Selective Effects of Paraherquamide on Ascaris Suum Nachr Subtypes

Iowa State University Capstones, Theses and Retrospective Theses and Dissertations Dissertations

1-1-2003

Selective effects of paraherquamide on Ascaris suum nAChR subtypes

Hai Qian Iowa State University

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Recommended Citation Qian, Hai, "Selective effects of paraherquamide on Ascaris suum nAChR subtypes" (2003). Retrospective Theses and Dissertations. 19544. https://lib.dr.iastate.edu/rtd/19544

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Hai Qian

A thesis submitted to the graduate faculty in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE

Major: Neuroscience

Program of Study Committee: Richard Martin, Major Professor Alan Robertson Walter Hsu Tim Day Douglas Jones

Iowa State University Ames, Iowa 2003

Graduate College Iowa State University

This is to certify that the master's thesis of

Hai Qian has met the thesis requirements of Iowa State University

--,

Signatures have been redacted for privacy 111

TABLE of CONTENTS LIST OF FIGURES vi

LIST of TABLES Vlll

ABSTRACT ix

CHAPTER 1. GENERAL INTRCJDUCTIUN 1.1. Introduction 1 1.2. Thesis organization 1

CHAPTER 2. LITERATURE REVIEW 2.1. Parasites 3 2.1.1. Parasitic nematodes: ~iscaris suum and 14scaris lumbricordes 2.2. Nervous system of 14scaris 5 2.3. The somatic muscle cell of Ascaris 7 2.3.1. Belly 2.3.2. Arm 2.3.3. Spindle 2.3.4. Membrane potential of the muscle cell 2.3.5. Spontaneous electrical activity of the muscle cell 2.4. Neurotransmitters and pharmacology of the neuromuscular system 10 2.4.1. Acetylcholine 2.4.2. GABA 2.5. Anthelmintics: levamisole and paraherquamide 12 2.5.1. Levamisole 2.5.2. Paraherquamide 2.6. Structure of nicotinic acetylcholine receptors 14 2.6.1. The structure of Torpedo nACh receptors 2.6.2. Structural changes during ligand binding 1V

2.6.3. The amino acid sequences of Torpedo nicotinic acetylcholine receptors

2.7. Subunits of nicotinic acetylcholine receptors 20 2.7.1. The subunits of nAChRs in the nematode C. elegans a. Levamiso e receptors b. DEG-3-like c. ACR-8-like 2.7.2. The subunits of nAChRs in vertebrates

2.8. Pharmacological subtypes of nematode nAChRs 24

CHAPTER 3. MATERIALS AND METHODS 3.1. Collection and maintenance of ~4scaris suum 26 3.2. The vesicle preparation 26 3.3. Single-channel recording 28 3.3.1. Instruments a. Amplifier b . Digitizer c. Instrument setup 3.3.2. Electrodes 3.3.3. Measuring the pipette resistance 3.3.4. Reducing background noise 3.3.5. A giga-ohm seal 3.3.6. Recording ion channel currents 3.3.7. Application of paraherquamide 3.3.8. Storage of data 3.4. Data processing 3 3 3.4.1. Software tools 3.4.2. Event list s 3.4.3. Single-channel conductance

3.4.4 • Popen 3.4.5. Mean open time CHAPTER 4. SINGLE-CHANNEL CURRENTS FROM ASCARIS SUUM LEVAMISOLE-ACTIVATED RECEPTORS 4.1. The single-channel conductances show more than one level 3 9 4.2. The mean open time is correlative to the single-channel conductance 45 4.3. Discussion 48

CHAPTER 5. TAE SELECTIVE EFFECTS OF PARAHERQUAMIDE ON THE LEVAMISOLE-ACTIVATED RECEPTORS LOCATED ON ASCARIS SUUM MUSCLE CELLS 5.1. Paraherquamide is an antagonist of the nAChR located on Ascaris scum 50 muscle cells 5.2. The inhibitory effects of paraherquamide are selective on the nAChR 53 subtypes 5.3. Discussion 57

CHAPTER 6. GENERAL DISCUSSION 6.1. The mean level-Popen of the three nAChR subtypes and the %contribution 58 of each subtype to the whole cell current induced by 30 ~,M levamisole at +75 mV 6.2. Numbers of the three nAChR subtypes on Ascaris suum muscle cells 60

6.3 . Channel-Popen of the three nAChR subtypes 61 6.4. The paraherquamide effects on the whole cell currents 62 6.5. Do the conductance subtypes correspond to pharmacological subtypes? 62

CHAPTER 7. CONCLUSIONS 64

REFERENCES 66

ACKNOWLEDGEMENTS 76 V1

LIST OF FIGURES Figure 2.1. The life cycle of nematode Ascaris lumbricoides 4 Figure 2.2. The anterior part of the nervous system S Figure 2.3 . Transverse section of Ascaris 6 Figure 2.4. Diagram of the dorsal and ventral nerve cord in one segment of Ascaris 6 Figure 2.5. Schematic diagram of the muscle cell and the neuromuscular junction 8 Figure 2.6. An example of Spontaneous electrical activities recorded from the bag 9 region of Ascaris muscle cells Figure 2.7. Chemical structure of acetylcholine 11 Figure 2.8. Chemical structure of gamma-aminobutryic acid (GABA) 12 Figure 2.9. Chemical structure of levamisole 13 Figure 2.10. The chemical structure of the paraherquamide A 14 Figure 2.11. Diagram of the nicotinic acetylcholine receptor 16 Figure 2.12. The ACh binding of nAChRs in top view 17 Figure 2.13. The secondary structure of the nicotinic acetylcholine receptor subunit 17 Figure 2.14. The amino acid sequences of the Torpedo nAChR subunits 19 Figure 2.15. The diagram of the tree of the C. elegans nAChR subunits 21 Figure 3.1. Schematic diagram of the instrument setup 3 0 Figure 3.2. An example of the events list 34 Figure 3.3 . The Popen of the levamisole-activated receptor 3 6 Figure 4.1. The single-channel currents recorded at four different membrane 40 potentials Figure 4.2. The single-channel currents observed at +75 mV membrane potential on 41 a shorter time scale Figure 4.3. The amplitude histogram 41 Figure 4.4 . The I- T~ plot 42 Figure 4.5 . The histogram of collected conductances from 3 0 patches 44 Figure 4.6. The open dwell-time at +75 mV 45 Figure 4.7. The scatter plot of the mean open time vs. single-channel conductance 47 Figure 4.8. The mean open time for each conductance level 47 Vll

Figure 4.9. Double-opening of single-channel current at +75 mV membrane 49 potential Figure S .1. The inhibitory effect of paraherquamide on the levamisole-activated single-channel currents of the ~4scaris suum nAChR 51 Figure 5.2. Paraherquamide reduces the patch-Popen of the levamisole-activated 52 receptor with aconcentration-dependant manner Figure 5.3. The selective effects of 1µM paraherquamide on the nAChR subtypes 54 Figure 5.4. The inhibitory effects of paraherquamide on the three subtypes 56 Figure 6.1. A model for calculating the contributions of the three subtypes nAChRs 60 to the transmembrane currents induced by 3 0 µM levamisole Vlll

LIST OF TABLES Table 2.1. Hierarchy of the Ligand Gated Ion Channel 15 Table 3.1. Composition of solutions used in experiments 27 Table 4.1. The conductances and reversal potentials of the levamisole-activated 43 receptor Table 4.2. The mean open times at +75 mV 46 Table 5.1. The effects of 3µM paraherquamide on the conductance and the mean 53 open time

Table 5.2. The effect of paraherquamide on the level-Pope s of the nAChR subtypes 5 5

Table 6.1. The level-Popen s observed from 30 patches 59 1X

ABSTRACT

Nematode parasites infest humans and animals causing morbidity and significant economic loss. Ascaris suum lives in the small intestine of the pig. Ascaris infestation of pigs can cause Ascariasis and poor growth rate. Another species, Ascaris lumbricoides infects humans. More than one billion people in the world suffer the Ascaris infestation (Crompton, 1984). Nicotinic anthelmintic compounds, which act on nematode nicotinic acetylcholine receptors (nAChRs), used to treat nematode infestation. However, the long-term repeated intake of one anthelmintic compound may cause the drug resistance. Robertson (1999) suggested that the resistance to anthelmintic compound levamisole may due to the loss of a levamisole-sensitive nAChR subtypes. Paraherquamide is a novel anthelmintic compound and is effective against strains of parasites that are resistant to the known broad-spectrum anthelmintic drugs (Shoop et al., 1990, 1991, 1992). The published data suggests that it behaves like a competitive antagonist of nematode nAChRs located on the nematode somatic musculature. In this research the effect of paraherquamide on the nematode Ascaris suum nAChRs was investigated at the single-channel level. Membrane vesicles were prepared from collagenase-treated Ascaris muscle flaps. The single-channel currents induced by 30 µM levamisole were recorded using inside-out patches isolated from the vesicles. The channels had conductances ranging from 18 pS to 52 pS. Paraherquamide was added to the bath chamber during recordings to give a final concentration of 0.3 -10 µM and its effects on the single-channel currents were

monitored. Paraherquamide reduced the probability of channel being open (Popen) in a

concentration-dependent manner. 1 µM paraherquamide selectively reduced the Popen values

of the 45 pS channel; it had less effect on the Popen of the 3 5 pS channel, and the smallest effect on the 21 p S channel. This selectivity illustrates that the 21, 3 5 and 4 5 p S channels are pharmacologically distinct and suggests structural differences. The present study demonstrates that paraherquamide is an antagonist of the nAChRs located on the somatic musculature of parasitic nematodes and confirms at the single-channel level the selective effect of paraherquamide on the Ascaris nAChRs subtypes. 1

CHAPTER 1. GENERAL INTRODUCTION

1.1. Introduction Parasite Ascaris is widely distributed all over the world. The infestation of Ascaris causes the disease Ascariasis in domestic animals and humans. The nicotinic anthelmintics such as levamisole and pyrantel are used to treat the Ascariasis (Aceves et al, 1970; Aubery et al., 1970; Harrow & Gration et al., 1985). These compounds have been shown as agonists of nicotinic acetylcholine receptors (nAChRs) on the Ascaris muscle cells. The mechanisms of these compounds have been studies in our laboratory for a long time. The pharmacological studies indicate the existence of three pharmacological nAChR subtypes, levamisole- preferring, nicotine-preferring and bephenium-preferring (Robertson et al., 2002). The discovery of the at least 27 nAChR genes in C. elegans indicates the existence of multiply biophysical nAChR subtypes in nematodes (Bargmann, 1998). The purpose of this study is to investigate the levamisole-activated nAChR located on the Ascaris muscle cells and the mechanism of a novel anthelmintic paraherquamide. In this thesis, I report that at least three nAChR subtypes are activated by anthelmintic levamisole at single-channel level. Each subtype has individual single-channel properties. Also I report the inhibitory effects of paraherquamide. Paraherquamide has been reported to behave as antagonist of the nAChR located on the Ascaris muscle cells. At the single-channel level, I found the selective effects of paraherquamide on the three nAChR subtypes. These observations are the first study of the mechanism of paraherquamide at the single-channel level. Compared to the paraherquamide study using muscle contraction experiments (Robertson et al. , 2002), I suggest a correlation between the pharmacological and biophysical nAChR subtypes.

1.2. Thesis Organization In chapter 2, the background information of the parasite Ascaris and the anthelmintics is reviewed as well as the molecular and pharmacological information of nAChRs. In chapter 3, I summarize the materials and methods for our single-channel experiments. It is worth to

note that in this chapter, I define three terms: patch Popen, level-Popen and channel-Popen. In 2 chapter 4, I report the levamisole-activated single-channel properties and suggest the existence of the three nAChR subtypes on the Ascaris muscle cells. In chapter 5, I report the inhibitory effects of paraherquamide on the levamisole-activated nAChRs and the selective effects of paraherquamide on the three nAChR subtypes. In chapter 6, the contribution of each nAChR subtype to the whole .levamisole-induced current is discussed, as well as the paraherquamide inhibitory effect on the whole-cell current. In chapter 7, the general conclusion, I summarize the observations and results of this study and suggest the further studies, which may testify this study and give better understanding of the nematode levamisole-activated nAChR and the mechanism of paraherquamide. 3

CHAPTER 2. LITERATURE REVIEW

2.1. Parasites "A parasite is an organism that must spend part of its development in or on another living organism which is known as the host. During the relationship, the host provides the parasite with food, shelter and the exact conditions necessary for the parasite's growth and survival." (Crompton, 1984). Parasites are classified as ectoparasites and endoparasites. Parasites that are exposed to the outer surfaces of hosts and can be seen directly with little or no dissection of the host are known as ectoparasites. Endoparasites are defined as those that are located inside the organs, cavities and tissue of the host. Common ectoparasites of humans and animals include lice, fleas and ticks. The common types of parasitic worms, one of the endoparasites, include platyhelminthes (flatworms), nematodes (roundworms) and acanthocephala (spiny-headed worms). Among these worms, nematodes usually have cylindrical shapes and separate sexes. Common nematodes include: roundworm (Ascaris lumbricoides, Ascaris suum), hookworm (Necator americanus, Ancylostoma duodenale), pinworm (Enterobius vermicularis), heartworm (Dirofilaria immitis), strongyles (Stronglyoides stercoralis), whipworm (Trichinella spiralis) and filarial worms ~uchereria bancrofti, Brugia malayi, Onchocerca volvulus, Loa loa, 1Vlansonella streptocerca, Mansonella perstans, Mansonella ozzardi) . They range in size from microscopic microfilaria to adult Ascaris sp. of up to 40 cm in length.

