Iowa State University Capstones, Theses and Graduate Theses and Dissertations Dissertations 2012 Parasitic nematode ion channels: improving understanding of pharmacology and genetic composition Samuel Buxton Iowa State University Follow this and additional works at: https://lib.dr.iastate.edu/etd Part of the Parasitology Commons, Pharmacology Commons, and the Toxicology Commons Recommended Citation Buxton, Samuel, "Parasitic nematode ion channels: improving understanding of pharmacology and genetic composition" (2012). Graduate Theses and Dissertations. 12965. https://lib.dr.iastate.edu/etd/12965 This Dissertation is brought to you for free and open access by the Iowa State University Capstones, Theses and Dissertations at Iowa State University Digital Repository. It has been accepted for inclusion in Graduate Theses and Dissertations by an authorized administrator of Iowa State University Digital Repository. For more information, please contact [email protected]. Parasitic nematode ion channels: improving understanding of pharmacology and genetic composition by Samuel Buxton A dissertation submitted to the graduate faculty in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Major: Toxicology Program of Study Committee: Richard J. Martin, Major Professor Alan P. Robertson, Major Professor Anumantha G. Kanthasamy Heather M. W. Greenlee Jeffrey K. Beetham Jacques Cabaret Iowa State University Ames, Iowa 2012 Copyright © Samuel Buxton, 2012. All rights reserved. ii TABLE OF CONTENTS ABSTRACT .............................................................................................................................. x CHAPTER 1 General Introduction ........................................................................................... 1 1.1 Introduction ..................................................................................................................... 1 1.2 Thesis Organization ........................................................................................................ 2 CHAPTER 2 Literature Review ............................................................................................. 4 2.1 Soil Transmitted Helminths ............................................................................................ 4 2.1.1 Ascaris spp. and Ascariasis ...................................................................................... 7 2.1.2 Oesophagostomum spp. and Oesophagostomiasis ................................................. 11 2.2 Nematode Muscular and Nervous Systems .................................................................. 14 2.2.1 Nematode Muscular System .................................................................................. 14 2.2.2 Nematode Nervous System .................................................................................... 17 2.2.3 Electrophysiology of Somatic Muscle ................................................................... 20 2.3 Nicotinic Acetylcholine Receptors, nAChR ................................................................. 23 2.3.1 Vertebrate nAChR ................................................................................................. 24 2.3.2 Caenorhabditis elegans AChR .............................................................................. 30 2.3.3 Parasitic nematodes AChR .................................................................................... 36 2.4 Voltage-activated calcium-dependent potassium channels .......................................... 41 iii 2.5 Anthelmintics and anthelmintic resistance ................................................................... 46 2.5.1 Emodepside ............................................................................................................ 46 2.5.2 Levamisole, pyrantel, tribendimidine .................................................................... 51 2.5.3 Anthelmintic resistance .......................................................................................... 53 2.6 Heterologous expression systems: Xenopus laevis oocytes .......................................... 57 CHAPTER 3 On the mode of action of emodepside: slow effects in Ascaris suum .............. 61 3.1 Abstract ......................................................................................................................... 61 3.2 Introduction ................................................................................................................... 62 3.3 Materials and Methods .................................................................................................. 64 3.3.1 Collection and Maintenance of worms .................................................................. 64 3.3.2 Sequence and gene expression analysis ................................................................. 65 3.3.3 Somatic muscle preparation ................................................................................... 66 3.3.4 Electrophysiology of Somatic Muscle ................................................................... 66 3.3.5 Time control experiments ...................................................................................... 70 3.3.6 Data analysis .......................................................................................................... 70 3.3.7 Materials ................................................................................................................ 72 3.4 Results ........................................................................................................................... 72 3.4.1 slo-1 and lat-1 homologous genes from A. suum expressed at adult stage ........... 72 3.4.2 Emodepside has an inhibitory effect on spiking .................................................... 76 iv 3.4.3 Effect of emodepside on membrane potential and input conductance .................. 79 3.4.4 Emodepside effect on membrane potential: role of NO and PKC ......................... 82 3.4.5 Effect of emodepside (1 µM) on voltage-activated K+ currents ............................ 83 3.4.6 Effect of emodepside (10 µM) on voltage-activated K+ currents .......................... 86 3.4.7 Ca2+ is required for effects of emodepside on K+ currents .................................... 89 3.4.8 Emodepside effects on K+ currents: role of NO and PKC ..................................... 89 + 3.4.9 The 4-aminopyridine-sensitive K current includes Ia ........................................... 95 3.4.10 Effect of iberiotoxin ............................................................................................. 96 3.4.11 Emodepside effects on voltage-activated Ca2+ currents ...................................... 98 3.5 Discussion ................................................................................................................... 100 3.5.1 Different mode of action ...................................................................................... 100 3.5.2 Emodepside is not a GABA receptor agonist ...................................................... 100 3.5.3 K+-dependent hyperpolarization by releasing inhibitory neuropeptides ............. 101 3.5.4 Latrophilin Receptors........................................................................................... 102 3.5.5 SLO-1 as a target for emodepside in C. elegans .................................................. 103 3.5.6 SLO-1 as a target for emodepside in Ascaris suum ............................................. 103 3.6 Acknowledgements ..................................................................................................... 105 3.7 Supplementary information ........................................................................................ 107 3.7.1 Diethylcarbamazine (DEC) potentiates emodepside effect ................................. 108 v Chapter 4 Levamisole-sensitive acetylcholine receptors of O. dentatum ............................. 112 4.1 Abstract ....................................................................................................................... 112 4.2 Author Summary ......................................................................................................... 113 4.3 Introduction ................................................................................................................. 114 4.4 Materials and Methods ................................................................................................ 116 4.4.1 Ethical Concerns .................................................................................................. 116 4.4.2 Accession numbers .............................................................................................. 117 4.4.3 Nematode Isolates ................................................................................................ 117 4.4.4 Molecular Biology ............................................................................................... 117 4.4.5 Electrophysiological studies in oocytes ............................................................... 118 4.4.6 Data analysis ........................................................................................................ 118 4.5 Results ......................................................................................................................... 119 4.5.1 Identification of unc-29, acr-8, unc-38 and unc-63 homologs………………….119 4.5.2 Four receptor subtypes reconstituted with four AChR subunit genes ................