Mutations in
ric-3 were isolated as suppressors of the neurodegeneration caused by the activating
u662 mutation in the nictotinic acetylcholine receptor (nAChR) DEG-3 1 and in a screen for mutants resistant to the acetylcholinesterase inhibitor aldicarb 2 .
ric-3 mutants are resistant to levamisole, an agonist of the body wall muscle nAChR. They also show a reduction in the rate of pharyngeal pumping that correlates with a reduced MC spike, indicating impaired cholinergic transmission. Thus,
ric-3 affects cholinergic transmission mediated by three distinct nAChRs with no apparent defects in transmission by other neurotransmitters. Immunocytochemistry showing DEG-3 accumulation in the cell bodies of
ric-3 worms suggests that RIC-3 is a postsynaptic component of the machinery that assembles or transports nAChRs. We have cloned
ric-3 and identified it as T14A8.1.
ric-3 is a novel gene coding for a highly charged protein predicted to contain 2 TM domains and an extensive coiled-coil region. Characterization of four cDNAs kindly received from Y. Kohara has shown the existence of two transcripts, coding for proteins of 363 and 378 amino acids. While no strong homology is found with known proteins,
ric-3 homologues are present in the Drosophila genome and in Xenopus and human EST databases. A rescuing RIC-3:GFP fusion construct shows RIC-3 expression in muscle and neurons. RIC-3: GFP fluorescence indicates apparent ER localization, and some presence in neuronal processes. The ER localization is consistent with a role in receptor assembly, although presence at the plasma membrane cannot be ruled out. RIC-3 coexpressed with the DEG-3/DES-2 receptor in Xenopus oocytes significantly enhances receptor activity, while having no effect on native oocyte receptors or on coexpressed glutamate receptors. A similar enhancement is observed when RIC-3 is coexpressed with the homomeric rat alpha 7 nAChR. This result is consistent with the possibility that RIC-3 confers cholinergic specificity on the machinery which assembles and transports membrane receptors. We will also present work using biochemistry in the Xenopus oocyte system to further elucidate the mechanism by which RIC-3 enhances nicotinic receptor activity. 1 Treinin, M. and M. Chalfie (1995). A mutated acetylcholine receptor subunit causes neuronal degeneration in C. elegans . Neuron 14:871-877. 2 Miller, K.G., A.Alfonso, M. Nguyen, J.A. Crowell, C.D Johnson, and J.B. Rand, (1996). A genetic selection for Caenorhabditis elegans synaptic transmission mutants. Proc. Natl. Acad. Sci. USA 93:12593-12598.