Acetylcholine receptors direct rapsyn clusters to the neuromuscular synapse in zebrafish

Clustering of nicotinic muscle acetylcholine receptors (AChRs) requires association with intracellular rapsyn, a protein with an intrinsic ability to self-cluster. Previous studies on sofa potato (sop), an AChR null line of zebrafish, have suggested that AChRs may play an active role in subsynaptic localization of rapsyn clusters. To test this proposal directly, we identified and cloned the gene responsible for the sop phenotype and then attempted to rescue subsynaptic localization of the receptor-rapsyn complex in mutant fish. sop contains a leucine to proline mutation at position 28, near the N terminus of the zebrafish AChR δ subunit. Transient expression of mutant δ subunit in sop fish was unable to restore surface expression of muscle AChRs. In contrast, expression of wild-type δ subunit restored the ability of muscle to assemble surface receptors along with the ability of fish to swim. Most importantly, the ability of rapsyn clusters to localize effectively to subsynaptic sites also was rescued in large part. Our results point to direct involvement of the AChR molecule in restricting receptor-rapsyn clusters to the synapse.