DAT presentation LECTURE
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Lecture for class on Membrane Proteins
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DAT presentation LECTURE
- 2. Dopamine Transporter Background Structure DAT Localization Dopamine synthesis Dopamine Signaling Alternate DAT substrates Na+ dependence of DAT Cl- dependence of DAT Cloning of DAT gene DAT knockout mouse Site-directed mutagenesis LeuTAa crystal structure Lipid Rafts and DAT Cholesterol Role Cholesterol Effect on Drugs
- 3. •Amino acid transporter •High homology to other neurotransmitter transporters Serotonin Transporter Glutamate Transporter Dopamine Transporter
- 5. •Neurotransmitter •Produced in the substantia niagra •Locomotion •Enhanced Signaling Reward Cognition Dopamine
- 6. Dopamine Signaling VMAT loads dopamine into vesicles
- 7. Dopamine Signaling Vesicle fusion results in dopamine release into the synapse Dopamine binds receptors Signal
- 8. DAT Terminates signaling by uptake of dopamine Dopamine Transporter removes dopamine from synapse Signal Signal Terminated
- 9. DAT: Alternate Substrates Cocaine MPPAmphetamine
- 10. Cocaine blocks reuptake of dopamine Signal Leads to prolonged signaling
- 11. (Loland 2001) Dopamine uptake Blocked by cocaine In COS-7 cells Cocaine blocks reuptake of dopamine
- 12. DAT: Transports other substrates Cocaine Amphetamine MPP AWESOME AWESOME NOT AWESOME
- 13. Amphetamines reduce surface DAT and increase dopamine release Signal Elevates dopamine signaling Blocks VMAT Reverse Transport of Dopamine through DAT
- 14. Amphetamines requires DAT for dopamine release (Jones 1998) Amphetamine treated Wildtype DAT Knockout DAT-/- Dopamine release due to amphetamine treatment is DAT dependent
- 15. Signal Leads to prolonged signaling Amphetamines reduces DAT surface expression
- 16. Amphetamines reduce DAT surface expression (Saunders 2000) Flourescently labeled DAT localizes to the cell membrane When treated with amphetamines Becomes internalized
- 17. DAT: Transports other substrates Cocaine MPPAmphetamine
- 18. Parkinson’s Disease •Involuntary movement •Distinctive gait •Loss of dopaminergic neurons •Chronic and progressive
- 19. Parkinson’s Disease •High incidence of PD in rural areas •MPTP used as an herbicide •Selectively transported into DA neurons via DAT
- 20. MPTP leads to selective dopaminergic cell death Signal Converted to MPP+ via MAO-B Interferes with complex I Cell death Loss of dopaminergic signaling
- 21. MPTP Requires DAT for Toxicity • Markers of DA neurons • Lost due to MPP exposure • Protected in DAT-/- (Gainetdenov 2002)
- 22. DAT: Transports other substrates Cocaine MPP AWESOME AWESOME NOT AWESOME Resulting in permanent damage Sometimes when making this You can actually get that Amphetamine
- 23. Na+/Cl dependent neurotransmitter transporter Dopamine Transporter How does DAT transport DA against its own gradient?
- 24. •P-Type ATPase (Plasma membrane) •Na+-K+ ATPase •Uses ATP Na+-K+ ATPase generates ion gradient ATP •3 Na+ OUT •2 K+ IN • Generates ion gradient ADP LOW Na+
- 25. Na+/Cl- Cotransporter Transports 2 Na+ 1Cl- 1 Dopamine Uses ion gradient LOW Na+ DOWN Na+ gradient
- 26. Dopamine Uptake is Cl- dependent •Rabbit striatial slices •Radioactive dopamine •Measuring dopamine uptake (Dilberto 1988)
- 27. Dopamine Uptake is Cl- dependent
- 28. Dopamine Uptake is Na+ dependent •Sprague Dawley rats •Isolated Striatum •Isolate synaptosomes •9.5mM-133.2 mM NaCl (Wheeler 1993)
- 29. Dopamine Uptake is sodium dependent Increasing Na+ Leads to faster DA uptake INCREASING Na+ CONCENTRATION
- 30. Dopamine Transporter Gene cloned • Use a cDNA library • Screen rat mRNAs • Use Xenopus Laevis oocytes (Shimada 1991 Science)
- 31. Finding DAT Gene: cDNA library screen Start with RNA from tissue of interest Make cDNA using poly AA primers cDNA
- 32. ampR ampRampR Finding DAT Gene: cDNA library screen Ligate into plasmid put into bacteria then grow on selection.
