High Affinity Binding of α-Latrotoxin to Recombinant Neurexin Iα

α-Latrotoxin is a potent neurotoxin from black widow spider venom that stimulates neurotransmitter release. α-Latrotoxin is thought to act by binding to a high affinity receptor on presynaptic nerve terminals. In previous studies, high affinity α-latrotoxin binding proteins were isolated and demonstrated to contain neurexin Iα as a major component. Neurexin Iα is a cell surface protein that exists in multiple differentially spliced isoforms and belongs to a large family of neuron-specific proteins. Using a series of neurexin I-IgG fusion proteins, we now show that recombinant neurexin Iα binds α-latrotoxin directly with high affinity (Kd ≈ 4 nM). Binding of α-latrotoxin to recombinant neurexin Iα is dependent on Ca2+ (EC50 ≈ 30 μM). Our data suggest that neurexin Iα is a Ca2+-dependent high affinity receptor for α-latrotoxin.

␣-Latrotoxin is a potent neurotoxin from black widow spider venom that stimulates neurotransmitter release. ␣-Latrotoxin is thought to act by binding to a high affinity receptor on presynaptic nerve terminals. In previous studies, high affinity ␣-latrotoxin binding proteins were isolated and demonstrated to contain neurexin I␣ as a major component. Neurexin I␣ is a cell surface protein that exists in multiple differentially spliced isoforms and belongs to a large family of neuron-specific proteins. Using a series of neurexin I-IgG fusion proteins, we now show that recombinant neurexin I␣ binds ␣-latrotoxin directly with high affinity (K d Ϸ 4 nM). Binding of ␣-latrotoxin to recombinant neurexin I␣ is dependent on Ca 2؉ (EC 50 Ϸ 30 M). Our data suggest that neurexin I␣ is a Ca 2؉ -dependent high affinity receptor for ␣-latrotoxin.
␣-Latrotoxin, a component of black widow spider venom, is one of the most potent excitatory neurotoxins known. ␣-Latrotoxin stimulates neurotransmitter release from vertebrate nerve terminals by triggering massive exocytosis of small synaptic vesicles (1,2). ␣-Latrotoxin-stimulated neurotransmitter release is accompanied by presynaptic membrane depolarization and the influx of Ca 2ϩ through ion channels induced by the toxin (3,4). Purified ␣-latrotoxin forms cation channels in black lipid membranes, leading to the hypothesis that the toxin may act as an ionophore although the channel characteristics differ from those observed in intoxicated PC12 cells (5)(6)(7). ␣-Latrotoxin binds to specific membrane receptors that are found only in the nervous system (8,9). Immunofluorescence localization of bound ␣-latrotoxin at the neuromuscular junction suggested that the binding sites are localized to the presynaptic plasma membrane (10). Together, these studies suggest that ␣-latrotoxin acts by binding to presynaptic receptors, which it either activates directly or which serves to target its insertion into the presynaptic plasma membrane.
The binding sites for ␣-latrotoxin in brain membranes are of low abundance (Ϸ0.3 nmol/g of protein) and of high affinity (Ϸ10 Ϫ9 M). Affinity purification of ␣-latrotoxin-binding proteins from brain resulted in the isolation of a protein fraction that bound ␣-latrotoxin with high affinity (11) and contained two classes of proteins (12)(13)(14): 1 a family of high molecular mass proteins (180 -220 kDa) that were shown by molecular cloning to be composed of variants of neurexin I␣, and a distinct low molecular mass protein (29 kDa) named neurexophilin. The purification of these proteins suggested that they represent components of the ␣-latrotoxin receptor. However, it was impossible to define the exact binding partner because direct binding of recombinant proteins to ␣-latrotoxin was not achieved.
Neurexin I␣ and its isoforms, II␣ and III␣, structurally resemble cell surface proteins (13,15). The neurexins are highly polymorphic due to extensive alternative splicing (16). Each neurexin gene not only generates ␣-neurexins but also ␤-neurexins that have a distinct N terminus but share the C-terminal sequences with ␣-neurexin (13,17). The discovery of neurexin I␣ as a component of the protein complex that binds ␣-latrotoxin with high affinity raised the question of whether neurexin I␣ represents an ␣-latrotoxin receptor or is only purified indirectly. We have now studied the interaction of ␣-latrotoxin with recombinant neurexins and determined the requirements for high affinity binding. Our data demonstrate that neurexin I␣ represents a high affinity, Ca 2ϩ -dependent cell surface binding molecule for ␣-latrotoxin.

