Jana Liewald, Ruta Almedom, Thorsten Schedletzky and Alexander Gottschalk. We have previously identified proteins associated with the levamisole receptor, one of two nicotinic acetylcholine receptors (nAChR) acting at the neuromuscular junction of Caenorhabditis elegans, after tandem affinity purification, mass spectrometry, and subsequent RNAi-based functional screening (Gottschalk et al., 2005). For proteins causing altered nicotine-sensitivity in vivo, genomic (deletion) mutants were obtained. Currently, we are analyzing these mutants electrophysiologically for the effects on the functional properties of nAChRs (and GABAA receptors) present at the neuromuscular junction. Therefore, body wall muscle cells of C. elegans are whole cell patch-clamped and inward currents are determined, induced by application of neurotransmitters (ACh, GABA) and other cholinergic agonists (levamisole, nicotine).. Preliminary data indicate that the
nra-2(
tm1453) mutant shows significantly reduced currents mediated by ACh and levamisole, while GABA-induced currents are like in wild-type animals. This is in agreement with other functional assays for sensitivity to such agonists carried out in our group:
nra-2(
tm1453) mutants show moderate resistance to paralysis induced by levamisole and nicotine, as well as significantly reduced levamisole receptor levels at the neuromuscular junction (found after in vivo fluorescence labeling). Sensitivity to muscimol, a GABAA receptor agonist, however, was not affected in
nra-2(
tm1453) animals.. Loss of the POLO-like kinase PLK-2, which had caused weak nicotine resistance in our paralysis assays (see also abstract by T. Schedletzky), did not show significantly reduced inward currents in body wall muscle cells after application of cholinergic agonists, though a trend towards reduced currents became apparent.. Other mutants studied are a membrane-binding Copine (NRA-1) and the receptor-tyrosine kinase signalling protein SOC-1. Loss of either protein causes reduced synaptic levamisole receptor levels, while
soc-1 mutants are also affected for synaptic GABAA receptor expression. Their electrophysiological evaluation is in progress and results will be presented at the meeting. References: Gottschalk, A., Almedom, R. B., Schedletzky, T., Anderson, S. D., Yates III, J. R., and Schafer, W. R. Identification and characterization of novel nicotinic receptor-associated proteins in Caenorhabditis elegans. EMBO J. 24 (2005), 2566-2578.