2.1.1. Parasitic nematodes: Ascaris suum and Ascaris lumbricoides Two Ascaris species are: A. lumbricoides in humans and A. suum in pigs. Ascaris are large nematodes: adult females are 20-40 cm in length and 0.3-0.6 cm in diameter, adult males are 15-30 cm in length and 0.2-0.4 cm in diameter. Adult females are easily recognized by their large ovaries which occupy the posterior ~60% of the worm. Figure 2.1 shows the life circle of A. lumbricoides. Adults are normally found in the lumen of the small intestine. A female is reported to release about 200,000 eggs per day, which are passed with the feces. Infective eggs contaminate food and water. Following ingestion of contaminated food or water, Ascaris larvae hatch in the host intestine. Larvae migrate through the intestinal wall 4 and then reach the lungs. After molting, L4 larvae migrate up the trachea and are swallowed. The L4 larvae then develop into adults in the small intestine. Infestation with Ascaris causes the disease Ascariasis. The host feels abdominal pain and nausea with disturbed function of the alimentary tract. When larvae arrive in the lungs, the host may have some respiratory symptoms.

Figure 2.1. The life cycle of nematode Ascaris lumbricoides. Adult A. lumbricoides are found in the lumen of the small intestine. The eggs produced by females are defecated with feces. Infective eggs contaminate food and water. Following ingestion of contaminated food or water, Ascaris larvae hatch in the host intestine. Larvae migrate through the intestinal wall and then reach the lungs. After molting, L4 larvae migrate up the trachea and are swallowed. L4 larvae then develop into adults in the small intestine (modified from http://www.thelifetree.com/roundworms.cfin).

Ascaris lumbricoides is distributed worldwide, particularly in developing countries. Between 800 million and 1.3 billion people are infected by this parasitic nematode (Crompton, 1984). Ascaris suum is a very common parasite infecting pigs and is also distributed worldwide. Infestation causes poor growth rate of pigs and consequently considerable economic loss. The only anatomical difference between these two species is that 5

Ascaris suum has a row of small teeth on the lips (Lapage, 1963). Although they are very similar, infection of man with Ascaris suum or pigs with Ascaris lumbricoides is rare (Urquart et al., 1987). Anthelmintic drugs are used to treat Ascaris infestations. One class of these drugs is the nicotinic anthelmintic compounds, such as levamisole and pyrantel. Application of these compounds to whole worms or to a muscle flap preparation induces spastic paralysis of Ascaris suum associated with muscle depolarization and contraction (Coles et al., 1975; Martin, 1982). Studies using the patch-clamp technique confirm that these compounds act as nACh receptor agonists (Pennington and Martin, 1990).

2.2. Nervous system of Ascaris The central nervous system of Ascaris is near the head and consists of a set of ganglia associated with a nerve ring (Figure 2.2). This ring receives the primary sensory input and gives rise to the cephalic muscle motor output. Two major nerve cords, dorsal and ventral, originate from the nerve ring (Figure 2.2 and 2.3). There is also one small lateral nerve cord on each side, named the dorsal lateral and ventral lateral nerve cords (reviewed by DeBell, 1965).

Mouth and lips"

2 mm

. Nerve ri ng

Dorsal nerve Dorsal lateral nerve: Ventral nerve

Figure 2.2. The anterior part of the nervous system. The nerve ring receives the primary sensory input and gives rise to the cephalic muscle motor output. The two major nerve cords, dorsal and ventral, originate from the nerve ring (modified from Crompton, 1980). 6

Dorsal nerve cord

Syncytium

~.-~~ ,~ Muscle bag ~~ ~~.~ Lateral canal Gy1 ~ f ~"=---~--~- Lateral line Perienteric fluid

~`''-- Hypodermis ~---~_ Cuticle

,_ 1 mm Ventral nerve cord

Figure 2.3. Transverse section of Ascaris. The dorsal and ventral nerve cords are indicated as well as the two small lateral nerve cords. The two lateral lines separate somatic muscles into ventral and dorsal layers

bC ~ Right _,,.,.,~ commissures ~'

Figure 2.4. Diagram of the dorsal and ventral nerve cord in one segment of Ascaris (modified from Martin et al. , 1996b). Seven anatomical types of motorneurons are present: dorsal excitatory (DEI, DE2 and DE3), dorsal inhibitory (DI), ventral inhibitory (VI), ventral excitatory (V 1 and V2). O Inhibitory motorneuron • Excitatory motorneuron D Inhibitory output ~ Excitatory output 7

The anatomical studies show that the cell bodies of motorneurons are located in the ventral nerve cord and connect with the dorsal nerve cord through one left-hand and three right-hand commissures (Figure 2.4). There are seven classes of motorneuron: dorsal excitatory DE1, DE2 and DE3; ventral excitatory V1 and V2; dorsal inhibitory DI and ventral inhibitory VI. The connection commissures are arranged in a pattern along the nematode body forming eve segments and each segment contains eleven motorneurons: one of DI, DE2 and DE3, two of DE1, VI, V1 and V2 (Stretton et al. , 1978, quoted by Martin, 1996).

2.3. The somatic muscle cell of Ascaris Two lateral lines separate somatic muscles into ventral and dorsal layers (Figure 2.3, Stretton, 1976; quoted by Del Castillo et al., 1989). Each somatic muscle cell has three parts (Figure 2.5): a spindle region of ~2 mm in length which is anchored to the hypodermis, a balloon-like bag of 200-250 µm in diameter which contains the nucleus, and arms which arises at the base of the bag region and extends to the nerve cord.

2.3.1. Belly The belly or bag region of the Ascaris muscle cell is in the pseudocoelomic space. The cortical region of the belly contains fibrillar bundles of cytoskeleton and a row of small mitochondria. Harris and Crofton (1957) suggested that the belly serves as an endoskeleton. The cytoplasm contains the nucleus and is filled with glycogen particles. These glycogen particles are depleted during starvation or exercise (Rosenbluth 1963).

2.3.2. Arm The ann is a thin process rising from the muscle belly. One muscle cell may have more than one and up to five arms (Rosenbluth, 1965b). When the arms arrive at the nerve cord, they separate into several processes in bundles with gaps of 400-500 µM. The arms overlap each other to form a structure called the "syncytium" (Del Castillo et al., 1989) (see Figures 2.5). Synapses are formed between the syncytium and the nerve cord. 8

Figure 2.5. Schematic diagram of the muscle cell and the neuromuscular junction. The muscle cell includes a spindle, a belly (or bag) and one or more arm regions. The neuromuscular junction is formed between the end of the arms (which forms a syncytium) and the nerve cord. n: nerve cord; h: hypodermis; c: cuticle (modified from Rosenbluth, 1973).

2.3.3. Spindle The spindle region contains the contractile apparatus of the muscle cell forming a long tube lying against the hypodermis (Rosenluth, 1965a). Despite the similarity of the contractile apparatus to that of the cross-strated muscle, the contraction and relaxation Ascaris muscle are slow and it has been suggested that this muscle has the biochemical characteristics of that in a "smooth" muscle (Rosenbluth, 1967).

2.3.4. Membrane potential of the muscle cell The electrical activity of the somatic cells of Ascaris has been studied. The average resting membrane potential of the belly region is about -30 mV (DeBell et al., 1963). The membrane potential varies with different bathing solutions. For example, the average value was -29.83 mV in diluted (30%) sea water and -34.5 mV in the perienteric fluid (Del Castillo et al., 1964a). One of the features of the resting membrane potential of Ascaris muscle cells 9 is the relative insensitivity to change in the composition of extracellular concentrations of Na+ and K+ (Brading and Caldwell, 1971). Del Castillo et al. (1964b) found the membrane potential was affected by changes in external C1- concentration and proposed that there is a Cl- battery in the membrane to maintain the potential. However, Brading and Caldwell (1971) doubted this suggestion and proposed a modified Goldman-Hodgkin-Katz equation:

RT PK[K + +Pcl[Cl ]; +x E = In ]o +PNa[Na'+]o F Px[K+]~ +PNQ[Na+]; +Pct~Cl ]o +y where they suggest that the relative insensitivity to K+ and Na+ is due to the two large additional components represented by x and y .They proposed an electrogenic ion transport mechanism: a carboxylic acid transport system associated with Na° transport.

2.3. S. Spontaneous electrical activity of the muscle cell

0 mV

Figure 2.6. An example of spontaneous electrical activities recorded from the bag region of Ascaris muscle cells. Each spontaneous activity has three phases: a slow wave, a spike and a transient post-spike hyperpolarization (modified from DeBell et al. , 1963).

The steady membrane potential is rhythmically interrupted by spontaneous depolarizations with a frequency of 1.5-7 per second (DeBell et al., 1963). The spontaneous activity (Figure 2.6) has three phases: slow waves lasting up to 0.2 sec, spikes of ~22 ms duration (DeBell et al., 1963) and transient post-spike hyperpolarization (Weisblat et al., 10

1976; reviewed by Del Castillo et al., 1989). DeBell et al. (1963) stimulated the belly region of the muscle cell with external electrodes and failed to induce propagated electrical activities, while electrical stimulation to the nerve cord gave rise to potential activities similar to the spontaneous activities. These observations suggest that the release of neurotransmitters from the presynaptic nerve cord induce postsynaptic potentials. These "pacemaker" potentials passively propagate from the arm regions of the muscle cells to the belly regions. Del Castillo et al. (1989) concluded that the amplitude of the slow waves and the frequency of the spikes were modulated by the interplay of neurotransmitters ACh (excitatory) and GABA (inhibitory).

2.4. Neurotransmitters and pharmacology of the neuromuscular system The two common neurotransmitters that modulate the electrical activity of Ascaris somatic muscle cells are acetylcholine (ACh) and y-aminobutyric acid (GABA). It had been thought that ACh and GABA receptors were exclusively located at the neuromuscular junction. However, Brading and Caldwell (1971) found that the bag region of the muscle cells also responded to the application of ACh and GABA (reviewed by Del Castillo et al. 1989). Subsequent experiments by Martin (1980, 1982) confirmed the presence of the extrasynapic ACh and GABA receptors. The physiological roles of these e~rasynaptic receptors remain to be understood. Harrow and Gration (1985) suggested that the extrasynaptic ACh receptor was the target site of nicotinic anthelmintic compounds such as levamisole, pyrantel and morantel.

2.4.1. Acetylcholine In 1949, Baldwin and Moyle observed that acetylcholine (Figure 2.7) produced muscle contraction (Baldwin and Moyle, 1949; reviewed by Del Castillo et al. 1989). Electrophysiology studies confirmed that acetylcholine induces muscle cell depolarization and an increase in cation conductance (Martin, 1982). The existence of cholinesterase in the excitatory motorneurons also suggests that acetylcholine is an excitatory neurotransmitter in Ascaris (Bueding, 1952; Lee, 1962; reviewed by Del Castillo et al., 1989). 11

O CH3 I I I + CH3 -C-OCH2 CH2 -N-CH3 CH3

Figure 2.7. The chemical structure of acetylcholine. Acetylcholine, which is synthesized from choline and acetyl-CoA, is one of the major neurotransmitters, Acetylcholine is hydrolyzed by acetylcholinesterase into choline and acetate after release into the synapses.

Nicotine also activates the ACh receptor and produces muscle contraction at low concentrations (Baldwin and Moyle, 1949). Dimethylphenylpiperazinium (DMPP), a selective ganglionic nicotinic agonist, was shown to be a very potent agonist of Ascaris acetylcholine receptors (Colghoun et al., 1990). Some anthelmintic compounds (levamisole, morantel and pyrantel) were also reported to be agonists of the Ascaris acetylcholine receptor (Natoff, 1969; Harrow & Gration, 1985; Colquhoun et al., 1990). The muscle contraction induced by acetylcholine was blocked by nicotinic antagonist d-tubocurarine (dTC) (Norton & De Beer, 1957; reviewed by Del Castillo et al., 1989). The specific ganglionic antagonists mecamylamine and methyllycaconitine are potent cholinergic antagonists in Ascaris (Natoff, 1969; Colquhoun et al., 1990). These observations suggest that the Ascaris nicotinic acetylcholine receptor is most similar to vertebrate ganglionic nicotinic receptors rather than the receptors in vertebrate muscles or CNS. However, hexamethonium, a nicotinic cholinergic antagonist often referred to as the ganglionic blocker, is less potent on Ascaris acetylcholine receptors than on ganglionic ones demonstrating they are not identical (Rozhkova et al. , 1980; Colquhoun et al. , 1990).

2.4.2. GABA Standen (1955) observed that the anthelmintic piperazine induced a reversible paralysis of Ascaris suum. Norton and De Beer (1957) originally suggested this anthelmintic behaves as an acetylcholine antagonist. But Del Castillo et al. (1964) found that the piperazine- induced paralysis could be mimicked by stimulating the nerve cord. This observation led to the proposal that piperazine acted like an endogenous inhibitory transmitter. GABA (Figure 2.8) was found to have similar effects to piperazine (Del Castillo et al., 1964b). Both GABA 12 and piperazine induce a marked hyperpolarization of the Ascaris muscle membrane, but GABA is 10 times more potent than piperazine (reviewed by Del Castillo et al., 1989). These observations demonstrate that GABA is the inhibitory neurotransmitter in Ascaris.

NH2 OII CH3CHCH2 - C - OH

Figure 2.8. The chemical structure of gamma-aminobutryic acid (GABA). GABA is an amino acid, which has been shown to be an inhibitory neurotransmitter in Ascaris.

2.5. Anthelmintics: levamisole and paraherquamide Nicotinic agonists, including levamisole, pyrantel, and morantel, are one of the major types of anthelmintic used to treat both adult and larval nematode infections of domestic animals. These compounds have been reported to cause depolarization and contraction of nematode muscle and produce spastic paralysis of parasites (Aceves et al. , 1970; ~ Aubery et al., 1970). Levamisole, pyrantel, and morantel act as agonists of nACh receptors on the muscle bag membranes of Ascaris suum. The relative potencies of levamisole, pyrantel, and morantel have been determined from concentration-conductance relationships on whole cells and found to be: morantel =pyrantel > levamisole > ACh (Harrow & Gration et al., 1985). These compounds also act as cholinergic receptor agonists in other invertebrates and vertebrates. The relative potencies of the drugs are different for receptors in the different species (Eyre, 1997). paraherquamide is a novel anthelmintic, which was observed to induce flaccid paralysis of nematodes. The published data suggests that it behaves like a competitive antagonist of nicotinic acetylcholine receptors (Robertson et al., 2002).