EXPERIMENTAL PROCEDURES
Construction and Transfection of Expression Vectors-Vectors directing expression of extracellular domains of neurexins fused to the Fc domain of human IgG were obtained by an adaptation of the method of Aruffo et al. (18) utilizing pCD5-IgG 1 as the starting vector as described (17,19) and the rat and bovine neurexin cDNAs (13,15,16). The vectors used in the current study encode the following residues and splice variants of neurexins (all numbers correspond to the numbering of the rat proteins in Refs. 13  pCMVIGNI␤-1 and -3 encode residues 1 to 300 from rat neurexin I␤ without or with an insert in splice site 4, respectively; and pCMVIG-NIII␣-2 encodes residues 1-1499 of rat neurexin III␣ with no insert in splice sites 1 and 5 and full inserts in 3 and 4. The proteins encoded by the vectors are depicted schematically in Fig. 1. Plasmid DNA was transfected into COS cells using DEAE-dextran (20), and expressed proteins were purified from the medium as described (17,19). As controls, media from COS cells transfected with salmon sperm DNA or with control IgG vector were used.
␣-Latrotoxin Binding to Recombinant Neurexins-␣-Latrotoxin was purified from the glands of Latrodectus mactans as described (12). Activity was assayed using release of labeled noradrenaline from synaptosomes. For some experiments, ␣-latrotoxin was iodinated using chloramine T (12). ␣-Latrotoxin was incubated in buffer A (50 mM Tris-HCl, pH 7.7, 2 g/liter bovine serum albumin, and 150 mM NaCl) containing the indicated concentrations of EGTA, Ca 2ϩ , and Mg 2ϩ with protein A-Sepharose beads to which the recombinant proteins had been attached. Incubations were for 15 min under vigorous shaking at room temperature. Beads were then washed with buffer A containing the * This study was supported by a fellowship from the Human Frontiers Science Program (to Y. H.) and by a grant from the Perot Family Foundation. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18  additions described in the figure legends, and bound proteins were analyzed by SDS-PAGE 2 followed by Coomassie Blue staining or immunoblotting and/or determination of radioactivity. For determinations of binding affinities, aliquots of the COS cell medium were spotted onto nitrocellulose, and binding of 125 I-labeled ␣-latrotoxin to the immobilized neurexins was analyzed as described (12).
Miscellaneous Procedures-SDS-PAGE, Coomassie staining, and immunoblotting were performed as described (14). Immunoreactive bands were detected by enhanced chemiluminescence (Amersham). The antibody against ␣-latrotoxin (X751) was raised against purified protein in rabbits.

RESULTS AND DISCUSSION
We constructed a series of fusion proteins of bovine neurexin I␣ with IgG in order to take advantage of the recent cloning of a large number of independent neurexin I␣ cDNAs (16). The neurexin I␣ IgG fusion proteins are depicted schematically in Fig. 1 together with the other IgG fusion proteins used for the current study. Incubation of neurexin I␣-IgG fusion protein immobilized on protein A-Sepharose with purified ␣-latrotoxin demonstrated stoichiometric and specific binding of ␣-latrotoxin only in the presence of Ca 2ϩ (Fig. 2, lanes 1-4). Binding was reversible since ␣-latrotoxin that was bound to neurexin I␣-IgG in the presence of Ca 2ϩ could be readily dissociated by EGTA (Fig. 2, lane 6). Thus, ␣-latrotoxin binds to the extracellular domains of recombinant neurexin I␣ in a Ca 2ϩ -dependent manner.
Previous studies using recombinant rat neurexin I␣ were unsuccessful in detecting binding. Therefore, we studied the potential dependence of binding on splice variants by analyzing a series of independent cDNAs. Four different neurexin I␣-IgG fusion proteins containing a variety of inserts in the first three splice sites of ␣-neurexins bound ␣-latrotoxin, whereas the recombinant proteins corresponding to neurexin III␣ and the previously studied rat neurexin I␣ did not (lanes 1-8 versus  13-16, Fig. 3). Furthermore, C-terminal truncations of cDNAs that bound ␣-latrotoxin as full-length protein abolished binding (lanes 9 -12), and the two splice variants of neurexin I␤ were also unable to bind (lanes 17-20, Fig. 3; see Fig. 1 for an overview of the structures of the neurexin-IgG fusion proteins). Thus, several recombinant neurexin I␣ proteins with different splice site variants bind ␣-latrotoxin. Both N-terminal ␣-specific sequences of neurexin I␣ and its C-terminal half are required for binding.
The nearly stoichiometric binding of ␣-latrotoxin to neurexin I␣ suggests a stable interaction of high affinity. To test this, the binding of radiolabeled ␣-latrotoxin to recombinant neurexin I␣ was measured (Fig. 4). A binding affinity of approximately 4 nM was determined, suggesting that neurexin I␣ is indeed a high affinity ␣-latrotoxin-binding protein. The affinity of recombinant neurexin I␣ was compared with that of the high affinity binding proteins that were purified by affinity chromatography on immobilized ␣-latrotoxin (11,12,14). Recombinant neurexin I␣ had an almost identical affinity as the purified protein, confirming that the ␣-latrotoxin binding observed in the purified receptor corresponds to neurexin I␣ (Fig. 4).
