The Role of the Synaptic Protein SNAP-25 in the Potency of Botulinum Neurotoxin Type A

Botulinum neurotoxin serotype A (BoNT/A) is distinguished from BoNT/E by longer duration of paralysis and greater potency. The proteolytic activity of BoNT/A in cultures of dissociated spinal cord neurons persists beyond 80 days, whereas BoNT/E activity persists for less than 1 day (Keller, J. E., Neale, E. A. Oyler, G., and Adler, M. (1999) FEBS Lett. 456, 137–142). This single quality of toxin activity can account for the differences observed in the duration of muscle block. In the present work we sought to understand the basis for the apparent greater potency of BoNT/A. BoNT/E cleaves a 26-amino acid fragment from the C terminus of the synaptic protein SNAP-25 whereas BoNT/A removes only nine residues creating a 197-amino acid fragment (P197) that is 95% the length of SNAP-25. We show that inhibition of neurotransmitter release by BoNT/E is equivalent to the damage caused to SNAP-25. However, synaptic blockade by BoNT/A is greater than the extent of SNAP-25 proteolysis. These findings can be explained if P197 produces an inhibitory effect on neurotransmitter release. A mathematical model of the experimentally determined relationship between SNAP-25 damage and blockade of neurotransmission supports this interpretation. Furthermore, neurotransmitter release following complete cleavage of SNAP-25 can be achieved by P197, but with about 5-fold less sensitivity to external Ca2+. In this case, vesicular release is restored by increasing intracellular Ca2+. These data demonstrate that P197 competes with intact SNAP-25, but is unable to initiate normal synaptic vesicle fusion in physiological concentrations of Ca2+. Botulinum neurotoxin serotype A (BoNT/A) is distinguished from BoNT/E by longer duration of paralysis and greater potency. The proteolytic activity of BoNT/A in cultures of dissociated spinal cord neurons persists beyond 80 days, whereas BoNT/E activity persists for less than 1 day (Keller, J. E., Neale, E. A. Oyler, G., and Adler, M. (1999) FEBS Lett. 456, 137–142). This single quality of toxin activity can account for the differences observed in the duration of muscle block. In the present work we sought to understand the basis for the apparent greater potency of BoNT/A. BoNT/E cleaves a 26-amino acid fragment from the C terminus of the synaptic protein SNAP-25 whereas BoNT/A removes only nine residues creating a 197-amino acid fragment (P197) that is 95% the length of SNAP-25. We show that inhibition of neurotransmitter release by BoNT/E is equivalent to the damage caused to SNAP-25. However, synaptic blockade by BoNT/A is greater than the extent of SNAP-25 proteolysis. These findings can be explained if P197 produces an inhibitory effect on neurotransmitter release. A mathematical model of the experimentally determined relationship between SNAP-25 damage and blockade of neurotransmission supports this interpretation. Furthermore, neurotransmitter release following complete cleavage of SNAP-25 can be achieved by P197, but with about 5-fold less sensitivity to external Ca2+. In this case, vesicular release is restored by increasing intracellular Ca2+. These data demonstrate that P197 competes with intact SNAP-25, but is unable to initiate normal synaptic vesicle fusion in physiological concentrations of Ca2+. Intoxication with botulinum neurotoxin (BoNT)1 in vivoleads to flaccid paralysis by blockade of acetylcholine release at the neuromuscular junction (1Burgen A.S.V. Dickens F. Zatman L.J. J. Physiol. 1949; 109: 10-24Crossref PubMed Scopus (356) Google Scholar, 2Brooks V.B. J. Physiol. 1956; 134: 264-277Crossref PubMed Scopus (88) Google Scholar, 3Brooks V.B. J. Physiol. 