CALCIUM CHANNEL BETA SUBUNITS AND SCA6-TYPE CALCIUM CHANNEL ALPHA SUBUNITS C-TERMINI REGULATE TARGETING AND FUNCTION OF PRESYNAPTIC CALCIUM CHANNELS IN HIPPOCAMPAL NEURONS By MIAN XIE Submitted in partial fulfillment of the requirents For the degree of Doctor of Philosophy Dissertation Adviser: Stefan Herlitze Department of Neuroscience CASE WESTERN RESERVE UNIVERSITY January, 2008 CASE WESTERN RESERVE UNIVERSITY SCHOOL OF GRADUATE STUDIES We hereby approve the thesis/dissertation of Mian Xie candidate for the PHD degree*. (signed) Lynn Landmesser (chair of the committee) Robert Miller Hisashi Fujioka Stefan Herlitze (date) 08/27/07 *We also certify that written approval has been obtained for any proprietary material contained therein. DEDICATION For my grandparents parents and my wife ii TABLE OF CONTENTS Title page…………………………………………………………………………………..i Dedication…………………………………………………………………………………ii Table of contents……………………………………………………………………........iii List of figures……………………………………………………………………………...v Acknowledgements………………………………………………………………………vii Abstract………………………………………………………………………………….viii Chapter 1: Introduction……………………………………………………………………1 Structure, biophysical property, and distribution of Voltage-gated Calcium Channels……………………………………………………………………………...2 Structure and function of Cavβ subunits……………………………………………..7 Cavβ subunits target VGCC in heterologous expression systems and neurons...........9 Presynaptic VGCC and synaptic transmission……………………………………..12 Channelopathy caused by alteration of P/Q- type channel C- terminus: SCA6........14 Research goals……………………………………………………………………...17 Chapter 2: Facilitation versus Depression in cultured hippocampal neurons determined 2+ by targeting of Ca channel Cavβ4 versus Cavβ2 subunits to synaptic terminal………..19 Introduction…………………………………………………………………………20 Material and Methods…………………………………………………………........22 Results………………………………………………………………………………29 Discussion…………………………………………………………………………..41 iii Figures…..…………………………………………………………………………..48 Chapter 3: The human P/Q-type C-terminus underlying SCA6 forms cytoplasmic aggregates, impairs synaptic transmission and increases synapse number………………79 Introduction…………………………………………………………………………80 Material and Methods…………………………………………………………........84 Results………………………………………………………………………………89 Discussion…………………………………………………………………………..98 Figures……………………………………………………………………………..105 Chapter 4: Discussion…………………………………………………………………..123 Research conclusions………………………………………………………….......124 Importance of VGCC Cavβ subunits in subunit-specific channel targeting………125 Cavβ subunits and short-term synaptic plasticity………………………………….128 Physiological and pathological consequences of expression different P/Q- type C-termini……………………………………………………………………..130 Remain questions and future directions…………………………………………...132 Figures……………………………………………………………………………..138 Chapter 5: Bibliography………………………………………………………………...145 iv List of Figures Chapter 2: Facilitation versus Depression in cultured hippocampal neurons determined by 2+ targeting of Ca channel Cavβ4 versus Cavβ2 subunits to synaptic terminals Figure 1…………………………………………………………………………49 Figure 2…………………………………………………………………………51 Figure 3…………………………………………………………………………54 Figure 4…………………………………………………………………………56 Figure 5…………………………………………………………………………58 Figure 6…………………………………………………………………………61 Figure 7…………………………………………………………………………63 Figure 8…………………………………………………………………………66 Figure 9…………………………………………………………………………68 Figure 10………………………………………………………………………..71 Figure 11………………………………………………………………………..73 Figure 12………………………………………………………………………..76 Chapter 3: The human P/Q-type C-terminus underlying SCA6 forms cytoplasmic aggregates, impairs synaptic transmission and increases synapse number Figure 1………………………………………………………………………..106 Figure 2………………………………………………………………………..108 Figure 3………………………………………………………………………..110 Figure 4………………………………………………………………………..112 v Figure 5………………………………………………………………………..114 Figure 6………………………………………………………………………..116 Figure 7………………………………………………………………………..118 Figure 8………………………………………………………………………..120 Chapter 4: Discussion Figure 1………………………………………………………………………..139 Figure 2………………………………………………………………………..141 Figure 3………………………………………………………………………..143 vi Acknowledgements This thesis would not have been possible without the support of many people. I would like to express my thanks and appreciation to all those who have helped along the way. Especially, I would like to sincerely thank my adviser and mentor Dr. Stefan Herlitze. Stefan has been an exceptional example of a successful, passionate and hard working scientist and provided me with his support, guidance and caring throughout the years. I would also like to thank other members in my committee, Dr. Lynn Landmesser, Dr. Robert Miller, and Dr. Hisashi Fujioka, for their support, guidance and participation in