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

Abstract

α-Latrotoxin triggers massive neurotransmitter release from nerve terminals by binding to at least two distinct presynaptic receptors, neurexin 1α and CIRL1/ latrophilin1 (CL1). We have now generated knockout (KO) mice that lack CL1 and analyzed them alone or in combination with neurexin 1α KO mice. Mice lacking only CL1, or both CL1 and neurexin 1α, were viable and fertile. Ca2+-independent binding of α-latrotoxin to brain membranes was impaired similarly in CL1 single and in CL1/neurexin 1α double KO mice (∼75% decrease) but not in neurexin 1α single KO mice. In contrast, Ca2+-dependent binding (∼2 times above Ca2+-independent binding) was altered in both CL1 (∼50% decrease) and neurexin 1α single KO mice (∼25% decrease) and was decreased further in double KO mice (∼75% decrease). Synaptosomes lacking CL1 exhibited the same decrease in α-latrotoxin-stimulated glutamate release in the presence and absence of Ca2+ (∼75%). In contrast, synaptosomes lacking neurexin 1α exhibited only a small decrease in α-latrotoxin-triggered release in the absence of Ca2+ (∼20%) but a major decrease in the presence of Ca2+ (∼75%). Surprisingly, synaptosomes lacking both CL1 and neurexin 1α displayed a relatively smaller decrease in α-latrotoxin-stimulated glutamate release than synaptosomes lacking only CL1 in the absence of Ca2+ (∼50 versus ∼75%), but the same decrease in the presence of Ca2+ (∼75%). Our data suggest the following two major conclusions. 1) CL1 and neurexin 1α together account for the majority (75%) of α-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 α-latrotoxin but not in triggering release. Together these data suggest that the two receptors act autonomously in binding of α-latrotoxin but cooperatively in transducing the stimulation of neurotransmitter release by α-latrotoxin.