Glutamate-gated chloride channel receptors and mechanisms of drug resistance in pathogenic species Mohammed Atif B. Pharmacy, M. Pharmacy (Pharmacology) A thesis submitted for the degree of Doctor of Philosophy at The University of Queensland in 2019 Queensland Brain Institute Dedicated to my beloved parents & my demised brother who I miss everyday ii Thesis Abstract Pentameric ligand-gated ion channels (pLGICs) are important therapeutic targets for a wide range of neurological disorders that include cognitive impairment, stroke, psychiatric conditions and peripheral pain. They are also targets for treating parasite infections and controlling pest species in agriculture, veterinary practice and human health. Here we focus on one family of the pLGICs i.e., the glutamate-gated chloride channel receptors (GluClRs) which are expressed at inhibitory synapses of invertebrates. Ivermectin (IVM) is one of the main drugs used to control pest species and parasites, and it works by activating GluClRs in nematode and arthropod muscle and nerves. IVM resistance is becoming a major problem in many invertebrate pathogens, necessitating the development of novel anti-parasitic drugs. This project started with the simple aim of determining the sensitivity to glutamate and IVM of GluClRs from two different pest species: the parasitic nematode Haemonchus contortus (HcoGluClRs) and the mosquito malaria vector Anopheles gambiae (AgGluClRs). In chapter 3, we found that the β homomeric GluClRs of H.contortus were insensitive to IVM (EC50> 10 µM), whereas α homomeric HcoGluClRs were highly sensitive (EC50 = 20 nM). Heteromeric αβ HcoGluClRs exhibited an intermediate sensitivity to IVM (EC50 = 135 nM). By contrast, the EC50 values for glutamate at α homomeric and αβ heteromeric receptors were not distinguishable; falling between 20-30 µM. GluClR-mediated IPSCs were significantly different for α relative to αβ receptors, with αβ GluClRs exhibiting a faster decay rate of 17 ms in contrast to α GluClRs which had a decay rate of 40ms. 5 nM IVM slowed the decay rates of IPSCs mediated by both isoforms with α GluClRs reaching a mean decay rate of 100 ms and αβ GluClRs decaying at a mean rate of 72ms. Evaluating the biophysical properties of single heteromeric αβ and homomeric β GluClRs revealed that different stoichiometric GluClR isoforms exhibited different open state amplitudes. The α GluClR had the biggest current (1.8 pA) followed by the αβ (1:1) GluClR (two different amplitudes – 1.2 pA, 90% and 0.7 pA, 10%) and the smallest being β homomer (0.4 pA). The β and αβ GluClRs had mean active durations of 5.5 ms and 43 ms, respectively, in response to 2 µM glutamate. This duration increased to 126 ms and 876 ms, respectively, when 2 µM glutamate was co-applied with 5 nM IVM. Collectively, the data from both the IPSCs and the single channel measurements show a correlation between shorter active periods; faster IPSC decay rates and decreased IVM sensitivity. iii For better drug optimisation, a quantitative understanding of activation and modulation mechanisms of the GluClR is necessary. For this purpose, in chapter 4 we evaluated the biophysical properties of the α homomeric HcoGluClR and a mutation to a position that confers IVM resistance in several pest species (TM3-36’) in presence of glutamate and IVM. We showed that the G36’A mutation decreased the duration of active periods and increased receptor desensitisation. As the results showed a common effect for both glutamate- and IVM-gated currents, we infer that the intrinsic properties of the receptor were affected by the G36’A mutation and not the IVM binding mechanism as previously proposed. In Chapter 5, we looked to characterize the effects of glutamate, IVM, lindane, fipronil and picrotoxin on 2 splice isoforms of GluClRs expressed by A. gambiae. The two splice isoforms AgGluCl-a1 and AgGluCl-b when evaluated as homomeric GluClRs for effects of glutamate and IVM disclosed something interesting. We found that the AgGluCl-b was sensitive to IVM, contrary to what was reported by (Meyers et al. 2015). Lindane and fipronil both block glycine receptors (Islam and Lynch 2012), which, like the GluClR, is an inhibitory member of pLGIC family. In addition, many insecticides target GluClRs, this prompted us to investigate how lindane and fipronil affected the two GluClR isoforms. Both compounds were effective at inhibiting glutamate-gated currents mediated by both isoforms. Glutamate-gated currents completely recovered in a time dependent manner for AgGluClR-a1. By contrast, only a partial recovery of currents was observed for AgGluClR-b, suggesting a greater affinity of lindane and fipronil at AgGluClR-b. This differential affinity implies different binding mechanisms between the two