Genetic analysis of alpha-latrotoxin receptors reveals functional interdependence of CIRL/latrophilin 1 and neurexin 1 alpha

alpha-Latrotoxin triggers massive neurotransmitter release from nerve terminals by binding to at least two distinct presynaptic receptors, neurexin la and CIRL1/ latrophilinl (CL1). We have now generated knockout (KO) mice that lack CL1 and analyzed them alone or in combination with neurexin la KO mice. Mice lacking only CL1, or both CL1 and neurexin 1alpha, were viable and fertile. Ca2+-independent binding of alpha-latrotoxin to brain membranes was impaired similarly in CL1 single and in CL1/neurexin 1alpha double KO mice (similar to75% decrease) but not in neurexin 1alpha single KO mice. In contrast, Ca2+ dependent binding (similar to2 times above Ca2+-independent binding) was altered in both CL1 (similar to50% decrease) and neurexin 1alpha single KO mice (similar to25% decrease) and was decreased further in double KO mice (similar to75% decrease). Synaptosomes lacking CL1 exhibited the same decrease in alpha- latrotoxin-stimulated glutamate release in the presence and absence of Ca2+ (similar to75%). In contrast, synaptosomes lacking neurexin 1alpha exhibited only a small decrease in alpha-latrotoxin-triggered release in the absence of Ca2+ (similar to20%) but a major decrease in the presence of Ca2+ (similar to75%). Surprisingly, synaptosomes lacking both CL1 and neurexin 1alpha displayed a relatively smaller decrease in alpha-latrotoxin-stimulated glutamate release than synaptosomes lacking only CL1 in the absence of Ca2+ (similar to50 versus similar to75%), but the same decrease in the presence of Ca2+ (similar to75%). Our data suggest the following two major conclusions. 1) CL1 and neurexin 1alpha together account for the majority (75%) of alpha-latrotoxin receptors in brain, with the remaining receptor activity possibly due to other CL and neurexin isoforms, and 2) the two receptors act additively in binding alpha-latrotoxin but not in triggering release. Together these data suggest that the two receptors act autonomously in binding of alpha-latrotoxin but cooperatively in transducing the stimulation of neurotransmitter release by alpha-latrotoxin