Mechanisms of presynaptic CaV2.2 (N-type) modulation by Allen W. Chan A thesis submitted in conformity with the requirements for the degree of Doctor of Philosophy Graduate Department of Physiology University of Toronto ©Copyright by Allen Chan 2008 ii Abstract Mechanisms of presynaptic CaV2.2 (N-type) modulation. Allen W. Chan Doctor of Philosophy, 2008 Department of Physiology University of Toronto Neurotransmitter release at presynaptic terminals is a complex process involving calcium ion influx through voltage-gated calcium channels (CaV). In addition to their role as entry points through which calcium influx may occur, CaV are now understood to be fundamental components of a common release-site complex that is highly adapted for modulation. Consistent with this model, I investigated mechanisms of modulating a presynaptic calcium channel, CaV2.2, via a heterotrimeric G-protein pathway. Using the patch-clamp technique, I demonstrated in chick dorsal root ganglion (DRG) neurons that the slow kinetics of G-protein inhibition of CaV2.2 via GTPγS were limited by the rate of GDP dissociation from the G-protein nucleotide binding site. In addition, I investigated the role of G-protein regulation of CaV2.2 currents evoked by action potential-like stimuli. Here, I characterized an inhibited current that was advanced in time with respect to uninhibited controls. These currents exhibited a shorter latency to current activation and faster deactivation. These findings may have important physiological ramifications on signal transduction and timing. iii In addition to G-protein regulation, presynaptic CaV2.2 have been demonstrated to exhibit a resistance to voltage-dependent inactivation (VDI), a property thought to be important in determining channel availability and synaptic excitability. I demonstrated a role for dynamic palmitoylation in conferring resistance to VDI in presynaptic terminals of the chick ciliary ganglion. Using tunicamycin, an inhibitor of palmitoylation, I induced a hyperpolarizing shift in the steady-state-inactivation (SSI) profile of presynaptic CaV2.2. Finally, I examined the role of a CaV interacting protein, Munc18, as a potential regulator of CaV. I probed for alterations in CaV2.2 function in DRG neurons that had been transfected with Munc18 or Munc18 siRNA. Despite the intimate interaction between Munc18 and CaV2.2, no major effects on the fundamental characteristics of CaV2.2 function were observed. However, a hyperpolarizing shift in the inactivation profile of CaV2.2 was determined in DRG neurons in which Munc18 was knocked down. It is not clear if this was a direct consequence of Munc18 perturbation. iv Acknowledgements I would like to extend my deepest gratitude to my supervisor, Dr. Elise Stanley. From the moment I began my studies, she provided an intense intellectually stimulating and challenging environment. She has taught me to be rigorous in all manners of scientific thought, to be mindful of the nuances of experimental limitations, and yet to also remain open-minded to the often unexpected possibilities that lay in science. Above and beyond her duties as a supervisor, I would like to also extend my sincerest gratitude to Dr. Stanley for her advice, her sympathy and her support during a time when a severe illness in my family threatened to derail my studies and my world as I knew it. What was unquestionably the darkest time in my life was made lighter with her help. I would also like to extend my appreciation to the members of my supervisory committee, Dr. Lyanne Schlichter and Dr. Frances Skinner. Their advice and encouragement throughout my studies have been invaluable and truly appreciated. Also, thank you to the members of my doctoral transfer committee, Dr. Harold Atwood, Dr. Jane Mitchell, and Dr. Shuzo Sugita. A special thanks to Dr. Qi Li, who has always been willing to help and provide technical support for almost every facet of my work in the laboratory. Thank you to Dr. Steven Owens, Dr. Evan Newell and Luigi Gentile for their advice, tutelage, and camaraderie in all aspects of the intricacies and frustrations of electrophysiology. Thank you to current v and past members of the laboratory, Dr. Rajesh Khanna, Dr. Terence Morris, Fiona Wong, and Sabiha Gardezi for the incalculable support, discussion, and friendship. Thank you to my parents who have supported me throughout these years and trusted that what I was doing was important. For financial support, I have been the beneficiary of fellowships from the University of Toronto. Also, a special thank you to the Ontario Student Opportunities Trust Fund (OSOTF) for awarding me the Margaret and Howard Gamble Research Grant, and to the Canadian Institutes of Health Research (CIHR) for their generous support in awarding me the doctoral Sir Frederick Banting and Dr. Charles Best Canada Graduate Scholarship. Finally, a special thank you to Erika without whose unconditional support, encouragement, and optimism, none of this would be possible. Her spirit has, and will always remain, an inspiration for me to do the best I can in life. vi Table of Contents Abstract ......................................................................................................................................ii Acknowledgements .................................................................................................................. iv Table of Contents ...................................................................................................................... vi List of Figures ............................................................................................................................ ix Abbreviations ............................................................................................................................ xi Chapter 1 : Introduction .......................................................................................................... 1 1.1 Introduction to presynaptic calcium channels ............................................................................ 1 1.2 Molecular structure of voltage‐gated calcium channels ............................................................. 3 1.2.1 Calcium channel α1 subunit ................................................................................................. 3 1.2.2 Calcium channel auxiliary subunits ...................................................................................... 8 1.3 G‐protein modulation of calcium channels ............................................................................... 11 1.3.1 G‐protein coupled receptor signaling ................................................................................ 11 1.3.2 Voltage‐dependent G‐protein modulation ........................................................................ 13 1.3.3 G‐protein interaction with syntaxin ................................................................................... 18 1.3.4 Voltage‐independent G‐protein modulation ..................................................................... 19 1.3.4.1 Voltage‐independent G‐protein modulation via tyrosine kinase signaling ................... 19 1.3.4.2 Voltage‐independent G‐protein modulation via PIP2 regulation .................................... 20 1.4 PKC phosphorylation ................................................................................................................. 22 1.4.1 Crosstalk between PKC & G‐protein signaling pathways ................................................... 22 1.5 Calcium channel regulation by synaptic proteins ..................................................................... 24 1.5.1 Syntaxin‐1/Munc‐18 ........................................................................................................... 24 1.6 Experimental models ................................................................................................................. 26 1.6.1 Chick ciliary ganglion calyx synapse ................................................................................... 26 1.6.2 Chick dorsal root ganglion neurons .................................................................................... 30 Chapter 2 : Materials and methods ....................................................................................... 32 2.1 Preparation of the ciliary ganglion synapse .............................................................................. 32 2.2 Preparation of dorsal root ganglion (DRG) neurons ................................................................. 33 vii 2.3 Patch‐clamp recording .............................................................................................................. 34 2.4 Drug treatments ........................................................................................................................ 35 2.5 DRG transfection ....................................................................................................................... 36 2.6 Statistical analysis ...................................................................................................................... 37 Chapter 3 : Kinetic analysis of G‐protein inhibition of CaV2.2 by G‐protein coupled receptor activation and direct G‐protein activation with GTPγS .......................................................... 38 3.1 Summary ................................................................................................................................... 38 3.2 Introduction ..............................................................................................................................