FUNCTIONAL PROPERTIES OF VERTEBRATE NON- NMDA EXCITATORY AMINO ACID RECEPTORS EXPRESSED IN XENOPUS LAEVIS OOCYTES A thesis submitted for the Degree of Doctor of Philosophy in the University of London, Faculty of Science. by Derek Bowie, B.Sc. (Hons.) Department of Pharmacology, School of Pharmacy, 29/39, Brunswick Square, London. WC1N 1AX. September 1991 1 ProQuest N um ber: U552896 All rights reserved INFORMATION TO ALL USERS The quality of this reproduction is dependent upon the quality of the copy submitted. In the unlikely event that the author did not send a com plete manuscript and there are missing pages, these will be noted. Also, if material had to be removed, a note will indicate the deletion. uest ProQuest U552896 Published by ProQuest LLC(2017). Copyright of the Dissertation is held by the Author. All rights reserved. This work is protected against unauthorized copying under Title 17, United States C ode Microform Edition © ProQuest LLC. ProQuest LLC. 789 East Eisenhower Parkway P.O. Box 1346 Ann Arbor, Ml 48106- 1346 ABSTRACT The Xenopus oocyte expression system was used to express non-N-methyl-D-aspartate (non- NMDA) receptors from mammalian (calf and rat) and avian (chick) brains. In each case, the properties of the expressed receptors were examined using two-electrode current and voltage clamp techniques and compared by examining their pharmacology using dose-response curve analysis (D/R) and constructing current-voltage (l-V) relationships using non-NMDA agonists. Fully-grown immature oocytes (stages V and VI) removed from female Xenopus laevis were microinjected with exogenous messenger ribonucleic acid (mRNA) and after a period of incubation (2-3 days), became responsive to a variety of agonists including central nervous system (CNS) transmitters. Responses evoked by bath application of non-NMDA receptor agonists including kainic acid (KA), domoic acid (Dorn) and 5-bromowillardiine (BrW) all produced large, non-desensitizing membrane currents, whereas the responses to a-amino-3-hydroxy-5-methyl-5-isoxazolepropionic acid (AMPA), quisqualic acid (QA) and L-glutamic acid (L-Glu) were much smaller and showed signs of desensitization. The rank order of agonist-potency determined from D/R curves was QA > AM PA > BrW > KA in all mRNA-injected oocytes. Non-NMDA receptors expressed from each mRNA preparation had a similar sensitivity to KA and conventional non-NMDA receptor antagonists did not differentiate between agonist-evoked responses in oocytes injected with each different mRNA. I-V relationships determined for each agonist were dependent on the mRNA preparation. In oocytes injected with calf or chick brain mRNA, the membrane current evoked by KA (Dorn and BrW) exhibited inward rectification at positive (>+20mV) membrane potentials. At positive potentials, the degree of rectification observed was dose-dependent but agonist-independent. In contrast, in oocytes injected with rat brain mRNA, the response to KA was relatively voltage-independent at negative potentials and exhibited outward rectification at positive potentials^The l-V relationship determined for AM PA (QA and L-Glu) was the same in all mRNA-injected oocytes exhibiting a similar voltage-sensitivity to the KA-response observed in oocytes injected with rat brain mRNA. The estimated reversal potentials (VREV « -6mV ± 2mV) for all non-NMDA receptor agonists were quite similar. In all mRNA-injected oocytes, the response to KA (Dorn and BrW) was antagonised by other excitatory amino acid (EAA) receptor agonists with the following rank order of inhibition; QA > AMPA « L-Glu > NMDA. In oocytes injected with rat brain mRNA, AMPA antagonised the KA-response in a competitive manner. Moreover, a comparison between the amplitude of KA and AMPA responses in the same oocyte exhibited a direct correlation consistent with both agonists acting on the same receptor-ionophore complex. Despite this, the responses evoked by KA and AMPA could still be pharmacologically separated with thiocyanate (SCN ) ions which selectively 2 antagonised only AMPA responses. In oocytes injected with calf or chick brain mRNA, the response to KA was antagonised by AMPA, QA and L-Glu in an apparently non-competitive manner. In some cases, the response to KA was still antagonised even in the absence of any measurable response to QA. A comparison between the amplitude of KA-responses with AMPA, QA or L-Glu in the same oocyte did not show a direct correlation. It is concluded that different types of non-NMDA receptor, equi-sensitive to KA, were successfully expressed in oocytes from mammalian and avian brain mRNA. In each case, the receptor exhibited similar pharmacological properties to nascent receptors in mammalian and avian neurones; however, novel aspects of receptor function were revealed from l-V relationships and antagonism studies with SCN‘ ions. The likelihood of non-NMDA receptor heterogeneity is considered. 