1954; 123: 501-515Crossref PubMed Scopus (45) Google Scholar). The proteolytic activity of the toxin responsible for causing paralysis resides within the 50-kDa light chain domain (4Bandyopadhyay S. Clark A.W. DasGupta B.R. Sathyamoorthy V. J. Biol. Chem. 1987; 262: 2660-2663Abstract Full Text PDF PubMed Google Scholar, 5Fujii N. Kimura K. Yokosawa N. Tsuzuki K. Oguma K. Toxicon. 1992; 30: 1486-1488Crossref PubMed Scopus (20) Google Scholar) and is directed against three synaptic proteins: synaptosomal-associated protein of 25-kDa (SNAP-25), vesicle-associated membrane protein (VAMP), or syntaxin (6Blasi J. Chapman E.R. Link E. Binz T. Yamasaki S. De Camilli P. Sudhof T.C. Niemann H. Jahn R. Nature. 1993; 365: 160-163Crossref PubMed Scopus (1033) Google Scholar, 7Binz T. Blasi J. Yamasaki S. Baumeister A. Link E. Sudhof T.C. Jahn R. Niemann H. J. Biol. Chem. 1994; 269: 1617-1620Abstract Full Text PDF PubMed Google Scholar, 8Blasi J. Chapman E.R. Yamasaki S. Binz T. Niemann H. Jahn R. EMBO J. 1993; 12: 4821-4828Crossref PubMed Scopus (476) Google Scholar, 9Yamasaki S. Baumeister A. Binz T. Blasi J. Link E. Cornille F. Roques B. Fykse E.M. Sudhof T.C. Jahn R. Niemann H. J. Biol. Chem. 1994; 269: 12764-12772Abstract Full Text PDF PubMed Google Scholar, 10Schiavo G. Shone C.C. Rossetto O. Alexander F.C. Montecucco C. J. Biol. Chem. 1993; 268: 11516-11519Abstract Full Text PDF PubMed Google Scholar, 11Schiavo G. Benfenati F. Poulain B. Rossetto O. Polverino de Laureto P. DasGupta B.R. Montecucco C. Nature. 1992; 359: 832-835Crossref PubMed Scopus (1433) Google Scholar). BoNT/A and -E both cleave SNAP-25 but at distinct sites (12Schiavo G. Santucci A. Dasgupta B.R. Mehta P.P. Jontes J. Benfenati F. Wilson M.C. Montecucco C. FEBS Lett. 1993; 335: 99-103Crossref PubMed Scopus (375) Google Scholar). Interestingly, paralysis from BoNT/A lasts for many months whereas blockade caused by BoNT/E lasts for relatively brief periods (13Sellin L.C. Kauffman J.A. Dasgupta B.R. Med. Biol. 1983; 61: 120-125PubMed Google Scholar, 14Adler M. Macdonald D.A. Sellin L.C. Parker G.W. Toxicon. 1996; 34: 237-249Crossref PubMed Scopus (43) Google Scholar). Two hypotheses were proposed to account for this difference: (a) BoNT/A remains catalytically active for a longer interval than BoNT/E or, alternatively (b) the catalytic activity of both toxins is very short-term but the major SNAP-25 fragment generated by BoNT/A (P197) persists in nerve terminals and interferes with neurotransmitter release (15Eleopra R. Tugnoli V. Rossetto O. De Grandis D. Montecucco C. Neurosci. Lett. 1998; 256: 135-138Crossref PubMed Scopus (164) Google Scholar). The first hypothesis has been substantiated by direct demonstration of BoNT/A persistence in primary mouse spinal cord cultures, electroporated chromaffin cells, and mammalian neuromuscular junction preparations in vivo (16Keller J.E. Neale E.A. Oyler G. Adler M. FEBS Lett. 1999; 456: 137-142Crossref PubMed Scopus (144) Google Scholar, 17Bartels F. Bergel H. Bigalke H. Frevert J. Halpern J. Middlebrook J. J. Biol. Chem. 1994; 269: 8122-8127Abstract Full Text PDF PubMed Google Scholar, 18Adler M. Keller J.E. Sheridan R.E. Deshpande S.S. Toxicon. 2001; 39: 233-243Crossref PubMed Scopus (79) Google Scholar). Evidence for the second hypothesis is indirect. Overexpressed P197 in an insulinoma Hit-T15 cell line inhibits insulin secretion (19Huang X. Wheeler M.B. Kang Y.H. Sheu L. Lukacs G.L. Trimble W.S. Gaisano H.Y. Mol. Endocrinol. 1998; 12: 1060-1070PubMed Google Scholar). BoNT/A action at the neuromuscular junction produces a strong immunogenic signal for P197 within nerve terminals, indicating that P197 resides for some time in the proper intracellular regions where it could exert an inhibitory effect on neurotransmission (20de Paiva A. Meunier F.A. Molgo J. Aoki K.R. Dolly J.O. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 3200-3205Crossref PubMed Scopus (547) Google Scholar, 21Raciborska D.A. Charlton M.P. Can. J. Physiol. Pharmacol. 