- 33. Finding DAT Gene: cDNA library screen •Library of each expressed RNA •Can generate RNA in vitro from each clone •Test independently
- 34. YourFavGT7 +IPTG T7Pol In E. coli Finding DAT Gene: cDNA library screen ampR Induce RNA expresion of single clone Purify RNA
- 35. Finding DAT Gene: cDNA library screen Xenopus Laevis Xenopus oocytes Large cell Durable Commonly used for patch clamp studies
- 36. Finding DAT Gene: cDNA library screen Inject mRNA into xenopus oocytes Translational machinery create protein
- 37. Finding DAT Gene: cDNA library screen Incubate with dopamine
- 38. Finding DAT Gene: cDNA library screen If expressed clone is dopamine transporter Radiolabeled dopamine will enter oocyte Oocytes washed and homogenized Radioactivity measured
- 39. DAT cDNA behaves like DAT Cocaine blocks uptake Uptake is Na+ dependent RNA results in DA uptake
- 40. Finding DAT Gene: cDNA library screen •Go back to bacterial plates •PCR insert •Sequence insert ampR DAT cDNA
- 41. GAT hydrophobicity plot 1 2 3 4 5 6 7 8 9 10 11 12 Indicates 12 Transmembrane domains
- 42. Dopamine Transporter Human Gene cloned • 1992 used rat sequence to do hybridization on human chromosomes to find human gene
- 44. DAT knockout mouse • Bound/Free uptake of labeled DA • Mouse striatial synaptosomes • DAT-/- • DAT-/+ • DAT+/+ Knockout unable to transport dopamine DAT-/- DAT-/+ DAT+/+
- 45. Decreased survival of DAT knockout mouse • DAT -/+ normal survival • DAT -/- poor survival • Most die by 10 weeks DAT-/- DAT-/+ DAT+/+
- 46. DAT knockout mouse DAT-/- DAT+/+ DAT-/+ Knockout mice are hyperactive
- 47. DAT -/- resistant to cocaine and amphetamine Homozygouse does not increase movement Heterozygous reduced increase in movement
- 48. Crystal Structure of LeuTAa • Bacterial orthologue of DAT • Aquifex aeolicus • Crystalized in complex with Leucine Two sodium ions
- 49. • PCR cloned genomic LeuTAa • Overexpressed protein in bacteria IPTG inducible • His-tag translational fusion with thrombin site T7 LeuTAa His Thrombin Site Crystal Structure of LeuTAa
- 50. • C41 cells express toxic proteins • Include seleno-methionine in media • Label protein with seleno-methionine heavy atom helps solve phase problem Crystal Structure of LeuTAa
- 51. • Isolated cell membranes • Solubilized in 40mM dodecylmaltoside • Passed over ion affinity chromatography column • Remove His-tag via thrombin digestion • Size exclusion column with 40mM o-noctyl-B- glucopyranoside (B-OG) • Dialyzed O/N with low salt and same B-OG Crystal Structure of LeuTAa
- 52. Crystal Structure of LeuTAa Hang Drop Vapour Diffusion SealedCrystals Remain •Concentratedprotein on slide •Flip upside down •Sealed container •Above reservoir •As it dries •Leaves precipitate to form crysta
- 53. Crystal Structure of LeuTAa X-ray Diffraction •PEG 550 cryoprotection •Liquid nitrogen flash freeze •Pass X-ray laser through cyrsta •Can rotate •Collect refraction data
- 54. Crystal Structure of LeuTAa Multiwavelength Anomalous Dispersion •Intensity of spot is measured •Gives wavelength amplitude •Not phase •Both are needed •Solution:MAD
- 55. Crystal Structure of LeuTAa Multiwavelength Anomalous Dispersion •Heavy atoms such as Selenium •Electrons absorb certain wavelengths •Re-emit after a delay •Causes a phase shift •Can back calculate to get initial phase
- 56. Crystal Structure of LeuTAa Stereogram
