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W2040479508 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+ (∼50versus ∼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. 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+ (∼50versus ∼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. α-Latrotoxin is a 130-kDa component of black widow spider venom that is initially synthesized as a 160-kDa precursor protein and is then processed proteolytically at the N and C terminus (1Kiyatkin N. Dulubova I. Grishin E. Eur. J. Biochem. 1993; 213: 121-127Crossref PubMed Scopus (57) Google Scholar, 2Dulubova I.E. Krasnoperov V.G. Khvotchev M.V. Pluzhnikov K.A. Volkova T.M. Grishin E.V Vais H. Bell D.R. Usherwood P.N. J. Biol. Chem. 1996; 271: 7535-7543Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar, 3Khvotchev M. Südhof T.C. EMBO J. 2000; 19: 3250-3262Crossref PubMed Google Scholar). α-Latrotoxin is a potent neurotoxin, causing massive release of neurotransmitter in nerve terminals by exocytosis (4Frontali N. Ceccarelli B. Gorio A. Mauro A. Siekevitz P. Tzeng M.C. Hurlbut W.P. J Cell Biol. 1976; 68: 462-479Crossref PubMed Scopus (178) Google Scholar, 5Tzeng M.C. Cohen R.S. Siekevitz P. Proc. Natl. Acad. Sci. U. S. A. 1978; 75: 4016-4020Crossref PubMed Scopus (47) Google Scholar). It seems that the toxin triggers release by first binding to neuronal surface receptors and then directly inserting into the presynaptic plasma membrane (3Khvotchev M. Südhof T.C. EMBO J. 2000; 19: 3250-3262Crossref PubMed Google Scholar). Most studies imply that the toxin is active in the absence of extracellular Ca2+ and directly stimulates the secretory apparatus (6Ceccarelli B. Hurlbut W.P. J. Cell Biol. 1980; 87: 297-303Crossref PubMed Scopus (182) Google Scholar, 7Capogna M. Gahwiler B.H. Thompson S.M. J. Neurophysiol. 1996; 76: 3149-3158Crossref PubMed Scopus (77) Google Scholar, 8Khvotchev M. Lonart G Südhof T.C. Neuroscience. 2000; 101: 793-802Crossref PubMed Scopus (39) Google Scholar). Two classes of cell surface receptors for α-latrotoxin have been identified. Neurexins are neuron-specific proteins with a single transmembrane domain (9Ushkaryov Y.A. Petrenko A.G. Geppert M. Südhof T.C. Science. 1992; 257: 50-56Crossref PubMed Scopus (545) Google Scholar, 10Sugita S. Khvochtev M. Südhof T.C. Neuron. 1999; 22: 489-496Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar). They function at least partly as cell adhesion molecules; one isoform, neurexin 1α, binds α-latrotoxin with high affinity in a Ca2+-dependent manner. CLs (CIRLs and latrophilins) 1CLCIRL/latrophilinKOknockout 1CLCIRL/latrophilinKOknockout are G-protein-coupled receptors with seven transmembrane domains and unusually large intra- and extracellular domains (11Lelianova V.G. Davletov B.A. Sterling A. Rahman M.A. Grishin E.V. Totty N.F. Ushkaryov Y.A. J. Biol. Chem. 1997; 272: 21504-21508Abstract Full Text Full Text PDF PubMed Scopus (238) Google Scholar, 12Krasnoperov V.G. Bittner M.A. Beavis R. Kuang Y. Salnikow K.V. Chepurny O.G. Little A.R. Plotnikov A.N., Wu, D. Holz R.W. Petrenko A.G. Neuron. 1997; 18: 925-937Abstract Full Text Full Text PDF PubMed Scopus (272) Google Scholar). Currently, three CL isoforms are known (13Sugita S. Ichtchenko K. Khvotchev M. Südhof T.C. J. Biol. Chem. 1998; 273: 32715-32724Abstract Full Text Full Text PDF PubMed Scopus (145) Google Scholar, 14Ichtchenko K. Bittner M.A. Krasnoperov V. Little A.R. Chepurny O. Holz R.W. Petrenko A.G J. Biol. Chem. 1999; 274: 5491-5498Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar). CL1 and CL3 are highly enriched in brain, whereas CL2 is expressed ubiquitously. Although CL1 and neurexin 1α appear to be the most important receptors for α-latrotoxin, most of the other neurexin and CL isoforms also constitute functional α-latrotoxin receptors (10Sugita S. Khvochtev M. Südhof T.C. Neuron. 