1999; 77: 679-688Crossref PubMed Scopus (37) Google Scholar). However, there has been no quantitative analysis to determine whether the concentrations of P197 generated by toxin activity are able to inhibit neurotransmitter release. BoNT/A and -E are unique among the BoNTs in that, under certain experimental conditions, elevated Ca2+ can partially relieve a toxin-induced blockade (22Molgo J. Dasgupta B.R. Thesleff S. Acta Physiol. Scand. 1989; 137: 497-501Crossref PubMed Scopus (24) Google Scholar, 23Cull-Candy S.G. Lundh H. Thesleff S. J. Physiol. 1976; 260: 177-203Crossref PubMed Scopus (159) Google Scholar, 24Lundh H. Cull-Candy S.G. Leander S. Thesleff S. Brain Res. 1976; 110: 194-198Crossref PubMed Scopus (22) Google Scholar). The effect of Ca2+ on reversing BoNT/A-induced paralysis has been reported for muscle preparations (25Molgo J. Lundh H. Thesleff S. Eur. J. Pharmacol. 1980; 61: 25-34Crossref PubMed Scopus (146) Google Scholar, 26Adler M. Capacio B. Deshpande S.S. Toxicon. 2000; 38: 1381-1388Crossref PubMed Scopus (26) Google Scholar), hippocampal brain slices (27Capogna M. McKinney R.A. O'Connor V. Gahwiler B.H. Thompson S.M. J. Neurosci. 1997; 17: 7190-7202Crossref PubMed Google Scholar, 28Capogna M. Gahwiler B.H. Thompson S.M. J. Neurophysiol. 1996; PubMed Scopus Google Scholar), A. J.A. DasGupta B.R. J. Biol. Chem. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar), and chromaffin T. Binz T. Niemann H. E. Neurosci. 1998; PubMed Scopus Google Scholar). The relationship between SNAP-25 and Ca2+ is The Ca2+ sensitivity observed in for BoNT/A or -E J.A. J. Biol. Chem. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar) is with neuromuscular or chromaffin cell preparations to the of SNAP-25 the toxin J. Thesleff S. Brain Res. PubMed Scopus Google Scholar, L.C. Thesleff S. Dasgupta B.R. Acta Physiol. Scand. 1983; PubMed Scopus Google Scholar, L. E. 1993; 262: PubMed Scopus Google Scholar). Furthermore, that the C terminus of SNAP-25, is by BoNT/A and to the of distinct in the neurotransmitter release T. Binz T. Niemann H. E. Neurosci. 1998; PubMed Scopus Google Scholar). SNAP-25 and P197 to and syntaxin the is less to than a SNAP-25 T. H. Yamasaki S. Binz T. Sudhof T.C. Niemann H. EMBO J. 1994; PubMed Scopus Google Scholar, T. Yamasaki S. S. Binz T. Niemann H. EMBO J. PubMed Scopus Google Scholar, O'Connor V. F. H. EMBO J. PubMed Scopus Google Scholar). the inhibitory of P197 within the of the hypothesis T. J.E. 1993; Full Text PDF PubMed Scopus Google Scholar, T. M. H. S. P. J.E. Nature. 1993; PubMed Scopus Google Scholar) that BoNT/A damage the of the to vesicle a of the from a to a vesicle fusion and neurotransmitter release S.M. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar, S. T. U. A. U. J. E. EMBO J. 2000; PubMed Google Scholar). SNAP-25 cleavage and Ca2+ it is that BoNT/A action on SNAP-25 neurotransmitter release. this it that Ca2+ between SNAP-25 and P197 with J.A. J. Biol. Chem. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar). there be for SNAP-25 and Ca2+ in the release L. E. 1993; 262: PubMed Scopus Google Scholar, S. T. U. A. U. J. E. EMBO J. 2000; PubMed Google Scholar, E. 1993; Full Text PDF PubMed Scopus Google Scholar, P. T. B. Brain Res. Mol. Brain Res. 1997; PubMed Scopus Google Scholar). been to between the of Ca2+ and direct of elevated intracellular Ca2+ on BoNT/A action (27Capogna M. McKinney R.A. O'Connor V. Gahwiler B.H. Thompson S.M. J. Neurosci. 1997; 17: 7190-7202Crossref PubMed Google Scholar, 28Capogna M. Gahwiler B.H. Thompson S.M. J. Neurophysiol. 1996; PubMed Scopus Google Scholar, A. R. G. M. M. 1999; PubMed Scopus Google Scholar, A. S. Eur. J. Neurosci. 