Contributions from Caenorhabditis elegans functional genetics to antiparasitic drug target identification and validation: nicotinic acetylcholine receptors, a case study

Following the complete sequencing of the genome of the free-living nematode, Caenorhabditis elegans, in 1998, rapid advances have been made in assigning functions to many genes. Forward and reverse genetics have been used to identify novel components of synaptic transmission as well as determine the key components of antiparasitic drug targets. The nicotinic acetylcholine receptors (nAChRs) are prototypical ligand-gated ion channels. The functions of these transmembrane proteins and the roles of the different members of their extensive subunit families are increasingly well characterised. The simple nervous system of C. elegans possesses one of the largest nicotinic acetylcholine receptor gene families known for any organism and a combination of genetic, microarray, physiological and reporter gene expression studies have added greatly to our understanding of the components of nematode muscle and neuronal nAChR subtypes. Chemistry-to-gene screens have identified five subunits that are components of nAChRs sensitive to the antiparasitic drug, levamisole. A novel, validated target acting downstream of the levamisole-sensitive nAChR has also been identified in such screens. Physiology and molecular biology studies on nAChRs of parasitic nematodes have also identified levamisole-sensitive and insensitive subtypes and further subdivisions are under investigation.     

α7 mutants mimicking atypical motifs (YxxCC of loop-C, and E to H at −1′ in TM2) in the C. elegans LEV-8 subunit affect nicotinic acetylcholine receptor function

The ACR-8-like group of C. elegans nicotinic acetylcholine receptor (nAChR) subunits contain unusual motifs in the ACh binding site and in the −1′ position of transmembrane region two (TM2). Using site-directed mutagenesis (SDM) we have introduced these motifs into chicken α7 as it has not been possible to express C. elegans nAChR in vitro. Oocytes expressing α7 with the C. elegans binding motif show a reduced affinity and efficacy for both ACh and nicotine. The blocking action of the anthelmintic drug levamisole is reduced. The TM2 motif resulted in a non-functional receptor. We conclude that the TM2 motif profoundly restricts cation movement through the α7 channel but does not confer anion permeability. The altered form of the ACh binding motif is likely to result in a receptor with altered pharmacology, adding potential functional diversity at synapses in the nervous system and neuromuscular junctions of C. elegans.     

Activity of novel nicotinic anthelmintics in cut preparations of Caenorhabditis elegans

The free-living nematode Caenorhabditis elegans is a useful model for studying the pharmacology of anthelmintics. Currently approved anthelmintics have various mechanisms of action, including activity at nematode nicotinic acetylcholine receptors (nAChRs). Classical anthelmintic agonists of these receptors (nicotine, levamisole, pyrantel and bephenium) caused intact specimens of C. elegans to undergo contracted paralysis. The nAChR antagonist mecamylamine paralysed intact worms and blocked the actions of the agonists. The time to onset of effects of these drugs was enhanced when worms bisected between the mid- and anterior-portions were tested. The novel anthelmintic nAChR antagonist derquantel (2-desoxoparaherquamide, 2-DOPH) was weakly active in intact specimens of C. elegans at concentrations of 50 μM over several days. No antagonism of the nAChR agonists was observed with this drug in intact worms. However, derquantel had direct and marked effects on motility in cut worms and blocked the effects of nAChR agonists in this preparation. A representative of the new amino-acetonitrile derivative (AAD) class of nAChR agonists was not antagonised by derquantel in cut C. elegans, suggesting that these two anthelmintics may not demonstrate unfavourable drug-drug interactions at the receptor level if used to treat livestock infected with parasitic nematodes. The permeability properties of the C. elegans cuticle may be more restrictive than those of adult parasites, calling into question primary anthelmintic screening strategies that rely on this model organism.     