2.5.1. Levamisole Levamisole is (-)-2,3,5,6-tetrahydro-6-phenylimidazo[2,1-b]thiazole (Figure 2.9). Following oral admission in sheep and goats, levamisole is rapidly absorbed from the 13 gastrointestinal tract and gives excellent results against both adults and larvae of most nematodes (Renoux, 1980).

Figure 2.9. Chemical structure of levamisole. Molecular Formula is C1iH1aNaS. Levamisole is a potent anthelmintic used to treat nematode infestation. This compound has been shown to act as an agonist of nACh receptors .

After uptake of levamisole, there is an initial contraction followed by a paralysis of whole Ascaris (Aceves et al., 1970). Levamisole was found to induce an irreversible contraction that could not be inhibited by atropine, tubocurarine, hexamethonium or piperazine (Coles et al., 1974, 1975). This observation suggested that levamisole might act at ACh receptors without inducing the release of ACh. Using intracellular current and voltage clamp techniques, Harrow and Gration (1985) showed that levamisole acted at ACh receptors located on the muscle bag membrane of Ascaris suum causing an increase in input conductance and depolarization of the muscle cells.

2. S. 2. Paraherquamide Paraherquamide is an oxindole alkaloid metabolite of Penicillium paraherquii. The first isolation of paraherquamide was back in 1981 (Yamazaki et al., 1981; cited by Zinser et al., 2002). Subsequently, Shoop et al. (1990) detected the anthelmintic activity of paraherquamide. Paraherquamide is effective against strains of parasites that are resistant to the known broad-spectrum anthelmintic drugs (Shoop et al., 1990, 1991, 1992). The paraherquamide family consists of a group of analogues (paraherquamide A-G); among them paraherquamide A (Figure 2.10) is most active. Recently, some research groups including Phamacia Upjohn investigated other new analogues, one of which, 2-deoxo-paxaherquamide was chosen for further study. Zinser et al. (2002) observed that paraherquamide and 2-deoxo- paraherquamide applied in vitro induced flaccid paralysis of nematode somatic muscle 14 without affecting ATP levels. Another research group also reported that these two compounds behaved as competitive antagonists of nAChRs in nematode Ascaris suum (Robertson et al., 2002).

Figure 2.10. The chemical structure of the paraherquamide A. The O* is missing in 2-deoxy-paraherquamide.

2.6. Structure of nicotinic acetylcholine receptors Nicotinic acetylcholine receptors belong to the large superfamily of ligand-gated ion channels (LGIC) which include 5-HT3, GABA, and glycine receptors (Cockcroft et al. 1992). The Hierachy of the ligand-gated ion channel family is shown in Table 2.1. Each LGIC is comprised of five subunits. The subunits surround a central pore. nAChRs, a superfamily of these LGIC, are distributed in nervous systems and somatic muscle cells. The subunits of nicotinic acetylcholine receptors (nAChRs) fall into two main categories: a and non-a subunits. The nAChR located on the electric organ of Torpedo californica was the first nAChR purified (Noda et al., 1982). 15

Table 2.1. Hierarchy of the ligand-gated ion channel (modified from Le Novere & Changeux, 1999).

Superfamily of nicotinicoid receptors family of nicotinic receptors subfamily of epithelial receptors subfamily of neuronal a-bungarotoxin sensitive receptors subfamily of muscle receptors subfamily of heteromeric neuronal receptors subfamily of heteromeric protostomian receptors family of serotonin receptors family of GABA receptors subfamily of GABAA receptors subfamily of GABA~ receptors family of variable agonist receptors subfamily of GABA receptors subfamily of glutamate receptors subfamily of glycine receptors Superfamily of excitatory glutamate receptors family of excitatory glutamate receptors subfamily of NMDA receptor subunits subfamily of AMPA receptor subunits subfamily of kainate receptor subunits subfamily of kainate binding proteins subfamily of delta subunits Superfamily of ATP receptors family of ATP receptors

2.6.1. The structure of Torpedo nACh receptors The Torpedo nAChR is a pentamer, made up of four subunits al, (31, y and S arranged with a 2:1:1:1 stoichiometry (Karlin, 1991). The three dimensional structure information of the receptor from electron microscopy at 4.6 A resolution shows that the five transmembrane proteins create a central pore (Figure 2.11) with an inner diameter of ~25 A. The pore becomes narrower at the transmembrane level, which is suggested to correspond to the gate of the channel (Miyazawa et al., 1999). Two cavities in the a-subunits located 30 A above the lipid bilayer surface are believed to be the ACh binding sites (Figure 2.11). The cavities connect by tunnels about 10-15 A to the central water-filled vestibule. 16

ACh

Na+

Extracellular f

Intracellular ----► ~t--i._.i-- .~~

Figure 2.11. Diagram of the nicotinic acetylcholine receptor, depicting the ion channel and the ACh binding sites (modified from Brejc et al., 2001).

The crystal structure of an ACh-binding protein reveals the ligand-binding domain of nACh receptors (Brejc et al., 2001). The ACh binding site, which is located at the subunit interfaces of a and the adjacent non-a subunits, is contributed by six loops (Figure 2.12): loops A, B and C from a subunit and loops D, E and F from the non-a subunits. All amino acid residues in the binding site are conserved between most of the nACh receptors except the loop E and F (Corringer et al. , 2000; Brejc et al. , 2001). The loop E and F regions have low sequence conservations in the nAChR family and this change may lead to variations in ligand binding affinity. For example, it has been shown that the binding affinities of the aJ~y and a/b are pharmacologically different (Sina, 1993; Sina et al., 1995), and mutation of the loop F was found to decrease the affinity for ACh (Martin et al., 1996a). 17

Figure 2.12. The ACh binding of nAChRs in top view. The ACh binding sites at the subunit interfaces contributed by loops A, B, and C from a subunits and loops D, E, and F from non-cx subunits (modified Corringer et al., 2000).

Ligand binding domain ~~ Extracellular

Intracellular

Figure 2.13. The secondary structure of the nicotinic acetylcholine receptor subunit (modified from Paterson and Nardberg, 2000). Each subunit of the nicotinic acetylcholine receptor consists of a large hydrophilic N-terminal domain, four hydrophobic transmembrane domains (M1-M4), a small highly variable hydrophilic domain between M3 and M4, and a hydrophobic C-terminal domain.

The protein structure of a nAChR subunit (Figure 2.13) consists of (1) a large hydrophilic N-terminal domain, (2) four hydrophobic transmembrane domains (M1-M4), (3) a small highly variable hydrophilic domain between M3 and M4 and (4) a hydrophobic G terminal domain of approximately 20 amino acids. It is suggested that the large hydrophilic N-terminal domain plays a very important role in the ligand binding. nAChRs contain five 18 subunits and each of the five M2 domains lines the ion channel pore (Galzi & Changeux, 1995). The nature of the charged amino acids on M2 determines whether the channel carries either cations or anions. According to circular dichroism measurements of the M1-M2-M3 segments (Corbin et al., 1998), infrared spectroscopy of the Torpedo receptor (Gorne- Tschelnokow et al., 1994) and secondary structure prediction (Le Novere et al., 1999), the ~20 amino acids transmembrane segments are mixed with an a-helix/(3-strand topology. The M2 transmembrane segment folds in an a-helix and contributes to the ion-channel pore. Experimental evidence indicates a common transmembrane topology shared by all subunits (reviewed by Hucho et al., 1996). The study of circular dichroism measurement indicates that the N-terminal extracellular domain contains an abundance of ~3-strands and may spontaneously fold to become stabilized into anative-like conformation (West et al., 1997).

2.6.2. Structural changes during ligand binding The computed model and structural model suggest that the N-terminal domain of nAChRs consists of a rigid core of ~3-strands, which reduces the structural flexibility of the extracellular region, while experimental data supports that the allosteric transitions mediated by the binding of ACh molecules are associated with the structural change of this region. One suggestion is that this change of the N-terminal domain may occur between the subunits but not within the interface region. Within the transmembrane domain, electron microscopy reveals that the binding of ACh molecules induce the M2 segments to bend near the middle of the membrane and twist around the central axis at the lower part. In conclusion, structural changes occur in both the N-terminal and transmembrane regions during the allosteric transitions. The global conformational change within the transmembrane region is associated with the channel opening (reviewed by Corringer et al., 2000).

2.6.3. The amino acid sequences of Torpedo nicotinic acetylcholine receptors The amino acid sequences of this muscle nAChR are the best known. A database of the amino acid sequence of (LGICs) is accessible via the worldwide web site: http://www.pasteur.fr/units/neubiomoULGIC.html. The a subunits contain vicinal cysteine residues near position 190 that form part of the acetylcholine binding site (Kao &Karlin, 19

1986). The amino acid sequences of the Torpedo acetylcholine receptor subunits are shown in Figure 2.14.

Torpedo alpha MILCSYWHVGLVLLLFSCCGLVLGSEHQTR~IANQLEN--~IKVIRQVEHHTHFVDITV~QQIQ Torpedo beta MENVRRMALGLi7VMMALALSGVGASVMQDTQLSVQFET--Y~-;PKVRQAQTVGDKVTVRVG~T~±1 Torpedo gamma MVT~`~TLLL I I CLALE V RS - ENEQGRQI E I~jLGD - -QDKR I I QAKTLDH I I DVTLI~'T~TN Torpedo delta ---MGNIHFVYLLISCLYYSGCSGVNEQERQINDQLIVNF~IKHVR~/KHNNEWNIALS~SN

Torpedo alpha QISVD~INQIVE'T~RLRQQ~I~iVmR~JPAD~GGOKKIy'7LQSDD, '~I,~YI 'AI VH Torpedo beta QLILNQKIEMTT7~iFLNLA~TI~iYmQ~DPAAQEGOKDI,~y'71QSSDVQQPDI S'E I TL Torpedo gamma QISLNQKEFALT'Ti~WIEIQ~NQiYmS~TTSE~EG~DLV~y'71QSELL~ QFEVAY Torpedo delta QI SLKQTD~TLTSi~4JMDHA~YQiH~T~TASE~SDQS I L~y'7LQPELVQI YHVAY

Torpedo alpha MTKLQLDY'I'~e KIM~TQP~FI~YQEQIVT TMKLGIWT©DGTKVSISPESDRP ;LS- Torpedo beta HVNV~7QHT~eAVSOQQS~YI~S0TQK1M TMVFKSYT~DTSEVTLQHALDAKGER- Torpedo gamma YANVQVYND~SMI'QLQP~YR~'TOP~T SLVFRSQT~NAHEVNLQLSAEEGE--- Torpedo delta FCNVQVRPNCeYVTOL~P~FROS~PQNuLY SLKFTALNQDAN~ITMDLMTDTIDGKD Ml Torpedo alpha ----TFMES CeE~1MKDYRGW~HWVYYT PDTPYLDITYHF~'IQ~y'71PLYFVVNVQI PC Torpedo beta -EVKEIVINKDAFTEN~eQWSIEHKPSR~y----NWRSDDPSYEDVTFYLQIQ~y'71~FYIVYTQ Torpedo gamma -AVEWIHIDPEDFTEIVCi'E~TIRHRPA~TYNWQLTKDDTDFQEI I FFLQIQ~y'71CmFYI INIQP.m Torpedo delta YPIEWIIIDPEAFTEIVCeEWEIIHKPAK~IYPDKFPNGTNYQDVTFYLQIR~K~FYVINFOTPC M2 M3 Torpedo alpha LQFOFQTC-,~VFYLPTDS~e - E~y7MTLS~7mSLTV~I,V I VELIQSQSSA~LQGI~MLQT~I FV I Torpedo beta IQIQIQAI~7FYLPPDACe-E~7MSLS~AVTV~LLLADKVQEQSLS~IQIR~LMQI~ILVP. Torpedo gamma VQIQS~VYFmP.QAQGQ~CTLSmVmAQT ImFL I AQKVQEQSLN~LQGI~L I ~iFV SN Torpedo delta VQIQFQASQAFY~ESCa' -E~ISTAmVm.~QAVmLLTSQRLQEQALA~LOGFC~TL~N~I~SLVZ

Torpedo alpha SSIIITWVI~TFi SQSmTMPQWVRKIQIDTIQNVMFFSTMKRASKEKQE Torpedo beta FSVILSWVLd~LHHOS~imTMPNWIRQIQIETL~PFLWIQRPVTTPSPDSKPTIIS~.AND Torpedo gamma LIVMNCVIVL~VSLQT~ImSLSEKIKHLQLGFLQKYLGMQLEPSEETPEKP--QPR$~2RSS-FCzI Torpedo delta GVIVNCGIVL~FHFQTQS~VLSTRVKQIQLEKLQRILHMSRADESEQPDWQNDLKLRRSSSVY

Torpedo alpha NK IFADDIDI SDI S GKQVTGEVI FQTPLI KN~DV~ySAIEGVKY Torpedo beta EYFIRKPAGDFVCPVDNARVAVQ-PERLFSEMKWHLNG--LTQPVTLPQDL~iEAVEAIKY Torpedo gamma MIKAEEYILKKPRSELMFEEQKDRHGLKRVNKMTSDIDIGTTVDLYKDLANFA~EI~ySCVEACNF Torpedo delta ISKAQEYFNIKSRSELMFEKQSERHGL--VPRVTPRIGFGNNNENIAASDQLHDEI~SGIDSTNY

Torpedo alpha ©AEHMKSDEESSNAAEEWKYVAMVI~HILLCVFMLICII~TSV~AGRLIELSQEG Torpedo beta QAEQLESASEFDDLKKD~QYVAMVA~RLFLYVFFVICSI®FSI©LDASHNVP,~DNPFA Torpedo gamma QAKSTKEQNDSGSEN~VLIGKVI~KACFWIALLLFSI®LAI©LTGHFNQV=°; F~FPGDPRK Torpedo delta ~TKQIKEKNAYDEEVGN~NLVGQTI~RLSMFIITPVMVL~IFI~lMGNFNHPFAKFFEGDPFDY

Torpedo alpha Torpedo beta Torpedo gamma VP Torpedo delta SSDHPRCA

Figure 2.14. The amino acid sequences of the Torpedo nAChR subunits. Regions of sequence identity between the subunits are indicated by black coloring. Regions of sequence high similarity between the subunits are indicated by dark coloring. Regions of sequence low similarity between the subunits are indicated by light coloring. The vicinal cysteine residues on the a subunit are indicated by red color. The positions of the four transmembrane domains (M1 — M4) are indicated. 20

2.7. Subtypes of nicotinic acetylcholine receptors Both in vertebrates and in invertebrates, the existence of multiple nAChR subtypes have been shown. Each subtype may have individual physiological and pharmacological properties, such as in the human brain different neuronal nAChR subtypes are distributed in different areas and involved in a number of physiological and behavioral processes (reviewed by Paterson & Nordberg, 2000). nAChRs are comprised of five subunits. Genetic analysis indicates the existence of multiple nAChR subunits both in vertebrates and in invertebrates. The different combinations of these subunits give rise to the large numbers of nAChR subtypes. The vertebrate nAChR subtypes are reviewed in this section, as well as the nematode nAChR subtypes. For the nematode nAChR subtypes, although a lot of information comes from genetic analysis and pharmacological studies, the physiological functions and modulation mechanisms remain to be fully understood.