1999; 22: 489-496Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar, 11Lelianova V.G. Davletov B.A. Sterling A. Rahman M.A. Grishin E.V. Totty N.F. Ushkaryov Y.A. J. Biol. Chem. 1997; 272: 21504-21508Abstract Full Text Full Text PDF PubMed Scopus (238) Google Scholar, 12Krasnoperov V.G. Bittner M.A. Beavis R. Kuang Y. Salnikow K.V. Chepurny O.G. Little A.R. Plotnikov A.N., Wu, D. Holz R.W. Petrenko A.G. Neuron. 1997; 18: 925-937Abstract Full Text Full Text PDF PubMed Scopus (272) Google Scholar, 13Sugita S. Ichtchenko K. Khvotchev M. Südhof T.C. J. Biol. Chem. 1998; 273: 32715-32724Abstract Full Text Full Text PDF PubMed Scopus (145) Google Scholar, 14Ichtchenko K. Bittner M.A. Krasnoperov V. Little A.R. Chepurny O. Holz R.W. Petrenko A.G J. Biol. Chem. 1999; 274: 5491-5498Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar, 15Davletov B.A. Krasnoperov V. Hata Y. Petrenko A.G. Südhof T.C. J. Biol. Chem. 1995; 270: 23903-23905Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar). Furthermore, consistent with the α-latrotoxin binding data, experiments on neurexin 1α KO mice showed that release triggered by α-latrotoxin does not require neurexin 1α in the absence of Ca2+ but does require neurexin 1α in the presence of Ca2+ (16Geppert M. Khvotchev M. Krasnoperov V. Goda Y. Missler M. Hammer R.E. Ichtchenko K. Petrenko A.G. Südhof T.C. J. Biol. Chem. 1998; 273: 1705-1710Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar).Despite extensive work that includes the identification of multiple receptors, the mechanism by which α-latrotoxin triggers release has remained elusive (reviewed in Ref. 17Südhof T.C. Annu. Rev. Neurosci. 2001; 24: 933-962Crossref PubMed Scopus (168) Google Scholar). Studies using a recombinant mutant of α-latrotoxin revealed that high affinity binding of α-latrotoxin to its receptors is essential but not sufficient to trigger neurotransmitter release (18Ichtchenko K. Khvotchev M. Kiyatkin N. Simpson L. Sugita S. Südhof T.C. EMBO J. 1998; 17: 6188-6199Crossref PubMed Scopus (72) Google Scholar). Expression of neurexin 1α or CL1 in PC12 cells highly sensitizes them to α-latrotoxin; this occurs even when deletion mutants of both receptors lacking intracellular sequences are expressed (10Sugita S. Khvochtev M. Südhof T.C. Neuron. 1999; 22: 489-496Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar, 13Sugita S. Ichtchenko K. Khvotchev M. Südhof T.C. J. Biol. Chem. 1998; 273: 32715-32724Abstract Full Text Full Text PDF PubMed Scopus (145) Google Scholar, 14Ichtchenko K. Bittner M.A. Krasnoperov V. Little A.R. Chepurny O. Holz R.W. Petrenko A.G J. Biol. Chem. 1999; 274: 5491-5498Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar). Because the mutant receptors lack intracellular sequences, they are presumably incapable of transducing a surface signal into the cell interior, suggesting that α-latrotoxin does not trigger release by activation of intracellular signal transduction pathways dependent on neurexin 1α and CL1. Moreover, thein vivo importance of the various proposed α-latrotoxin receptors is largely unclear. The fact that deletion of neurexin 1α severely impaired the α-latrotoxin response in the presence but not in the absence of Ca2+ confirms the importance of neurexin 1α as an α-latrotoxin receptor, although it is also puzzling. Specifically, because α-latrotoxin-triggered release is similar in the presence and absence of Ca2+ (8Khvotchev M. Lonart G Südhof T.C. Neuroscience. 