A critical problem in neurobiology is understanding the nature of the biochemical machinery responsible for synaptic vesicle release at presynaptic nerve terminals. Certain proteins involved in this process were identified recently; they are associated with synaptic vesicles, or on the terminal membrane, or in the cytoplasm. We focus on nerve terminal proteins targeted by alpha-latrotoxin, the neurotoxic component of black widow spider venom, one of the strongest known stimulators of neurotransmitter release. The high-affinity, alpha-latrotoxin-binding sites are localized in the nerve terminal1s cytoplasmic membrane. We hypothesize that stimulation of neurotransmitter release by alpha-latrotoxin can be explained by the toxin1s interaction with the protein components of the presynaptic docking-fusion complex either directly or via a toxin1s presynaptic membrane receptor. Restricted location of alpha-latrotoxin receptors together with the strong neurosecretory action of the toxin implicates these receptors as major components of the neurotransmitter release machinery.

Unexpectedly, we discovered that two neuronal receptors of alpha-latrotoxin exist which are integral membrane proteins that bind the toxin with the same high affinity. However, they are structurally completely different and one of them, neurexin Ia, interacts with alpha-latrotoxin only in the presence of Ca2+. Our data suggest that the other, Ca2+-independent receptor (CIRL) is critically involved in the extracellular stimulation of neurosecretion by alpha-latrotoxin whereas the role of neurexin Ia may be in the development of the long-term neurodegenerative effects.

CIRL is a novel orphan G-protein-coupled receptor, a member of the secretin receptor family. In contrast with other known serpentine receptors, CIRL consists of two subunits that are the result of endogenous proteolytic cleavage of a precursor polypeptide. CIRL is found in brain but not in other tissues. Syntaxin and synaptotagmin, components of the fusion complex, co-purify with CIRL on an alpha-latrotoxin affinity column and form stable complexes with this receptor in vitro suggesting its role in the direct regulation of exocytosis. Currently, we are trying to identify the endogenous ligand(s) of CIRL and to elucidate its intracellular signaling pathway.

Our future efforts will be aimed at the structural and functional characterization of CIRL, neurexin Ia and their endogenous ligands by the combination of biochemical, physiological and genetic approaches.