2.7.1. The subunits of nAChRs in the nematode C. elegans More than 40 genes that predict to encode nicotinic receptor subunits have been identified in the C. elegans genome, and 27 of them have been shown to be authentic nAChR homologs (Bargmann, 1998). These genes can be grouped into five groups (Figure 2.15): four groups, UNC-38 -like, ACR-16-like, DEG-3 -like and ACR-8-like mainly encode a subunits; the UNC-29-like group encodes non-a subunits (Mongan et al., 1998, 2002). The UNC-38 -like group consists of three a subunits, UNC-63, ACR-6 and UNC-38, which are most similar to insect a subunits. The ACR-16-like group consists of nine, ACR-7, 9, 10, 11, 14, 15, 16, 19 and 21, which are similar to the vertebrate a7 neuronal subunit. The LTNC-29-like group consists of four non-a subunits, ACR-2, 3, LEV-1 and UNC-29, which are similar to insect non-a and vertebrate skeletal muscle non-a subunits. The DEG-3 -like group includes eight, ACR-5, 20, 22, 23, DES-2, DEG-3 and ACR-17, 18. The ACR-8-like group includes three, ACR-8, 12 and 13. The DEG-3-like and ACR-8-like groups appear to be unique to nematodes. 21

ACR-22 ACR-23 DES-2 C ACR-20 ACR-5 DEG-3 DEG-3 ACR-18 ACR-17

ACR-21 ACR-19 ACR-16 - ACR-11 ACR-9 ACR-16 ACR-14 ACR-15 - ACR-10 - ACR-7

ACR-12 ACR-13 ACR-8 ACR-8

UNC-63 ACR-6 UNC-38 UNC-38

ACR-2 ACR-3 UNC-29 UNC-29 LEV-1

Figure 2.15. The diagram of the tree of the C. elegans nAChR subunits. These 27 subunits are distinguished to hve groups: DEG-3-like, ACR-16-like, ACR-8-like, UNC-3 8-like and UNC-29-like. The a subunits are shown in black and the non-a subunits are shown in red . a. Levamisole receptors Although these genes encoding nematode nAChRs have been identified in C. elegans, only a few of the genes have been studied at the functional level. The most extensively studied functional nACh receptor in nematodes is the levamisole receptor that is the target of nicotinic anthelmintic levamisole (Eyre, 1970). Treatment of nematodes with levamisole can induce spastic paralysis of the muscle cells and stimulation of egg laying (Lewis et al., 1980a, 1987). Studies of levamisole-resistant mutants make it possible to identify the genes that affect the function of the levamisole receptor (Lewis et al., 1980b). Six genes have been reported to have a dramatic effect the levamisole-resistance (Fleming et al., 1993, 1997). 22

Among these genes, lev-1 and unc-29 encode non-oc subunits; unc-38 and unc-63 encode a subunits; the gene unc-SO does not encode a nAChR subunit but is required for acetylcholine receptor function (Schafer, 2002); the product of unc-74 remains unknown. Co-expression of unc-29 and lev-1 with unc-38 or unc-63 in Xenopus oocytes leads to the observation of levamisole-activated currents, suggesting that these genes encode the subunits of levamisole receptors. UNC-38 and UNC-29 subunits are necessary for nAChR functions. It is also observed that mutants of unc-38 and unc-29 do not retain normal motor behavior but are extremely resistant to levamisole. This observation suggests that levamisole and the neurotransmitter acetylcholine may target different nAChR subtypes (Fleming et al., 1997). The functions of levamisole receptors located at the neuromuscular junction have been reported to be involved in the body movement of nematodes, but the levamisole receptor null mutations only partially impair the nematode movement. It is believed that other nAChR subtypes that are levamisole-insensitive are present at the neuromuscular junction, and also contribute to the excitatory neurotransmission between motor neurons and muscle cells (Richmond and Jorgensen, 1999). b. DEG-3-like Besides the subunits of levamisole receptors, the other nAChR subunits studied at the physiological level are DEG-3 and DES-2. The studies were based on the loss-of-function and gain-of-function mutants of DEG-3 and DES-2. The loss-of-function mutant of DEG-3 resulted in sensory neuron degeneration (Treinin & Chalfie, 1995). The gain-of-function mutant of DEG-3 causes cation currents to trigger cell death, and the loss-of-function mutant of DES-2 suppressed this phenotype (Treinin et al., 1998). When DEG-3 and DES-2 were expressed in Xenopus oocytes, it was observed that the nACh receptors prefer to be activated by choline rather than acetylcholine (Yassin et al., 2001). Confocal images indicate that the DEG-3/DES-2 receptor is not located at synapses. Although the roles of DEG-3 and DES-2 subunits in normal nervous system function remain unclear, it has been suggested that the DEG-3/DES-2 nAChR is a chemoreceptor for choline. 23 c. ACR-8-like It is worth noting that in all of the acr-8 genes, the highly conserved acidic glutamic acid at the -1' position is replaced by basic histidine in the pore-lining 1~~I2 region, implying that acr-8 genes might encode anion selective rather than cation selective channels (Mongan et al., 1998). Consistently, experimental evidence indicates the existence of nicotinic anion channels containing ACR-8-like subunits in neurons of the invertebrate Aplysia California (Kehoe &McIntosh, 1998). The ACR-8 containing receptor is an ACh-gated chloride channel and physiological role remains unknown. This AChCI receptor responds to the agonists nicotine and cytosine with rapid desensitization, and the response to nicotine and cytosine can be antagonized by a-conoto~n.

2.7.2. The subunits of nAChRs in vertebrates In chick, rat and human, multiple genes that encode nAChR subunits have been identified using molecular cloning studies. The subunits encoded by these genes can be grouped into three classes that are consider with their evolutionary development and their pharmacological and physiological properties: muscle subunits (al, (31, S, E and y), standard neuronal subunits (a2-a6 and Q2-(34) that form nAChRs in a(3 combinations, and subunits (a7-a9) capable of forming homomeric nAChRs (Colquhoun &Patrick, 1997; Le Novere & Changeux, 1995; McGehee &Role, 1995). In the third classification, only the a7 subunit (not a8 or a9) is observed distributing in mammalian central nervous systems. Peripheral nAChRs found at the neuromuscular junction are made up of two al, one (3, one s and one y subunits. In vertebrate nerve systems, the neuronal nAChRs may have many diverse combinations according to an a2f33 stoichiometry with the possibility of more than one a subunit type in one nicotinic receptor (Conroy et al., 1992). In addition, a7, a8 and a9 subunits are known to form functional homo-oligomers (Couturier et al., 1990). However, heterologous expression of these subunits in Xenopus oocytes and mammalian cells has established certain rules to limit the subtypes of nAChRs that may functionally exist: (1) a2, a3 or a4 subunits can form functional nAChRs combining with (32 and (34; (2) The combination of only a5 and (33 subunits cannot form functional nAChRs but they have to express with at least two other types of subunits (Lukas et al., 1999); (3) a6 subunits have 24 been shown to form functional heteromeric nAChRs when co-expressed with j34 or at least two other types of subunits including X33 (Kuryatov et al., 2000); (4) a7, a8 and a9 are distinguished by their ability to form homomeric nAChRs in expression systems (Couturier et al., 1990; Keyser et al., 1993; Elgoyhen et al., 1994; Gotti et al., 1994). Evidence indicates that the a7 subunit can also form more complex combinations (Girod et al., 1999; Yu & Role, 1998). In native systems, only a few of major combinations of nAChRs have been identified. In the mammalian brain, most nAChRs contain either a4R2 or a7 subunits (Charpantier et al., 1998). The a4 j32-containing nAChRs is the major subtype existing in the central nervous system (CNS). Expression studies in Xenopus oocytes proposed the stoichiometry (a4)2(~32)3 (Cooper et al., 1991). However, other combinations may exist in vivo (Lukas et al., 1999). Another major subtype of nAChRs existing in CNS and peripheral nervous system is comprised of a7 subunits (Chen &Patrick, 1997). The a7 subunit is generally thought to form homomeric nAChRs. But in the chick brain and retina, it has been reported that a7 subunits also combined with a8 subunits to form heteromeric subtypes (Keyser et al., 1993; Gotti et al., 1994; Yu &Role, 1998). The a8 homomeric and heteromeric subtypes have been observed in avian brain and tissues (Couturier et al., 1990; Keyser et al., 1993; Elgoyhen et al. , 1994; Gotti et al. , 1994) .

2.8. Pharmacological subtypes of nematode nAChRs Richmond and Jorgensen (1999) reported the presence of nicotinic-sensitive and levamisole-sensitive cholinergic receptors on muscle cells of the nematode C. elegans. The levamisole-sensitive receptor requires the expression of unc-38 and unc-29. The electrophysiological response of the receptor was insensitive to 5µM dihydro-~i-erythroidine (DH(3E). The unc-38 and unc-29 knockout mutant of C. elegans showed the existence of another cholinergic receptor that was sensitive to nicotine but not to levamisole and was selectively blocked by DH(3E. These two classes of cholinergic receptors in the muscle of C. elegans are pharmacologically distinguishable. Using the competitive antagonist model, Robertson et al. (2002) also reported the existence of three pharmacological subtypes of nAChRs on nematode muscle cells, named 25 levamisole-preferring, nicotine-preferring and bephenium-preferring receptors. The antagonist paraherquamide could distinguish nicotine-preferring from levamisole-preferring and bephenium receptors, while 2-deoxy-paraherquamide could distinguish bephenium- preferring from the other two. Single-channel recordings of nAChRs on the nematode Oesophagostomum dentatum reveal the presence of four biophysical subtypes (Robertson et al., 1999). This evidence suggested the presence of multiple subtypes of nAChRs in nematodes and different ligands can be selective for the different receptor subtypes. 26

CHAPTER 3. MATERIALS AND METHODS

3.1. Collection and maintenance of Ascaris suum The Ascaris suum were collected from the IBP Meat Packing Plant, Storm Lake, Iowa. During transport, the worms were kept in Locke's solution (Table 3.1) at 32°C. On arrival at the laboratory, the worms were transferred to clear glass beakers filled with Locke's solution and the cylinders were maintained in a water bath at 32°C in a fume hood. To prolong the life of these parasites, a small number of parasites were kept in each container. The waste products were removed and the Locke's solution was changed daily. In this condition the parasites can be kept for one week. Mature, active Ascaris about 10-20 cm in length were selected for experiments. In general small worms and very large worms were rejected for experimentation.

3.2. The vesicle preparation A cylindrical muscle section was cut from the anterior part (about 3-5 cm from head) of the parasite and was dissected along one of the lateral lines (see Figure 2.3). The muscle flap was pinned with cuticle side down onto a 35 x 10 mm plastic dish lined with Sylgard (Sylgard 184 silicone elastomer kit, Dow Corning Corporation, Midland, USA). Using a dissecting microscope (Nikon SMZ-U), the gut of the parasite was carefully removed with forceps. The gut of a healthy Ascaris scum was yellow and could be removed in one piece. The muscle flap preparation was washed with maintenance solution (Table 3.1) several times to remove fragments of the gut. The maintenance solution was then replaced with collagenase solution (Table 3.1). After collagenase-treatment for 4-8 minutes (depending on the condition of the muscle flap) at 37°C, the muscle preparation was washed with maintenance solution 6-10 times. After washing, the muscle preparation was incubated in maintenance solution at 37°C for 50-60 minutes. Small membranous vesicles, 10-50 µm in diameter, grew out from the membrane of the muscle cells. In general, longer enzyme- treatment and incubation produced more vesicles and resulted in easier giga-ohm seal formation (see 3.3.5. A giga-ohm seal. However, it appeared to reduce the number of active 27 nicotinic acetylcholine receptors in the membrane patches, so we treated muscle flap preparations with collagenase as short as possible. These membranous vesicles were transferred to a recording chamber (RC-26Z, Warner Instrument Corp.). The bottom coverslip size for the chamber is 22 x 50 mm (CS-22/50, Warner Instrument Corp.). The maintenance solution in the chamber was gently washed out with bath solution and the vesicles in the bath solution were used for recording for 2-3 hours at room temperature. The vesicle preparation has been described previously by Martin et al. (1990).