1998; PubMed Scopus Google Scholar). This has been to the present to the for intracellular Ca2+ in the inhibitory action of BoNT/A in mouse spinal cord The findings demonstrate that a mathematical between the of P197 by BoNT/A and inhibition of neurotransmitter indicating that the of P197 within neurons to SNAP-25 from to and were from were from membrane from of BoNT/A and -E toxin were from with reported of and of BoNT/E by for at with in by of and for at (16Keller J.E. Neale E.A. Oyler G. Adler M. FEBS Lett. 1999; 456: 137-142Crossref PubMed Scopus (144) Google Scholar). were and at a of toxin to concentrations in the were of and for BoNT/A and were from the and cord cell cultures were L.C. Neale E.A. J. 1992; PubMed Scopus (43) Google Scholar, B.R. Neale E. M. J. Neurophysiol. PubMed Scopus Google Scholar). spinal were from at day dissociated with and on at a of were for at in an of of of with and a of A. J. A. B. A and of the R. Scholar). were with with for in by were with and for analysis or neurotransmitter release. by in with 1 and 1 The to in a for to and at to were with of at for and by of protein were by the of Nature. PubMed Scopus Google Scholar). were H. G. 1987; PubMed Scopus Google Scholar), and to membrane with a and (16Keller J.E. Neale E.A. Oyler G. Adler M. FEBS Lett. 1999; 456: 137-142Crossref PubMed Scopus (144) Google Scholar). neurotransmitter release determined L.C. Halpern Montecucco C. J.E. Neale E.A. J. Biol. Chem. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar). were with for at and with a of release by of and Ca2+ to at release determined by from cultures in the of and a of the of were and of were analysis of data with of BoNT/E of spinal cord cell cultures in cleavage of SNAP-25 in a the of the for BoNT/E cleavage of SNAP-25 is of synaptic that release is to the extent SNAP-25 cleavage 1 In cultures, the extent of release is to in the external in the of and and of the release is achieved with external Ca2+ this the for the BoNT/E of release from to for the 1 and release. cord cultures were in BoNT/A or -E at 1 for at release of with toxin a to with an for Ca2+ of and a of release for from to of the and to cultures Ca2+ with an of and release Ca2+ The for this release of the release from cultures no Ca2+ release in the of Ca2+. were from and the of The model in 1 C the effect of BoNT/E on the release In this SNAP-25 is to with for neurotransmitter release with an by release in a is to the of SNAP-25 SNAP-25 is by increasing concentrations of neurotransmitter release to a SNAP-25 has been to with and T. Yamasaki S. S. Binz T. Niemann H. EMBO J. PubMed Scopus Google Scholar, O'Connor V. F. H. EMBO J. PubMed Scopus Google Scholar). However, data that in this intact with very to SNAP-25. Furthermore, increasing Ca2+ physiological concentrations the to findings with neuromuscular preparations M. Macdonald D.A. Sellin L.C. Parker G.W. Toxicon. 1996; 34: 237-249Crossref PubMed Scopus (43) Google Scholar). data with BoNT/A for SNAP-25 and blockade of neurotransmitter release SNAP-25 cleavage with an of and release is with from to Ca2+ concentrations from to in the release Ca2+ to or to that with Ca2+ Ca2+ concentrations than a greater of inhibition to SNAP-25 cleavage the for neurotransmitter release were the with the for SNAP-25 a of C between SNAP-25 and P197 and on the release In this model P197 to the SNAP-25. elevated Ca2+ has been to BoNT/A inhibition in brain slices (27Capogna M. McKinney R.A. O'Connor V. Gahwiler B.H. Thompson S.M. J. Neurosci. 1997; 17: 7190-7202Crossref PubMed Google Scholar), A. J.A. DasGupta B.R. J. Biol. Chem. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar), and neuromuscular preparations J. N. Thesleff S. J. Physiol. 