Rescue of Exocytosis in Botulinum Toxin A-poisoned Chromaffin Cells by Expression of Cleavage-resistant SNAP-25

Abstract

Botulinum neurotoxin (BoNT) types A and B selectively block exocytosis by cleavage of SNAP-25 and synaptobrevin, respectively; in humans, many months are required for full recovery from the resultant neuromuscular paralysis. To decipher the molecular basis for such prolonged poisoning, intoxication in adreno-chromaffin cells was monitored over 2 months. Exocytosis from BoNT/B-treated cells resumed after 56 days because of the appearance of intact synaptobrevin. However, inhibition continued in BoNT/A-treated cells, throughout the same interval, with a continued predominance of cleaved SNAP-25-(1–197) over the intact protein. When recovery from poisoning was attempted by transfection of the latter cells with the gene encoding full-length SNAP-25-(1–206), no restoration of exocytosis ensued even after 3 weeks. To ascertain if this failure was because of the persistence of the toxin's protease activity, the cells were transfected with BoNT/A-resistant SNAP-25 constructs; importantly, exocytosis was rescued. C-terminal truncation of the toxin-insensitive SNAP-25 revealed that residues 1–201, 1–202, 1–203 afforded a significant return of exocytosis, unlike shorter forms 1–197, −198, −199, or −200; accordingly, mutants M202A or L203A of full-length SNAP-25 rescued secretion. These findings give insights into the C-terminal functional domain of SNAP-25, demonstrate the longevity of BoNT/A protease, and provide the prospect of a therapy for botulism. Botulinum neurotoxin (BoNT) types A and B selectively block exocytosis by cleavage of SNAP-25 and synaptobrevin, respectively; in humans, many months are required for full recovery from the resultant neuromuscular paralysis. To decipher the molecular basis for such prolonged poisoning, intoxication in adreno-chromaffin cells was monitored over 2 months. Exocytosis from BoNT/B-treated cells resumed after 56 days because of the appearance of intact synaptobrevin. However, inhibition continued in BoNT/A-treated cells, throughout the same interval, with a continued predominance of cleaved SNAP-25-(1–197) over the intact protein. When recovery from poisoning was attempted by transfection of the latter cells with the gene encoding full-length SNAP-25-(1–206), no restoration of exocytosis ensued even after 3 weeks. To ascertain if this failure was because of the persistence of the toxin's protease activity, the cells were transfected with BoNT/A-resistant SNAP-25 constructs; importantly, exocytosis was rescued. C-terminal truncation of the toxin-insensitive SNAP-25 revealed that residues 1–201, 1–202, 1–203 afforded a significant return of exocytosis, unlike shorter forms 1–197, −198, −199, or −200; accordingly, mutants M202A or L203A of full-length SNAP-25 rescued secretion. These findings give insights into the C-terminal functional domain of SNAP-25, demonstrate the longevity of BoNT/A protease, and provide the prospect of a therapy for botulism. /B, /C, and /E, botulinum neurotoxins type A, B, C, and E, respectively chloramphenicol acetyltransferase cellubrevin Chinese hamster ovary dithiothreitol glutathioneS-transferase human growth hormone immunoglobulin type G light chain large dense core vesicle low ionic strength medium neuromuscular junction polyacrylamide gel electrophoresis radioimmunoassay synaptobrevin synaptosomal-associated protein with molecular mass of 25 kDa the products of SNAP-25 proteolysis by BoNT/A and BoNT/C, respectively solubleN-ethylmaleimide-sensitive factor attachment protein receptor tetanus toxin enzyme-linked immunosorbent assay Botulinum neurotoxin type A (BoNT/A)1 is being employed successfully for the therapy of dystonias and dysphonias because it potently and selectively inhibits acetylcholine release at the neuromuscular junction (NMJ), thereby resulting in paralysis that lasts for several months (Refs. 1Angaut-Petit D. Molgo J. Comella J.X. Faille L. Tabti N. Neuroscience. 1990; 37: 799-808Crossref PubMed Scopus (114) Google Scholar and 2Sloop R.R. Cole B.A. Escutin R.O. Neurology. 