Presynaptic calcium channels and α3-integrins are complexed with synaptic cleft laminins, cytoskeletal elements and active zone components.

Pre- and postsynaptic elements of developing chemical synapses provide signals that organize differentiation of post- and presynaptic structures, respectively. At the skeletal neuromuscular junction (NMJ), one prominent organizer of nerve terminals is laminin 421, a heterotrimer of α4, β2 and γ1 subunits. Laminin 421 is synthesized by muscle fibers and inserted into the basal lamina that occupies the synaptic cleft between nerve and muscle membranes. In mature muscle, the α4, and β2 subunits are highly concentrated in the synaptic cleft, along with the α5 subunit, while the γ1 subunit is also present in laminin 211 (α2/β1/γ1), a major component of the basal lamina that coats the remainder of the muscle fiber surface (Patton et al., 1997). In mice with a targeted mutation of the laminin β2 (lamb2) gene, presynaptic maturation is dramatically disrupted and homozygous mutants die by the time of weaning (Nishimune et al., 2004; Noakes et al., 1995). Mice with a targeted mutation of the lama4 gene are outwardly healthy, but exhibit disruption of the precise apposition between presynaptic neurotransmitter release sites (active zones) and postsynaptic invaginations (junctional folds) (Patton et al., 2001). The more severe phenotype of lamb2 compared to lama4 mutants presumably reflects the fact that laminin α4 is present only in laminin 421, whereas laminin β2 is present in three distinct laminin heterotrimers, laminins 221, 421 and 521 (Patton et al., 1997); thus, considerable laminin β2 is present at synapses in lama4 mutants. Together, these results suggest that the laminin α4 and β2 chains play distinct roles in the development on the NMJ, and raise the question of what receptors that they interact with on the nerve terminal. We recently showed that one receptor is the voltage-gated calcium channel (VGCC), which was already known to play a central role in neurotransmitter secretion: the VGCC opens in response to membrane depolarization, allowing the influx of calcium ions that trigger neurotransmitter release (Kandel, et al., 2000). In addition, interaction of the pore-forming VGCC α subunit (Cavα) with synaptic laminin is required for formation and stabilization of active zones in early postnatal life (Nishimune, et al., 2004). It remains to be determined, however, how Cavα links to active zones. Here, we address this issue by isolating and identifying intracellular proteins associated with the VGCC in nerve terminals. To this end, we used the Torpedo electric organ. This tissue contains nicotinic cholinergic synapses that are similar to NMJs in many respects, but are far larger and more abundant. Synapses in the electric organ occupy most of one surface of slender electroplaque cells, whereas NMJs occupy only ~0.1% of the surface of large, cylindrical muscle fibers. Consequently, the concentration of synaptic molecules such as postsynaptic acetylcholine receptors (AChRs) is >100X higher in electric than in muscle. Indeed, many key components of NMJs were originally purified from electric organ; these include AChRs, acetylcholinesterase, agrin, rapsyn, and several synaptic vesicle proteins (Hall, 1992). Likewise, use of electric organ allowed us to isolate a VGCC-associated complex in amounts sufficient to identify several of its major components by mass spectroscopy. Our results suggest that the presynaptic Cavα anchors a complex of cytoskeletal and active zone-associated proteins. Our previous studies demonstrated that the presynaptic Cavα subunit binds directly to laminin β2 in the laminin 421 heterotrimer (Nishimune et al., 2004). As noted above, however, the distinct phenotypes of targeted lama4 and lamb2 mutants suggest distinct roles for laminins β2 and α4, implying the existence of a laminin α4 binding protein on nerve terminals. In the second part of this study, we used electric organ to seek such a protein. We focused on integrins, which are the best-studied laminin receptors in both neural and non-neural tissues, and are known to interact with laminin α subunits (Suzuki, et al., 2005). Among several integrin subunits present at the NMJ (Martin et al., 1995), integrin α3 was of particular interest because it is concentrated near active zones in frog nerve terminals (Cohen, et al., 2000), and might explain the mislocalization of active zones in mouse lama4 mutants (Patton, et al., 2001). Indeed, we show that laminin 421 interacts with integrin α3 and that this integrin, in turn, interacts intracellularly with at least one component of the VGCC-associated complex. Thus, our results define two paths by which laminin 421 in the synaptic cleft can provide developmentally important signals to motor axons.