1989; PubMed Scopus Google Scholar), we that neurotransmitter release the Ca2+ in of that achieved normal Ca2+ normal is with the This model to the relationship between SNAP-25 cleavage and of release. (a) that P197 with with the or the SNAP-25, and (b) that at of the of the SNAP-25 and P197 the of SNAP-25 in the of the in is to this the of the for neurotransmitter release to SNAP-25 is The data for neurotransmitter release with Ca2+ the at Ca2+ cultures show a greater in release we the of on release following to a of BoNT/A or BoNTs at 1 cleave SNAP-25 1 A and release from cultures toxin is Ca2+ with an for Ca2+ of and a of about release from cultures is Ca2+ with an of and a that is of release release for both and cultures is Ca2+ and the of release is by toxin with an of Proc. Natl. Acad. Sci. U. S. A. 1998; PubMed Scopus Google Scholar, M.P. J. Physiol. PubMed Scopus Google Scholar). release from cultures is at of Ca2+ This that the of intracellular Ca2+ from can some to but to with from 1 and the of intracellular Ca2+ from release by in the and of and a partially release to BoNT/A with BoNT/A release to of release. of in with Ca2+ the effect of release to of determine the of Ca2+ that to this release with in the of or cultures with to intracellular Ca2+ O. A.W. J. Pharmacol. 1989; PubMed Scopus (37) Google Scholar, H. O. J. Biol. Chem. 1989; Full Text PDF PubMed Google Scholar). The of release with external Ca2+ with and the of to the release that Ca2+ to of the BoNT/A-induced block. this cultures were with 1 BoNT/A or BoNT/E for and neurotransmitter release in the of in with external Ca2+ produces a of the BoNT/A at Ca2+ concentrations with complete in the of partially with release at of cultures experimental conditions, neurons are about and less to than has on reversing BoNT/E neurons release and demonstrate sensitivity to Ca2+. In the of release of to However, the for Ca2+ is with for the release achieved in the of at the cultures that were with the Ca2+ sensitivity the extent of release In this we the relationship between blockade of neurotransmitter release and cleavage of SNAP-25 caused by BoNT/A and -E to in BoNT/A to greater potency than BoNT/E M. Macdonald D.A. Sellin L.C. Parker G.W. Toxicon. 1996; 34: 237-249Crossref PubMed Scopus (43) Google Scholar, J. Dasgupta B.R. Thesleff S. Acta Physiol. Scand. 1989; 137: 497-501Crossref PubMed Scopus (24) Google Scholar, 26Adler M. Capacio B. Deshpande S.S. Toxicon. 2000; 38: 1381-1388Crossref PubMed Scopus (26) Google Scholar, J. Thesleff S. Brain Res. PubMed Scopus Google Scholar). BoNTs a in synaptic vesicle and of SNAP-25. However, it that in to SNAP-25, BoNT/A action has a inhibitory The of SNAP-25 cleavage and of release in this to the of an inhibitory a cleavage fragment by BoNT/A. BoNT/A action on SNAP-25 produces a a in SNAP-25 and of a fragment that synaptic vesicle The of this fragment could be the 197-amino acid P197, or the fragment from the C of SNAP-25 synaptic vesicle in chromaffin J.A. M. S. FEBS Lett. PubMed Scopus Google Scholar, S. J. M. J. Biol. Chem. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar). with to the and with of and S. J. M. J. Biol. Chem. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar, A. S. J.A. Adler M. M. FEBS Lett. 1998; PubMed Scopus Google Scholar). A of the C terminus to inhibit release to extent G.W. P. N. DasGupta B.R. Dolly J.O. 1997; PubMed Scopus Google Scholar). The of length with potency that the cleavage fragment generated by BoNT/A with neurotransmitter release. inhibitory of P197 by of P197 an Hit-T15 cell line and inhibition of insulin secretion (19Huang X. Wheeler M.B. Kang Y.H. Sheu L. Lukacs G.L. Trimble W.S. Gaisano H.Y. Mol. Endocrinol. 