipasha Guptaroy L'Aurelle Johnson David Lund, Margaret E. Gnegy Mu Fa Zhou Amy Newman

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