2000; 101: 793-802Crossref PubMed Scopus (39) Google Scholar), it would have been expected that because neurexin 1α only binds to α-latrotoxin in the presence of Ca2+, CL1 should be sufficient to elicit a complete α-latrotoxin response. Thus the fact that the neurexin 1α KO had an effect at all is puzzling, even though as predicted, a large effect was only observed in the presence of Ca2+. Together these data raise exciting new questions. For example, is CL1 responsible for the ability of α-latrotoxin to trigger neurotransmitter release in the absence of Ca2+ under conditions where α-latrotoxin release is not severely impaired in neurexin 1α KO mice? Are neurexin 1α and CL1 truly the primary α-latrotoxin receptors, and do they function independently of each other or cooperatively?In the current study, we have addressed these questions by generating KO mice that lack CL1. By mating these mice with neurexin 1α KO mice, we established double KO mice, which are deficient in both of the two major known α-latrotoxin receptors. We then examined the effect of the various knockouts on α-latrotoxin binding to brain membranes and on neurotransmitter release triggered by α-latrotoxin from isolated nerve terminals (synaptosomes). Our results demonstrate that CL1 is the major Ca2+-independent α-latrotoxin receptor in glutamatergic nerve terminals, mediating the exocytosis of ∼75% of released glutamate. Surprisingly, nerve terminals lacking CL1, neurexin 1α, or both respond to α-latrotoxin in the presence of Ca2+ in an indistinguishable manner, suggesting that CL1 and neurexin 1α cooperate at the cell surface to release glutamate in response to α-latrotoxin.DISCUSSIONα-Latrotoxin is a potent excitatory toxin in black widow spider venom that triggers massive neurotransmitter release. α-Latrotoxin stimulates secretion of classical transmitters contained in small clear synaptic vesicles (e.g. GABA, glutamate, and acetylcholine) in the presence and absence of Ca2+. In contrast, transmitters and neuropeptides contained in dense core vesicles (e.g. norepinephrine and neuropeptides) are secreted in response to α-latrotoxin only in the presence of Ca2+(see Refs. 8Khvotchev M. Lonart G Südhof T.C. Neuroscience. 2000; 101: 793-802Crossref PubMed Scopus (39) Google Scholar and 17Südhof T.C. Annu. Rev. Neurosci. 2001; 24: 933-962Crossref PubMed Scopus (168) Google Scholar and references cited therein). α-Latrotoxin acts by binding to specific high-affinity cell surface receptors (5Tzeng M.C. Cohen R.S. Siekevitz P. Proc. Natl. Acad. Sci. U. S. A. 1978; 75: 4016-4020Crossref PubMed Scopus (47) Google Scholar), and inserting partially into the plasma membrane (3Khvotchev M. Südhof T.C. EMBO J. 2000; 19: 3250-3262Crossref PubMed Google Scholar). α-Latrotoxin forms pores in the plasma membrane, but the pores alone do not appear to explain α-latrotoxin action because cadmium blocks the pore conductance (29Lishko V.K. Sichenko E.A. Storchak L.G. Gimmerl'reikh N.G. Biokimiia. 1990; 55: 1578-1583PubMed Google Scholar) but enhances α-latrotoxin action (18Ichtchenko K. Khvotchev M. Kiyatkin N. Simpson L. Sugita S. Südhof T.C. EMBO J. 1998; 17: 6188-6199Crossref PubMed Scopus (72) Google Scholar). The precise mechanism of action of α-latrotoxin has remained unclear. One surprising discovery was that α-latrotoxin binds to at least two distinct high affinity receptors on neurons (9Ushkaryov Y.A. Petrenko A.G. Geppert M. Südhof T.C. Science. 1992; 257: 50-56Crossref PubMed Scopus (545) Google Scholar, 10Sugita S. Khvochtev M. Südhof T.C. Neuron. 1999; 22: 489-496Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar, 11Lelianova V.G. Davletov B.A. Sterling A. Rahman M.A. Grishin E.V. Totty N.F. Ushkaryov Y.A. J. Biol. Chem. 1997; 272: 21504-21508Abstract Full Text Full Text PDF PubMed Scopus (238) Google Scholar, 12Krasnoperov V.G. Bittner M.A. Beavis R. Kuang Y. Salnikow K.V. Chepurny O.G. Little A.R. Plotnikov A.N., Wu, D. Holz R.W. Petrenko A.G. Neuron. 