Table 3.1. Composition of solutions used in experiments. In the maintenance, bath and pipette solutions, EGTA, which is a chelating agent of Cat+, was added to reduce the concentration of Cat+ and inhibit the activity of Cat+-dependent Cl"channels. The bath and pipette solutions are Na+-and K+-free to prevent the activity of Na+ channels and K+ channels . The maintenance and collagenase solutions were incubated at 37°C for about one hour before vesicle preparation. The bath and pipette solutions were at room temperature. Collagenase solution has no EGTA to promote enzyme activity.

Solution Locke's Maintenance Collagenase Bath Pipette Compound NaCI (~ 155 35 35 Na acetate (mM) 105 105 CsCI(m1V~ 35 140 Cs acetate (mM) 105 KCl (m1V~ 5 2 2 MgC12 (m1Vn 2 2 2 2 CaCl2 (mM) 2 NaHCO3 (~ 1.5 HEPES (mM) 10 10 10 10 D-glucose (mM) 5 3 3 L-ascorbic acid (mM) 2 2 EGTA (mM) 1 1 1 Collagenase (mg/ml) 1 Levamisole (µM) 30 pH n/a 7.2 (NaOI~ 7.2 (NaOI~ 7.2 (CsOi~ 7.2 (CsOI~ 28

3.3. Single-channel recording The single-channel recording technique, which was developed by the Nobel Prize winners Erwin Neher and Bert Sakmann, is a very powerful technique for studying the electrophysiological properties of ion channels. The development of a high resistance seal (up to 10 giga-ohm) between a glass electrode pipette and cell membrane dramatically reduces the background noise and gives the possibility of recording and measuring the signal of asingle-channel current. Back in 1981, Hamill et al. described the improved patch-clamp technique to record AChR-channel currents with cell-attached and cell-free configurations (Hamill et al. , 1981). In my study, I used this technique to record the single-channel currents activated by levamisole from the vesicle preparation.

3.3.1. Instruments The main parts of the experimental equipment include an amplifier, a digitizer, and a PC computer. The current signal is collected by an electrode and then sent to the amplifier that amplifies the tiny signal and filters the background noise. The digitizer is an A/D converter that converts the signals from analog to digital format. The computer receives the digital signals from the digitizer, and provides data storage. An oscilloscope is also used to monitor the recording. a. Amplifier The ~opatch 200B (Anon Instruments Inc., CA) is a new generation of patch-clamp amplifier with an ultra-low noise performance and a capacitive feedback mechanism. This amplifier can be used for both whole-cell and single-channel recording. The CV203BU headstage is used for patch-clamp. This headstage is acurrent-to-voltage (I-~ converter. In other words, the voltage output is proportional to the current input. Compared to the conventional headstage, the CV203BU uses capacitor instead of resistor feedback. The capacitor mode has a reduced noise and better linearity. The noise reduction is particularly significant in the frequency band of interest for single channel recordings (10 Hz — 10 kHz). The disadvantage of this headstage is that the capacitive component must be reset when it approaches the supply limits. The frequency of resets depends on the current that passes through the headstage. With the 29 larger current at lower resistance seals, the capacitor of the headstage discharges frequently producing a significant number of reset spikes. However, these 50 ~,s spikes are easily identified and impossible to confuse with "real" single-channel events. The passive membrane response to a voltage step consists of a transient and asteady- state component. The transient is due to the membrane and pipette capacitances. The capacitance compensation in the amplifier is used to eliminate the transient during a voltage step. The steady-state component is eliminated by the "leak subtraction" control. In order to compensate for the offset of the liquid-liquid and liquid-metal junction potentials in the electrode and bath, the "pipette offset" control is used to adjust the pipette command potential by about 5 mV. The internal filter of the Axopatch 200B amplifier is a 4-pole lowpass Bessel filter. The attenuation of signals and noise above the —3 dB frequency is 80 dB/decade. The lowpass Bessel filter control allows selecting a bandpass of 1 kHz, 2 kHz, 5 kHz, 10 kHz or 100 kHz. In my experiments, the bandpass was set at 2 kHz. When a signal containing a step change passes the lowpass filter, the 10-90% rise time (tlo-9o) is given by:

t 10-90 ti ~• 3 5 /f 3, where f_3 is the —3 dB frequency in Hertz. For example, the rise time (tlo-9o) of a 2 kHz filter is approximately 0.175 ms. b. Digitizer The Digidata 1320A (Axon Instruments Inc., CA) is a low-noise digitizer with 16-bit data acquisition system. The maximum sampling rate of this digitizer is 500 kHz. In our experiments, we set the sampling rate at 25 kHz. The digitizer communicates with the computer by a SCSI interface. c. Instrument setup The recording chamber was mounted on the stage of an inverted microscope (TMS, Nikon, magnification 200-400x), which was placed on an anti-vibration table (Micro-g, Technical Manufacturing Corp., MA). The anti-vibration table was covered by an aluminium Faraday cage to reduce the electrical interference from the outside. The headstage was 30 mounted on a micromanipulator (Narishige Group, Japan), which was placed on the table, and connected to the amplifier. The amplifier, digitizer, oscilloscope and other instruments were mounted on a rack. The output interface of the amplifier was connected to the analog input interface of the Digidata 1320A (Figure 3.1). The digital output of the digitizer in turn connected to the PC computer by SCSI cable.

Oscilloscope

Amplifier Digitizer Computer

Bath solution

vesicle

Figure 3.1. Schematic diagram of the instrument setup. Channel currents recorded by the electrode is sent to the amplifier. The output interface of the amplifier was connected to the digitizer which digitizes the signals. A computer is used to record and analyze the single-channel currents. Also an oscilloscope is needed to monitor during experiments .

3.3.2. Electrodes The patch electrode was made from capillary glass (inner diameter =1.15 mm, outer diameter =1.55 mm, length=75 mm, Garner Glass Company, CA). The pipette was heated and pulled by a two-stage vertical puller (PB-7 or PP-830, Narishige Group, Japan). In the first pull, the capillary was thinned in a 7-10 mm region to a minimum diameter of 20 µm. In the second pull, the capillary was broken at the thinned part into two pipettes. In general the diameter of the pipette tip was 1-2 µm and had a resistance of ~S MSZ. The anterior ~1 cm of the electrode was covered with Sylgard (Sylgard 184 silicone elastomer kit, Dow Corning Corporation, Midland, USA) to reduce background noise. The very tip (250 µm) remained uncoated to allow seal formation. To form a giga-ohm seal, the tip of the electrode was heat 31 polished using amicro-forge (MF-900, Narishige Group, Japan). The electrode pipette was filled with pipette solution and mounted on the pipette holder.

3.3.3. Measuring the pipette resistance The tip of the electrode filled with pipette solution was dipped into the bath solution. In "Track" mode, the baseline was reset to zero using the "pipette offset" control of the amplifier. Then a 5 mV voltage pulse was applied to the electrode. The current through the electrode to the bath ground was measured and the pipette resistance was calculated using Ohm's law, R = V / I. In general, electrodes with resistances of 3-5 MS2 were used for experiments.

3.3.4. Reducing background noise Noise came from several sources including: mechanical vibration; electrical noise from the environment; electrical noise from the instruments, such as amplifier; and background noise from the interface between the electrode and bath solution. To reduce the mechanical vibration, the microscope and the manipulator were mounted on an anti-vibration table. To reduce the electrical noise, a large aluminum cage was used to cover the anti-vibration table and the instruments mounted on the table. To reduce the background noise from the glass- solution interface, the electrode pipette was coated with Sylgard. The instruments were grounded well to reduce the electrical noise, as well to prevent electrical shock.

3.3.5. Agiga-ohm seal A fresh pipette was used for each seal. The pipette was placed in the electrode holder and the tip of the electrode was dipped into the bath solution. 5 mV voltage pulse with was then applied to the electrode. The duration of each pulse was 8 ms. The electrode was moved smoothly and placed on the vesicle surface by micromanipulator. After the electrode tip contacted the surface of a membranous vesicle, a gentle suction was applied through a 1 ml syringe, and held for several seconds or longer. Agiga-ohm seal might develop. The current through the membrane was measured, and the seal resistance between the pipette and membrane was calculated by R = T~ / I. Only patches with a seal resistance > 1 GSZ were use or experiments. 32

3.3.6. Recording ion channel currents In "V-Clamp" mode, the single-channel currents were measured at different membrane potentials between —75 mV and +75 mV. It is believed that the Ascaris suum nAChR is a non-selective cation channel (Pennington &Martin, 1990), so the chloride permeability is negligible. Therefore, the predicted reversal potential under my experimental conditions was calculated using the Goldman-Hodgkin-Katz equation:

RT 1'cs [CS + ]o + Pcr [Cl ~ ~ ,,, RT Pcs [Cs+ ] o E — In _ ~ In , F Pcs [Cs ]1 + Pcl [Cl ] o F Pcs [Cs ] j where E is the resting membrane potential, R is the gas constant, T is the absolute temperature, F is the Faraday constant, Pcs is the permeability of the channel to Cs+, [Cs+]o is the extracellular concentration of Cs+, [Cs+]; is the intracellular concentration of Cs+. Pcr is the permeability of the channel to Ct. [Ct]o is the extracellular concentration of Ct. [Ct]1 is the intracellular concentration of Ct. The Cs+ concentration of both the pipette and bath solutions was 140 rrLM, so the predicted reversal potential: RT Pcs [Cs + ] o RT 140Pcs E = In = In = 0 mV. F Pcs [Cs + ] I F 140Pcs

The binding of the levamisole with the nicotinic acetylcholine receptor induced the opening of the receptor-associated ion channel. At negative membrane potentials, Cs+ ions flowed into the vesicle. At positive membrane potential, the direction of Cs+ ion flow was outward . The reversal potential for Cl" under the experimental conditions is about —30 mV. The cation channels associated with the nicotinic receptors are distinguished from anion (C1") channels because of the different reversal potentials.

3.3.7. Application of paraherquamide paraherquamide was dissolved in dimethylsulfoxide (DMSO), as it is insoluble in water. After 1-2 minutes of control recording, paraherquamide solution was added into the bath solution to give a final concentration of between 0.1 and 10 ~M. The final concentration of DMSO was less than 0.3% because high concentrations of DMSO are known to disrupt 33 membranes. The single-channel currents were measured after the addition of paraherquamide and compared to the control recording from the same vesicle to calculate the effects of paraherquamide.

3.3.8. Storage of data The signal from the amplifier was converted from analog to digital format by the digitizer and sent to the computer. The recording was stored on the hard disk of the computer and copied to CD as backup.

3.4. Data processing The data was stored in the computer as ABF files. The first step of data processing was to idealize the recording into "events lists" characterizing the amplitudes and durations of events (both openings and closings). The channel properties, such as channel conductance,

Popen and mean open time, were analyzed by measuring these "events lists" .

3.4.1. Software tools The software pClamp 8 was the data acquisition tool. I used "gap-free" mode to acquire data and monitored in real time during acquisition. Fetchan 6 was used to identify events from recordings and to convert the raw data to "events lists". pSTAT 6 performed the data analysis and generated a wide variety of histograms for analysis. This software also provided many statistical functions and equations to fit the histograms of events with idealized curves. I also used Clampfit 8, GraphPad Prizm 3.00 and Origin 7.0 for statistical analysis.

3.4. Z. Events lists The original recording cannot be analyzed by pClamp 8. Before the analysis, the original data must be idealized by Fetchan 6. Figure 3.2 shows an original recording and the idealized events list for it. If the channel activity to be detected was assumed to consist of rectangular current pulse with random duration but fixed amplitude Ao, the threshold is set at

Ao and the events whose amplitudes are larger than 1/ZAo are considered as channel open levels. The idealized data (Figure 3.2.C) then is analyzed using pSTAT, or exported to be analyzed by Clampfit 8, GraphPad Prizm 3.00 and Origin 7.0. 34

C. Leve E Awe t [ ins l f~Mp I t (pR) StdAev ~~~# ~ ~ l aiased t i ~e Conner

. 8 : . . . `, 2.680 0.115.17 8.17513 15~a~ : .88 1 5 X88 3 , 8~ ~~.1 8.7903? °1'~►3. . e 2 : 7fi880 -~ ...~4s . 6.17199 159 :2C3 ;1. 0.8 f8f~ :1~9 A . l'IOfiA x. 1.'96 0 2.6#808._ 0 ' X947_ ._ 6.11878 1~2<. 76 1 3:4 80 3.59342 6.39238 <15~5:.48... .._.. 8 8 :1~~$ Ei : X6741 6.3J23La >>„'1'. ~3E3 1 1.~808g' <' 3377?1 8.342.91 'I~6.9`.g4 0 6.120®p 6 ~~2 0 , 2~~1._ '1~ __:' _... 1 1.68808 3.1 ___. _5 8.62014_ . 157$:44 _. 1 0 .` _ _.... 00 _ Z .16675 8.61295 1587`:_.... 48 0 3.2~g0 . 8.84...... 1 0.21X2 "~ : 72 1 0.928@ 2 : 52t4`t 8.68' 0 1598 . ~6 0 1.2. 80 6.287 8.13921 1591, S~f _...... _ . ____ _ e 3.i~Bee __ x.48113 6:z~3~4 x.16 ___ a 1.128e0_. ~ . 79}71__ ~ .17625 1612.

