Our proposal is aimed at elucidating how the extracellular matrix localizes the neurosecretory apparatus in the nerve terminal during synaptogenesis at the neuromuscular junction (NMJ). Nerve regeneration studies at NMJ suggest that synaptic basal lamina components tell the retuming axon where to locate neurotransmitter release machinery, including synaptic vesicle clusters and active zones, the sites of neurotransmitter release. Genetic evidence suggests that synaptic laminins containing alpha4 or Beta2 chains are the critical basal lamina ligands which localize the neurosecretory apparatus. Electron microscopic studies suggest that the transmembrane anchor for the neurotransmitter release machinery is a voltage-gated calcium channel. We have found a transmembrane link between a synaptic laminin, containing alpha4, Beta2, and gamma1chains, and the voltage-gated calcium channel at the electric organ synapse, which is homologous to the NMJ. Using detergent-solubilized electric organ synaptosomes, we have immuno-isolated a protein complex containing this laminin, as well as the calcium channel, the cytoskeletal protein spectrin, and several unidentified proteins. Cross-linking studies suggest that the alpha4Beta2gamma1 laminin is directly bound to the calcium channel in the protein complex. Further, we find that this laminin binds preferentially to cultured cells expressing the calcium channel. We hypothesize that alpha4Beta2gamma1 laminin in the synaptic basal lamina attaches to calcium channel, which in turn joins to the cytosolic neurosecretory apparatus. The alpha4Beta2gamma1 laminin might act to organize the presynaptic secretory apparatus the way agrin organizes the postsynaptic membrane. To test our hypothesis we will ask the following questions: Does the calcium channel act as the receptor for the alpha4Beta2gamma1 laminin? Is it the only receptor? Are there other alpha4Beta2gamma1 laminin-binding proteins in the complex? What kinds of proteins are the unidentified components of the calcium channel/ alpha4Beta2gamma1 laminin protein complex ? Could they be signal transduction proteins, or new active zone components? Will clustering of calcium channels on the surface of motor neuron axons cause the clustering of other components of the neurosecretory apparatus, such as synaptic vesicles? This work investigates a fundamental question of neurobiology and may have clinical relevance to nerve regeneration.