1997; 18: 925-937Abstract Full Text Full Text PDF PubMed Scopus (272) Google Scholar, 13Sugita S. Ichtchenko K. Khvotchev M. Südhof T.C. J. Biol. Chem. 1998; 273: 32715-32724Abstract Full Text Full Text PDF PubMed Scopus (145) Google Scholar, 14Ichtchenko K. Bittner M.A. Krasnoperov V. Little A.R. Chepurny O. Holz R.W. Petrenko A.G J. Biol. Chem. 1999; 274: 5491-5498Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar, 15Davletov B.A. Krasnoperov V. Hata Y. Petrenko A.G. Südhof T.C. J. Biol. Chem. 1995; 270: 23903-23905Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar). These receptors, neurexin 1α and CL1, share no sequence similarity and have no obvious common properties. Furthermore, neurexin 1α binds to α-latrotoxin only in the presence of Ca2+ (10Sugita S. Khvochtev M. Südhof T.C. Neuron. 1999; 22: 489-496Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar, 15Davletov B.A. Krasnoperov V. Hata Y. Petrenko A.G. Südhof T.C. J. Biol. Chem. 1995; 270: 23903-23905Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar), whereas CL1 binding is Ca2+-independent (11Lelianova V.G. Davletov B.A. Sterling A. Rahman M.A. Grishin E.V. Totty N.F. Ushkaryov Y.A. J. Biol. Chem. 1997; 272: 21504-21508Abstract Full Text Full Text PDF PubMed Scopus (238) Google Scholar, 12Krasnoperov V.G. Bittner M.A. Beavis R. Kuang Y. Salnikow K.V. Chepurny O.G. Little A.R. Plotnikov A.N., Wu, D. Holz R.W. Petrenko A.G. Neuron. 1997; 18: 925-937Abstract Full Text Full Text PDF PubMed Scopus (272) Google Scholar). When the two different receptors for α-latrotoxin were discovered, three possible explanations for the existence of double receptors were raised. The first explanation was that neurexin 1α is responsible for dense-core vesicle exocytosis, whereas CL1 mediates release of classical neurotransmitters. However, the finding that neurexin 1α is widely expressed in most neurons but largely absent from chromaffin cells (9Ushkaryov Y.A. Petrenko A.G. Geppert M. Südhof T.C. Science. 1992; 257: 50-56Crossref PubMed Scopus (545) Google Scholar) and that KO of neurexin 1α impairs α-latrotoxin-triggered release of classical neurotransmitters in the presence of Ca2+invalidated this possibility (16Geppert M. Khvotchev M. Krasnoperov V. Goda Y. Missler M. Hammer R.E. Ichtchenko K. Petrenko A.G. Südhof T.C. J. Biol. Chem. 1998; 273: 1705-1710Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar). A second explanation was that neurexin 1α is simply not a functional α-latrotoxin receptor. Again, this possibility was ruled out by the demonstration that the ability of α-latrotoxin to trigger neurotransmitter release is impaired in neurexin 1α-deficient neurons (16Geppert M. Khvotchev M. Krasnoperov V. Goda Y. Missler M. Hammer R.E. Ichtchenko K. Petrenko A.G. Südhof T.C. J. Biol. Chem. 1998; 273: 1705-1710Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar), and by the finding that expression of neurexin 1α conferred an enhanced α-latrotoxin response onto neuroendocrine PC12 cells (10Sugita S. Khvochtev M. Südhof T.C. Neuron. 1999; 22: 489-496Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar). A third possible explanation for the presence of two α-latrotoxin receptors was that neurexin 1α and CL1 are co-receptors, which act as heteromultimers, analogous to some G-protein-linked receptors (reviewed in Ref. 30Bockaert J. Pin J.P. EMBO J. 1999; 18: 1723-1729Crossref PubMed Scopus (1218) Google Scholar). This possibility, however, was made improbable by the demonstration that each receptor separately binds to α-latrotoxin with the requisite high specificity and affinity expected of a physiological receptor (10Sugita S. Khvochtev M. Südhof T.C. Neuron. 