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Mechanisms of Epileptogenesis Due to Mutations of Voltage-Gated Sodium Channel SCN1B By Who moved my protein? Mechanisms of Epileptogenesis due to Mutations of Voltage-Gated Sodium Channel SCN1B by Gustavo A. Patino A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy (Neuroscience) in The University of Michigan 2010 Doctoral Committee: Professor Lori L. Isom, Chair Professor Miriam H. Meisler Professor Michael D. Uhler Associate Professor Geoffrey G. Murphy Associate Professor Jack M. Parent DEDICATION To my family: My loving parents, my amazing brother and my wonderful wife Aubrey ii ACKNOWLEDGEMENTS This work would not have been possible without the help of so many people, both in the personal and professional aspects of my life, who believed in me and encouraged me at every point. I am grateful to all of you. First and foremost, thanks to my family: To my wonderful wife Aubrey who has been so patient and loving with me during this endeavor. My parents who have always been there for me, and even when in another country always made sure I knew I was not alone in any situation. My brother who is always making sure I am doing good and never hesitated to help me with any of my projects. I would like to thank my advisor, Dr. Lori L. Isom for her mentorship on becoming a scientist and balancing work and family successfully. Also, for her continuous support in all my ideas and projects over years. Without it, so many of my achievements at Michigan would not have happened. Also, thanks to all the members of the Isom lab that through the years helped me in so many projects and made the lab a great environment in which to work and thrive intellectually. I am grateful to all the members of my dissertation committee: Dr. Miriam Meisler, Dr. Geoffrey Murphy, Dr. Jack Parent, and Dr. Michael Uhler. I could always count on them to help me with experimental techniques, explain difficult concepts, make sense of some of my data and support various applications to grants and courses throughout the years. Many teachers also guided me in the years before coming to Michigan and help me form the basis of the scientist I have become so far. I want to mention especially: Dr. Edgar Osuna, Dr. Jaime Toro, Dr. Manuel Yepes, Dr. Aristides Duque, Dr. Carlos Mayor, Dr. Mario Bernal, Dr. José Félix Patiño, Dr. Alfredo Rubiano, Dr. Eugenio Matijasevic, Dr. Eduardo Londoño, Dr. John Duperly, Dr. Jose Rafael Toro and Helena Groot de Restrepo. After my arrival to Michigan many professors again made sure that I would find here a home in which to learn and thrive. Besides Dr. Isom and the members of my thesis committee I would like to mention first Dr. Peter Hitchcock and Dr. Roger Albin, for iii supporting my admission into the program. Dr. Albin also introduced me to life in research, helped focused my research interests and continually advised me on career and life issues. Also I want to thank Dr. Andrew Lieberman, Dr. Jeff Martens, Dr. Mario Delmar, Dr. Mark Russell, Dr. Richard Hume, Dr. Richard Neubig, Dr. Kate Barald, Dr. Steve Fisher, Dr. Gary Huffnagle, Dr. Raymond Ruddon, and Dr. Victoria Booth. Thank you to Dr. Kent Berridge too, for agreeing to be my mentor for the Graduate Teaching Certificate program. Finally, to all my friends near and far, who have shared the good times and supported me during any hardships. I am indebted to you all, for all the laughter, the tears, the confidences and the quiet moments. iv Table of Contents Dedication…………………………………………………………………………………………ii Acknowledgements……………………………………………………………………………...iii List of figures…………………………………………………………………………………….vii List of Tables…………………………………………………………………………………..…ix Chapter I. Electrophysiology and beyond: Multiple roles of Na+ channel β subunits in development and disease…………………………………………………….…….1 β subunits are immunoglobulin (Ig) superfamily CAMs……………….......2 β subunits are expressed in excitable and non-excitable tissues…..…….5 β subunits interact with multiple cytoskeletal, cell adhesion, signal transduction, and extracellular matrix proteins…………..…………..……...7 What do β subunits do?...........................................................................10 What is the role of β subunits in disease…………..……………………....16 Conclusions and future directions………..………………………………....25 Bibliography……………..……………………………………………………..27 II. A functional null mutation of SCN1B in a patient with Dravet Syndrome…….36 Introduction…………………………………………………………………….36 Materials and methods………………………….……………………………37 Results………………………………………………………………………….50 Discussion……………………………………………………………………..67 Acknowledgements………...…………………………………………………74 Bibliography…………………..…………………………….………………….76 v III. Voltage-Gated Na+ channel β1B: a secreted cell adhesion molecule involved in human epilepsy……………………………….