The Caenorhabditis elegans unc-63 gene encodes a levamisole-sensitive nicotinic acetylcholine receptor alpha subunit

The anthelmintic drug levamisole causes hypercontraction of body wall muscles and lethality in nematode worms. In the nematode Caenorhabditis elegans, a genetic screen for levamisole resistance has identified 12 genes, three of which (unc-38, unc-29, and lev-1) encode nicotinic acetylcholine receptor (nAChR) subunits. Here we describe the molecular and functional characterization of another levamisole-resistant gene, unc-63, encoding a nAChR alpha subunit with a predicted amino acid sequence most similar to that of UNC-38. Like UNC-38 and UNC-29, UNC-63 is expressed in body wall muscles. In addition, UNC-63 is expressed in vulval muscles and neurons. We also show that LEV-1 is expressed in body wall muscle, thus overlapping the cellular localization of UNC-63, UNC-38, and UNC-29 and suggesting possible association in vivo. This is supported by electrophysiological studies on body wall muscle, which demonstrate that a levamisole-sensitive nAChR present at the C. elegans neuromuscular junction requires both UNC-63 and LEV-1 subunits. Thus, at least four subunits, two alpha types (UNC-38 and UNC-63) and two non-alpha types (UNC-29 and LEV-1), can contribute to levamisole-sensitive muscle nAChRs in nematodes.     

Genes affecting the activity of nicotinic receptors involved in Caenorhabditis elegans egg-laying behavior

Egg-laying behavior in Caenorhabditis elegans is regulated by multiple neurotransmitters, including acetylcholine and serotonin. Agonists of nicotinic acetylcholine receptors such as nicotine and levamisole stimulate egg laying; however, the genetic and molecular basis for cholinergic neurotransmission in the egg-laying circuitry is not well understood. Here we describe the egg-laying phenotypes of eight levamisole resistance genes, which affect the activity of levamisole-sensitive nicotinic receptors in nematodes. Seven of these genes, including the nicotinic receptor subunit genes unc-29, unc-38, and lev-1, were essential for the stimulation of egg laying by levamisole, though they had only subtle effects on egg-laying behavior in the absence of drug. Thus, these genes appear to encode components of a nicotinic receptor that can promote egg laying but is not necessary for egg-laying muscle contraction. Since the levamisole-receptor mutants responded to other cholinergic drugs, other acetylcholine receptors are likely to function in parallel with the levamisole-sensitive receptors to mediate cholinergic neurotransmission in the egg-laying circuitry. In addition, since expression of functional unc-29 in muscle cells restored levamisole sensitivity under some but not all conditions, both neuronal and muscle cell UNC-29 receptors are likely to contribute to the regulation of egg-laying behavior. Mutations in one levamisole receptor gene, unc-38, also conferred both hypersensitivity and reduced peak response to serotonin; thus nicotinic receptors may play a role in regulating serotonin response pathways in the egg-laying neuromusculature.     

A highly conserved cytoplasmic cysteine residue in the α4 nicotinic acetylcholine receptor is palmitoylated and regulates protein expression

Nicotinic acetylcholine receptor (nAChR) cell surface expression levels are modulated during nicotine dependence and multiple disorders of the nervous system, but the mechanisms underlying nAChR trafficking remain unclear. To determine the role of cysteine residues, including their palmitoylation, on neuronal α4 nAChR subunit maturation and cell surface trafficking, the cysteines in the two intracellular regions of the receptor were replaced with serines using site-directed mutagenesis. Palmitoylation is a post-translational modification that regulates membrane receptor trafficking and function. Metabolic labeling with [(3)H]palmitate determined that the cysteine in the cytoplasmic loop between transmembrane domains 1 and 2 (M1-M2) is palmitoylated. When this cysteine is mutated to a serine, producing a depalmitoylated α4 nAChR, total protein expression decreases, but surface expression increases compared with wild-type α4 levels, as determined by Western blotting and enzyme-linked immunoassays, respectively. The cysteines in the M3-M4 cytoplasmic loop do not appear to be palmitoylated, but replacing all of the cysteines in the loop with serines increases total and cell surface expression. When all of the intracellular cysteines in both loops are mutated to serines, there is no change in total expression, but there is an increase in surface expression. Calcium accumulation assays and high affinity binding for [(3)H]epibatidine determined that all mutants retain functional activity. Thus, our results identify a novel palmitoylation site on cysteine 273 in the M1-M2 loop of the α4 nAChR and determine that cysteines in both intracellular loops are regulatory factors in total and cell surface protein expression of the α4β2 nAChR.     