1999; 22: 489-496Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar, 11Lelianova V.G. Davletov B.A. Sterling A. Rahman M.A. Grishin E.V. Totty N.F. Ushkaryov Y.A. J. Biol. Chem. 1997; 272: 21504-21508Abstract Full Text Full Text PDF PubMed Scopus (238) Google Scholar, 12Krasnoperov V.G. Bittner M.A. Beavis R. Kuang Y. Salnikow K.V. Chepurny O.G. Little A.R. Plotnikov A.N., Wu, D. Holz R.W. Petrenko A.G. Neuron. 1997; 18: 925-937Abstract Full Text Full Text PDF PubMed Scopus (272) Google Scholar, 13Sugita S. Ichtchenko K. Khvotchev M. Südhof T.C. J. Biol. Chem. 1998; 273: 32715-32724Abstract Full Text Full Text PDF PubMed Scopus (145) Google Scholar, 14Ichtchenko K. Bittner M.A. Krasnoperov V. Little A.R. Chepurny O. Holz R.W. Petrenko A.G J. Biol. Chem. 1999; 274: 5491-5498Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar, 15Davletov B.A. Krasnoperov V. Hata Y. Petrenko A.G. Südhof T.C. J. Biol. Chem. 1995; 270: 23903-23905Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar); thus the receptors do not function as heteromultimers in binding to α-latrotoxin. Furthermore, both receptors were shown separately to cause a similar sensitization of α-latrotoxin-triggered release in transfected PC12 cells, and no increase in release was observed upon co-expression of both receptors (10Sugita S. Khvochtev M. Südhof T.C. Neuron. 1999; 22: 489-496Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar). Together these findings document that other explanations have to be found for the unusual presence of two receptors for this toxin, and for the uncommon properties of how this toxin works. In an attempt to address this, we have now generated KO mice that lack the second receptor, CL1, analyzed the action of α-latrotoxin in these KO mice, and compared the changes in α-latrotoxin response in these mice to those observed in KO mice that lack neurexin 1α alone or both CL1 and neurexin 1α.Our data demonstrate that single and double KO mice that lack CL1 and/or neurexin 1α are viable and fertile, although they probably exhibit more subtle neuronal and behavioral phenotypes that have not been analyzed in the present experiments. Furthermore, we show that α-latrotoxin binding to brain membranes from these animals is decreased in a pattern that largely follows expectations based on previous studies (11Lelianova V.G. Davletov B.A. Sterling A. Rahman M.A. Grishin E.V. Totty N.F. Ushkaryov Y.A. J. Biol. Chem. 1997; 272: 21504-21508Abstract Full Text Full Text PDF PubMed Scopus (238) Google Scholar, 12Krasnoperov V.G. Bittner M.A. Beavis R. Kuang Y. Salnikow K.V. Chepurny O.G. Little A.R. Plotnikov A.N., Wu, D. Holz R.W. Petrenko A.G. Neuron. 1997; 18: 925-937Abstract Full Text Full Text PDF PubMed Scopus (272) Google Scholar, 15Davletov B.A. Krasnoperov V. Hata Y. Petrenko A.G. Südhof T.C. J. Biol. Chem. 1995; 270: 23903-23905Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar, 16Geppert M. Khvotchev M. Krasnoperov V. Goda Y. Missler M. Hammer R.E. Ichtchenko K. Petrenko A.G. Südhof T.C. J. Biol. Chem. 1998; 273: 1705-1710Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar): Total binding of α-latrotoxin to wild type membranes in the presence of Ca2+ was two times higher than the binding observed in the absence of Ca2+ as described (16Geppert M. Khvotchev M. Krasnoperov V. Goda Y. Missler M. Hammer R.E. Ichtchenko K. Petrenko A.G. Südhof T.C. J. Biol. Chem. 1998; 273: 1705-1710Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar); CL1 accounts for the majority of both the Ca2+-dependent and -independent binding whereas neurexin 1α only contributes to Ca2+-dependent