……………………….82 Introduction…………………………………………………………………….82 Materials and methods……………………………………………….………83 Results………………………………………………………………………….94 Discussion….…………………………………………………..…………….113 Acknowledgements………………………………………………………….116 Bibliography……………………………………………………...…………..118 IV. Conclusions………………………………………..………………………………122 Bibliography…………………………………...……………………………..129 vi List of Figures Figure I.1. Subunit structure of VGSCs…………………..…………………………………...2 I.2. Localization of human epilepsy and cardiac arrhythmia mutations in VGSC β subunits……..……………………………………………………………………21 II.1. Specificity of anti-β1intra antibody………..……………….……………………..40 II.2. SCN1B homozygous mutation found in Dravet Syndrome…………………..52 II.3. p.R125C and β1WT have no effect on the properties of sodium current in HEKrNav1.1 cells…………………………………………………………………54 II.4. p.R125C does not modulate sodium current expressed by Nav1.2 in SNaIIa cells…………………………………………………………………….....56 II.5. p.R125C is poorly expressed at the cell surface at physiological temperatures………………………………………………………………...……58 II.6. Cell surface expression of p.R125C is rescued at low temperatures……….60 II.7. p.R125C modulates sodium currents expressed by Nav1.2 in Xenopus oocytes………………………………………………………………………….....62 II.8. Time to death due to status epilepticus of Scn1b+/- mice is similar to Scn1b+/+ mice.............................………………………………………………..65 II.9. β1 is expressed in hippocampal CA3 bipolar neurons….…………..………..68 III.1. A SCN1B mutation that affects β1B is found in two pedigrees of idiopathic epilepsy………………………………………………………………..…………88 III.2. β1B is a secreted protein…………………………………………..……………97 III.3. Rat and mouse β1B are predicted to be secreted proteins………………….99 III.4. The unique C-terminal region of β1B is not conserved between species..100 III.5. β1B is a glycoprotein…………………………………………………………...101 III.6. Association of β1B with Nav1.1, Nav1.3 or Nav1.5 is not detectable………104 III.7. β1B is the predominant SCN1B splice variant during human fetal brain Development…………………………………………………………………...106 vii III.8. β1B stimulates neurite outgrowth of CGNs………………………………….108 III.9. β1B p.G257R is a trafficking deficient mutant……………………………….110 III.10. Confirmation of expression of β1B or p.G257R in pTRACER in 1610- β1V5 cells or 1610- β1BV5 cells…………………………………………….112 viii List of Tables Table I.1. Length of signal peptides and identity of the first aminoacid after it in VGSC β subunits……………………………………………………………………………4 I.2. VGSC β subunit protein-protein interactions…………………………………….9 I.3. Human diseases associated with β subunits…………………………………..19 II.1. Biophysical parameters of sodium current in HEKrNav1.1 cells compared with HEKrNav1.1 cells co-expressing β1WT or p.R125C………………….…55 II.2. Biophysical parameters of sodium current in SNaIIa cells compared with SNaIIa cells co-expressing β1WT or p.R125C……………………………..…57 II.3. Biophysical parameters of sodium currents expressed by Nav1.2 alone or Nav1.2 co-expressed with β1WT or p.R125C in Xenopus oocytes…………63 II.4. Seizure parameters in Scn1b+/+ and Scn1b +/- mice…………………………..64 II.5 Analysis of evoked action potentials in CA1 or CA3 hippocampal slices from Scn1b+/+ and Scn1b-/- mice…………………………………………………66 II.6 Characteristics of SCN1B mutants associated with epilepsy………………...72 III.1. PCR primers used to amplify regions of the human SCN1B gene…………84 III.2. Touch-Down PCR amplification conditions…………………………………...85 III.3. Identified human SCN1B variants……………………………………………...96 ix Chapter I Electrophysiology and beyond: Multiple roles of Na+ Channel β subunits in development and disease (Portions of this chapter have been published in Neurosci Lett 2010, doi:10.1016/j.neulet.2010.06.050) Voltage-gated Na+ channel (VGSC) β subunits are not “auxiliary.” These multifunctional molecules not only modulate Na+ current, but also function as cell adhesion molecules (CAMs) – playing roles in cellular aggregation, migration, invasion, neurite outgrowth, and axonal fasciculation [9, 10]. β subunits are integral members of VGSC signaling complexes at nodes of Ranvier, axon initial segments, and cardiac intercalated disks, regulating action potential propagation through critical intermolecular and cell-cell communication events. At least in vitro, many β subunit cell adhesive functions occur both in the presence and absence of pore-forming VGSC α subunits [64, 65, 70], and in vivo β subunits are expressed in excitable as well as non-excitable cells [3, 7, 18, 24, 30, 33, 54, 85] [11, 80](O’Malley, submitted), thus β subunits may play important functional roles on their own, in the absence of α subunits. VGSC β1 subunits are essential for life, and appear to be especially important during brain development [6, 9, 17]. Mutations in β subunit genes result in a variety of human neurological and cardiovascular diseases (summarized in
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