Purification and identification of G protein-coupled receptor protein complexes under native conditions

G protein-coupled receptors (GPCRs) constitute the largest family of membrane receptors and are of major therapeutic importance. The identification of GPCR-associated proteins is an important step toward a better understanding of these receptors. However, current methods are not satisfying as only isolated receptor domains (intracellular loops or carboxyl-terminal tails) can be used as "bait." We report here a method based on tandem affinity purification coupled to mass spectrometry that overcomes these limitations as the entire receptor is used to identify protein complexes formed in living mammalian cells. The human MT(1) and MT(2) melatonin receptors were chosen as model GPCRs. Both receptors were tagged with the tandem affinity purification tag at their carboxyl-terminal tails and expressed in human embryonic kidney 293 cells. Receptor solubilization and purification conditions were optimized. The method was validated by the co-purification of G(i) proteins, which are well known GPCR interaction partners but which are difficult to identify with current protein-protein interaction assays. Several new and functionally relevant MT(1)- and MT(2)-associated proteins were identified; some of them were common to both receptors, and others were specific for each subtype. Taken together, our protocol allowed for the first time the purification of GPCR-associated proteins under native conditions in quantities suitable for mass spectrometry analysis.     

Functional genomics of the nicotinic acetylcholine receptor gene family of the nematode, Caenorhabditis elegans

Nicotinic acetylcholine receptors (nAChRs) are ligand-gated ion channels that bring about a diversity of fast synaptic actions. Analysis of the Caenorhabditis elegans genome has revealed one of the most-extensive and diverse nAChR gene families known, consisting of at least 27 subunits. Striking variation with possible functional implications has been observed in normally conserved motifs at the acetylcholine-binding site and in the channel-lining region. Some nAChR subunits are particular to neurons whilst others are present in both neurons and muscles. The localization of subunits in non-synaptic regions suggests novel roles for nAChRs. Genetic and heterologous expression studies have identified a subset of nAChR subunits that are important drug targets while the study of mutants has identified genes functionally-linked to nAChRs. Future studies using C. elegans offer the prospect of increasing our understanding of the functional diversity of a complex nAChR gene family as well as addressing the role of nAChRs and associated proteins in human disorders.     

The prototoxin lynx1 acts on nicotinic acetylcholine receptors to balance neuronal activity and survival in vivo

Nicotinic acetylcholine receptors (nAChRs) affect a wide array of biological processes, including learning and memory, attention, and addiction. lynx1, the founding member of a family of mammalian prototoxins, modulates nAChR function in vitro by altering agonist sensitivity and desensitization kinetics. Here we demonstrate, through the generation of lynx1 null mutant mice, that lynx1 modulates nAChR signaling in vivo. Its loss decreases the EC(50) for nicotine by approximately 10-fold, decreases receptor desensitization, elevates intracellular calcium levels in response to nicotine, and enhances synaptic efficacy. lynx1 null mutant mice exhibit enhanced performance in specific tests of learning and memory. Consistent with reports that mutations resulting in hyperactivation of nAChRs can lead to neurodegeneration, aging lynx1 null mutant mice exhibit a vacuolating degeneration that is exacerbated by nicotine and ameliorated by null mutations in nAChRs. We conclude that lynx1 functions as an allosteric modulator of nAChR function in vivo, balancing neuronal activity and survival in the CNS.     

Nitrosamines as nicotinic receptor ligands

Nitrosamines are carcinogens formed in the mammalian organism from amine precursors contained in food, beverages, cosmetics and drugs. The potent carcinogen, NNK, and the weaker carcinogen, NNN, are nitrosamines formed from nicotine. Metabolites of the nitrosamines react with DNA to form adducts responsible for genotoxic effects. We have identified NNK as a high affinity agonist for the alpha7 nicotinic acetylcholine receptor (alpha7nAChR) whereas NNN bound with high affinity to epibatidine-sensitive nAChRs. Diethylnitrosamine (DEN) bound to both receptors but with lower affinity. High levels of the alpha7nAChR were expressed in human small cell lung cancer (SCLC) cell lines and in hamster pulmonary neuroendocrine cells (PNECs), which serve as a model for the cell of origin of human SCLC. Exposure of SCLC or PNECs to NNK or nicotine increased expression of the alpha7nAChR and caused influx of Ca(2+), activation of PKC, Raf-1, ERK1/2, and c-myc, resulting in the stimulation of cell proliferation. Signaling via the alpha7nAChR was enhanced when cells were maintained in an environment of 10-15% CO(2) similar to that in the diseased lung. Hamsters with hyperoxia-induced pulmonary fibrosis developed neuroendocrine lung carcinomas similar to human SCLC when treated with NNK, DEN, or nicotine. The development of the NNK-induced tumors was prevented by green tea or theophylline. The beta-adrenergic receptor agonist, isoproterenol or theophylline blocked NNK-induced cell proliferation in vitro. NNK and nicotine-induced hyperactivity of the alpha7nAChR/RAF/ERK1/2 pathway thus appears to play a crucial role in the development of SCLC in smokers and could be targeted for cancer prevention.     