binding; and the deficits in binding observed in the individual knockouts are additive in the double KO (Fig. 3). However, our data reveal surprising effects of the knockouts on the ability of α-latrotoxin to trigger neurotransmitter release. These effects cannot be explained in terms of α-latrotoxin binding alone. Specifically, we found the following.1) In the absence of Ca2+, deletion of CL1 depresses the majority of α-latrotoxin induced glutamate release, confirming the hypothesis that CL1 constitutes the major Ca2+-independent α-latrotoxin receptor (11Lelianova V.G. Davletov B.A. Sterling A. Rahman M.A. Grishin E.V. Totty N.F. Ushkaryov Y.A. J. Biol. Chem. 1997; 272: 21504-21508Abstract Full Text Full Text PDF PubMed Scopus (238) Google Scholar, 12Krasnoperov V.G. Bittner M.A. Beavis R. Kuang Y. Salnikow K.V. Chepurny O.G. Little A.R. Plotnikov A.N., Wu, D. Holz R.W. Petrenko A.G. Neuron. 1997; 18: 925-937Abstract Full Text Full Text PDF PubMed Scopus (272) Google Scholar). However, we also observed a small but significant effect of the neurexin 1α KO on release that may reflect a participation of neurexin 1α in the release process itself. This effect was small, explaining why it was not observed in a previous study (16Geppert M. Khvotchev M. Krasnoperov V. Goda Y. Missler M. Hammer R.E. Ichtchenko K. Petrenko A.G. Südhof T.C. J. Biol. Chem. 1998; 273: 1705-1710Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar), but was reproducible at different α-latrotoxin concentrations (Fig. 6 A). Curiously, in the absence of Ca2+ α-latrotoxin was more potent in triggering release from synaptosomes lacking both CL1 and neurexin 1α than from synaptosomes lacking only CL1 (Fig. 5 A); this effect was independent of the α-latrotoxin concentration (Fig.6 A).2) In the presence of Ca2+, we observed a very simple effect of each single KO and of the double CL1/neurexin 1α KO on glutamate release: No matter which receptors were deleted, release was strongly inhibited, with the impairment in α-latrotoxin-triggered release being equal for all three genotypes.Overall, these results allow two major conclusions. First, they unequivocally establish that both CL1 and neurexin 1α are physiologically the most important α-latrotoxin receptors. Minor receptor activity remains in the absence of CL1 and neurexin 1α, which may be explained by the presence of CL2 and CL3, and of neurexins 1β, 2α, 2β, 3α, and 3β that are known to function at least partly as α-latrotoxin receptors (10Sugita S. Khvochtev M. Südhof T.C. Neuron. 1999; 22: 489-496Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar, 13Sugita S. Ichtchenko K. Khvotchev M. Südhof T.C. J. Biol. Chem. 1998; 273: 32715-32724Abstract Full Text Full Text PDF PubMed Scopus (145) Google Scholar, 14Ichtchenko K. Bittner M.A. Krasnoperov V. Little A.R. Chepurny O. Holz R.W. Petrenko A.G J. Biol. Chem. 1999; 274: 5491-5498Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar) and have not been deleted in the CL1 and neurexin 1α knockouts. Second, these results demonstrate that the two types of α-latrotoxin receptors do not function independently of each other in vivo, despite their autonomous α-latrotoxin binding activities. Multiple lines of evidence support this conclusion. For example, both in the presence and the absence of Ca2+, the effects of the CL1 and neurexin 1α knockouts on α-latrotoxin-triggered release were not additive, although their effects on α-latrotoxin binding were. In the presence of Ca2+, both receptors are equally required, and the impairment observed in the double KO is no more severe than that of each single KO, suggesting that under these more physiological conditions the two receptors are essential components of