SCREENING OF FUNCTIONAL NOREPINEPHRINE TRANSPORTER INSENSITIVE TO COCAINE INHIBITION AND GENERATION OF KNOCK-IN MOUSE DISSERTATION Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of the Ohio State University By Hua Wei, M.S. ***** The Ohio State University 2009 Dissertation Committee: Approved by Howard Haogang Gu, PhD, Advisor ___________________________ Lane Jackson Wallace, PhD Advisor Michael Xi Zhu, PhD Biochemistry Graduate Program James Willam Dewille, PhD ABSTRACT This dissertation consists of three parts. The first part explores the possibility of screening a functional but cocaine insensitive norepinephrine transporter and generation of a cocaine insensitive knock-in mouse line, the second part attempts to identify residues in mouse norepinephrine transporter (NET) transmembrane domain 3 (TMD3) that would differentiate norepinephrine and dopamine uptake, while the third part discusses that two extracellular cysteines in dopamine transporter form a disulfide bond, which is vital for the transporter’s function. Cocaine blocks dopamine transporter (DAT), NET and serotonin transporters (SERT) in the brain and increases extracellular neurotransmitter concentration in various brain regions. It is not known how each of these contributes to complex cocaine addictive effects. Genetically modified mice with a single one of these transporter removed still prefer cocaine, suggesting that none of them is absolutely required for cocaine rewarding effect. We have generated one unique knock-in mouse line, carrying a cocaine insensitive DAT. This mouse line does not show cocaine preference when given cocaine, which showed that DAT is necessary for cocaine rewarding effects. However, how NET is involved in cocaine addictive effect is still unknown. Based on the previous study, I performed several round of random mutagenesis around ii residuesF105 and F150 in transmembrane regions 2 and 3 region. One triple mutation (F101C-A105G-N153T, mNETCGT) that retains wild type uptake activity for substrates but displays decreased cocaine affinity by 37 fold was found. Interestingly, this mutation also decreases desipramine affinity by 24 fold. These results reveal a number of residues in transmembrane regions 2 and 3 that are important for inhibition by different drugs. To study the exact role of NET involved in cocaine addiction, a knock-in mouse line carrying the above functional and cocaine insensitive NET was generated by replacing wild type NET with mNETCGT. Feeder cell free ES cells (E14Tg2A) were introduced to generate a unique mouse line. This mouse line will be used to study the role of NET in mediating the addictive action of cocaine and the therapeutic effect of desipramine. Recent research revealed that NET also plays an important role in the regulation of DA homeostasis. Hence, delineating residues of NET differentially involved in DA and NE uptake would provide potential intervention for treatment of drug abuse, depression or other related psychiatric disorders. Our study identified for the first time residues in the TM3 region of mNET that are more critical for NE uptake than DA. Dopaminergic neurotransmission is terminated by removal of extracellular dopamine via DAT, which belongs to a Na+/Cl- dependant neurotransmitter superfamily. Information about the structure and function relationship of transporters allows us to better understand the molecular mechanism of these transporters and therapeutic drug iii design. Cysteine residues in transporters form inter- or intra- molecular disulfide bonds which may be critical for proper protein folding, trafficking, surface expression, stability and uptake function. Replacing two extracellular cysteines with alanine in Drosophila melanogaster DAT (dmDAT) abolished transporter uptake activity and surface expression. It has been proposed that these two cysteines form a disulfide bond. However, there was no evidence for the existence of such a disulfide bond. Thus, Dr. Gu generated one functional dmDAT mutant with all cysteines replaced by other residues except two extracellular loop cysteines (EL2), and this mutant was analyzed for this EL2 disulfide bond function. iv DEDICATION Dedicated to my parents, grandparents, and the rest of my family. v ACKNOWLEDGMENTS First, I would like to acknowledge my advisor Dr. Howard Gu who has always been supportive, helpful and patient throughout my time in the lab. Without his guidance and support, I would not be able to accomplish my doctoral degree. I am very grateful to Drs. Lane Jackson Wallace, Michael Xi Zhu, and James Willam DeWille for intellectual supports as well as serving on my committee. I wish to thank my colleagues, Rong Chen, Michael Tilley, Dawn Han, Erik R. Hill, Brian O’Neill, Brad Martin and Bart Naughton for their great helpfulness and friendship. Working with them made my research in the lab rather enjoyable. I am so thankful to faculty members and students at the Ohio State University Pharmacology Department and Biochemistry Program for their kindness and assistance. Finally, I would like to thank my family for their love, encouragement and support in my life. vi VITA 1975………………………………………….