Functional Impact of 14 Single Nucleotide Polymorphisms Causing Missense Mutations of Human α7 Nicotinic Receptor

The α7nicotinic receptor (nAChR) is a major subtype of the nAChRs in the central nervous system, and the receptor plays an important role in brain function. In the dbSNP database, there are 55 single nucleotide polymorphisms (SNPs) that cause missense mutations of the human α7nAChR in the coding region. In this study, we tested the impact of 14 SNPs that cause missense mutations in the agonist binding site or the coupling region between binding site and channel gate on the receptor function. The wild type or mutant receptors were expressed or co-expressed in Xenopus oocytes, and the agonist-induced currents were tested using two-electrode voltage clamp. Our results demonstrated that 6 mutants were nonfunctional, 4 mutants had reduced current expression, and 1 mutants altered ACh and nicotine efficacy in the opposite direction, and one additional mutant had slightly reduced agonist sensitivity. Interestingly, the function of most of these nonfunctional mutants could be rescued by α7nAChR positive allosteric modulator PNU-120596 and agonist-PAM 4BP-TQS. Finally, when coexpressed with the wild type, the nonfunctional mutants could also influence the receptor function. These changes of the receptor properties by the mutations could potentially have an impact on the physiological function of the α7nAChR-mediated cholinergic synaptic transmission and anti-inflammatory effects in the human SNP carriers. Rescuing the nonfunctional mutants could provide a novel way to treat the related disorders.     

A C. elegans model of nicotine-dependent behavior: regulation by TRP-family channels

Nicotine, the primary addictive substance in tobacco, induces profound behavioral responses in mammals, but the underlying genetic mechanisms are not well understood. Here we develop a C. elegans model of nicotine-dependent behavior. We show that worms exhibit behavioral responses to nicotine that parallel those observed in mammals, including acute response, tolerance, withdrawal, and sensitization. These nicotine responses require nicotinic acetylcholine receptor (nAChR) family genes that are known to mediate nicotine dependence in mammals, suggesting functional conservation of nAChRs in nicotine responses. Importantly, we find that mutant worms lacking TRPC (transient receptor potential canonical) channels are defective in their response to nicotine and that such a defect can be rescued by a human TRPC channel, revealing an unexpected role for TRPC channels in regulating nicotine-dependent behavior. Thus, C. elegans can be used to characterize known genes as well as to identify new genes regulating nicotine responses.     

Interacting Genes Required for Pharyngeal Excitation by Motor Neuron Mc in Caenorhabditis Elegans

We studied the control of pharyngeal excitation in Caenorhabditis elegans. By laser ablating subsets of the pharyngeal nervous system, we found that the MC neuron type is necessary and probably sufficient for rapid pharyngeal pumping. Electropharyngeograms showed that MC transmits excitatory postsynaptic potentials, suggesting that MC acts as a neurogenic pacemaker for pharyngeal pumping. Mutations in genes required for acetylcholine (ACh) release and an antagonist of the nicotinic ACh receptor (nAChR) reduced pumping rates, suggesting that a nAChR is required for MC transmission. To identify genes required for MC neurotransmission, we screened for mutations that cause slow pumping but no other defects. Mutations in two genes, eat-2 and eat-18, eliminated MC neurotransmission. A gain-of-function eat-18 mutation, ad820sd, and a putative loss-of-function eat-18 mutation, ad1110, both reduced the excitation of pharyngeal muscle in response to the nAChR agonists nicotine and carbachol, suggesting that eat-18 is required for the function of a pharyngeal nAChR. Fourteen recessive mutations in eat-2 fell into five complementation classes. We found allele-specific genetic interactions between eat-2 and eat-18 that correlated with complementation classes of eat-2. We propose that eat-18 and eat-2 function in a multisubunit protein complex involved in the function of a pharyngeal nAChR.     

