We are studying the molecular changes underlying adaptation of C. elegans to nicotine and other cholinergic agonists. Therefore, we have assessed the dynamics of expression of nAChRs and their localization to the plasma membrane that are occuring in worms subjected to nicotine. Worms expressing translational GFP fusions of Lev-1 and Unc-29, two subunits of the main muscle ("levamisole"-) receptor, show an overall strong reduction of their expression levels in muscles and neurons after nicotine treatment. This was also found for Des-2::GFP, a mainly neuronal nAChR; in contrast, expression of the GABAA receptor subunit Unc-49 remained unchanged. Thus the effect of nicotine on expression levels is specific for nAChRs, but may be independent of their agonist specificity, since Des-2 was shown to be more sensitive for choline rather than acetylcholine [Yassin et al. (2001), MCN 17, 589-99]. Expression of GFP driven by the promoters of the levamisole-receptor subunits Unc-29, Unc-38, Lev-1 and Unc-63, was not influenced by nicotine treatment; neither was GFP-expression driven by p
acr-5. Thus, downregulation is not achieved at the transcriptional level, but must be controled either at the level of de-novo translation of receptors, or by enhancing a turnover mechanism that quickly degrades existing and newly synthesized nAChRs. To study only those receptors exposed at the plasma membrane, we developed a novel method for immunostaining of cell surface-exposed proteins in live C. elegans. Worms expressing Lev-1 or Unc-38 with epitope tags at their (extracellular) C-termini, were injected into the pseudocoelom with dilute solutions of fluorescently labeled antibodies specific for these epitope tags. The anti-bodies readily accessed the extracellular epitopes, but did not stain intracellular receptors. After 5-6 hours, the scavenger cells had filtered unbound antibodies from the pseudocoelomic fluid, eliminating background staining. We could thus clearly visualize sites containing surface-exposed nAChRs in live worms. These sites are most likely postsynaptic densities of neuromuscular junctions and neuronal synapses, since they are juxtaposed, but strictly not overlapping, with sites containing presynaptic VAMP::GFP. Further experiments showed that co-expressed Unc-29::GFP and epitope tagged Lev-1 and Unc-38 are found in the same punctate sites along the ventral and dorsal nervecords, in the nerve-ring and on nose muscles, confirming genetic evidence that these proteins are part of the same receptor. However, we found Unc-38 also in a secondary nerve bundle running in parallel to the main ventral cord, that contained neither Lev-1 nor Unc-29. This indicates functions of Unc-38 in receptors different from the levamisole receptor. Consistently, we found Unc-38 in many more neurons than Lev-1 or Unc-29. Surprisingly, over-night nicotine treatment, that leads to behavioural adaptation and overall downregulation of the receptors, did not result in any gross change in the numbers and density of nAChRs exposed at those postsynaptic sites. This was true even after 48 hours on nicotine and was independent of the sequence of nicotine exposure and antibody injection. Since nAChRs are not removed from postsynaptic sites, we propose that adaptation to nicotine is mainly achieved by desensitization of these receptors. The reason for the global receptor reduction may thus be to prevent insertion of new, non-desensitized receptors into the plasma membrane. Further, we studied whether mutations in proteins known to be involved in nAChR expression, maturation and clustering have effects on the expression of receptors at the cell surface. We studied a deletion mutant of C. elegans rapsyn, a mutant of
kin-8/cam-1, the strongest C. elegans MuSK homolog, agrin-RNAi worms,
ric-3 mutants, and a nicotine-hypersensitive mutant isolated in our lab,
nic-1. In untreated worms, only
ric-3 mutants showed significantly different receptor densities at NMJs, as compared to wild type: those animals had strongly reduced levels of receptors at the plasma membrane, in agreement with the proposed function of
ric-3 in receptor maturation. Ric-3 animals were also strongly resistant to nicotine. The other mutants tested showed no difference, even though
kin-8 mutants were also moderately nicotine resistant. When we subjected the worms to nicotine, we observed that the mutants did not show significant changes, with the exception of
nic-1 animals. These worms exhibited an increase in receptor numbers, that could explain the hypersensitivity of the
nic-1 mutants to nicotine.