University of North Dakota UND Scholarly Commons Theses and Dissertations Theses, Dissertations, and Senior Projects January 2015 Understanding The trS ucture-Function Relationships Between Monoamine Neurotransmitter Transporters And Their ogC nate Ions And Ligands Bruce Felts Follow this and additional works at: https://commons.und.edu/theses Recommended Citation Felts, Bruce, "Understanding The trS ucture-Function Relationships Between Monoamine Neurotransmitter Transporters And Their Cognate Ions And Ligands" (2015). Theses and Dissertations. 1769. https://commons.und.edu/theses/1769 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]. UNDERSTANDING THE STRUCTURE-FUNCTION RELATIONSHIPS BETWEEN MONOAMINE NEUROTRANSMITTER TRANSPORTERS AND THEIR COGNATE IONS AND LIGANDS by Bruce F. Felts Bachelor of Science, University of Minnesota 2009 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 August 2015 Copyright 2015 Bruce Felts ii TABLE OF CONTENTS LIST OF FIGURES………………………………………………………………………... xii LIST OF TABLES……………………….………………………………………………… xv ACKNOWLEDGMENTS.…………………………………………………………….… xvi ABSTRACT.………………………………………………………………………….……. xviii CHAPTERS I. INTRODUCTION.………………………………………………………………… 1 The Solute Carrier Super-family of Proteins………………………………. 1 The Neurophysiologic Role of MATs……………………………………... 2 Monoamine Transporter Structure…………………………………………. 5 The Substrate Binding Pocket……………………………………… 10 The S1 binding site in LeuT………………………………... 11 The S1 binding site in MATs………………………………. 13 The S2 binding site in the extracellular vestibule………….. 14 Ion Binding Sites in MATs………………………………………… 17 The Na+ binding sites………………………………………. 17 The Cl– binding site………………………………………... 20 Chemiosmotic Coupling and the Translocation Mechanism………………. 22 The Alternating Access Mechanism of Substrate Transport………. 22 Kinetic and Electrogenic Properties of MATs……………………... 26 v Pharmacological Intervention and MATs…………………...……………... 28 II. A CONSERVED ASPARAGINE RESIDUE IN TRANSMEMBRANE SEGMENT ONE OF THE SEROTONIN TRANSPORTER DICTATES CHLORIDE-COUPLED NEUROTRANSMITTER TRANSPORT……………… 32 Introduction………………………………………………………………… 32 Methodology……………………………………………………………….. 34 Site-directed Mutagenesis and Construction of Mutant Plasmids…. 34 5-HT and NE Transport Measurements……………………………. 34 Spontaneous 5-HT Efflux………………………………………….. 36 Total and Cell Surface Expression Protein Analysis………………. 36 Evaluation of Cysteine Accessibility………………………………. 37 hSERT Expression in Xenopus laevis Oocytes……………………. 38 Simultaneous Measurement of 5-HT Uptake and 5-HT-induced Currents…………………………………………………………….. 39 hSERT Molecular Modeling……………………………………….. 39 Molecular Dynamics and Free Energy Perturbation (FEP) Simulations………………………………………………………… 41 Results……………………………………………………………………… 42 hSERT N101 Mutation Eliminates Cl− Dependence of 5-HT Uptake……………………………………………………………… 42 N101 Dictates Cl−-Dependent Conformational Changes in TM1 and EL4…………………………………………………………….. 44 hSERT N101 Dictates Ion Selectivity of 5-HT-independent Charge Flux………………………………………………………………… 48 N101 Dictates Cl− Dependence of 5-HT-induced Currents………... 52 vi N101 Dictates hSERT Coupling and Stoichiometry………………. 54 Molecular Modeling Suggests a Mechanism for N101 Participation in Ion-Coupled 5-HT Transport……………………………………. 56 Effect of Cl− and Different Mutations on Ion and Solute Binding to the Transporter……………………………………………………... 60 Validation of the Partnership Between N101 and S336 in Cl−- Dependent 5-HT Transport………………………………………… 61 Discussion………………………………………………………………….. 67 III. THE TWO SODIUM SITES IN THE SEROTONIN TRANSPORTER PLAY DISTINCT ROLES IN THE ION COUPLING AND ELECTROGENICITY OF TRANSPORT……………………………………………………………………… 74 Introduction………………………………………………………………… 74 Methodology……………………………………………………………….. 77 Site-Directed Mutagenesis…………………………………………. 77 5-HT Uptake Analysis……………………………………………... 77 Protein Expression Analysis……………………………………….. 78 Cysteine Accessibility Analysis……………………………………. 79 Two-Electrode Voltage Clamp Analysis…………………………... 79 cRNA preparation………………………………………….. 79 Oocyte preparation…………………………………………. 79 Electrophysiological recordings in X. laevis oocytes……… 80 Whole Cell Patch Clamp…………………………………………… 80 Molecular Dynamic (MD) Simulations of hSERT·5-HT·Ion Complexes………………………………………………………….. 81 Results……………………………………………………………………… 84 vii N101 Mutation Specifically Modifies Cation Dependence, Allowing Ca2+ to Functionally Replace Na+ for 5-HT Transport….. 84 Na+, but Not Ca2+, Imparts Conformational Changes in Native hSERT, whereas Both Na+ and Ca2+ Can Promote 5-HT-Induced Conformational Changes in the N101A Mutant…………………… 87 hSERT N101 Mutants Display a Loss of Potency for Na+ to Drive 5-HT Transport…………………………………………………….. 