Voltage-Gated Sodium Channel b1 Subunit Processing by BACE1 and g-secretase: Regulatory Mechanisms and Downstream Signaling by Alexandra A. Bouza A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy (Pharmacology) in the University of Michigan 2020 Doctoral Committee: Professor Lori L. Isom, Chair Professor Pierre Coulombe Research Assistant Professor James Offord Professor Yoichi Osawa Professor Manoj Puthenveedu Alexandra A. Bouza [email protected] ORCID iD: 0000-0002-7610-5517 © Alexandra A. Bouza 2020 Acknowledgements First, I would like to thank my mentor, Dr. Lori Isom, for her incredible support and guidance throughout the duration of my graduate studies. I feel very fortunate to have been trained in the Isom lab with a mentor who is so passionate about not only research, but graduate education. I would also like to thank the additional members of my thesis committee: Drs. Pierre Coulombe, Jim Offord, Yoichi Osawa, and Manoj Puthenveedu. The insight and advice during both committee and individual meetings greatly helped advance my thesis project and my own progression as a scientist. In particular, I would like to thank Dr. Jim Offord for sharing his over 30 years of experience with me each day in the lab to help turn a chemist into a molecular biologist/pharmacologist. Next, I would like to thank all current and previous members of the Isom lab. Thank you for providing the most kind and supportive environment to learn and thank you all for your great passion for food and a good happy hour. I would also like to thank the many collaborators for their contributions to this thesis as well as those who included me in their own work. To Julie Philippe, thank you for loving b1 palmitoylation as much as I do. Without you, the b1 palmitoylation project truly would not have been possible. Nnamdi Edokobi and Dr. Luis Lopez-Santiago, thank you for completing the many of the electrophysiological experiments presented in this thesis. I so appreciate the large amount of time you committed to moving my project forward. Dr. Gemma Carvill, thank you for your advice and analysis on the b1 proteomic study presented. Thank you to the incredible University ii of Michigan core facilities: Advanced genomics, proteomics and peptide synthesis, and bioinformatics. In particular, thank you to Dr. Becky Tagett, Dr. Dana King, and Dr. Henriette Remmer for your contributions to my project. I would also like to thank Dr. Louis Dang, Preethi Swaminathan, and Tracy Qiao for including me in your work on patient-derived induced Pluripotent Stem Cells. Finally, I would like to thank our collaborators in Belgium, Dr. Alec Aeby and Claudine Sculier, for allowing me the opportunity to biochemically characterize a SCN1B- linked disease variant. I am also grateful to the very kind and helpful Department of Pharmacology administration for their support in all aspects of graduate school outside of the lab, of which there are many, including: Lisa Garber, Josh Daniels, Ingrid Shriner-Ward, Dar-Weia Liao, Chereen Mroz, Denise Gakle, Dennis Ondreyka, Audrey Morton-Dziekan, and Elizabeth Oxford. I would also like to thank everyone in the entire department as well as Wolverine Pathways that contributed to making Pathways to Pharmacology a reality. Pathways is one of my most fond memories of my graduate school experience. In particular, I would like to thank Julie Philippe, Andrea Pesch, Lisa Garber, and Elizabeth Oxford for their extensive contributions to this initiative. I would also like to express my gratitude for the funding sources that have supported my graduate work including the Program in Biomedical Sciences, Rackham Predoctoral Fellowship, Systems and Integrative Biology training grant (T-32-GM008322), the National Heart, Lung, and Blood Institute F31 (1F31-HL144047-01), and a R01 to Dr. Lori Isom (NIH R01-NS076752-05). iii Finally, I would like to thank my family and friends for all of their support throughout life and during my graduate career. To my parents, Rick and Janet Bouza, thank you for your constant and unwavering care for the last 27 years and of particular importance, on my journey to becoming a scientist. To my brother, Tony Bouza, thank you for being a great role model to look up to my entire life. To my sister-in-law, Mariel Bouza, thank you for always providing a good laugh and the leftovers from family dinner. To my godmother, Marilyn Lasseter, thank you for traveling to every single important event which has happened in my life to be there for me. To my Chandler family, Tom, Mary, Tommy, and Farley, thank you for being so welcoming, loving, and kind over the last 5 years. Finally, to my fiancé, Ben, thank you for listening at the dinner table while I drew the results of my failed experiments on