my research. Thanks to all former and present members in Herlitze lab for your kind and generous help. Thanks for my friends and family, especially my wife, for your love and support. There is no way I could have don this without your help. vii Calcium Channel Beta Subunits and SCA6-type Calcium Channel Alpha Subunits C-Termini Regulate Targeting and Function of Presynaptic Calcium Channels in Hippocampal Neurons Abstract by MIAN XIE Ca2+ channel β subunits determine the transport and physiological properties of high voltage activated Ca2+ channel complexes, and the poly-glutamination within the C-terminus (CT) of the P/Q-type Ca2+ channel α subunit is linked with Spinocerebellar ataxia type 6 (SCA6). In the first part of this study we analyzed the distribution of the Cavβ subunit family members in hippocampal neurons and correlated their synaptic distribution with their involvement in transmitter release. We found that exogenously expressed Cavβ4b and Cavβ2a subunits distribute in clusters and localize to synapses, while Cavβ1b and Cavβ3 are homogenously distributed. According to their localization Cavβ2a and 4b subunits modulate the synaptic plasticity of autaptic hippocampal neurons, i.e. Cavβ2a induces depression, while Cavβ4b induces paired-pulse facilitation followed by synaptic viii depression during longer stimuli trains. The induction of paired-pulse facilitation by Cavβ4b is correlated with a reduction in the release probability and cooperativity of the transmitter release. These results suggest that Cavβ subunits determine the gating properties of the presynaptic Ca2+ channels within the presynaptic terminal in a subunit specific manner and may be involved in the organization of the Ca2+ channel relative to the release machinery. We also examined whether the poly-glutamination of P/Q-type channel CT can increase the channel stability and induce a gradual accumulation of a CT degradation product. We demonstrated that the poly-glutaminated CT degradation product distributes in cytoplasmic aggregates in cultured neurons as found in SCA6 patients and has drastic physiological effects on synaptic function and synapse assembly. Our results show that the CTs induce a change in the Ca2+ dependence of transmitter release correlated with a reduced vesicular release probability, which causes synaptic depression during repetitive high frequency stimulations. The CT containing the SCA6 mutation also caused an increase in the number of synapses. Our results predict that CT degradation products derived from the P/Q-type channels in SCA6 patients would reduce synaptic strength in each synaptic terminal, but increased the overall synapse formation per neuron. The increased synapse formation may give a mechanistic explanation for the survival of nucleo-olivary pathways in SCA6, which is not observed in other SCAs. ix Chapter 1: Introduction 1 Voltage gated Ca2+ Channel (VGCC) complexes mediate the voltage-dependent Ca2+ influx in subcellular compartments. They trigger diverse processes including neurotransmitter release, dendritic action potentials, excitation-contraction and excitation-transcription coupling. Targeting of biophysically defined VGCC complexes to the correct subcellular structures is therefore critical to proper cell and system function. Minute changes in targeting of VGCC complexes are therefore related to the modulation of the channel functions within the subcellular structure, and alterations of the proper targeting of Ca2+ channels can lead to channelopathy such as SCA6. Structure, biophysical property, and distribution of Voltage-gated Calcium Channels VGCC complexes are composed of the pore forming α subunit (Cavα), the ancillary β (Cavβ) and α2δ subunits, and these subunits are necessary to the function of the channels (Leveque et al., 1994; McEnery et al., 1991). Although γ subunits in skeletal muscle and their brain homologous are also important to the channel function, their assembly is not required to form functional channels (Catterall, 2000; Curtis and Catterall, 1984; Herlitze and Mark, 2005). The structure of the Cavα subunit consists of four transmembrane domains, each of which contains six transmembrane segments. Inside each domain there is a segment considered function as the voltage sensor and a hairpin structure that most likely forms the pore of the channel (Catterall, 2000; Herlitze et al., 2003). The domains are connected 2 to each other by peptide loops, which are targets for intracellular protein-protein interactions and therefore important for channel targeting, modulation, sorting and clustering. The loop I-II and the C- terminus of the Cavα subunit are believed to have the sites for the binding of Cavβ subunits (Bichet et al., 2000; Dolphin, 2003; Sandoz et al., 2004). According