1997; 49: 189-194Crossref PubMed Scopus (160) Google Scholar; and reviewed in Ref. 3Cava T.J. Eur. J. Neurol. 1995; 2: 57-60Google Scholar). Seven serotypes of BoNTs (A to G) are produced by Clostridium botulinum, whereas only a single form of tetanus toxin (TeTX) is synthesized by Clostridium tetani; the latter blocks transmitter release at central inhibitory nerve terminals (4Pellizzari R. Rossetto O. Schiavo G. Montecucco C. Philos. Trans. R. Soc. Lond. B. Biol. Sci. 1999; 354: 259-268Crossref PubMed Scopus (221) Google Scholar). These homologous, but immunologically distinct, proteins share many properties; each active neurotoxin consists of a heavy chain and a light chain (LC) linked by a disulfide bond and non-covalent interactions. The heavy chain is required for high affinity binding to specific neuronal ecto-acceptors, subsequent internalization, and translocation of the LC into the cytosol, where the latter blocks synaptic vesicle exocytosis (reviewed in Ref. 4Pellizzari R. Rossetto O. Schiavo G. Montecucco C. Philos. Trans. R. Soc. Lond. B. Biol. Sci. 1999; 354: 259-268Crossref PubMed Scopus (221) Google Scholar). The LCs of BoNTs and TeTX are Zn2+-dependent endoproteinases that exhibit remarkable substrate selectivities, targeting single bonds (except for BoNT/C, see below) in one of three SNARE (soluble N -ethylmaleimide-sensitive factor attachment protein receptor) proteins proposed to constitute the core components of the vesicle docking/fusion complex that mediates the regulated exocytosis of neurotransmitters (5Söllner T. Bennett M.K. Whiteheart S.W. Scheller R.H. Rothman J.E. Cell. 1993; 75: 409-418Abstract Full Text PDF PubMed Scopus (1577) Google Scholar). Synaptosomal-associated protein with a molecular mass of 25 kDa (SNAP-25) is proteolyzed by BoNT/A, /C, and /E at separate sites near the C terminus: Gln197-Arg198, Arg198-Ala199, and Arg180-Ile181 (6Schiavo 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 (377) Google Scholar, 7Vaidyanathan V.V. Yoshino K. Jahnz M. Dörries C. Bade S. Nauenburg S. Niemann H. Binz T. J. Neurochem. 1999; 72: 327-337Crossref PubMed Scopus (184) Google Scholar, 8Binz T. Blasi J. Yamasaki S. Baumeister A. Link E. Südhof T.C. Jahn R. Niemann H. J. Biol. Chem. 1994; 269: 1617-1620Abstract Full Text PDF PubMed Google Scholar), respectively. Syntaxin 1A/B is also cleaved by BoNT/C and synaptobrevin (Sbr) by BoNT/B, /D, /F, /G, and TeTX (reviewed in Ref. 4Pellizzari R. Rossetto O. Schiavo G. Montecucco C. Philos. Trans. R. Soc. Lond. B. Biol. Sci. 1999; 354: 259-268Crossref PubMed Scopus (221) Google Scholar). Unlike syntaxin 1A/B and Sbr, which each possess a transmembrane anchor (9Niemann H. Blasi J. Jahn R. Trends Cell Biol. 1994; 4: 179-185Abstract Full Text PDF PubMed Scopus (291) Google Scholar), SNAP-25 is attached to the membrane through thioester-linked palmitate modifications of one or more of its four centrally located cysteines (10Lane S.R. Liu Y.C. J. Neurochem. 1997; 69: 1864-1869Crossref PubMed Scopus (104) Google Scholar). BoNT/A- or /E-truncated SNAP-25 remain membrane-bound, but release is inhibited because of the destabilization of the ternary complex even though assembly and disassembly can still occur (11Hayashi T. McMahon H.J. Yamasaki S. Binz T. Hata Y. Südhof T.C. Niemann H. EMBO J. 1994; 13: 5051-5061Crossref PubMed Scopus (661) Google Scholar, 12Pellegrini L.L. O'Connor V. Lottspeich F. Betz H. EMBO J. 1995; 14: 4705-4713Crossref PubMed Scopus (127) Google Scholar). The precise basis of their blockade of neuroexocytosis has yet to be elucidated. While the mechanism of BoNT-induced inhibition has received much attention, the molecular processes involved in recovery of the NMJ from poisoning, which can take up to 4 months depending on the serotype used, remains poorly understood. In humans, the extent of recovery from the neuromuscular paralysis caused by BoNT/A or /B was found to be ∼6 and ∼66%, respectively, at 49 days post-injection (2Sloop R.R. Cole B.A. Escutin R.O. Neurology. 