a napkin to get your opinion. I am so grateful to have experienced every single day of graduate school together. iv Table of Contents Acknowledgements ii List of Tables vi List of Figures vii Abstract ix Chapter 1 Introduction 1 Summary 1 The basics of the voltage-gated sodium channel β subunits 2 Modulation of the ion channel pore by β subunits 9 The β subunits as cell adhesion molecules 14 The role of β subunits in pathophysiology 20 Regulated Intramembrane Proteolysis (RIP) 36 Conclusion 44 Chapter 2 VGSC b1 Subunit Processing by BACE1 and g-secretase Regulates Excitability by Ion Channel Gene Transcription 45 Summary 45 Introduction 47 Results 51 Discussion 86 Materials and Methods 96 Chapter 3 VGSC b1 Palmitoylation Promotes its Plasma Membrane Localization 107 Summary 107 Introduction 109 Results 113 Discussion 134 Materials and Methods 137 Chapter 4 Discussion and Future Directions 148 Summary and Significance 148 Future Directions 150 Overall Conclusions 165 Materials and Methods 166 Bibliography 168 v List of Tables Table 1.1: VGSC genes and their encoded proteins. .................................................................... 3 Table 2.1: b1-ICD peptide has no effect on Nav1.5 transient or persistent sodium current density. ................................................................................................................................................. 63 Table 2.2: b1-ICD peptide has no effect on Nav1.5 voltage-dependence of activation or inactivation. .............................................................................................................................. 63 Table 2.3: Voltage-dependence of inactivation of peak potassium currents recorded from dissociated ventricular cardiomyocytes. .................................................................................... 75 Table 2.4: Voltage-dependence of inactivation of end potassium currents. ................................ 77 Table 2.5: Voltage-dependence of inactivation of Ito f, Ito s, IK slow, and Iss. .................................. 80 Table 4.1: b1 binding partners which may implicate the b1-ICD in chromatin remodeling as a mechanism by which it modulates transcription. ..................................................................... 158 Table 4.2: Selection of SCN1B patient variants from ClinVar. ................................................. 164 vi List of Figures Figure 1.1: Cartoon diagram of the voltage-gated sodium channel (VGSC) ................................. 6 Figure 1.2: Cartoon diagram of β1/β1B topology. ....................................................................... 8 Figure 1.3: b1 participates in homophilic and heterophilic cell adhesion ................................... 17 Figure 1.4: β subunits are sequentially cleaved by α-secretase and/or the β-site amyloid precursor protein-cleaving enzyme 1 (BACE1) and subsequently by γ-secretase in the lumen of the membrane. ................................................................................................................................ 19 Figure 1.5: Disease-linked β subunit mutations ......................................................................... 35 Figure 1.6: Cartoon diagram of RIP. ......................................................................................... 38 Figure 2.1 β1 subunits are substrates for BACE1 and γ-secretase intramembrane cleavage. ...... 54 Figure 2.2: β1-ICD-V5 localizes to the nucleus. ........................................................................ 56 Figure 2.3: Expression of the β1-ICD alters VGSC gene expression. ......................................... 60 Supplemental Fig. 2.1: b1-ICD has no effect on sodium current in heterologous cells. .............. 61 Figure 2.4: The β1-ICD modulates gene transcription in vitro and in vivo. ................................ 67 Supplemental Fig. 2.2: Principal Component Analysis (PCA) for RNA-Seq experiments ......... 68 Supplemental Fig. 2.3: ClueGO network analysis of RNA-seq data sets. ................................... 69 Figure 2.5: b1-ICD regulates potassium channel genes and potassium currents in cardiac ventricular myocytes. ................................................................................................................ 74 Figure 2.6: Comparison of current density and voltage-dependence of inactivation of peak and end potassium currents. ............................................................................................................. 76 Figure 2.7: Comparison of current density, rate of decay, and availability of individual components of the potassium current. ....................................................................................... 79 Figure