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This electronic thesis or dissertation has been downloaded from Explore Bristol Research, http://research-information.bristol.ac.uk Author: Gurung, Sonam Title: Kainate Receptors in various forms of plasticity General rights Access to the thesis is subject to the Creative Commons Attribution - NonCommercial-No Derivatives 4.0 International Public License. A copy of this may be found at https://creativecommons.org/licenses/by-nc-nd/4.0/legalcode This license sets out your rights and the restrictions that apply to your access to the thesis so it is important you read this before proceeding. Take down policy Some pages of this thesis may have been removed for copyright restrictions prior to having it been deposited in Explore Bristol Research. However, if you have discovered material within the thesis that you consider to be unlawful e.g. breaches of copyright (either yours or that of a third party) or any other law, including but not limited to those relating to patent, trademark, confidentiality, data protection, obscenity, defamation, libel, then please contact [email protected] and include the following information in your message: •Your contact details •Bibliographic details for the item, including a URL •An outline nature of the complaint Your claim will be investigated and, where appropriate, the item in question will be removed from public view as soon as possible. Kainate Receptors in various forms of plasticity Sonam Gurung August 2018 A dissertation submitted to the University of Bristol in accordance with the requirements for award of the degree of Doctor of Philosophy by advanced study in the School of Biochemistry, Faculty of Life Sciences. Word Count:46,182 Abstract Kainate receptors (KARs) are glutamate-gated ion channels that regulate neuronal excitability and network function in the brain. They can signal through both canonical ionotropic and a non-canonical metabotropic pathway. They regulate neuronal excitability by undergoing plasticity themselves while also regulating plasticity of other receptors. Most KARs contain the subunit GluK2 and the precise properties of these GluK2-containing KARs are determined by additional factors including ADAR2-mediated mRNA editing of a single codon that changes a genomically encoded glutamine (Q) to an arginine (R), which affects their assembly, trafficking and channel properties. I initially set out to study the role of KARs in various forms of plasticity including long term potentiation (LTP) and long term depression (LTD) of AMPA receptors (AMPAR) and homeostatic scaling of KARs themselves. I was unable to reliably replicate previous data showing transient KAR stimulation leads to their increased surface expression which in turn increases AMPAR surface expression. Interestingly however, I discovered that sustained KAR stimulation decreased AMPAR surface expression leading to AMPAR LTD. In addition, I identified that KARs undergo homeostatic plasticity whereby they upscale and downscale in response to changes in the network activity. My findings show that ADAR2-dependent Q/R editing of GluK2 is dynamically regulated during homeostatic plasticity elicited by the suppression of synaptic activity. This suppression of synaptic activity decreases ADAR2 levels by enhancing their proteasomal degradation, which selectively reduces the numbers of GluK2 subunits that are edited. This loss of editing results in increased KAR oligomerisation and ER exit to increase the surface expression of GluK2-containing KARs. Furthermore, I show that partial ADAR2 knockdown phenocopies and occludes TTX-induced scaling of KARs. These data indicate that activity-dependent regulation of ADAR2 levels and GluK2 Q/R editing provides a mechanism for KAR homeostatic plasticity. i Author’s Declaration I declare that the work in this dissertation was carried out in accordance with the requirements of the University's Regulations and Code of Practice for Research Degree Programmes and that it has not been submitted for any other academic award. Except where indicated by specific reference in the text, the work is the candidate's own work. Work done in collaboration with, or with the assistance of, others, is indicated as such. Any views expressed in the dissertation are those of the author. SIGNED: ………………………………………DATE: ………………. ii Acknowledgements First and foremost, thank you Jeremy for the ‘incredible’ supervision! You have been an amazing supervisor and thank you for believing in me more than I did. You have helped me build my confidence. I have learned a lot and enjoyed my PhD throughout. Thank you, Kev, for teaching me pretty much everything throughout my PhD. All my achievements and everything I learned was all due to your amazing supervision and patience and I am very grateful for it. Thank you, Ash, for all your help, especially my scaling project; a project I thoroughly enjoyed working with and for my new-found love for ADARs! Thank you, Suko, for everything, the lab would not have functioned without you and I have learned a lot from you. Thank you to Dan and Ruth for your advice and help throughout my PhD. To Laura, Alex, Vanilla, Jodie, Caroline and Zsombor, thank you for all the tea breaks and sharing our woes together and all the science advice! Vanilla and Luis, my dissection buddies. Those neurones would not have scaled, if it hadn’t been for the beautiful cells, we dissected every week! Alex again thank you so much for the macros that saved me so much time on analysing hundreds of nuclei! Thank you, Phil, for helping me settle in the lab when I first started as an undergrad and Chun for your supervision during my rotation months. Everybody in Henley and Hanley lab, past and present, thank you! Also thank you to my rotation supervisors: Ash Toye and Chrissy Hammond. I got an opportunity to be part of completely different and interesting projects with you both and I enjoyed being part of both of your labs. Thank you my Wellcome course mates: Rob, Lea, Mel and Amy for providing me with all the support, inspiration and fun times I will cherish very much. Finally, I want to thank my family. My sisters, Sophia and Sarina, for providing me with all the support throughout my life, not just my PhD that I always feel lucky to have and my parents for all the love, belief, support and the food! To end, I would say everything I have learned and achieved throughout my PhD was due to all the positive support I had around me, so thank you to everyone who helped me throughout my PhD! iii Table of Contents General Introduction ............................................................... 1 1.1 Synaptic transmission ....................................................................... 2 1.1.1 Synapses ................................................................................... 2 1.1.2 Chemical synapses and transmission ........................................ 2 1.1.3 Excitatory and inhibitory transmission ........................................ 3 1.1.4 Measuring synaptic transmission ............................................... 4 1.2 Ionotropic glutamate receptors ......................................................... 5 1.2.1 AMPARs ..................................................................................... 6 1.2.2 Kainate Receptors .................................................................... 10 1.3 Synaptic plasticity ........................................................................... 25 1.3.1 Hebbian plasticity ..................................................................... 25 1.3.2 Homeostatic plasticity ............................................................... 30 1.4 Adenosine to Inosine RNA editing by ADARs ................................. 42 1.4.1 Adenosine Deaminase Acting on RNA (ADAR) ....................... 42 1.4.2 ADAR structure ........................................................................ 43 1.4.3 ADAR isoforms ......................................................................... 45 1.4.4 ADAR tissue expression and cellular localisation ..................... 46 1.4.5 ADAR substrate preference and specificity .............................. 47 1.4.6 Potential co-factor involvement in the deamination .................. 48 1.4.7 ADAR3 lacks catalytic activity .................................................. 48 1.5 Q/R editing of glutamate receptor by ADARs .................................. 49 1.5.1 ADAR2 in Q/R editing of KARs and AMPARs .......................... 50 1.5.2 Effects of Q/R editing on glutamate receptor properties ........... 52 1.5.3 Q/R editing of GluA2 is important for survival ........................... 54 iv 1.5.4 Developmental and plasticity role of Q/R editing in AMPARs ... 55 1.5.5 Developmental and plasticity role of Q/R editing in KARs ........ 56 1.5.6 GluK2 Q/R editing and seizures ............................................... 57 1.6 Other forms of Glutamate receptor RNA editing ............................. 58 1.7 Glutamate receptor RNA editing in diseases .................................. 60 1.8 Objectives ....................................................................................... 61 Materials and methods .......................................................... 65 2.1 Materials ......................................................................................... 66 2.1.1 Chemicals ................................................................................ 66 2.1.2 Bacterial reagents ...................................................................
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