Mutations in ric-3 (resistant to inhibitors of cholinesterase) suppress the neuronal degenerations caused by a gain of function mutation in the Caenorhabditis elegans DEG-3 acetylcholine receptor. RIC-3 is a novel protein with two transmembrane domains and extensive coiled-coil domains. It is expressed in both muscles and neurons, and the protein is concentrated within the cell bodies. We demonstrate that RIC-3 is required for the function of at least four nicotinic acetylcholine receptors. However, GABA and glutamate receptors expressed in the same cells are unaffected. In ric-3 mutants, the DEG-3 receptor accumulates in the cell body instead of in the cell processes. Moreover, co-expression of ric-3 in Xenopus laevis oocytes enhances the activity of the C.elegans DEG-3/DES-2 and of the rat a-7 acetylcholine receptors. Together, these data suggest that RIC-3 is speci®cally required for the maturation of acetylcholine receptors.

and the §Instituto de Neurociencias, Alicante, Spain In Caenorhabditis elegans, the ric-3 gene is required for the maturation of multiple nicotinic acetylcholine receptors (nAChRs), whereas other neurotransmittergated channels expressed within the same cells are unaffected by the presence of RIC-3. Here we show that RIC-3 is a member of a conserved gene family with representatives in both vertebrates and invertebrates. All members of this family have two transmembrane domains followed by a coiled-coil domain. Expression of the human ric-3 homolog, hric3, like the C. elegans ric-3, enhances C. elegans DEG-3/DES-2, rat ␣7, and human ␣7 nAChR-dependent whole-cell current amplitudes in Xenopus leavis oocytes, thus demonstrating functional conservation. However, hric3 also reduces human ␣4␤2 and ␣3␤4 nAChR-dependent whole-cell current amplitudes. Thus, hric3 shows differential effects on human nAChRs unlike the observed uniform effect of ric-3 on C. elegans nAChRs. Moreover, hric3 totally abolished currents evoked by 5-HT 3 serotonin receptors, whereas it barely modified ␣1 glycine receptor currents. With this caveat, RIC-3 belongs to a conserved family of genes likely to regulate nAChR-mediated transmission throughout evolution. Analysis of transcripts encoded by the hric3 locus shows that it encodes for multiple transcripts, likely to produce multiple hric3 isoforms, and that hric3 is expressed in neurons and muscles, thus enabling its interactions with nAChRs in vivo.Nicotinic acetylcholine receptors are widely expressed ligand-gated ion channels that mediate fast synaptic excitation and have additional roles including modulation of synaptic release (1). The nAChRs 1 are homomers or heteromers composed of five subunits. Each subunit traverses the membrane four times and is posttranslationally modified by both disulfide bond formation and glycosylation. Maturation of nAChRs, leading to production of fully assembled and functional receptors on the plasma membrane, is a complex, time-consuming, and poorly characterized process (2-4). Recently, we identified a Caenorhabditis elegans gene, ric-3, likely to be an important player in the maturation of nAChRs. In C. elegans, RIC-3 is required for cholinergic transmission mediated by nAChRs in neurons and in muscles but not for synaptic transmission mediated by other ligand-gated ion channels, even when these are expressed within the same cells as the nAChRs. RIC-3 activity is required within the cells that express the nAChRs and is likely to affect the processes of receptor folding or assembly within the endoplasmic reticulum (5).RIC-3 is a protein with two transmembrane domains followed by coiled-coil domains. When first identified, this protein showed no similarity to any characterized protein. Only one homolog was identified, the Drosophila CG9349 gene that is similar in both sequence and overall predicted structure. However, the demonstrated ability of RIC-3 to enhance whole-cell current amplitudes resulting from expression of both the C. elegans DEG-3/DES-2 and th...

No abstract

The membrane topology of a2/6 subunit was investigated utilizing electrophysiological functional assay and specific anti-a2 antibodies. (a) cRNA encoding a deleted a216 subunit was coinjected with alC subunit of the L-type calcium channel into Xenopus oocytes. The truncated form, lacking the third putative TM domain (c~21~ATMIII), failed to amplify the expressed inward currents, normally induced by a~c coinjected with intact c~2/6 subunit. Western blot analysis of a216ATMIII shows the appearance of a degraded o~2 protein and no expression of the full-size two-TM truncated-protein. The improper processing of a2/6ATMIII suggests that the a216 is a single TM domain protein and the TM region is positioned at the 6 subunit. (b) External application of anti-a2 antibodies, prepared for an epitope within the alternatively spliced and 'intracellular' region, inhibits depolarization induced secretion in PC12, further supporting an external location of the a2 subunit and establishing 6 subunit as the only membrane anchor for the extraceHular a2 subunit.

These results confirm the presence of antibodies against the L-type calcium channel in the majority of sera from ALS patients, supporting their role in the pathogenesis of ALS.