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Characterization of Dopamine Transporter Amino Terminal Palmitoylation and Neurotoxic Substrate Activity

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Characterization of Dopamine Transporter Amino Terminal Palmitoylation and Neurotoxic Substrate Activity University of North Dakota UND Scholarly Commons Theses and Dissertations Theses, Dissertations, and Senior Projects January 2019 Characterization Of Dopamine Transporter Amino Terminal Palmitoylation And Neurotoxic Substrate Activity Daniel Jacob Stanislowski Follow this and additional works at: https://commons.und.edu/theses Recommended Citation Stanislowski, Daniel Jacob, "Characterization Of Dopamine Transporter Amino Terminal Palmitoylation And Neurotoxic Substrate Activity" (2019). Theses and Dissertations. 2867. https://commons.und.edu/theses/2867 This Dissertation is brought to you for free and open access by the Theses, Dissertations, and Senior Projects at UND Scholarly Commons. It has been accepted for inclusion in Theses and Dissertations by an authorized administrator of UND Scholarly Commons. For more information, please contact [email protected]. CHARACTERIZATION OF DOPAMINE TRANSPORTER AMINO TERMINAL PALMITOYLATION AND NEUROTOXIC SUBSTRATE ACTIVITY by Daniel Jacob Stanislowski Bachelor of Science, University of North Dakota, 2012 A Dissertation Submitted to the Graduate Faculty of the University of North Dakota in partial fulfillment of the requirements for the degree of Doctor of Philosophy Grand Forks, North Dakota December 2019 Copyright 2019 Daniel J. Stanislowski ii PERMISSION Title: Characterization of dopamine transporter amino terminal palmitoylation and neurotoxic substrate activity Department: Biochemistry and Molecular Biology Degree: Doctor of Philosophy In presenting this dissertation in partial fulfillment of the requirements for a graduate degree from the University of North Dakota, I agree that the library of this University shall make it freely available for inspection. I further agree that permission for extensive copying for scholarly purposes may be granted by the professor who supervised my dissertation work or, in his absence, by the Chairperson of the department or the dean of the School of Graduate Studies. It is understood that any copying or publication or other use of this dissertation or part thereof for financial gain shall not be allowed without my written permission. It is also understood that due recognition shall be given to me and to the University of North Dakota in any scholarly use which may be made of any material in my dissertation. Daniel J. Stanislowski December, 2019 iv TABLE OF CONTENTS LIST OF FIGURES ............................................................................................... ix LIST OF TABLES ............................................................................................... xiii ACKNOWLEDGEMENTS ................................................................................... xiv ABSTRACT ...................................................................................................... xviii CHAPTER 1. INTRODUCTION ............................................................................. 1 Dopamine ............................................................................. 1 Preface – The Modern Soma ..................................... 1 Pavlov, Skinner, Learning, Behavior, and…Advertising? ..................................................... 5 Synthesis, Exocytotic Release, and Receptors ....... 12 The Dopamine Transporter ................................................ 17 Structure and Mechanism of DA Transport .............. 17 Regulation ............................................................... 24 DAT-Mediated DA Efflux .......................................... 32 DAT Disease Association ........................................ 32 S-Palmitoylation ................................................................. 36 Overview .................................................................. 36 The Acyl-Biotinyl Exchange ..................................... 41 DAT Palmitoylation .................................................. 43 2. DAT N-TERMINAL PALMITOYLATION AND THIS MODIFICATION’S EFFECT ON DOPAMINE EFFLUX ................. 47 v Abstract .............................................................................. 47 Introduction ......................................................................... 48 Results ............................................................................... 52 Acyl-Biotinyl Exchange Analysis of Cys to Ala Point Mutant Palmitoylation .............................................. 52 Palmitoylation of Single Intracellular Cys rDATs ...... 53 Palmitoylation of DATs with or without the Putative Palmitoylation Sites ................................................. 56 In Vivo Palmitoylation of the rDAT N-terminal Peptide .................................................................... 56 Cyanogen Bromide (CNBr) Digestion and ABE Analysis of the rDAT N-terminus ............................. 58 CNBr Proteolytic Mapping of Cys to Ala rDATs Expressed in LLCPK1 Cells ..................................... 62 Analysis of Cys90 Palmitoylation ............................. 65 Surface Expression Analysis of Palmitoylation- Deficient rDATs........................................................ 65 DA Uptake Saturation Kinetics of Palmitoylation Deficient rDATs........................................................ 69 Global Palmitoylation Inhibition with 2BP Effect on DA Uptake ..................................................................... 75 Palmitoylation Deficient rDATs Display Altered DA efflux ........................................................................ 77 Global Palmitoylation Inhibition by 2BP Effect on DA Efflux ........................................................................ 81 Discussion .......................................................................... 82 Methods.............................................................................. 98 Cell Culture and Site Directed Mutagenesis ............ 98 Transfection ............................................................. 98 SDS-PAGE and Western Blotting ............................ 99 Plasma Membrane Preparation ............................. 100 Synaptosome Preparation ..................................... 100 Asp-N Endoproteinase Proteolysis ........................ 101 vi CNBr Chemical Proteolysis ................................... 101 Acyl-Biotinyl Exchange .......................................... 102 2BP DA Uptake Assay……………………………….104 DA Efflux Assay ..................................................... 104 DA Uptake Saturation Analysis .............................. 105 Surface Biotinylation .............................................. 105 3. THE EFFECTS OF THE PARKINSON-INDUCING NEUROTOXINS MPP+ AND 6-OHDA ON THE DOPAMINE TRANSPORTER’S MODIFICATION AND FUNCTION .............................................. 107 Abstract ........................................................................... 107 Introduction ....................................................................... 109 Results ............................................................................. 114 6-OHDA and MPP+ Competitively Inhibit DAT- mediated DA Uptake .............................................. 114 6-OHDA and MPP+ Induce Downregulation of DAT Activity ................................................................... 116 Incubation of 6-OHDA and MPP+ on DAT-Expressing Cells Affects DA Uptake and Requires Functional DATs ...................................................................... 118 Neurotoxin Effects on DAT-Mediated Reverse DA Transport ............................................................... 120 Inhibiting PKC Reduces Neurotoxin-Induced DA Efflux ...................................................................... 123 Neurotoxin Effect on DAT Phosphorylation ........... 129 Neurotoxin Effect on DAT Palmitoylation ............... 132 Discussion ........................................................................ 134 Methods............................................................................ 139 Cell Culture ............................................................ 139 Generation of DAT-Expressing LLCPK1 Cells ....... 139 SDS-PAGE and Western Blotting .......................... 140 Neurotoxin Dose-Response Assay on Inhibition of DA Uptake ................................................................... 141 DA Uptake Downregulation Assay ......................... 141 vii Neurotoxin Pre-incubation DA Uptake Assay ........ 142 Neurotoxin DA Efflux Assay ................................... 143 Dopamine Transporter Phosphorylation Assay ..... 143 Plasma Membrane Preparation ............................. 145 Acyl-Biotinyl Exchange .......................................... 145 4. DISCUSSION .............................................................................. 148 Limitations of the Work Presented in this Dissertation ..... 148 Future Studies .................................................................. 149 Summary Conclusions...................................................... 150 5. REFERENCES............................................................................ 152 viii LIST OF FIGURES Chapter 1: Figure Page 1. Pavlovian and Skinnerian conditioning compared ..................................... 6 2. Major dopamine pathways in the brain ..................................................... .8 3. Firing of DA neurons encodes reward prediction error .............................. 9 4. How does this sell bacon cheeseburgers?!? ........................................... 11 5. Synthesis and release of DA ...................................................................
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