The levamisole activated acetylcholine receptor in C.elegans is now known to constitute four major subunits- LEV-1, UNC-29, UNC-38 and UNC-63. Mutants for these genes are strongly resistant to the anthelminthic, levamisole. However, regulators of receptor subunit function have not all been fully characterised. Mutations in lev-9 have been shown to confer partial resistance to both levamisole and nicotine, but do not affect levamisole binding in vitro (Lewis et al ., 1987). Thus, it has been suggested that lev-9 is a possible modulator of levamisole receptor function. We have cloned lev-9 and shown that it encodes a molecule homologous to the transmembrane glycoprotein, alpha sarcoglycan. In humans, the sarcoglycans are a major component of the dystrophin glycoprotein complex (DGC) and are associated with autosomal recessive forms of limb girdle muscular dystrophy. LEV-9 is mainly expressed in the muscles of the head, pharynx, body and vulva. Using fluorescently labeled a -bungarotoxin, we have shown that lev-9 exhibits decreased surface receptor expression compared to wild type. Preliminary results using in vivo calcium imaging from vulval muscles in dissected worm preparations also suggest a difference between lev-9 and wild type in the rate of calcium influx, in response to nicotine. Further experiments using fluorescent labeling to determine how LEV-9 affects the levamisole receptor surface expression and electron microscopic comparison of lev-9 and wild types are in progress. P> P> Lewis JA, et al . (1987) J. Neurosci . 7(10), 3059-71

DB Sattelle et al. (1999) International C. elegans Meeting "A novel C. elegans gene encodes a nicotinic acetylcholine receptor a subunit is capable of forming a functional hetero-oligomeric receptor in vitro when co-expressed with lev-1 and unc-29"

HA Baylis, E Culetto, DB Sattelle, K Matsuda, JT Fleming, MD Squire, JA Lewis, NP Mongan

[

International C. elegans Meeting,

1999]

Nicotinic acetylcholine receptors (nAChRs) mediate the fast action of the neurotransmitter acetylcholine at cholinergic synapses in the nematode C. elegans . An extensive and diverse family of nAChRs has been described in this organism (1). Genetic screens for mutants resistant to the cholinergic anthelmintic drug levamisole (2, 3) led to the molecular characterization of nAChRs subunit genes unc-29 , lev-1 and unc-38 (4). Of the eleven levamisole resistant genes so far characterized genetically, the molecular identity of five, unc-63 , unc-74 , lev-8 , lev-9 and lev-10 , remains to be determined. A new nAChR has been cloned using cross hybridization with unc-38 and unc-29 cDNAs and RT-PCR. This nAChR is a putative a subunit as it has the typical YxCC motif in loop C of the acetylcholine binding site. We have shown that this subunit is encoded by unc-63 . Three different mutant alleles of unc-63 have also been sequenced. The allele x37 confers on worms high resistance to levamisole and a very slow locomotion. Sequencing shows that a stop codon in the extracellular loop interrupts the open reading frame in the x37 allele. Worms with two rare alleles b404 and x26 have a slight levamisole resistance and nearly normal movement. The sequencing of b404 allele shows a deletion in the large intracellular loop whereas the x26 allele have a C to Y mutation in the highly conserved dicysteine loop in the N-terminal region. We used voltage clamp electrophysiology to study heterologous expression in Xenopus oocytes injected with cDNA expression constructs for lev-1 , unc-29 and unc-63 . This recombinant nAChR gives a robust current which is dose-dependent to acetylcholine (EC 50 = 30 microM). This value agrees well with EC 50 obtained for native nematode nAChRs. This recombinant heteromeric receptor is blocked reversibly by mecamylamine. (1) Mongan et al. (1998) Receptors & Channels 6, 218-228. (2) Brenner, S. (1974) Genetics 77, 71-94. (3) Lewis, J.A. et al. (1980) Genetics 95, 905-928. (4) Fleming, J.T. et al. (1997) J. Neurosci 17, 5843-5857.

Aamna Kaul et al. (2003) International Worm Meeting "Hyperactivation of glutamate-gated chloride channels with ivermectin results in necrosis."

Joseph Dent, Aamna Kaul

[

International Worm Meeting,

2003]

Ivermectin is a widely used antiparasitic drug. It kills worms by activating glutamate-gated chloride channels (GluCls), which belong to the family of ligand-gated anion channels that includes the GABA and glutamate receptors (Cully et al., 1994; Dent et al., 2000). The chloride permeability that ivermectin induces in excitable cells tends to prevent excitation. For example, ivermectin targets a GluCl expressed in the pharyngeal muscle to inhibit muscle contraction and prevent eating (Dent et al., 1997). The worms linger for several days in the presence of ivermectin before they starve to death. However, we have found that the lethal effects of ivermectin on C. elegans become irreversible after only a few hours of exposure. When L1 worms were exposed to 20ng/ml for 5 hours and then washed, they gradually developed large vacuoles in their pharyngeal muscle over the next several days. A mutant strain that lacks ivermectin receptors shows little or no necrosis when treated. Ivermectin is hydrophobic and it irreversibly opens GluCls expressed in Xenopus oocytes. So it is possible that ivermectin persists in membranes and continues to activate GluCls. Furthermore, it has been shown that hyperactive cation channels can induce excitotoxic necrosis (Driscoll and Chalfie, 1991). Why, though, would an inhibitory channel have a similar effect when hyperactivated? We are trying to address this question by looking at whether mutations known to inhibit excitotoxicity also inhibit the necrotic effects of ivermectin. Cully DF, Vassilatis DK, Liu KK, Paress PS, Van der Ploeg LHT, Schaeffer JM, Arena JP. Nature 371: 707-711 1994 Dent JA, Smith MM, Vassilatis DK, Avery L. PNAS USA 97: 2674-2679 2000 Dent JA, Davis MW, Avery L. EMBO Journal 16: 5867-5879 1997 Driscoll, M and Chalfie, M. Nature 349: 588-593 1991

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