1997; 49: 189-194Crossref PubMed Scopus (160) Google Scholar). Recently, the sequence of events involved in the protracted resumption of neurotransmission in BoNT/A poisoned motor endplates has been revealed by monitoring synaptic function in individual, identified nerve endings of living animals (13de 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 (551) Google Scholar). This showed that the transient appearance of functional nerve sprouts mediates a partial return of neuromuscular function, with full recovery relying on the originally poisoned nerve terminals reacquiring synaptic activity. However, it is still unclear how the of the persistence of toxin the of the To insights into the molecular basis of the but recovery this was in adreno-chromaffin exocytosis from the latter many with synaptic release from in SNAP-25, Sbr, and syntaxin 1A/B are and been to be required for release of their large dense core Dolly J.O. Eur. J. PubMed Scopus Google Scholar, Dolly J.O. PubMed Scopus Google because cleavage of by BoNT/A, /B, or blocks on cells that persistence of BoNT/A or TeTX was for prolonged inhibition of release F. H. H. J. J. J. J. Biol. Chem. 1994; 269: Full Text PDF PubMed Google Scholar). it was to the of recovery of exocytosis in cells to the of paralysis of NMJ in poisoned with BoNT/A and B (2Sloop R.R. Cole B.A. Escutin R.O. Neurology. 1997; 49: 189-194Crossref PubMed Scopus (160) Google Scholar). The of the and full-length SNARE be throughout the of intoxication in cells, a that be at the this of BoNT/A poisoning to be attempted through the of encoding forms of to exocytosis in this with the up to 3 continued of the This was by the with SNAP-25 mutants that were to be to This which that residues in SNAP-25 at or its in regulated whereas SNAP-25 inhibits the BoNT/A was 1995; PubMed Scopus Google Scholar). and /E were by C. C. for and and B. DasGupta and to N. DasGupta B.R. Dolly J.O. 1997; PubMed Scopus Google Scholar). specific for SNAP-25-(1–197) was a from T. A. N. and D. for and encoding and its were by L. of this radioimmunoassay and and were cells were from and Dolly J.O. Eur. J. PubMed Scopus Google Scholar). required for transfection were J. PubMed Scopus Google Scholar). days after the cells were with a low ionic strength medium at for in the or of BoNT/A or after were at Dolly J.O. Eur. J. PubMed Scopus Google Scholar). after with or BoNT/A or /B, the cells were with J. PubMed Scopus Google and in at with this in the or of 2 of the medium the cells were and for a Dolly J.O. Eur. J. PubMed Scopus Google Scholar). for release of were from the in the of to the which were a of the the The gene was into or the and or were into by chain or the When were the These or in the SNAP-25 gene were by of glutathioneS-transferase were and by affinity PubMed Scopus Google Scholar). of the proteins was with and to of BoNT/A at for 4 The were and with the residues of Dolly J.O. Eur. J. PubMed Scopus Google Scholar). was by and the were to appearance of the was of from were of for and the toxin of intact SNAP-25 in were of full-length and were of that for the intact protein and the of intact This was to the of intact SNAP-25 in each of the A intact SNAP-25 in cells, which SNAP-25, were transfected with and were in with and for were to with the Dolly J.O. Eur. J. PubMed Scopus Google Scholar, Dolly J.O. PubMed Scopus Google with and residues of human were to and by the the after in with or BoNT/A, the cells were transfected with the with acetyltransferase a or the SNAP-25 gene or the 1995; PubMed Scopus Google Scholar). to days after from intact cells was for and a In transfected cells were with in a N. DasGupta B.R. Dolly J.O. 1997; PubMed Scopus Google and for to over the subsequent was by the of and was were and for was for To the of how BoNTs their inhibitory for prolonged cells were for with BoNT/A or B a to the of high affinity Dolly J.O. Eur. J. PubMed Scopus Google Scholar). and of were at and 56 days was employed of or which because it much more release to of the with only by the latter still the same for SNAP-25 and Dolly J.O. Eur. J. PubMed Scopus Google Scholar). cells that been with BoNT/A or /B showed inhibition of release of and respectively, to that for SNAP-25 was monitored whereas