V . VWVO M . lw~~~1J l! . ~~BiML -. rt~/~~.4~.~ 0 21,~64~ #~ . 05 9 6.17 3 ~,fi :96 1 0.8 88'' 1.2 79 6.17753 ;...... lb'~5`:~2 S ~i l :l , 8.~ H .11514 E~ .1974Ei `<,~`. 98 1 2.8'888' 3.14382 6 , 51118 147>:~6 8 < . 7 . X80 0.1403E,< 0 , X8 4 X8 :,.7

Figure 3.2. An example of the events list. A, asingle-channel recording; B, the idealized events lists for A; C, the spreadsheet of the events list. The level 0 means the closed state of ion channels and the level 1 is the open state. The dwell time and amplt (amplitude) were exported to Clampfit for further analysis. 35

3.4.3. Single-channel conductance The single-channel currents were measured at different membrane potentials between — 75 mV and +75 mV. At each membrane potential, the current amplitude histogram including the baseline level was fitted with Gaussian distributions: ~2s12 n e — (x — x1)2 .f ()t = ~ A =1 ~ . 2~z i r where Ai is the proportion of each fitting component, xl is the mean of each component corresponding to the peak of the Gaussian distribution and ~l is the standard deviation. The difference between the peak of the open level and closed level is the single-channel current at a given membrane potential. The conductance was calculated according the equation g = I / V, where g was the conductance, I was the single-channel current and V was the membrane potential. Between —75 mV and +75 mV, the relationship of I / Tl was linear with a reversal potential near zero (see CHAPTER 4). The slope of I/Tl plot is the channel conductance and was calculated using linear regression.

3.4. Q. Popen The probability of a channel being open (Popen) provides a quantitative description of the activity of the channel vs. time. Using the patch-clamp technique, electrodes record channel currents from membrane patches. Because it is impossible to know exactly the number of channels on individual patch, here we define patch Popen and level-Popen as well as channel-Popen. If we assume only one channel present in Figure 3.3. A, then the channel-Popen is calculated by

t l +t 2 +t 3 +. . .+t n channel — Popen — ~ t~

where tl + t2 + t3 + • • • + to is the total open time and to is the time interval. However, the number of the active channels is unknown exactly, then the Popen calculated by above- mentioned equation was patch Popen indeed. In some individual patches, more than one current amplitude level was observed at certain membrane potentials (Figure 3.3.B). For such patches, we analyzed not only the patchPopen but also the level-Popen, Popen for 36

tl t ~ t3 to

T i~M i~ ~' w0n ~~ !~

t0 2 pA

50 ms

Large conductance evets, small conductance evets, The level Popen of the large level is Popen(L~ The level Popen of the small level is Popen(s) ~ ~ u~~ 1 ~L ~' , ~S

2 pA

10 ms

Figure 3.3. The Popen of the levamisole-activated receptor. A, the patch-Popen of the receptors on an individual patch is calculated by the total open time (tl +tz + t3 + tn) and the time interval to, patch-Popen — (tl +t2 + t3 + t n)/t o. B, two conductances present in this patch. For the large currents, Popen(L) is calculated only from those large conductance events. For the small currents, Popen(s) is calculated only from those small conductance events. C = no channel open; O =channel open. 37 individual conductance levels. For each conductance level, the sum of current open dwell- times is divided by the recording time interval to give the level-Popen. The level-Popen is not the channel-Popen but the Popen of all the channels with same conductance level in individual patches. The number of the channels with same conductance level is also .unknown. We assume the channels with same conductance level have the same channel-Popen, then

channel-Popen =levelPopen l N where N is the number of the channels with the same conductance level. For example, in Figure 3.3B, two conductance levels present in this patch. The channel-Popen for the large conductance level is:

channel-Popen(L) =level-Popen(L) / N(L) channel-Popen(S) =level-Popen ) / N(s), where N(L) and N(s) are the numbers of the large channels and the small channels respectively.

3.4. S. Mean open time The mean open time, which indicates the mean duration. of channel open events, was analyzed by fitting the open dwell-time histogram with the Exponential Probability equation using least squares non-linear regression:

f (t) — ~n P zj—~ e — t / Zi i=1 where P; is the proportion of each fitting component, and ~1 is the time constant for each component estimating the mean dwell-time. Because we did not find significant statistical evidence that the histogram of mean open time is fitted with two exponential curves better than one exponential curve, we used a single exponential curve for all the analysis to compare the results of different experiments. The fitted curve follows the equation:

f(t)—Pz— —1 e — t/Z where P is the proportion and i is the time constant estimating the mean open time. When the pulses of channel currents pass the lowpass Bessel filter, the respondent signals are delayed. The delay is dependent on the dead time of the filter. The dead time is the time taken for a pulse when filtered to reach 50% of the original rectangular pulse. 38

During our experiments, the filter frequency was set at 2 kHz. The dead time of the filter is about 0.1 ms. The channel currents with adwell-time less than 0.1 ms are undetectable. The minimum resolution of the dwell-time is twice. the dead time. In other words, the channel events with dwell-time less than 0.2 ms cannot be detected correctly in theory. When analyzing the dwell time of the currents, the analysis software pStat automatically set the fitted limit from the peak of the dwell-time histogram and ignored the small number events that have short open times (usually less than 0.4 ms). 39

CHAPTER 4. SINGLE-CHANNEL CUI~►RENTS FROM ASCA~IS sUUM LEVAMISOLE-ACTIVATED RECEPTORS Levamisole is an agonist of cholinergic receptors located on the muscle cells of the nematode Asca~is suum. At the single-channel level, this compound has been shown to bind with the nAChRs and open non-selective cation channels. In addition, it behaves as an open channel blocker at higher concentrations (Martin et al., 1990; Pennington &Martin, 1990). In this study, we investigated the single channel properties of the levamisole-activated receptor. The individual single-channel conductances ranged between 18 and 52 pS with three separable conductance levels. The mean open time was correlated to the conductance levels. We interpreted the results as evidence of three nAChR subtypes existing on the A. suum muscle cells.

4.1. The single-channel conductances show more than one level In the inside-out patch-clamp configuration, the nicotinic acetylcholine receptors were activated by 30 µM levamisole filled in the electrode pipette. The single-channel currents were recorded at different membrane potentials between —75 and +75 mV (Figure 4.1). To block Na+ channel, Cs+ was used in both bath and pipette solutions. Non-selective cation channels associated with nAChRs are permeable to Cs+. To identify these single-channel currents were induced by levamisole, electrode pipettes filled with levamisole-free solution were used to test on the same vesicles and no channel opening was observed. One, two or more amplitude levels were observed in individual patch recordings (Figure 4.2). The amplitudes of the events were idealized and sorted into histograms. The amplitude histograms were fitted with Gaussian distributions (Figure 4.3) to give the current amplitude at each membrane potential. Between —75 mV and +75 mV, the I-T~ relationships were linear, and the slopes of the I-V plots gave the channel conductances (Figure 4.4). Table 4.1 shows the conductances and the reversal potentials from 30 patches. The conductances had a wide range from less than 20 pS to more than 50 pS, and the mean of the reversal potentials was close to zero (1.618 ~ 0.656 mV, mean ~ S.E.M.; N = 47). Among these 30 patches, 16 showed a single conductance level, 11 showed 2 conductance levels, and 3 showed 3 40 conductance levels. The conductance values from these 30 patches were sorted in one histogram and the conductance histogram was fitted with three Gaussian distributions (Figure 4.5). The three peaks, 21, 35 and 45 pS suggest the presence of three separate conductance levels.

1 sec

Figure 4.1. The 3 0 µM levamisole-induced single-channel currents recorded at four different membrane potentials, -75 mV, -5 0 mV, +5 0 mV and +75 mV. 41

1 pA

2 ~ Ills

Figure 4.2. The single-channel currents observed at +75 mV membrane potential on a shorter time scale. The presence of two different amplitude levels is highlighted by the dashed lines (OS =small level, OL =large level, C = no channels open).

300 -

250 - (N) 200 -

150 - Observation of 100 -

Number 50 -

1 r 1.0 1.5 2.0 2.5 3.0 3.5 4.0 5.0 Amplitude (pA)

Figure 4.3. The amplitude histogram. Three amplitude levels with peaks at: 2.12 t 1.47 pA, 2.84 ~ 0.05 pA and 3.83 t 0.01 pA (mean ~ S.E.M.) are present at membrane potential +75 mV. The three amplitude peaks correspond to three conductances 27, 37 and 47 pS (see Figure 4.4). 42

I (pA) 5.0-

2.5-

-50 50

v (m~

-2.5

-5.0 -

Figure 4.4. The I-Y plot, where 1 is the single-channel current and V is the membrane potential. The I-V relationships were fitted with linear regression and the slopes of the lines gave the three conductances, 27 f 1 pS (r2 = 0.9994), 37 f 2 pS (r2 = 0.9952) and 47 f 1 pS (r~ = 0.9990) (mean f S.E.M.). 43

Table 4.1. The conductances and reversal potentials of the levamisole-activated receptor. [levamisole] = 30 µM. Observed from 30 patches. Among these 30 patches, 16 showed a single conductance level, 11 showed 2 conductances, and 3 showed 3 conductances. The mean value of the reversal potentials is 1.618 f 0.656 mV (N = 47).

Conductance f S.E.M. Reversal Conductance t S.E.M. Reversal Patch # Patch # (pS) potential (pS) potential

30 t 1.6 ,...., ss 19 27 t 1 -4 3 23t 0 3.6 37 f 2 -5.1 47 t 1 -2.8 5 20 f 1 -0.1

7 23f0 2.8 21 37 t 2 -2.1

29 t 2 5.8 23 35 t 2 -2.6

33 f 1 6.1 s: ':~~~.~::

25 28f1 35 t 1~, ~: 13 1812 -2.4 2711 2.3 27 3811 3.4

15 1810 -3.3 29 4711 4.5 3711 -3.3 35 f 1 3.9

17 26 t 2 44

21 pS 5- 35 pS (N) 4-

3- Observation of 2- Number 1-

0 r ~ r r r ~ r ~ r ~ r ~ r r r 16 20 24 28 32 36 40 44 48 52 Conductance (pS)

Figure 4.5. The histogram of collected conductances from 30 patches. The conductance histogram was fitted with three Gaussian distributions and gave three peaks, 21 pS, 35 pS and 45 pS. Sample size N = 47. According to the three Gaussian distributions, we suggest that the conductances between 18-30 pS belong to the 21-pS level, the conductances between 31-40pS belong to the 35-pS level, and the conductances between 41-52 belong to the 45-pS level. 45

4.2. The mean open time is correlative to the single-channel conductance In the 30 patches shown in Table 4.1, the mean open times at +75 mV were analyzed. However, some of the recording had no enough channel open events for the analysis of mean open time. The mean open times of 13 patches which showed enough events were listed in Table 4.2. The open dwell-time histogram of single-channel currents was fitted with a single exponential curve (Figure 4.6).

100

(N) 80

60 Observation

of 40

Number 20

l!,~, ~ Ihl~llld~ll~lll ullil~., Ifiil ~n~~ni iiu ~i~iii6 ~~i~~ ► im~i~■~i~,n~i~•~mi•e•i~~~.ri•~~m•~~~~■~ 4 6 10 Dwell time (ms)

Figure 4.6. The open dwell-time at +75 mV was fitted with one exponential curve to give the mean open time z= 0.79 ms. (fitted limit > 0.32 ms). 46

Table 4.2. The mean open times at +75 mV. Observed from 13 patches.

Conductance (pS) Mean open time (ms) at +75 mV 1 20 0.874

25 0.252 33 0.515

19 0.501 37 1.291

44 0.848

9 25 0.718 45 1.468

11 35 0.723

13 28 0.565 35 1.342

Statistical analysis indicated that the correlation between the mean open time and single-channel conductance was significant (correlation coefficient r = 0.6841, p < 0.05) (Figure 4.7). When the mean open times are separated to three groups according to the three conductance levels suggested in Figure 4.8, the mean values of the mean open time are 0.610 ms for the 21-pS level, 1.041 ms for the 35-pS level and 1.298 ms for the 45-pS level. The means are significantly different (one-way ANOVA, p < 0.05). The mean open time of the 21-pS is significantly different with that of the 35-pS (t-test, p < 0.05) and 45-pS (t-test, p 0.005). 47

r ■ ■ i ~ ~ ■ ~ ■

0 I 1 I I 1 0 10 20 30 40 50 60 Conductance (pS)

Figure 4.7. The scatter plot of the mean open time vs. single-channel conductance. The correlation coefficient of the mean open time and conductance was 0.68 and significantly non-zero (p < 0.05). The dashed squares indicate the three conductance groups, 21 pS (range 18-30 pS), 35 pS (range3l-40 pS) and 45 pS (range 41-52 pS) (see Figure 4.5).

p < 0.005 (ms) Time Open Mean

21-pS 35-pS 45-pS conductance level (pS)

Figure 4.8. The mean open time for each conductance level with the standard error shown on each bar. The mean open time of the 21-pS group is significantly different to the 35-pS (t-test, p < 0.05) and 45-pS groups (t-test, p < 0.005). 48

4.3. Discussion The levamisole-activated single-channel conductance of the nematode nAChR had a wide range from less than 20 pS to more than 50 pS. The wide range of conductance values coupled with the accuracy of the conductance calculations (S.E.M. < 3 pS, r2 > 0.95 for all the experiments) made it unlikely that there was a single conductance population. Therefore our observations were comprised of samples from more than one population. Indeed, three peaks were observed from the conductance histogram. Our experimental results also demonstrated the presence of multiple conductance levels within a single patch; since the experimental conditions were identical within individual patches, this was compelling evidence for multiple populations with discrete conductances. We conclude that the single- channel currents of the levamisole-activated receptor represent at least three conductance levels with the mean values of 21 pS, 35 pS and 45 pS. We believe that the three conductances belong to different biophysical nicotinic acetylcholine receptors for several reasons: (1) The transition status between two conductance levels has not been observed in any of the patches that show two or three conductances; (2) Double openings with two different conductances were observed (Figure 4.9 A & B). (3) Only one conductance was observed from some patches. These observations indicate that the three conductances are independent. It seems unlikely that they are from one receptor with different open status. In the C. elegans genome, 27 nicotinic receptor subunit genes have been shown to exist, 20 a and 7 non-a subunits. Heteromeric nAChRs comprise two a and three non-a subunits, while homomeric nAChRs comprise five a subunits. Studies indicate that more than one a and non-a subunits contribute to the levamisole receptors in C. elegans (Fleming et al., 1997; Richmond &Jorgensen, 1999). The different combinations and arrangements of these subunits give rise to the possibility of a large number of nAChR subtypes. We interprete our observations as evidence that at least three subtypes of nAChRs exist on the somatic muscle cells of the nematode Ascaris suum. 49

2 pA

5 ms

0 2 ~~

~ +' 2 pA

10 ms

Figure 4.9. Double-opening of single-channel current at +75 mV membrane potential. A, Double opening with 37-pS on 47-pS. B, Double opening with 47-pS on 37-pS. (O, =the first opening, 0 2 =the second opening, C = no channels open) 50

CHAPTER 5. THE SELECTIVE EFFECTS OF PARAHERQUAMIDE ON THE LEVAMISOLE-ACTIVATED RECEPTORS LOCATED ON ASCARIS SUUM MUSCLE CELLS Paraherquamide has been suggested to be an antagonist of nematode nicotinic acetylcholine receptors (Robertson et al., 2002). In this study, the single-channel currents activated by 30 µM levamisole were recorded from membrane vesicles. After 1-2 minutes of control recording, drug (paraherquamide + DMSO or DMSO only) solution was added into the recording chamber and diffused in the bath solution to give a final concentration between 0 -10 µM. The inhibitory effects of paraherquamide on the levamisole-activated nAChR located on Ascaris scum muscle cells were observed at the single-channel level, and each subtype (described in chapter 4) showed individual paraherquamide sensitivities.