3 FUNCTIONAL PROPERTIES OF VERTEBRATE NON-NMDA EXCITATORY AMINO ACID RECEPTORS EXPRESSED IN XENOPUS LAEVIS OOCYTES ABSTRACT 2-3 LIST OF FIGURES 9-12 LIST OF TABLES 12 CHAPTER 1: GENERAL INTRODUCTION 14-31 (i) Historical emergence of excitatory amino acid receptor subtypes. (ii) Ionic membrane conductances activated by non-NMDA receptor agonists. (iii) Radioligand binding and autoradiographic approaches to non-NMDA receptors. (iv) The Xenopus laevis oocyte as a model to study functional EAA receptors. (v) Aims of the present investigation. CHAPTER 2: MATERIALS & METHODS 32-59 (i) Overview. (ii) Extraction and purification of messenger RNA from mammalian and avian brains. (iii) Maintenance of female Xenopus laevis. (iv) Removal and injection of Xenopus oocytes. (v) Electrophysiological recordings. (vi) Drugs used in this study. 4 CHAPTER 3: RECEPTOR CHARACTERISATION: AGONIST AND ANTAGONIST PHARMACOLOGY 60-91 INTRODUCTION 61-62 RESULTS 63-68 (i) Expression of non-NMDA receptors from mammalian and avian brain mRNA: two-types of agonist-response. (ii) Receptor characterisation with non-NMDA receptor antagonists. DISCUSSION 68-77 (i) Agonist-responses may be expressed from a heterogeneous mixture of mRNA. (ii) Efficacy of non-NMDA receptor agonists. (iii) Non-NMDA receptor antagonists did not differentiate between agonist-responses. CHAPTER 4: NON-NMDA RECEPTOR DESENSITTZATION 92-118 INTRODUCTION 93-95 RESULTS 95-99 (i) Receptor desensitization: covert and overt forms. (ii) Concanavalin-A reduced the overt form of desensitization. DISCUSSION 100-109 (i) Receptor desensitization: covert and overt forms. (ii) Overt receptor desensitization was agonist dependent. (iii) Differential desensitization on the same receptor-ionophore. (iv) Mechanism of receptor desensitization. (v) The rebound inward tail current may result from agonist blockade of the channel. 5 (vi) Overt desensitization to kainate was species-dependent. (vii) Concanavalin-A relieved desensitization and increased membrane oscillations. CHAPTER 5: CURRENT-VOLTAGE ANALYSIS OF NON-NMDA RECEPTORS 119-172 INTRODUCTION 120-122 RESULTS 122-133 (i) Non-NMDA agonist responses are differentiated by their voltage sensitivity. (ii) Determination of current-voltage relationships. (iii) Two types of kainate current were species- dependent. (iv) Characterisation of the inward rectification produced by kainate. (v) Outward rectification of kainate responses in oocytes injected with calf and chick brain mRNA (vi) I-V relationships for QA AMPA and L-Glu. (vii) Comparing different kainate responses with AMPA in the same oocyte. DISCUSSION 133-147 (i) Are different I-V characteristics the result of heterologous mRNA expression? (ii) Comparison with previous studies. (iii) Inward rectification of membrane currents are ubiquitous. (iv) Different kainate responses in a physiological context. (v) Possible mechanisms to account for inward rectification. (vi) Anomalous outward rectification to kainate: seasonal variability and calcium permeability? 6 (vii) Do kainate and AMPA activate the same ion channel? CHAPTER 6: INTERACTIONS BETWEEN NON-NMDA RECEPTOR AGONISTS 173-205 INTRODUCTION 174-176 RESULTS 176-181 (i) Kainate responses are antagonised by other non-NMDA receptor agonists. (ii) Competitive and non-competitive antagonism of KA responses is species-dependent. (iii) Further characterisation of non-competitive antagonism of KA responses. (iv) The expression of non-NMDA receptor agonist responses. DISCUSSION 181-188 (i) The occurrence of competitive and non­ competitive antagonism. (ii) An interaction between full and partial agonists may explain competitive antagonism. (iii) Possible mechanisms to account for non­ competitive antagonism. (iv) Different types of antagonism may suggest heterogeneity of non-NMDA receptors. CHAPTER 7: THIOCYANATE IONS SELECTIVELY ANTAGONISE AMPA RESPONSES 206-224 INTRODUCTION 207-209 RESULTS 209-211 (i) SCN' selectively antagonised responses to AMPA in a non-competitive manner. 7 DISCUSSION 211-216 (i) Comparison with ligand binding studies. (ii) Mechanisms for antagonism of AMPA responses by SCN'. (iii) Discrete binding sites for non-NMDA receptor agonists on the same receptor/ion channel CHAPTER 8: GENERAL DISCUSSION 225-232 ACKNOWLEDGEMENTS 233 REFERENCES 235-275 PUBLICATIONS 276 8 LIST OF FIGURES 2. l M a in ten a n c e o f fem a le xenopus laevis. 48-49 2.2 R em oval o f x en o pu s oocytes u n d e r anaesthesia . 50-51 2 .3 M icroinjection o f ex o g en o u s mRNA in to XENOPUS OOCYTES. 52-53 2 .4 I dentification and im pa lem en t o f xenopus laevis OOCYTES. 54-55 2 .5 E lectrophysiological r eco r d in g c o n d it io n s . 56-57 2 .6 Cu r r en t and voltage clam p c ir c u it d e t a il s . 58-59 3. l R e sp o n s e s to n o n -nm da r e c e pto r a g o n ist s . 78-79 3.2 N o rm a lised d o se -r e spo n se curve to k a in a te IN OOCYTES INJECTED WITH CALF BRAIN mRNA. 80-81 3 .3 K ainate was e q u i -e ffe c t iv e in all mRNA INJECTED OOCYTES.