intact and cellubrevin a L. Link E. Binz T. Niemann H. Jahn R. Südhof T.C. 1993; PubMed Scopus Google were of exhibit no the of their cleavage by BoNT/A or The of SNAP-25 in the membrane was monitored specific for the latter intact SNAP-25 but with the of BoNT/A N. DasGupta B.R. Dolly J.O. 1997; PubMed Scopus Google Scholar, D. 1997; PubMed Scopus Google Scholar). of the membrane of the cells showed that the inhibition of at was by or of intact SNAP-25 or with the SNAP-25 being at this only a of SNAP-25 C was in cells to the in accordingly, the of was that on However, at 56 of intact SNAP-25 were in the poisoned cells, to a significant of recovery of exocytosis to of this in the cells to that to be to recovery from BoNT/A In a much recovery from blockade by was The high of inhibition of in cells, on of only inhibition at This resumption of regulated exocytosis was with of intact to a with that in cells on the it was that of of the full-length SNAP-25 in cells regulated exocytosis by with the a to this of SNAP-25 in cells to be this be by into cells that and of a of the cells showed that SNAP-25 was and the same the in cells 2 with it was also with but by specific for 2 of transfected cells to BoNT/A in the of with and the appearance of 2 the of the protein to the To the prospect of release through the of SNAP-25 into cells, the protein was with The which is from cells, with in for exocytosis 1995; PubMed Scopus Google Scholar). cells transfected with encoding and the of and this was inhibited in cells with BoNT/A 2 the of for cells with a encoding SNAP-25 the of from such of SNAP-25 to the extent of inhibition because of BoNT/A poisoning 2 These that persistence of BoNT/A protease for days after intoxication the blockade of to SNAP-25 mutants to BoNT/A to be and To the of the of the toxin being poisoned cells, forms of full-length SNAP-25 that were to BoNT/A but be to exocytosis were and at in SNAP-25, located on of the bond the or of and A. PubMed Scopus Google were because of SNAP-25 that the is for cleavage by BoNT/A J. Chem. 1997; PubMed Scopus (114) Google Scholar). the gene was by and the proteins were products that their to proteolysis be in Seven SNAP-25 mutants were single or at the in 3 was by affinity and that the and The cleavage of SNAP-25 by BoNT/A was which the of full-length substrate and SNAP-25 mutants to BoNT/A 3 the for the cleavage of each was from 3 B and in at the but at the proteins to BoNT/A with the of with or of SNAP-25 by and respectively In of the to caused the the in to at the to or to proteolysis by type A with the latter to be the in at the and the sequence to in SNAP-25 C. A. D. L. D. J. Chem. 1993; Scholar), or caused to BoNT/A and respectively; was at of SNAP-25 exocytosis from inhibition by of and SNAP-25 mutants to cleavage by of BoNT/A to of SNAP-25 in the of intact to the A were to the A which was to intact SNAP-25 in This was to the of BoNT/A required to of the or SNAP-25 from 3 to proteolysis by BoNT/A to by the BoNT/A cleavage of SNAP-25 by the for to of intact to the A were to the A which was to intact SNAP-25 in This was to the of BoNT/A required to of the or SNAP-25 from 3 by the BoNT/A cleavage of SNAP-25 by the for to in a the was for several its high to a single was more a to be to exocytosis and in cells, and in the of it being cleaved the be with because of the of was in the a to the the of and its of to BoNT/A were in was found to be at a to that of SNAP-25, from the on with full-length SNAP-25 or its C-terminal 2 of the transfected cells to BoNT/A the with and no be with 2 that was but cleaved by BoNT/A in cells, encoding several such SNAP-25 mutants were into of mutants in cells their to with transfected with SNAP-25 or protein which is involved in exocytosis 2 of each SNAP-25 in BoNT/A-treated cells which release was rescued function, to the for 2 of BoNT/A SNAP-25 can give restoration of regulated in cells poisoned days were to demonstrate that the of in BoNT/A cells was a of the of BoNT/A a to into cells the is for this that it the BoNT/A its in cells SNAP-25, poisoning with BoNT/A caused inhibition of release to the