1998; 12: 1060-1070PubMed Google Scholar). P197 has been to with the and syntaxin in a to SNAP-25 T. H. Yamasaki S. Binz T. Sudhof T.C. Niemann H. EMBO J. 1994; PubMed Scopus Google Scholar, O'Connor V. F. H. EMBO J. PubMed Scopus Google Scholar). on the for BoNT/A E. F. T. G. Frevert J. A. U. J. Bigalke H. Pharmacol. PubMed Scopus Google Scholar), of P197 in nerve terminals in vivo generated the hypothesis that P197 to SNAP-25 and with the normal vesicular for neurotransmitter release for or months (20de Paiva A. Meunier F.A. Molgo J. Aoki K.R. Dolly J.O. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 3200-3205Crossref PubMed Scopus (547) Google Scholar, 21Raciborska D.A. Charlton M.P. Can. J. Physiol. Pharmacol. 1999; 77: 679-688Crossref PubMed Scopus (37) Google Scholar). Two direct that BoNT/A catalytic activity persists within neurons with a greater than (16Keller J.E. Neale E.A. Oyler G. Adler M. FEBS Lett. 1999; 456: 137-142Crossref PubMed Scopus (144) Google Scholar, 18Adler M. Keller J.E. Sheridan R.E. Deshpande S.S. Toxicon. 2001; 39: 233-243Crossref PubMed Scopus (79) Google Scholar). on the of P197, at that it at a to SNAP-25 (16Keller J.E. Neale E.A. Oyler G. Adler M. FEBS Lett. 1999; 456: 137-142Crossref PubMed Scopus (144) Google Scholar). The duration of the catalytic action of BoNT/A the of SNAP-25 cleavage in the extent of the persistence of BoNT/A activity a of P197 to account for in nerve terminals (20de Paiva A. Meunier F.A. Molgo J. Aoki K.R. Dolly J.O. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 3200-3205Crossref PubMed Scopus (547) Google Scholar, 21Raciborska D.A. Charlton M.P. Can. J. Physiol. Pharmacol. 1999; 77: 679-688Crossref PubMed Scopus (37) Google Scholar). Furthermore, nerve the of paralysis S.G. Lundh H. Thesleff S. J. Physiol. 1976; 260: 177-203Crossref PubMed Scopus (159) Google Scholar, J. Pharmacol. Google Scholar, 1976; PubMed Scopus Google Scholar), toxin within nerve terminals where of P197 in the of vesicular on muscle from the of P197 and of SNAP-25 could muscle paralysis to of SNAP-25 by D.A. Trimble W.S. Charlton M.P. Eur. J. Neurosci. 1998; PubMed Scopus Google Scholar). The model in C that cleavage of the SNAP-25 in a of neurotransmitter release. This is on of P197 and SNAP-25 for of the fusion and the relationship is in D. H. Chapman E.R. Blasi J. Jahn R. Res. PubMed Scopus Google Scholar) reported that cleavage of of SNAP-25 in preparations caused a complete of in with model and experimental In to the observed for BoNT/E 1 the relationship of SNAP-25 cleavage to BoNT/A the of BoNT/A (20de Paiva A. Meunier F.A. Molgo J. Aoki K.R. Dolly J.O. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 3200-3205Crossref PubMed Scopus (547) Google Scholar) but BoNT/E within nerve SNAP-25 both BoNT/A and of SNAP-25 within of the for SNAP-25 be to the The model that is of and the hypothesis T. J.E. 1993; Full Text PDF PubMed Scopus Google Scholar, T. M. H. S. P. J.E. Nature. 1993; PubMed Scopus Google Scholar) the of or J.A. J. Biol. Chem. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar, G. G. J.E. Proc. Natl. Acad. Sci. U. S. A. 1997; PubMed Scopus Google Scholar, G. Sudhof T.C. J. Biol. Chem. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar). spinal cord cultures a in neurotransmitter release no greater than the extent of SNAP-25 The direct relationship between SNAP-25 cleavage and neurotransmitter blockade that, in intact SNAP-25 by BoNT/E with the release release from spinal cord neurons is less to Ca2+ than release from neurons is in the of release The could a were with preparations M. Gahwiler B.H. Thompson S.M. J. Neurophysiol. 1996; PubMed Scopus Google Scholar, A. S. Eur. J. Neurosci. 1998; PubMed Scopus Google Scholar) and are with in vivo that the of the that a from BoNT/A from BoNT/E M. Macdonald D.A. Sellin L.C. Parker G.W. Toxicon. 1996; 34: 237-249Crossref PubMed Scopus (43) Google Scholar). release from neurons could be This release with an for external Ca2+ of 5-fold than the concentrations of Ca2+ BoNT/A release and at release of the release from The effect of Ca2+ on the release is with intact chromaffin and hippocampal neurons T. Binz T. Niemann H. E. Neurosci. 