ACETYLCHOLINE RECEPTOR CLUSTERING IS MEDIATED BY L-TYPE CALCIUM CHANNELS Chris Dulla, Rebecca Milholland, and Herman Gordon

At the neuromuscular junction, the acetylcholine receptor (AChR), a postsynaptic, transmembrane molecule, is one of the key components of synaptic formation, electrical signal transduction, and synaptic plasticity. The aggregation of AChRs on the postsynaptic membrane is absolutely crucial to the formation of healthy synapses. Aggregation results from innervation in vivo, and can be induced in culture models using a number of different manipulations. Agrin, a neural factor, causes the formation of clusters in vitro. Agrin not only causes the aggregation of AChRs, but also causes rapid tyrosine phosphorylation of the Muscle Specific Kinase (MuSK), a transmembrane protein that is colocalized with the AChR clusters and is essential in the AChR clustering signal cascade. Elevated extracellular calcium levels mimic the effects of agrin on both clustering and on MuSK phosphorylation, but calcium’s role in the signaling pathway has not been understood. We have found using C2 cultured mouse myotubes that the effect of elevated calcium is mediated by a previously uncharacterized L-type calcium channel found on the post synaptic surface. An L-type calcium channel blocker, verapamil, blocks spontaneous AChR clustering, as well as agrin induced clustering. In concert, an L-type calcium channel agonist, BAY K 8644, induces the formation of clusters in a manner that mimics the frequency and morphology of agrin induced clustering. Furthermore, we have found that agrin’s ability to induce MuSK phosphorylation is independent of the blockade of L-type calcium channels. This strongly suggests that the role of L-type calcium channels is downstream of MuSK in the signaling cascade that causes AChR clustering. Nicotine induced, as well as calcium induced AChR clustering are both blocked by L-type calcium channel blockade, suggesting that L-type calcium channels could play a central role in the majority of mechanisms that are responsible for AChR cluster formation.