in BoNT/A cells the was rescued. cells were and to to the SNAP-25 N. DasGupta B.R. Dolly J.O. 1997; PubMed Scopus Google the restoration of was and with inhibition of The to exocytosis transfection of a but SNAP-25 in BoNT/A poisoned cells that the of the toxin for up to a 2 To BoNT/A active for the cells were with SNAP-25 and 3 after this to restoration of the of inhibition to the for cells transfected with and it that the protease of the toxin active and cleaved the the extent of from cells 3 but SNAP-25 was with that from cells transfected with the same or When SNAP-25, which been to be in cells 2 was into cells, it release its in cells the of that toxin it that the intact SNAP-25 can be from the SNARE complex by this mediates the inhibition of exocytosis by it was to how many of the C-terminal residues of SNAP-25 to be to its in this was a of mutants in B. on the SNAP-25 C-terminal domain C. Bennett M.K. Biol. PubMed Scopus Google it in the core residues form a in which to The of the of residues that can be from the C-terminal of SNAP-25, its to exocytosis, where this functional domain In this it is that the of of cleavage with and to be to BoNT/A or inhibited in or cells to that with full-length In a of was with or in of BoNT/A-resistant mutants rescued release in BoNT/A cells The of to exocytosis to a significant but that the the of SNAP-25 to function in exocytosis, which only in this this to be the because the of the of into To that residues to are for exocytosis, the was to in full-length the full of of the BoNT/A-resistant only exocytosis in cells but it rescued in poisoned cells it that SNAP-25 residues can exocytosis or the four a M. A. S. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: PubMed Scopus Google that to in SNAP-25 exocytosis in cells, this was of into cells caused no inhibition of of its BoNT/A-resistant exocytosis in cells and afforded of after poisoning This to the of SNAP-25 mutants with in the In this recovery of regulated release was 56 days after intoxication of cells with BoNT/B, by the of intact This to the of paralysis caused by of the toxin into (2Sloop R.R. Cole B.A. Escutin R.O. Neurology. 1997; 49: 189-194Crossref PubMed Scopus (160) Google and also with that found for cells poisoned with TeTX F. H. H. J. J. J. J. Biol. Chem. 1994; 269: Full Text PDF PubMed Google Scholar), that the of and TeTX are the of of intact the of In the of cells poisoned with type A the resumption of exocytosis was much with only a partial recovery being in 2 this is to for cells F. H. H. J. J. J. J. Biol. Chem. 1994; 269: Full Text PDF PubMed Google and human NMJ (2Sloop R.R. Cole B.A. Escutin R.O. Neurology. 1997; 49: 189-194Crossref PubMed Scopus (160) Google Scholar). it was that this prolonged inhibition is because of the of intact SNAP-25 and persistence of its the serotypes that BoNT/A remains active for the of SNAP-25 is that of for the that BoNT/A is active for 3 after poisoning because of SNAP-25 in cells to a whereas this was a the continued of is a factor in the prolonged inhibition of at in this a by the that a recovery of in cells F. H. H. J. J. J. J. Biol. Chem. 1994; 269: Full Text PDF PubMed Google Scholar). However, it is the continued BoNT/A the persistence of that recovery because into human of which a of with type A, a of recovery of the toxin R. V. Rossetto O. D. Montecucco C. Lett. PubMed Scopus Google Scholar). has been proposed that the cleavage of from the C-terminal of by of the thereby to a more recovery of The of poisoning, which is in of is by the the of toxin and the of SNAP-25 that this is in motor nerve endings cells, of in the nerve terminals by the of the recovery proposed in R. V. Rossetto O. D. Montecucco C. Lett. PubMed Scopus Google Scholar). In of that BoNT/A active for more a was for release on the of SNAP-25 mutants that were to BoNT/A but of of a of mutants revealed that the is for cleavage of full-length SNAP-25 by BoNT/A, been for C-terminal of SNAP-25 J. Chem. 1997; PubMed Scopus (114) Google Scholar). the of this at were to a high to by BoNT/A of SNAP-25 V.V. Yoshino K. Jahnz M. Dörries C. Bade S. Nauenburg S. Niemann H. Binz T. J. Neurochem. 