- 57. Protein Sequence of NSST
- 58. Structure of Dopamine Transporter
- 59. Two Main Binding Pockets
- 60. Identifying Amino Acids necessary for DA Transport Site-directed mutagenesis Mutate specific AA Test DA uptake in cell culture Asp 79 Misense Mutants WT Suggests Asp79 binds dopamine Asp79 mutants significantly lower DA uptake
- 62. Identifying Cl- interacting amino acids Neurotransmitter symporters all Cl- dependent Bacteria orthologues Cl- independent Identify amino acids that are shared in neurotransmitter transporters different in bacterial transporters Site directed mutagenesis Screen for Cl- independence
- 63. Cl- Binding Site
- 64. Na+ Binding Site 1
- 65. Na+ Binding Site 2
- 66. Localization of DAT • Lipid Rafts • Lateral Diffusion • Role of cholesterol • Restricted Mobility • Important for signaling
- 67. Measuring Lateral Mobility FRAP • Fluorescence recovery after photobleaching • Fluorescently tagged membrane protein
- 68. Measuring Lateral Mobility FRAP • Fluorescence recovery after photobleaching • Fluorescently tagged membrane protein • Photobleach a small area • Causes loss of flourescence
- 69. Measuring Lateral Mobility FRAP • Fluorescence recovery after photobleaching • Fluorescently tagged membrane protein • Photobleach a small area • Causes loss of flourescence • If the protein can move freely Flourescence recovers
- 70. Measuring Lateral Mobility FRAP • Fluorescence recovery after photobleaching • Fluorescently tagged membrane protein • Photobleach a small area • Causes loss of flourescence • If the protein movement is restricted Photobleaching remains
- 71. DAT does not recover photobleach when in neuronal cells Suggests immobile in this cell type (Adkins 2007)
- 72. Immobility of DAT is cholesterol dependent • mBCD: causes cholesterol depletion • Cytochalasin D interferes with actin DAT anchored by cholesterol and cytoskeleton
- 73. DAT in both membrane and non- membrane rafts Present in both raft and non-raft fractions • Na/K ATPase Non-raft marker • GM1 Raft marker
- 74. Cholesterol necessary for dopamine uptake (Adkins 2007)
- 75. Cholesterol modulates conformation of DAT • Synaptosomes • Transfected HEK cells • Added cholesterol solubilized in MBCD (Hong 2010)
- 76. Cholesterol enhances cocaine analogue binding (Hong 2010) Radiolabelled cocaine analogue No effect of MBCD Cholesterol increases binding
- 77. Cholesterol does not alter DAT levels (Hong 2010) • Overall expression NOT altered • Surface expression NOT altered
- 78. Cholesterol increases surface availability of Cys (Hong 2010) • Maleimide-PEO2 biotin • Membrane impermeable • Biotinylate outside residues • High levels of cholesterol Increased biotinylation
- 79. Cholesterol increases surface binding of DAT
- 80. Treatment with wsCholesterol decreases DA uptake Increased wsCholesterol leads to decreased DA uptake Conflicts with previous report No change due to MGCD
- 81. wsCholesterol and cocaine increase DAT surface Cys availability
- 82. Critiques: How do we know cholesterol getting into membrane • Adding water soluble cholesterol Cholesterol MBCD • Used only HEK cell line • Did not then check membrane fractions for higher cholesterol content • Other paper suggests depletion …..but they didn’t check either Interpretation may be backward
- 83. Say no to drugs Say yes to cheeseburgers