……..Born, Jiangxi, P.R.China 1992-1996…………………………………………..B.S. Animal Nutrition China Agricultural University 1998-2001…………………………….........M.S. Biochemistry and Molecular Biology China Agricultural University 2001-present………………………………..…..….Graduate Research Associate the Ohio State University PUBLICATIONS Wei H, Hill ER, Gu HH, Functional mutations in mouse norepinephrine transporter reduce sensitivity to cocaine inhibition. neuropharmacology doi:10.1016/j.neuropharm.2008.09.008 Chen R(a), Wei H (a), Hill ER, Chen L, Jiang LY, Han DD, Gu HH, Direct evidence that two cysteines in the dopamine transporter form a disulfide bond. (a) These two authors contribute equally to this manuscript. Mol Cell Biochem. 2007. 298:41-8 Chen R, Tilley MR, Wei H, Zhou F, Zhou FM, Ching S, Quan N, Stephens RL, Hill ER, Nottoli T, Han DD, Gu HH, Abolished cocaine reward in mutant mice with cocaine- insensitive dopamine transporter. Proc Natl Acad Sci USA. 2006, 103:9333-8. Chen R, Wu X, Wei H, Han DD, Gu HH, Molecular cloning and functional characterization of the dopamine transporter from Eloria noyesi, a caterpillar pest of cocaine-rich cocoa plants, Gene. 2006, 366:152-60. FIELD OF STUDY Major Field: Biochemistry Program vii TABLE OF CONTENTS Abstract………………………………………………………………………………...ii Acknowledgments……………………………………………………………….…….vi Vita………………………………………………………………………….……........vii List of Tables…………………………………………………………………..………xii List of Figures…………………………………………………………..…….….……xiii List of Abbreviations………………………………………………….…….…..……..xv Chapters 1. Introduction…………………………………………………….……….…..……........1 1.1Norepinephrine …………………………………………………….………….…..1 1.1.1 The noradrenergic system ……………………………………….…….…….1 1.1.2 Molecular characterization of NET …………………….……….……..…….3 1.1.3 Regulation of NET …………………………………………….……….…....6 1.1.4 Structure-function relationship of NET ………………….………………....12 1.1.5 Norepinephrine and cocaine addiction……………………………………....14 1.2 Figures……………………………………………………………...……….…....18 2. Functional mutations in mouse norepinephrine transporter reduce cocaine inhibition ………………………………………………………………………………………….20 2.1 Abstract…………………………………………………….....…………………..20 2.2 Introduction…………….………………………………………..……..…………21 viii 2.3 Material and method………………………………………………..…………..…23 2.4 Results…………………………………………………………..……...…………26 2.4.1 Specific mutations………………………………...……………………..…..26 2.4.2 First round of random mutagenesis…………………………………...…..…26 2.4.3 Second round of random mutagenesis……………………………………….28 2.4.4 Third round of mutagenesis………………….……………………..……..…29 2.4.5 Characterization of the triple mutant F101C-A105G-N153T………..…..…29 2.5 Discussion……………………...…………………………………………….…...30 2.6 Summary………………………………………………………..…..…………….33 2.7 Tables……………………………………………………….……….……….…...35 2.8 Figures………………………………………………………….……………....…38 3. Cocaine insensitive norepinephrine transporter knock-in mouse model for studying in vivo NET contribution to cocaine addcition………….…………..………………….…42 3.1 Abstract……………………………………………..…….…...…....…..….…….42 3.2 Introduction……………...……………………………………..………..….........43 3.3 Methods and results…………………...……………….…..…….……….…....…45 3.3.1 Materials……………………………………………..……………………..45 3.3.2 Preparation of the targeting vector and generation of recombinant ES cells46 3.3.3 ES cell culture…………………………………………………..…………..47 3.3.4 ES cell passage and expansion………………………………...…….....…..47 ix 3.3.5 Target vector DNA preparation...…………………………………………..48 3.3.6 Electroporation and isolation of ES cell lines ……………………….........48 3.3.7 Picking G418 and ganciclovir resistant colonies and ES cell screening…..49 3.3.8 F0 chimera mice generation…………….…………..……………..……….50 3.3.9 F1 chimera mice germline transmission……….…………….………..…....51 3.3.10 Genomic DNA purification…….………………...……………..…...……51 3.3.11 F2 cre removed mice generation and genotyping..……………………….52 3.4 Discussion and summary…………….…………...…………………………..…....53 3.5 Tables…………………….……………….…………………………………...…..54 3.6 Figures……………………………………………………………………….…….55 4. Residues in the transmembrane III of the norepinephrine transporter affecting dopamine and norepinephrine uptake.…………………………………….…..….…….63 4.1 Abstract………………………………………….………………….…….……...63 4.2 Introduction…………………………………………...…………….……..……..64 4.3 Material and methods……………………………..……………………..……….66 4.4 Results……………………………………………………………………....……70 4.4.1 Screening