5.1. Paraherquamide is an antagonist of the nAChR located on Ascaris suum muscle cells For testing the effects of paraherquamide on the A. suum nAChR at the single-channel level, paraherquamide solution was added into the chamber using pipettes. During the addition of drug solution, mechanical disturbance of the bath solution usually reduced the patch open of the channel currents. Since patch Popen was so sensitive to the mechanical disturbance and the sensitivity is different for each individual vesicle, we compared two patches from the same vesicle: one patch was tested by a control drug-free solution, and the second patch was tested by adding the drug solution (Figure 5.1). The interference of mechanical vibration was compensated by comparing the effects of the additions of the control and test solutions. Then the paraherquamide effect on the patch Popen (Normalized

Popen) was determined by:

• _P open(+d) I P open(—d ) Normalized open — ~ P open(+c) /P open(—c) where Popen(+d) 1S the patch ropers of the channel currents after drug applications; Popen(d) is the patch ' open before drug applications; Popen(+c) 1S the patch Popen ai~er applications of 51 control drug-free solutions; and Popen(-c> is the patch-Popen before applications of control drug- . free solutions.

A. Additon of control drug-free solution 0.0605 0.0715 Popen(-c) — 1 Popen(+c)

Addition of drug solution [para] = 10 µM P open(-d) — 0.0773 Popen(+~t) — 0.0291

1 pA I 2000 ms

Figure 5.1. The inhibitory effect of paraherquamide on the levamisole-activated single-channel currents of the Ascaris suum nAChR. A, The control recording by adding bath solution (drug-free control); B, The 10 µM paraherquamide test from the same vesicle. Observed at +75 mV . P open(-c) and Popens d) are the patch-Popens before the addition of solutions; Popen(+c) and Popen(+d) are the patch-Popens after the addition of solutions. [levamisole] = 30 µM.

Paraherquamide reduced the patch-Popen of levamisole-activated channel currents in a concentration-dependant manner (Figure 5.2). Compared to the control, 0.3 µM paraherquamide reduced the patch-Popen to 95% (n = 3), 1µM to 81% (n = 10), 3µM to 45 52

(n = 6) and 10 µM to 27 % (n = 5). The inhibitory effect of paraherquamide was fitted with the dose-response curve: Top —Bottom Y =Bottom + 1 + 10-~Logreso-x> where the variable "Bottom" is the normalized Popen at the bottom plateau; "Top" is the normalized Popen at the top plateau, and IC50 is the paraherquamide concentration when the inhibitory response is halfway between "Bottom" and "Top". The IC50 is 2.1 µM(Figure 5.2 B).

A. 2

0 DMSO 0.3 1 3 10 [para] µM

B. 2 Popes Normalized

0 0.1 0.3 1 3 10 30 [pars] µM

Figure 5.2. Paraherquamide reduced the patch-P°pen of the levamisole-activated receptor with aconcentration-dependant manner (mean + S.E.M.). A, The inhibitory effects of paraherquamide of different concentrations. The effects of 3µM and 10 µM were significantly different with that of DMSO control (p < 0.05). B, The relationship of the normalized Popen vs. paraherquamide concentration was fitted with the dose-response curve and gave an IC50 of 2.1 µM. 53

To determine whether paraherquamide has effects on the conductance and the mean open time, we analyzed the current amplitude and the mean open time for each conductance level at +75 mV before and after the application of 3µM paraherquamide (Table 5.1). The amplitude and the mean open time before and after the application of 3µM paraherquamide have no significant difference (see the legend of Table 5.1). These results indicate that paraherquamide is an antagonist of nicotinic acetylcholine receptor located on the Ascaris suum muscle cells and reduces the patch-Popes without significant effects on the conductance and the mean open time.

Table 5.1. The effects of 3µM paraherquamide on the conductance and the mean open time. The amplitude at +75 mV has no significant difference before and after the application of 3µM paraherquamide (paired t-test, p = 0.37, N = 5), while the mean open time at +75 mV also has no significant difference (paired t-test, p = 0.49, N = 5).

Amplitude ± Amplitude ± Mean open time ± Patch Conductance Mean open time + S.E.M. S.E.M. S.E.M. S.E.M. # ~pS) before Sara ~~A) after ara A before ara ms ) after ara ms r>r•r ~: ~ : r 'rtercr .., ,,,.,,, .~ r ,,.,, , P ,.,.,r, , . (. ,. ;;;c;r oc: cc, r~rr, rcr ^rr „ .^ „t. r.r~rr; ^,A.,.,, < rr r r^,.CC. r1G . rr. , r :: ryr',,:',,r.ir. ,.~. ^~ c ",. ~..,•^.,,, ^.r:.^r..^.c;rc ;: ~~:. rr%%'r': r'f'r''r''f~r.~. r rrr:.:r:. r r rrrr`frriri c r.:r: . .^cr~r:.r: r. ',. r. ,: rc:rc, :; cc.~ s . c. ,. ,.r r.r , .r. r. r. r. ,: ". ,. r. r. . ~^. . .c,c; 'r;:;rr": c. , . c., .cc.r .%.~.~;r;~r,;rrc. ,, . r, c. , ~~ , ~, .r.c. r. r. r. r. c c Cr..4. ~, , ~ , r r ~ r : ~(r~lr~C ~: ' r .,c,. . ,.rrrr. : x ';r. C. r. (. r. (. r. rr. sC. r ,c. c. ;,G . ,r F c. ,. C C ....,r;, r. ,. r. r,,", . r ;cc•c.. ,fr. ~,~,'.' . .'r..'~ r,.r,.r, , ,r .., rc'r. . .r.rr.r•.r.r.r..c. rc.rc ..c' . r. r ~ ".;i: :r~:~' ,. r. r. ,. r. r. r. :c.r( ,.r ,~f rrr,r r' , . ,r~[~ ^:% .' . c". ;,~.^;., , . ~c r c c r c c .. , . , , r, , ,>, ^,r^ c, , " ,,.,.c:':c; .ur~ u .,.,.;..^;r;c•.,....;.,.>.,,;r,c~;c;, ~, ,r., ^, ^ c."r,r;c,r.'. r.,c> .,r.,r. c,rc;c; ,;^ c.,'.7R,'r~~^,,,r . , ,.. ~; ~;. ~' . '^r.,r, ;: r ; ^:- ~~~;1;).AAAa. .A. . _Y~ .AS.A.f~adAAASX:>: r 1,A::~t,,. , _. .A.,AAA,.~.,>A,..,:AXfSA..,fAAAA,.. , ,S~AA : ,1,1A,1A,;,: 1„5,,,: _A~. . .AAA,)Ml AAÂA~,~. ~a^—A~~:,;.A AAAA,.A,~,. . ~".~;~::'^ 'd c.. „ ~r rr ^c., .O^ ,..~.;... .,,~.. .~.. ;~~~^ r ~0~ ~ SFr' ~,.F. ;,.. . , r ,,,c , rr r.. ..,c„ ,r,.^^ ,,,~„. F . ^,.,r,..rr,r,. , t~y :.î•,r ',r~.rt' r•~,; ';Ccr.~cc, S' , ;r~,rS FGr . •:•r r r, , „ ~;,,f;~;~,r J ti. ~;( ,,r.,,rc , r , r ^.^ c ,,^'c r r. ,. . r: c r: .,c,r,c,c fr rr.nrr.', r c',rrrf:r:.%^c. ^ ..,. ;r,r,.^^rc~~,'^rirFr , %,ca l r3%. , ,r;rc,crr'c , , ,., î•~'r crr r. ' ^r. r'. ^^ ,' „ .. . rrc :^ ~, ,. :.^' ,r,.,.,. fit,.r , "., r rr ^ ...... , .. .:r:c•, ...... , „ .,.,; -, , . ,.,:::::r:::.:.::•'• • ...... r.r~i. .c, c.... ' . .. ._ ...... '>i~rr,_":,. r.,.,':.,....,.:•:r,,,rr ,"~...r.r. .... r,r" r;:;.";, r::r 2 19 1.394 f 0.086 1.283 t 0.089 0.262 f 0.836 0.204 t 0.798 37 2.659 f 0.018 2.269 f 0.042 0.966 t 0.084 0.739 t 0.110 .. ,;,,.;.r'r ,. ,. : o r:.c: :~: ":~:~ :~ ~ - ~ccr. yrr.r . ;. ,. c. ,. r. ,. r, ,.r .o ;, c, r. r. r•r:.r.r.r.r;r rc , r , . . c. „-.^~'r. . ~• 7~?.;r ' r'%• ' cs, , c.c:cc•r , c rcr~cc rc>rc c , , ~i:r .., . r.. . r .. .r . . fi.,r,r. , ,r rrr ~r.,.;.,r.~ r. '.,.~(.r. . r ;rjFc', ' r. r. ,. r. . ^,r: ...... (r ",...... ,r;C~,r.. ,...... ,,,,,,,,, ,r;s:r,,,.,,:;G.G.r::r:~::...... ,,,,, . . ,,,,,,,,,,,,,,,,,,,,, ...r~...... ,e,,,,,,..s„~ ;. ,.~ ...... :.:r:r., ,. F..~ .GEC"::rCC. rr. , ~' ;X

5.2. The inhibitory effects of paraherquamide are selective on the nAChR subtypes Multiple subtypes of the levamisole-activated receptor have been observed from individual patches, based on single-channel conductances. When paraherquamide was present in the bath solution, the level-Popen of each subtype was reduced by different amounts (Figure 5.3). The selective inhibitory effects of 1µM paraherquamide on different subtypes were analyzed (Table 5.2). The values of Popen+~/Popen(-) and the conductances had a significant correlation (correlation coefficient r = -0.6649, p < 0.05) (Figure 5.4 A), where the Popen(-) and Popen(+) are the level-Popens of the channel currents before and after the paraherquamide application. This observation suggests that each nAChR subtype may have individual paraherquamide sensitivity. We analyzed the sensitivity for each subtype (Figure 5.4 B). The 45-pS subtype was most sensitive to 1µM paraherquamide with Popen+~lPopen(-) — 54

0.3 5 (N=4), the 3 5 -pS subtype was less sensitive with Popen(+)/Popen() = 0.43 (N=2) and the

21-pS subtype was least sensitive' ' with Popen(+)lP open() — 1.2 ( N— 4 ) . The sensitivities of 21- p S and 45-pS subtypes were significantly different (t-test, p < 0.05).

i 2 pA

2000 ms

B C ~o 47 pS 2so - 60 - 240 -

(N) 50 - Z_ c 200 - 1 0 40 - 47 pS t ~ 160 -

Observation 37 pS 30 - O 37 pS of 0 120 - -~'` a~ 20 - ~ so -

Number z 10- 27 pS ~ 40 -

0 f ' .%►;~ T ` ~ ( ~ 1 T 0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 Amplitude (pA) Amplitude (pA)

Figure 5.3. The selective effects of 1µM paraherquamide on the nAChR subtypes. Observed at +75 mV, [levamisole] = 30 µM. A & B, three subtypes with conductances of 27 pS, 37 pS and 47 pS have been observed in this patch. C, 1µM paraherquamide reduced the Popens of three subtypes. The level-Popen of the 27-pS had no significant change from 0.00027 to 0.00031, the level-Popen of the 37-pS was reduced from 0.00807 to 0.00545 (68%), and the level-Popen of the 47-pS was reduced from 0.04079 to 0.02010 (49%). The channel open events in B were observed from a 1-min period and the events in C were observed from a 4-min period. Because paraherquamide reduced the frequency of channel open, alonger-period recording was analyzed to collect enough channel open events. 55

Table 5.2. The effect of paraherquamide on the level-Popens of the nAChR subtypes. Observed at +75 mv. Popen~~ is the level-Popen before the addition of paraherquamide and ropen(+) is the level-Popen after the addition of 1µM paraherquamide. The value of Popen(+) /Popen(-) indicates the paraherquamide effects on different nAChR subtypes. [Paraherquamide] = 1µM, [levamisole] = 30 µM. In recording #3, because of the existence of background noise, the 31-pS level could not be analyzed.

Recordin• # Conductance •S P,

2 25 0.00011 0.00022 1.9615 45 0.01084 0.00281 0.2592

4 31 0.00070 0.00292 0.00141 0.4826 52 ~~~~~~~: r

~,r'~,i~,ri

;. c a~ r O 2 i ■ 0.