89 Mutation at N101 Diminishes the Ability of the Transporter to Concentrate 5-HT…………………………………………………... 92 Ca2+ Decreases the Apparent Affinity of 5-HT in both hSERT and the N101 Mutants…………………………………………………... 92 Mutations at Na2 Site Fail to Alter Cation Selectivity…………….. 93 The Na1 Site Does Not Contribute to the Substrate-induced or Leak Currents in hSERT…………………………………………… 96 Whole Cell Clamp of the N101A Mutant in HEK293 Cells Suggests That Cl− Is the Main Charge Carrier When External Na+ Is Substituted by Ca2+…………………………………………. 100 hSERT N101 Mutants Appear to Function as both Active and Passive Transporters……………………………………………….. 101 Molecular Dynamics Simulations Suggest a Mechanism for Ca2+ Gain-of-Function Phenotype…………………………………. 103 Discussion………………………………………………………………….. 109 IV. ANTAGONIST-INDUCED CONFORMATIONAL CHANGES IN DOPAMINE TRANSPORTER EXTRACELLULAR LOOP TWO INVOLVE RESIDUES IN A POTENTIAL SALT BRIDGE…………………………………………………... 118 Introduction………………………………………………………………… 118 Methodology……………………………………………………………….. 122 Tissue Preparation and Proteolysis………………………………… 122 viii Immunoblot Analysis………………………………………………. 123 Quantification of DAT Proteolysis………………………………… 123 Deglycosylation Analysis………………………………………….. 124 Asp-N Activity Assay……………………………………………… 124 [3H]CFT Binding and [3H]DA Uptake…………………………….. 124 SCAM Analysis……………………………………………………. 125 Molecular Modeling………………………………………………... 127 Results……………………………………………………………………… 128 Endoproteinase Asp-N Digestion of rDAT………………………… 128 Uptake Blockers Reduce Asp-N Proteolysis………………………. 130 Asp-N Treatment Disrupts DAT Function………………………… 132 SCAM Analysis of EL2 Residues…………………………………. 134 Molecular Modeling………………………………………………... 140 Discussion………………………………………………………………….. 143 V. THE FORMATION OF A PUTATIVE SALT BRIDGE AT THE EXTERNAL GATE OF THE SEROTONIN TRANSPORTER IS IMPORTANT FOR AMPHETAMINE TRANSLOCATION AND THE INDUCTION OF SUBSTRATE EFFLUX…………………………………………………………… 148 Introduction………………………………………………………………… 148 Methodology……………………………………………………………….. 151 Site-Directed Mutagenesis…………………………………………. 151 [3H]5-HT Uptake Assay……………………………………………. 151 Surface Expression Analysis via Surface Biotinylation…………… 153 ix Whole-Cell Radioligand Binding Assay…………………………… 153 Two-electrode Voltage Clamping of Xenopus Oocytes…………... 153 cRNA preparation………………………………………….. 153 Oocyte preparation…………………………………………. 154 Electrophysiological recordings in X. laevis oocytes……… 154 Results……………………………………………………………………… 155 Disruption of hSERT External Gate Abolishes Transport…………. 155 Species-Scanning Mutagenesis of the External Gate Yields Little Effect on 5-HT Transport or Protein Trafficking…..…………….... 155 The Acidic TM10 Gating Residue is Important for the Ability of the Transporter to Mediate MDMA-Induced Efflux………………. 158 Mutation at the Outer Gate does not Significantly Alter MDMA Potency……………………………………………………………... 161 The Generation of MDMA-Induced Current by SERT is Dependent on the Presence of the Acidic TM10 Gating Residue…. 164 Discussion………………………………………………………………….. 168 VI. NOVEL AZIDO-IODO PHOTOAFFINITY LIGANDS FOR THE SEROTONIN TRANSPORTER BASED ON THE SELECTIVE SEROTONIN REUPTAKE INHIBITOR (S)-CITALOPRAM………………………………………………….. 177 Introduction………………………………………………………………… 177 Methodology……………………………………………………………….. 182 Whole-cell Competition Uptake Assay……………………………. 182 hSERT Photoaffinity Labeling…………………………………….. 182 Results……………………………………………………………………… 183 Pharmacological Properties of NYDU-2-24, VK-03-51 and VK- 03-83……………………………………………………………….. 183 x Photoaffinity Labeling Experiments with hSERT…………………. 184 Discussion………………………………………………………………….. 189 REFERENCES…………………………………………………………………….. 192 xi LIST OF FIGURES Figure Page(s) 1. Role of MATs in Synaptic Transmission…………………………………………. 3 2. Basic structure of a MAT…………………………………………………………. 7 3. X-ray crystal structure and topology of the SLC6 bacterial homologue, LeuT…... 8 4. The S1 binding site of LeuT, dDAT and hSERT…………………………………. 12 5. The location of the S1 and S2 binding sites in LeuT……………………………… 15 6. The ion binding sites in LeuT, dDAT and hSERT………………………………... 19 7. The alternating access model of transport………………………………………… 23 8. Three distinct structural conformations of LeuT provide a general outline of the alternating access mechanism of transport………………………………………... 25 9. Chemical structures of classic MAT substrates and inhibitors………………...….. 29 10. Kinetic analysis and Cl– dependence of 5-HT uptake in hSERT N101