Nicotinic acetylcholine receptors (nAChRs) are ligand-gated ion channels mediating excitation of muscles and neurons. At the neuromuscular junction (NMJ), nAChRs are clustered opposite of motoneuron release sites, a process regulated in vertebrates by receptor tyrosine kinases (RTKs), e.g. MuSK. We and others showed that in C. elegans, CAM-1 and EGL-15 RTKs affect NMJ clustering of nAChRs (1, 2). Recently, the RTK cytoplasmic adaptor protein Dok-7, that interacts with MuSK, was shown to be essential for nAChR clustering in mice (3). We biochemically co-purified a similar adaptor protein, SOC-1, with the levamisole receptor (LevR; 1). Like Dok-7, SOC-1 has an N-terminal pleckstrin homology (PH) domain and C-terminal SH2 domain binding sites. We found that SOC-1 influences postsynaptic LevR levels, and that
soc-1(
n1789) mutants are resistant to cholinergic agonists (levamisole and nicotine). In addition,
soc-1 mutants were muscimol- (GABA agonist) resistant, and synaptic expression of UNC-49 GABAA receptors was also reduced, indicating functions of SOC-1 beyond nAChR clustering. Cholinergic phenotypes were rescued by expression of SOC-1::GFP in muscles, indicating cell-autonomous function. SOC-1::GFP localized to the plasma membrane, including muscle arms, consistent with its interaction with the LevR. By electrophysiology,
soc-1(
n1789) mutants showed reduced levamisole- or nicotine-evoked currents in muscle. However, ACh-, as well as GABA- and muscimol-evoked currents were normal. The latter findings could indicate dispersed clustering of GABAARs, rather than reduced surface expression, which we try to confirm by evoking endogenous GABA release using the light-activated cation channel ChR2 (4). In addition, we aim to decode the protein interaction network orchestrating postsynaptic organization, which should include SOC-1 and the LevR. To this end, we purify SOC-1-associated proteins from muscle cells by tandem affinity purification (TAP). Most of the SOC-1::TAP protein is soluble. Initial purification from the soluble fraction yielded a number of proteins which will be identified by mass spectrometry. Furthermore, purification of SOC-1::TAP from the membrane fraction is underway, possibly identifying associated membrane proteins like the LevR, or RTKs acting upstream of SOC-1. Candidate RTKs affecting cholinergic agonist sensitivity were also identified by screening available mutants. 1) Gottschalk et al. (2005) EMBO J 2) Francis et al. (2005) Neuron 3) Okada et al. (2006) Science 4) Nagel et al. (2005) Curr Biol.