The experiment in Fig. 2 suggested that ␣-latrotoxin binding to neurexin I␣ may be Ca 2ϩ -dependent. To investigate this further, we studied the effect of different Ca 2ϩ concentrations on binding in the presence of a saturating concentration of Mg 2ϩ (Fig. 5). Mg 2ϩ alone was unable to trigger binding. Ca 2ϩ acted in a concentration-dependent manner with an EC 50 of Ϸ35 M and with a single apparent binding site. This result suggests that ␣-latrotoxin and/or neurexins contain a structural Ca 2ϩ binding site which has to be occupied in order for the two proteins to interact. Since previous studies demonstrated  (lanes 1, 3, and 5) or with the bovine neurexin I␣-IgG expression vector pCMVIGbN1␣-1 (lanes 2, 4, and 6) was incubated with 5 g of purified ␣-latrotoxin in the presence of 10 mM EGTA (lanes 1 and 2) or 1 mM Ca 2ϩ (lanes 3-6). Beads were washed with 50 mM Tris, pH 7.7, 1 M NaCl in the presence of either 1 mM Ca 2ϩ (lanes 1-4) or 10 mM EGTA (lanes 5 and 6). Bound proteins were analyzed by SDS-PAGE and Coomassie Blue staining. Bovine serum albumin (BSA) was present in all buffers to block nonspecific binding. Immunoglobulin G (IgG) was bound to the protein A from the serum used for cell culture. Since neurexin I␤ constructs express much better than the ␣ constructs, no immunoglobulin heavy chain (IgG-HC) is detected in the lanes with neurexin I␤-IgG constructs because fewer protein A-beads were used. that neuroligin 1, the ligand for ␤-neurexins, also requires Ca 2ϩ for binding (19), it is tempting to speculate that the extracellular domains of neurexins contain structural Ca 2ϩ binding sites that are required to keep the molecule in an active conformation.
The goal of the current study was to investigate the candidacy of neurexin I␣ as the ␣-latrotoxin receptor. This receptor is interesting because binding to it may mediate the ability of ␣-latrotoxin to trigger massive neurotransmitter release. Synaptotagmin, a nerve terminal Ca 2ϩ sensor (21), co-purifies with this receptor on an ␣-latrotoxin column, suggesting a possible role of the ␣-latrotoxin receptor in regulating synaptic vesicle fusion with the plasma membrane (22). The current study demonstrates that the extracellular domains of neurexin I␣ bind ␣-latrotoxin with high affinity in a Ca 2ϩ -dependent manner. This binding is specific since it was observed with only a subset of neurexin I␣-IgG fusion proteins and not with control proteins or other IgG fusion proteins.
The affinity of the interaction between neurexin I␣ and ␣-latrotoxin agrees well with the ␣-latrotoxin concentrations required for toxic actions (1)(2)(3)(4). However, the Ca 2ϩ dependence of the interaction is puzzling, even though the Ca 2ϩ concentration required for binding is low. Although the ␣-latrotoxin receptor purified by affinity chromatography also requires Ca 2ϩ for binding, ␣-latrotoxin binding to brain membranes is decreased but not abolished in the absence of Ca 2ϩ (23). Furthermore, ␣-latrotoxin is capable of triggering neurotransmitter release in the absence of extracellular Ca 2ϩ if Mg 2ϩ is present. Thus, it is possible that a second high affinity binding protein for ␣-latrotoxin exists that is distinct from neurexin I␣ and binds the toxin in the absence of Ca 2ϩ . Since Scatchard plots of ␣-latrotoxin binding demonstrated only a single class of binding sites, any putative receptor would have to bind ␣-latrotoxin with the same affinity as neurexin I␣. Alternatively, a neurexin isoform may exist that does not require Ca 2ϩ for ␣-latrotoxin binding. Future experiments will have to address these possibilities. FIG. 4. Affinity of ␣-latrotoxin for neurexin I␣-IgG. Immobilized neurexin I␣-IgG (squares, IG-Nx) or ␣-latrotoxin binding proteins purified by affinity chromatography on ␣-latrotoxin (diamonds, LTR) (11,12,14) were incubated with radiolabeled ␣-latrotoxin at the indicated concentrations. The amount of bound and free ␣-latrotoxin was determined and analyzed in a Scatchard plot as shown. Note that the affinity of recombinant neurexin is virtually identical with that of the purified protein complex.