Aggregation of sodium channels induced by a postnatally upregulated isoform of agrin

Agrin is involved in signaling the formation of high concentrations of acetylcholine receptors (AChRs) at the neuromuscular junction (NMJ). There are multiple isoforms of agrin attributable to alternative splicing, and these isoforms are differentially expressed during development and between tissues. The ability to cluster AChRs varies among the agrin isoforms. Sodium channels (NaChs) are also concentrated at the NMJ. We have tested various agrin isoforms for their ability to induce formation of clusters of NaChs. We grew cocultures of dissociated adult rat muscle fibers with chinese hamster ovary (CHO) cells that had been transfected with different isoforms of agrin. Using immunocytochemical techniques, we determined that after 1 d in culture, CHO cells synthesizing the neuronally expressed isoform with an eight amino acid insert (Agrin8) were able to form NaCh clusters at sites of contact between the CHO cell and muscle cell. Clusters of NaChs could be formed anywhere along a muscle fiber, but more clusters were detected close to the endplate where the endogenous level of NaChs was higher. None of the other neuronal-specific agrin isoforms was able to cluster NaChs. Because Agrin8 is the only agrin isoform that is upregulated at birth when NaChs begin to cluster at the NMJ, we conclude that Agrin8 expression by motor neurons is a signal for NaCh clustering at the NMJ during normal development.