Function and Mechanism of Polarized Targeting of Neuronal Membrane Proteins Dissertation Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University By Joshua A. Barry, B.S. Graduate Program in Molecular, Cellular and Developmental Biology The Ohio State University 2013 Committee: Chen Gu, Advisor Anthony Brown Tsonwin Hai Peter Mohler i Copyright by Joshua A. Barry 2013 ii ABSTRACT A neuron’s ability to survive and function properly requires many factors including a multitude of proteins. Membrane proteins are a specific class that are expressed within the plasma membrane and function in a variety of roles from action potential firing to neurotransmitter reuptake. To perform this variety of functions requires proper localization of these proteins, however, the exact functional relevance and underlying mechanisms that regulates this targeting is still an area of intense research. In this thesis I examined the functional role that NgCAM had on inducing bundling of axons or dendrites via regulation by domain deletion or phosphorylation. I also examined how the polarized targeting of the splice variants of Kv3.1 could affect the maximal spiking frequency of neurons. Then I explored how Kv3.1 could induce clustering and activation of its motor kinesin 1/KIF5. Finally, I looked at the role that metal binding sites, specifically zinc, plays on the localization and activity of Kv3.1. My various areas of research are all linked by the shared idea that the proper localization of membrane proteins can regulate a neuron’s function. ii DEDICATION This work is dedicated to my mom and dad, and my two sisters Jillian and Emma. – thanks for supporting me and keeping me going iii ACKNOWLEDGEMENTS The time spent in graduate school has been a life changing experience for me. When I was in high school I knew I wanted to be a scientist. Then I went to college and earned a B.S. in biology, but it wasn’t until I started graduate school that I learned what a true scientist is. Scientists don’t just sit at the bench and discover answers to questions they have. Every day is a learning experience, with the successes and failures of experiments that molded me into the competent scientist I am today. But I did not do all this work by myself. I would like to thank the following people for all their support. First, I must say thank you to Dr. Chen Gu. He has stood behind me one-hundred percent as I stumbled my way through learning how to become a better scientist. When I started in his lab I had a basic textbook understanding about molecular biology, but he gave me the tools to build a foundation of molecular biology techniques that I will always carry with me and help me pursue my career as a scientist. I would also like to thank the enormous help of the past and present members of the Gu lab. Dr. Mingxuan Xu taught me all I know about molecular biology. Dr. Yuanzheng Gu taught me about electrophysiology and how to generate the hippocampal neuron culture that I used almost every week. Peter Jukkola, the other graduate student in Dr. Gu’s lab, taught me how the molecules I break apart and study at the molecular iv level function in the whole brain. These three people are not only coworkers but also friends and I will miss them. I would like to thank our collaborators: Dr. Robert McDougel and Dr. David Terman for their help in generating the computer simulations of fast spiking neurons. I would like to thank Andrew Dangel for his help in using the Surface Plasmon Resonance machine to study protein-protein high affinity binding. And finally Dr. Chandra Shrestha for her help on the competition assay between KIF5 and Kv3.1 and other laboratory work. Also, I would like to thank my committee members Dr. Tsonwin Hai, Dr. Anthony Brown and Dr. Peter Mohler for being on my committee and helping me on this journey to receive my PhD. v Vita October 10, 1981 ....................................................................Born – Madison, Wisconsin June 2004 ...............................................................................B.S., Biology Mount Union College 2004-2013 ..............................................................................Graduate Research Associate The Ohio State University Publications 1. Barry, J., Xu, M., Gu, Y., Dangel, A., Shrestha, C., and Gu, C. (2013) Activation of Conventional Kinesin Motors in Clusters by Shaw Voltage-Gated Potassium Channels. Journal of Cell Science 126, 2027-2041 Published online March 2013. 2. Gu, Y., Barry, J., and Gu, C. (2013) Kv3 channel assembly, trafficking and activity are regulated by zinc through different binding sites. Journal of Physiology 591, 2491-2507 Published online Feb. 18th, 2013. 3. Barry, J., and Gu, C. (2012) Coupling Mechanical Forces to Electrical Signaling: Molecular Motors and the Intracellular Transport of Ion Channels. The Neuroscientist 19, 145-159 Published online Aug. 24th, 2012. 4. Gu, Y.Z., Barry, J., McDougel, R., Terman, D., and Gu, C. (2012) Alternative splicing regulates Kv3.1 polarized targeting to adjust the maximal spiking frequency. The Journal of Biological Chemistry 287, 1755-1769. 5. Gu, C., and Barry, J. (2011) Function and mechanism of axonal targeting of voltage-sensitive potassium channels. Progress in Neurobiology (review article) 94, 115-132. 6. Xu, M., Gu, Y.Z., Barry, J., and Gu, C. (2010) Kinesin I transports tetramerized Kv3 channels through the axon initial segment via direct binding. Journal of Neuroscience 30, 15987-16001. 7. Barry, J., Gu, Y., and Gu, C. (2010) Polarized targeting of L1-CAM regulates axonal and dendritic bundling in vitro. European Journal of Neuroscience 32, 1618-1631. vi 8. Baumann, A., Barry, J., Wang, S., Fujiwara Y., Wilson, T.G. (2010) Paralogous genes involved in juvenile hormone action in Drosophila melanogaster. Genetics 185, 1327-1336. 9. Barry, J., Wang, S., Wilson, T.G. (2008) Overexpression of Methoprene-tolerant, a Drosophila melanogaster gene that is critical for juvenile hormone action and insecticide resistance. Insect Biochemistry and Molecular Biology 38, 346-353. Fields of Study Major Field: Molecular, Cellular and Developmental Biology Program vii TABLE OF CONTENTS 2013 ................................................................................................................................................................II ABSTRACT ..................................................................................................................................................II DEDICATION ............................................................................................................................................ III ACKNOWLEDGEMENTS ....................................................................................................................... IV VITA ............................................................................................................................................................ VI LIST OF TABELS ....................................................................................................................................... X LIST OF FIGURES ................................................................................................................................... XI CHAPTER 1 .................................................................................................................................................. 1 INTRODUCTION ...................................................................................................................................... 1 NEURON POLARITY ...................................................................................................................................... 1 MOTOR PROTEINS: KINESINS ....................................................................................................................... 2 CELL ADHESION MOLECULES ....................................................................................................................... 3 ION CHANNELS ............................................................................................................................................ 4 Potassium Channels ............................................................................................................................... 6 TRANSPORT OF ION CHANNELS BY ADAPTOR PROTEINS ............................................................................. 14 AMPA receptors ................................................................................................................................... 14 NMDA receptors ................................................................................................................................. 16 GABAA Receptors................................................................................................................................ 21 Voltage-gated potassium channel (Kv3) and Kinesin 1 ..................................................................... 24 CLC-5 (Chloride/Proton antiporter) and Kinesin 2 ........................................................................... 25 REGULATION OF ION CHANNEL AND KINESIN INTERACTION ................................................................ 27 ADAPTOR PROTEIN WITH MULTIPLE BINDING PARTNERS ...................................................................... 28 CHANNEL OLIGOMERIZATION AND KINESIN TRANSPORT......................................................................