The Ror receptor tyrosine kinase CAM-1 is required for ACR-16-mediated synaptic transmission at the C. elegans neuromuscular junction

Nicotinic (cholinergic) neurotransmission plays a critical role in the vertebrate nervous system, underlies nicotine addiction, and nicotinic receptor dysfunction leads to neurological disorders. The C. elegans neuromuscular junction (NMJ) shares many characteristics with neuronal synapses, including multiple classes of postsynaptic currents. Here, we identify two genes required for the major excitatory current found at the C. elegans NMJ: acr-16, which encodes a nicotinic AChR subunit homologous to the vertebrate alpha7 subunit, and cam-1, which encodes a Ror receptor tyrosine kinase. acr-16 mutants lack fast cholinergic current at the NMJ and exhibit synthetic behavioral deficits with other known AChR mutants. In cam-1 mutants, ACR-16 is mislocalized and ACR-16-dependent currents are disrupted. The postsynaptic deficit in cam-1 mutants is accompanied by alterations in the distribution of cholinergic vesicles and associated synaptic proteins. We hypothesize that CAM-1 contributes to the localization or stabilization of postsynaptic ACR-16 receptors and presynaptic release sites.     

Nicotine enhances intracellular nicotinic receptor maturation: a novel mechanism of neural plasticity?

Nicotine addiction, the primary cause of tobacco consumption, is mediated through nicotine binding to brain nicotinic acetylcholine receptor (nAChRs). Upon chronic exposure, nicotine elicits a cascade of events, starting with nAChR activation and desensitization, followed by a long term up-regulation that corresponds to an increase in the number of the high affinity nAChRs, a paradoxical process that occurs in the brain of smokers. Recent investigation of the maturation and trafficking of the major brain alpha4beta2 nAChR demonstrates that up-regulation is initiated in the endoplasmic reticulum soon after protein translation. The data thus far accumulated provide evidence that nicotine elicits up-regulation by promoting maturation of nAChR precursors that would otherwise be degraded. This "maturational enhancer" action of nicotine probably contributes to the long term effect of chronic nicotine, and suggests a novel mechanism of neuronal plasticity through an yet unknown endogenous substance which would modulate the receptor expression under physiological conditions.     

The nicotine‐mediated decline in l‐dopa‐induced dyskinesias is associated with a decrease in striatal dopamine release

l ‐dopa‐induced dyskinesias (LIDs) are a side effect of Parkinson's disease therapy that is thought to arise, at least in part, because of excessive dopaminergic activity. Thus, drugs that regulate dopaminergic tone may provide an approach to manage LIDs. Our previous studies showed that nicotine treatment reduced LIDs in Parkinsonian animal models. This study investigates whether nicotine may exert its beneficial effects by modulating pre‐synaptic dopaminergic function. Rats were unilaterally lesioned by injection of 6‐hydroxydopamine (6‐OHDA) (2 × 3 ug per site) into the medial forebrain bundle to yield moderate Parkinsonism. They were then implanted with minipumps containing vehicle or nicotine (2.0 mg/kg/d) and rendered dyskinetic with l ‐dopa (8 mg/kg plus 15 mg/kg benserazide). Lesioning alone decreased the striatal dopamine transporter, nicotinic receptor (nAChR) levels, and nAChR‐mediated ³H‐dopamine release, consistent with previous results. Nicotine administration reduced l ‐dopa‐induced abnormal involuntary movements throughout the course of the study (4 months). Nicotine treatment led to declines in the striatal dopamine transporter, α6β2* nAChRs and various components of α6β2* and α4β2* nAChR‐mediated release. l ‐dopa treatment had no effect. These data suggest that nicotine may improve LIDs in Parkinsonian animal models by dampening striatal dopaminergic activity.