isoforms. Picrotoxin, a classical pore blocker, equally blocked glutamate currents for both the isoforms. In chapter 6, we screened libraries of natural marine extracts against the hippocampal-dominant α5 GABAAR isoform (α5β3γ2L) using an automated yellow fluorescent protein-based screening assay to identify fractions with selective activity. Hits were confirmed using electrophysiology. Although the hits were potent, further screening of the compounds was not proceeded due to low compound availability. In conclusion, we demonstrated that reduced IVM sensitivity is due to shorter active periods and faster decay rates. This conclusion was supported by the findings from chapter 3 that showed that heteromeric population of αβ HcoGluClRs were less sensitive to the potentiating effects of IVM compared to α HcoGluClRs. The briefer single receptor periods iv seen in β-containing GluClRs correspond closely with, faster IPSC decay rates. All the results point towards β HcoGluClRs being responsible for reduced IVM sensitivity. v Declaration by author This thesis is composed of my original work, and contains no material previously published or written by another person except where due reference has been made in the text. I have clearly stated the contribution by others to jointly-authored works that I have included in my thesis. I have clearly stated the contribution of others to my thesis as a whole, including statistical assistance, survey design, data analysis, significant technical procedures, professional editorial advice, financial support and any other original research work used or reported in my thesis. The content of my thesis is the result of work I have carried out since the commencement of my higher degree by research candidature and does not include a substantial part of work that has been submitted to qualify for the award of any other degree or diploma in any university or other tertiary institution. I have clearly stated which parts of my thesis, if any, have been submitted to qualify for another award. I acknowledge that an electronic copy of my thesis must be lodged with the University Library and, subject to the policy and procedures of The University of Queensland, the thesis be made available for research and study in accordance with the Copyright Act 1968 unless a period of embargo has been approved by the Dean of the Graduate School. I acknowledge that copyright of all material contained in my thesis resides with the copyright holder(s) of that material. Where appropriate I have obtained copyright permission from the copyright holder to reproduce material in this thesis and have sought permission from co- authors for any jointly authored works included in the thesis. Mohammed Atif vi Publications during candidature Peer-reviewed journal publications Atif M, Estrada-Mondragon A, Nguyen B, Lynch JW, Keramidas A (2017) Effects of glutamate and ivermectin on single glutamate-gated chloride channels of the parasitic nematode H. contortus. (Chapter 4) PLoSPathog 13(10): e1006663. https://doi.org/10.1371/journal.ppat.1006663. Submitted manuscripts included in this thesis Atif M, Smith JJ, Estrada-Mondragon A, Xiao X, Capon RJ, Lynch JW and Keramidas A (2018) GluClR-mediated inhibitory postsynaptic currents reveal targets for ivermectin and potential mechanisms of IVM resistance (Chapter 3) (In press – PLoS pathogens). Presentations 1. ASB 2017; Annual Meeting of the Australian society for Biophysics, Sydney, Australia. Atif M, Lynch JW and Keramidas A. Modulating ivermectin sensitivity at glutamate- gated chloride channels of H.contortus and A.gambiae (Poster) 2. SfN 2018; Society for Neuroscience, San Diego, USA. Atif M, Smith JJ, Lynch JW and Keramidas A. Modulating ivermectin sensitivity at glutamate-gated chloride channels of H. contortus and study of inhibitory postsynaptic currents mediated by α and αβ HcoGluCl receptors (Poster). 3. ANS 2018; 38th Annual scientific Meeting of the Australasian Neuroscience Society, Brisbane, Australia Atif M, Smith JJ, Lynch JW and Keramidas A. Modulating ivermectin sensitivity at glutamate-gated chloride channels of H. contortus and study of inhibitory postsynaptic currents mediated by α and αβ HcoGluCl receptors (Poster). vii Publications included in this thesis Atif M, Estrada-Mondragon A, Nguyen B, Lynch JW, Keramidas A (2017) Effects of glutamate and ivermectin on single glutamate-gated chloride channels of the parasitic nematode H. contortus. (Incorporated as Chapter 4) PLoSPathog 13(10): e1006663. https://doi.org/10.1371/journal.ppat.1006663. Contributor Statement of contribution Author Mohammed Atif (Candidate) Electrophysiological recordings and data analysis (40%). Figure preparations (20%). Subsequent draft editing (20%) Author Argel Estrada-Mondragon Molecular study