~.. C ■ ■ ~O 1 ■ ,r i~ i ----~- ~~ ■ ■ ~ ~~ ~ i ~ i~~~ ~■ ~ I ~ I I I 1 10 20 30 40 50 60 conductance (pS)

2 PoPen() / ~ + t 1 pen

o P

0 21-pS 35-pS 45-pS nACh R subtype

Figure 5.4. The inhibitory effects of paraherquamide on the three subtypes were different. A, The scatter plot of Popen~+)/Popen() vs. conductances . The correlation coefficient was —0.66 and significantly non-zero (p < 0.05). The dashed squares indicate the three subtypes, 21-pS, 35-pS and 45-pS. B, The three nAChR subtypes had different sensitivities to paraherquamide. The mean ~ S . E. M. for 45 -pS was 0.35 ~ 0.09 (N = 4), for 35 -pS was 0.43 ~ 0.25 (N = 2) and for 25 -pS was 1.2 ~ 0.31 (N = 4) . The mean values of Popen~+)/Popen~~ for 21-p S and 35 -pS were significantly different (t-test, p < 0.05). [paraherquamide] = 1 µM, [levamisole] = 30 µM. Observed at +75 mV. 57

5.3. Discussion

We analyzed the patch Popen of each minute for individual patches and observed that

after the forming of giga-ohm seals, the patch P open of individual patches decreased gradually with time. This decrease may due to the "run-down" of the levamisole-activated receptor.

However, we analyzed the patch Popen of each minute for six recordings and the statistical analysis indicated that this decrease was not statistically significant (ANNA, p = 0.5, N=6) during a period of six minutes that was longer than our experimental period. To determine

the inhibitory effect of paraherquamide on the patch Popen of the levamisole-activated

channel currents, we measured the patch Popen of two patches from same vesicles in same

periods (usually 4-9 minutes after drug addition). The patch Popen from these two patches were compared and normalized by:

. P open(+d) (P open(—d )

Normalized Popen - P open(+c) (P open(—c) This calculation was used: (1) to compensate for the mechanical disturbance effect on the

° open and (2) to control difference between vesicles. We assumed individual patches from the same vesicles behaved in a similar manner. 58

CHAPTER 6. GENERAL DISCUSSION

6.1. The mean level-ropers of the three nAChR subtypes and the %contribution of each subtype to the whole cell current induced by 30 µM levamisole at +75 mV

Table 6.1 gives the level-Popen of each individual subtype presented in 30 patches (the same population as Table 4.1). Because it is impossible to know the exact number of the levamisole-activated receptors, we cannot measure the channel-Popen (see section 3.4.4). The Popen listed in the table is not the channelPopen but the level-Popen present on each patch. Among these 30 patches, 21 patches showed the existence of the 21-pS subtype. The mean level-Popen of the 21-pS subtype is the probability of the 21-pS channel level being open on individual patch (the patches without channel being observed are ignored because they did not contribute to the whole cell current), which is calculated by:

level-Popen (21J 0.0023 + 0.0250 + 0.0604 + • • • + 0.0155 n-21 _ = 0.0095 . 30 30 Where the level-Popen(21) is the level-Popen of the 21-pS subtype observed on each individual patch. Thus, the mean level-Popen of the 35-pS subtype (N = 14 from 30 patches) is:

levelPopen n-14 (35~ 0.1773+0.0133+0.0019+• • •+0.0001 - _ = 0.0097 30 30 and the mean level-Popen of the 45-pS subtype (N= 10 from 30 patches) is:

level-Popen (45) 0.0107 + 0.0018 + 0.0408 + . . . + 0.0005 n-10 _ = 0.0047 . 30 30 Where the level-Popen(3s) is the level-Popen of the 3 5-p S subtype and the level-Popen(4s) i s the levelPopen of the 45-pS subtype observed on each individual patch. 59

Table 6.1. The level-Popens observed from 30 patches. The mean level-Popen of the 21- pS (range 18-30 pS) is 0.0095 (N = 21); the mean level-Popen of the 35-pS (range3l- 40 pS) is 0.0097 (N = 14); and the mean level-Popen of the 45-pS (range 41-52 pS) is 0.0047 (N = 10).

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We assumed that the mean level-Popens from these 3 0 patches represent the level-Popens of the three nAChR subtypes on Ascaris suum muscle cells. The %contribution of each subtype to the levamisole-induced whole cell current is then calculated by:

x mean level-Popen(21) g(21) = 27% , mean levelP peri(21) X g(21) +mean level-Ppen 35 x g(35) +mean ZeveZP pen 45 x g(45 ) ( ) ( )

x mean level-Popen(35) g(35 ) = 45% , mean level-Popen(21) x g(21) +mean levelPopen 35 x g(3s) +mean level-Popen x g(4s ) ( ) ( 4s )

mean level-Popen(45) x g(45 = 28% . mean levelPopen (21) x g(21) +mean level-Popen 35 x g(35) +mean level-Popen 45 x g(4s ) O O 60

Where g is the mean single-channel conductance for each level. In summary, three subtypes of nicotinic acetylcholine receptors located on the Ascaris suum are activated by 30 µM levamisole at +75 mV. The % of the total current carried by the 21-pS subtype is 27%. The % of the total current carried by the 35-pS subtype is 45%. The of the total current carried by the 45-pS subtype is 28%.

6.2. Numbers of the three nAChR subtypes on Ascaris suum muscle cells The numbers of observation for each conductance were sorted in the histogram (Figure 4.5). These 47 observations were observed from 30 individual patches. The conductance histogram was fitted with the sum of three Gaussian distributions to give the mean conductance value for each subtypes (21, 35 and 45 pS) and the proportion of the number of channels of each subtypes (21-pS, 61 %; 35-pS, 29% and 45-pS, 10%). We assumed that the 30 patches are representative of the whole muscle cell membrane. The numbers of the three subtypes would then be in the proportions of 21-pS : 35-pS : 45-pS = 6.1 :2.9: 1.0 (Figure 6.1).

Proportions of the 21-pS : 35-pS : 45-pS = 6.1 :2.9: 1.0

21-pS

Mean level P open — 0.0095 of the total current: 27%

35-pS Mean level Popen — 0.0097 of the total current: 45~%

45 -pS Mean level Popen — 0.0047 of the total current: 28% Figure 6.1. A model for calculating the contributions of the three subtypes nAChRs to the transmembrane currents induced by 30 µM levamisole. The 21-pS subtype has a mean level-Popen of 0.0095 and contributes 27% of the transmembrane current; the 35-pS subtype has a mean level-Popen of 0.0097 and contributes 45% of the current; and the 45-pS subtype has a mean level-Popen of 0.0047 and contributes 28% of the current. The three nAChR subtypes are distributed on the muscle cell with a proportion of 6.1 :2.9: 1.0 (21-pS : 35-pS : 45-pS). 61

6.3. Channel-Popen of the three nAChR subtypes We know the relationship of the level-Popen and the channel-Popen for each conductance level is channel-Popen =level-Popen ~ N (see 3.4.4. Popen), then the channelPopen of the 21-pS, 3 5-pS and 45-pS subtypes can be calculated by:

channel - Popen(21) =level-Popen 1 ~ (21) ~ 21 ~

channel - Popen(3s) —level--Popen 35 1 ~ (35 ) ~ ~

channel - Popen(4s) — leVeZP open 45 N (45 ) ~ ~ Therefore,

level-Popen 35 1 ~ (35) channel - Popen(3s ) levelPopen 1V (21) ~21 ~

levelPopen~45~ 1 ~ (4s) channel - Popen(4s) levelPopen~21~ 1V (21)

From the calculation in section 6.1, we know that the mean level-Popen for 21-pS subtype is 0.0095, the mean level-Popen for the 3 5-pS is 0.0097, and the mean level-Popen for 4 5 -p S is 0.0047. We also know that the numbers of 21-p S, 3 5-p S and 4 5-p S follow the ratio of 6.1 :2.9: 1.0 from the calculation in section 6.3. Therefore, mean 6.1 level-Popen 35 channel X x ~ 2 x Pop en (35) — channel-Popen 21 channel-Popen 21 2.9 mean ~ ~ ~ ~ level-Popen 21

mean 6.1 level-Popen 45 channel x ~ x Popen 4s — X channel-Popen 21 3 channel-Popen 21 ~ ~ 1.0 mean level-Popen ~ ~ ~ ~ ~21 ~ In a brief summary, the 4 5 -p S nAChR subtype has the highest channel-Popen and the 21- pS subtype has the lowest channel-Popen. The ratio of the channel-Popen is abOUt 3:2: 1 (45- pS : 35-pS : 21-pS). 62

6.4. The paraherquamide effects on the whole cell currents The mean level-Popen of the 45-pS subtype is 0.0047. 1µM paraherquamide reduces this

Popen to 3 5 ± 9%. The mean levelPopen after 1 µM paraherquamide then is 0.0047 x 3 5% = 0.0016. The mean level-Popen of the 3 5-pS subtype is 0.0097. 1µM paraherquamide reduces this Popen to 43 ± 25%. The mean level-Popen after 1 µM paraherquamide then is 0.0097 x 43% = 0.0042 . The mean level-Popen of the 21-pS subtype is 0.0095. This Popen was changed to 120 ± 61%. The mean level-Popen after 1 µM paraherquamide then is 0.0095 x 120% = 0.0114. Therefore, 1µM paraherquamide would reduce the whole levamisole-induced current by:

(level x +level x Popen 21 g(21) Popen 35 g(3s) +levelPopen 4s x g(45)) after para 1 ~ ~ ~ ~ ~ ~ =39% x (levelPopen x g(21) +levelPopen 35 g(3s) +level-Popen x g(45) )before pars ~ 2i ~ ( ~ ~ 4s ~

The observations on muscle contraction show (Robertson et al., 2002) that 0.3 µM paraherquamide reduces the muscle flap contraction to 30 µM levamisole by ~45% and 3µM paraherquamide reduces the contraction by ~75%. The two sets of experiments suggest a similar level of antagonism by paraherquamide.

6.5. Do the conductance subtypes correspond to pharmacological subtypes? Robertson et al. (2002) have reported the existence of three pharmacological subtypes of Ascaris suum nAChRs, nicotine-, levamisole- and bephenium-preferring subtypes. Richmond and Jorgensen (1999) have reported the existence of levamisole-sensitive and nicotine-sensitive nACh receptors in the nematode C. elegans. These observations suggest the e~stence of multiple nAChR subtypes on the nematode muscle cells. We observed in this study that three subtypes of nicotinic acetylcholine receptors were located on the Ascaris scum muscle cells and activated by levamisole. There are two possibilities: (1) all of the three conductance subtypes are levamisole-preferring; (2) the three conductance subtypes belong to two or three of the pharmacological subtypes. It seems unlikely that all of the three conductance subtypes activated by 30 µM levamisole axe levamisole-preferring. One of the conductance subtypes may belong to nicotine-preferring 63 subtype. The levamisole-activated single-channel currents of the 45-pS subtype have higher conductances and channel-Popen than the two others. The 45-pS had the most sensitivity to paraherquamide. Paraherquamide can distinguish nicotine-preferring from levamisole- preferring receptors using the muscle contraction experiment (Robertson et al., 2002) and the levamisole-preferring subtype is more sensitive than the nicotine-preferring subtype. Therefore, we suggest that the 45-pS subtype corresponds to the levamisole-preferring pharmacological subtype and the 21-pS conductance subtype corresponds to the nicotine- preferring pharmacological subtype. The 3 5 -p S subtype had medium channel conductances and mean open time. The higher sensitivity of the 3 5-pS to paraherquamide suggest that it is unlikely to be nicotine-preferring subtype. So, it is either the bephenium-preferring or another levamisole-preferring nAChR subtype. 64

CHAPTER 7. CONCLUSIONS

In this study we demonstrated the existence of three nAChR subtypes located on the Ascaris swum muscle cells. These three subtypes of nicotinic acetylcholine receptors were separated by their channel conductance and mean open time. The mean conductance values of these subtypes were 21 p S, 3 S p S and 45 p S . Paraherquamide, anovel anthelmintic compound, is an antagonist of the nicotinic acetylcholine receptor located on Ascaris suum muscle cells. This compound reduced the

patch Popen of the levamisole-activated ion channels. No significant effect on the channel conductance and the mean open time was observed. The potent inhibitory effect of paraherquamide was concentration dependant and was fitted with adose-dependant curve between 0.3 µM and 10 µM with IC 5 0 2.1 µM.

The inhibitory effects of paraherquamide on levelP open were selective and dependent on the different subtypes of the levamisole-activated receptor. The 45-pS subtype was most sensitive to paraherquamide and the 21-pS had the least sensitivity. The sensitivities of Z1-pS and 45-pS subtypes were significantly different. These results are consistent with the observations that three pharmacological subtypes of cholinergic receptors exist on Ascaris suum muscle and paraherquamide can distinguish nicotine-preferring from levamisole- preferring receptors (Robertson et al., 2002). These studies are the first evidence of a correlation of the biophysical and the pharmacological nAChR subtypes. We conclude that the relative numbers of the three nAChR subtypes located on Ascaris muscle cells is 6.1 :2.9: 1.0 (21-pS : 3 5-pS : 45-pS). The 21-pS subtype is predicted to contribute 27% of the whole-cell current; the 35-pS subtype contributes 45% and the 45-pS subtype contributes 28%. Paraherquamide inhibits these channels with different potencies. After the application of 1µM paraherquamide, the whole-cell current is predicted to be reduced by 39%. This conclusion is consistent with the muscle contraction observation at a similar level. Further study on the molecular and genetic information may be provide more information to understand the Ascaris nAChRs, or further electrophysiological and 65

pharmacological study on the nematode C. elegans may provide comparable information about the levamisole-activated nAChR. 66

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ACKNOWLEDGMENTS

I would like to thank my major professor, Professor Richard Martin, and supervisor Dr. Alan Robertson. They provide me the opportunity to work in the laboratory. I very appreciate their advice and helpful discussions. I would like to thank my family for supporting me for many years.