the same machinery that mediates the action of α-latrotoxin. In the absence of Ca2+, we also observed a non-additive effect which, however, went in the opposite direction. Although we do not currently understand the molecular basis for these intriguing observations, they clearly demonstrate that the two receptors act autonomously in α-latrotoxin binding, but interdependently in α-latrotoxin action in neurotransmitter release.Apart from these major conclusions, however, our data raise important questions that we cannot currently answer. Why does the neurexin 1α KO decrease α-latrotoxin-triggered release in the absence of Ca2+, even though the decrease is small, but increase release on the background of the CL1 KO? Because neurexin 1α does not bind α-latrotoxin without Ca2+, the most plausible explanation is that neurexin 1α is part of the machinery by which α-latrotoxin binding to CL1 triggers neurotransmitter release, as described above. As part of this machinery, neurexin 1α has incongruous effects on α-latrotoxin action: It is required for full activation of release by α-latrotoxin binding to Ca2+-independent receptors when all of these receptors are present, but slows down release when the major Ca2+-independent receptor (CL1) is absent. A possible explanation for this finding that would also be consistent with the equal requirement for both receptor types in the presence of Ca2+ is that the precise stoichiometry of Ca2+-dependent and Ca2+-independent receptors (i.e. neurexins and CLs) is important, especially in the absence of Ca2+. Thus creating an excess of one over the other inhibits, whereas deleting both reactivates. However, precise definition of this hypothesis will require further insight into how precisely the two receptors types cooperate in α-latrotoxin action, the next experimental challenge in this interesting field. α-Latrotoxin is a 130-kDa component of black widow spider venom that is initially synthesized as a 160-kDa precursor protein and is then processed proteolytically at the N and C terminus (1Kiyatkin N. Dulubova I. Grishin E. Eur. J. Biochem. 1993; 213: 121-127Crossref PubMed Scopus (57) Google Scholar, 2Dulubova I.E. Krasnoperov V.G. Khvotchev M.V. Pluzhnikov K.A. Volkova T.M. Grishin E.V Vais H. Bell D.R. Usherwood P.N. J. Biol. Chem. 1996; 271: 7535-7543Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar, 3Khvotchev M. Südhof T.C. EMBO J. 2000; 19: 3250-3262Crossref PubMed Google Scholar). α-Latrotoxin is a potent neurotoxin, causing massive release of neurotransmitter in nerve terminals by exocytosis (4Frontali N. Ceccarelli B. Gorio A. Mauro A. Siekevitz P. Tzeng M.C. Hurlbut W.P. J Cell Biol. 1976; 68: 462-479Crossref PubMed Scopus (178) Google Scholar, 5Tzeng M.C. Cohen R.S. Siekevitz P. Proc. Natl. Acad. Sci. U. S. A. 1978; 75: 4016-4020Crossref PubMed Scopus (47) Google Scholar). It seems that the toxin triggers release by first binding to neuronal surface receptors and then directly inserting into the presynaptic plasma membrane (3Khvotchev M. Südhof T.C. EMBO J. 2000; 19: 3250-3262Crossref PubMed Google Scholar). Most studies imply that the toxin is active in the absence of extracellular Ca2+ and directly stimulates the secretory apparatus (6Ceccarelli B. Hurlbut W.P. J. Cell Biol. 1980; 87: 297-303Crossref PubMed Scopus (182) Google Scholar, 7Capogna M. Gahwiler B.H. Thompson S.M. J. Neurophysiol. 1996; 76: 3149-3158Crossref PubMed Scopus (77) Google Scholar, 8Khvotchev M. Lonart G Südhof T.C. Neuroscience. 2000; 101: 793-802Crossref PubMed Scopus (39) Google Scholar). Two classes of cell surface receptors for α-latrotoxin have been identified. Neurexins are neur" @default.
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