1999; 72: 327-337Crossref PubMed Scopus (184) Google Scholar). it is for that of BoNT/A-resistant forms of SNAP-25 into intact cells this recovery a for the of residues at the BoNT/A cleavage of SNAP-25 or BoNT/A-resistant mutants to the cells, by the extent of release SNAP-25 has been to be to the membrane in cells Wilson M.C. Proc. Natl. Acad. Sci. U. S. A. 1995; PubMed Scopus Google C. Niemann H. M. S. K. J. 1999; PubMed Scopus Google Scholar). SNAP-25 complex with the of syntaxin 1A/B and to its in exocytosis, it can be that SNAP-25 the of intact or SNAP-25 in SNARE complex The that was found to the of exocytosis for the of BoNT/A-resistant SNAP-25 in In light of it is that and in SNAP-25, which were in the are for In this and provide for being a high of in of a SNAP-25 on regulated exocytosis in cells, but it the inhibition of of the cells to C. Niemann H. M. S. K. J. 1999; PubMed Scopus Google Scholar). a of SNAP-25 has revealed that of residues to be for the of ternary and the of this of SNAP-25 to its function in regulated Scheller R.H. Cell. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar). The full extent of this was human which is only to was in this exocytosis which was to after of the cells K. A. Niemann H. U. A. R. S. J. Biol. Chem. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar). The is the to the of SNAP-25 by the of BoNT/A-resistant mutants to exocytosis in intact the of the of and SNAP-25, it was to the domain for exocytosis to by This is because on SNAP-25 that the C-terminal core domain in such a that it to the with C. Bennett M.K. Biol. PubMed Scopus Google Scholar). The of to is significant because it that the cleavage by BoNT/C this was but been because BoNT/C syntaxin 1A/B and SNAP-25 V.V. Yoshino K. Jahnz M. Dörries C. Bade S. Nauenburg S. Niemann H. Binz T. J. Neurochem. 1999; 72: 327-337Crossref PubMed Scopus (184) Google Scholar, Dolly J.O. PubMed Scopus Google Scholar, Montecucco C. J.E. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). the truncation of SNAP-25 by BoNT/C is inhibitory in its of its cleavage of syntaxin This with the which that found in the C-terminal domain of SNAP-25 and by BoNT/C, in with the of the SNAP-25 and syntaxin 1A/B C. Bennett M.K. Biol. PubMed Scopus Google Scholar). The that only residues are for the of SNAP-25 in exocytosis, with residues to and SNAP-25 the the BoNT/A the full-length for in was for being by that at or their to secretion. However, the of this M. A. S. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: PubMed Scopus Google showed that only three of C-terminal are for SNAP-25 residues 1–203 are for This be in one of several a for exocytosis in cells a block of exocytosis was only after with only a partial inhibition being in the latter with inhibition being in the the domain more the of for regulated However, at the such to be this a in the of the C-terminal of SNAP-25 to with syntaxin 1A/B which is with 2 and the of a at the of SNAP-25 in the M. A. S. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: PubMed Scopus Google its to exocytosis this truncation was A that findings that is a single which its to and M. A. S. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: PubMed Scopus Google that the of each of three residues after exocytosis to a extent the is with the the of 1–201, 1–202, or 1–203 to exocytosis is The of the of human are caused by intoxication with BoNT/A, /B, or /E (reviewed in Ref. M. PubMed Scopus Google Scholar), and at the only on the of this is the been by motor and of botulism. for are because of the and of BoNTs in the of (2Sloop R.R. Cole B.A. Escutin R.O. Neurology. 1997; 49: 189-194Crossref PubMed Scopus (160) Google Scholar). In of this and the is to for botulism. such that is being with is the of J. Chem. 1997; PubMed Scopus (114) Google Scholar). and the of gene therapy to release by in motor of BoNT/A that this is a because the regulated is required for the of SNAP-25 to the the which SNAP-25 to the membrane from the S. Biol. Cell. PubMed Scopus Google is in cells by to the for and the of this B. F. A. D. T. and S.