1998; PubMed Scopus Google Scholar, Proc. Natl. Acad. Sci. U. S. A. 1998; PubMed Scopus Google Scholar, E. J. Physiol. 1992; PubMed Scopus Google Scholar, P. J. Biol. 1994; PubMed Scopus Google a of neurotransmitter can be complete cleavage of SNAP-25 the of Ca2+ for this to is than physiological Ca2+ that with BoNT/A the of P197 to SNAP-25 to Ca2+ the extent of Ca2+ that under normal physiological the we to determine whether elevated Ca2+ the of BoNT/A H. Cull-Candy S.G. Leander S. Thesleff S. Brain Res. 1976; 110: 194-198Crossref PubMed Scopus (22) Google Scholar, A. J.A. DasGupta B.R. J. Biol. Chem. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar, F. A. 1983; PubMed Scopus (43) Google Scholar). the of vesicular release in The apparent for Ca2+ in the and of with of an effect on Ca2+ sensitivity and of a of release that the P197 release by Ca2+ is that the action of an of SNAP-25 could account for the observed synaptic release. this were the case, and SNAP-25 were BoNT/A the for release but the observed for Ca2+ the for neurons with The Ca2+ sensitivity following BoNT/A be to of fusion P197, for vesicle in nerve BoNT/A and neurotoxin of synaptic at active 1976; PubMed Scopus Google Scholar, E.A. M. L.C. J. Biol. 1999; PubMed Scopus Google Scholar). These unable to with the a for the of synaptic to and T. Binz T. Niemann H. E. Neurosci. 1998; PubMed Scopus Google Scholar, 1976; PubMed Scopus Google Scholar, T. E. J. Neurosci. 1997; 17: PubMed Google Scholar). we show that an intact with BoNT/A Ca2+ to release. This in be to in the of synaptic vesicle in and release sites caused by or from SNAP-25 cleavage J.A. J. Biol. Chem. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar). spinal cord cultures were to the first that catalytic activity of BoNT/A but BoNT/E for many months following (16Keller J.E. Neale E.A. Oyler G. Adler M. FEBS Lett. 1999; 456: 137-142Crossref PubMed Scopus (144) Google Scholar). This is with in vivo that BoNT/A but BoNT/E paralysis (13Sellin L.C. Kauffman J.A. Dasgupta B.R. Med. Biol. 1983; 61: 120-125PubMed Google Scholar, 18Adler M. Keller J.E. Sheridan R.E. Deshpande S.S. Toxicon. 2001; 39: 233-243Crossref PubMed Scopus (79) Google Scholar). In the present Ca2+ release of neurotransmitter with a of in the of is with M.P. J. Physiol. PubMed Scopus Google Scholar, E. J. Physiol. 1992; PubMed Scopus Google Scholar). release following to BoNT/A and to a to in vivo with of toxin the muscle of M. Macdonald D.A. Sellin L.C. Parker G.W. Toxicon. 1996; 34: 237-249Crossref PubMed Scopus (43) Google Scholar, J. Dasgupta B.R. Thesleff S. Acta Physiol. Scand. 1989; 137: 497-501Crossref PubMed Scopus (24) Google Scholar, 23Cull-Candy S.G. Lundh H. Thesleff S. J. Physiol. 1976; 260: 177-203Crossref PubMed Scopus (159) Google Scholar). data demonstrate that the of P197 generated by BoNT/A is to activity in a Furthermore, the of P197 to inhibit release is to against SNAP-25 in the release In of this of chromaffin the of secretion by F. Bergel H. Bigalke H. Frevert J. Halpern J. Middlebrook J. J. Biol. Chem. 1994; 269: 8122-8127Abstract Full Text PDF PubMed Google Scholar), indicating that of BoNT/A SNAP-25 to This that the effect of P197 is brief BoNT/A activity indicating P197 has a brief to BoNT/A. a model we that paralysis by (a) is to catalytic activity of this toxin within nerve terminals and (b) is caused by of SNAP-25 and of P197 that competes with the intact SNAP-25 to the block. We are to Adler, and Oyler for the of this We for in the in the J. for in and data we for and of the spinal cord cell cultures and with the present botulinum neurotoxin synaptosomal-associated protein of SNAP-25 SNAP-25 vesicle-associated membrane protein