Two families of cell-adhesion molecules, predominantly presynaptic neurexins and postsynaptic neuroligins, are important for the formation and functioning of synapses in the brain, and mutations in several genes encoding these transmembrane proteins have been found in autism patients. However, very little is known about how neurexins are targeted to synapses and which mechanisms regulate this process. Using various epitope-tagged neurexins in primary hippocampal neurons of wild-type and knock-out mice in vitro and in transgenic animals in vivo, we show that neurexins are trafficked throughout neurons via transport vesicles and the plasma membrane insertion of neurexins occurs preferentially in the axonal/synaptic compartment. We also observed that exit of neurexins from the ER/Golgi and correct targeting require their PDZ-binding motif at the C terminus, whereas two presumptive ER retention signals are inactive. The ubiquitous presence of neurexin-positive transport vesicles and absence of bassoon colabeling demonstrate that these carriers are not active zone precursor vesicles, but colocalization with CASK, RIM1␣, and calcium channels suggests that they may carry additional components of the exocytotic machinery. Our data indicate that neurexins are delivered to synapses by a polarized and regulated targeting process that involves PDZ-domain mediated interactions, suggesting a novel pathway for the distribution of neurexins and other synaptic proteins.

␣-Neurexins constitute a family of neuronal cell surface molecules that are essential for efficient neurotransmission, because mice lacking two or all three ␣-neurexin genes show a severe reduction of synaptic release. Although analyses of ␣-neurexin knock-outs and transgenic rescue animals suggested an involvement of voltage-dependent Ca 2ϩ channels, it remained unclear whether ␣-neurexins have a general role in Ca 2ϩ -dependent exocytosis and how they may affect Ca 2ϩ channels. Here we show by membrane capacitance measurements from melanotrophs in acute pituitary gland slices that release from endocrine cells is diminished by Ͼ50% in adult ␣-neurexin double knock-out and newborn triple knock-out mice. There is a reduction of the cell volume in mutant melanotrophs; however, no ultrastructural changes in size or intracellular distribution of the secretory granules were observed. Recordings of Ca 2ϩ currents from melanotrophs, transfected human embryonic kidney cells, and brainstem neurons reveal that ␣-neurexins do not affect the activation or inactivation properties of Ca 2ϩ channels directly but may be responsible for coupling them to release-ready vesicles and metabotropic receptors. Our data support a general and essential role for ␣-neurexins in Ca 2ϩ -triggered exocytosis that is similarly important for secretion from neurons and endocrine cells.