The Role of the Synaptic Protein SNAP-25 in the Potency of Botulinum Neurotoxin Type A
2001; Elsevier BV; Volume: 276; Issue: 16 Linguagem: Inglês
10.1074/jbc.m010992200
ISSN1083-351X
AutoresJames E. Keller, Elaine A. Neale,
Tópico(s)Hereditary Neurological Disorders
ResumoBotulinum 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; 75: 2017-2028Crossref PubMed Scopus (95) Google Scholar), synaptosomes (29Banerjee A. Kowalchyk J.A. DasGupta B.R. Martin T.F.J. J. Biol. Chem. 1996; 271: 20227-20230Abstract Full Text Full Text PDF PubMed Scopus (140) Google Scholar), and chromaffin cells (31Xu T. Binz T. Niemann H. Neher E. Nat. Neurosci. 1998; 1: 192-200Crossref PubMed Scopus (287) Google Scholar). The molecular relationship between SNAP-25 and Ca2+ is not well understood, however. The altered Ca2+ sensitivity observed in PC12 cells for either BoNT/A or -E (30Gerona R.R. Larsen E.C. Kowalchyk J.A. Martin T.F. J. Biol. Chem. 2000; 275: 6328-6336Abstract Full Text Full Text PDF PubMed Scopus (196) Google Scholar) is consistent with earlier observations using neuromuscular or chromaffin cell preparations prior to the discovery of SNAP-25 as the toxin substrate (32Molgo J. Thesleff S. Brain Res. 1984; 297: 309-316Crossref PubMed Scopus (39) Google Scholar, 33Sellin L.C. Thesleff S. Dasgupta B.R. Acta Physiol. Scand. 1983; 119: 127-133Crossref PubMed Scopus (64) Google Scholar, 34von Ruden L. Neher E. Science. 1993; 262: 1061-1065Crossref PubMed Scopus (272) Google Scholar). Furthermore, electrophysiological results indicate that the C terminus of SNAP-25, which is directly affected by BoNT/A and -E, contributes to the initiation of two distinct Ca2+-sensitive steps in the neurotransmitter release process (31Xu T. Binz T. Niemann H. Neher E. Nat. Neurosci. 1998; 1: 192-200Crossref PubMed Scopus (287) Google Scholar). SNAP-25 and P197 each bind to VAMP and syntaxin although the P197-containing complex is less stable to detergent solubilization than a complex containing SNAP-25 (35Hayashi T. McMahon H. Yamasaki S. Binz T. Hata Y. Sudhof T.C. Niemann H. EMBO J. 1994; 13: 5051-5061Crossref PubMed Scopus (661) Google Scholar, 36Hayashi T. Yamasaki S. Nauenburg S. Binz T. Niemann H. EMBO J. 1995; 14: 2317-2325Crossref PubMed Scopus (227) Google Scholar, 37Pellegrini L.L. O'Connor V. Lottspeich F. Betz H. EMBO J. 1995; 14: 4705-4713Crossref PubMed Scopus (127) Google Scholar). Examining the possible inhibitory role of P197 within the framework of the SNARE hypothesis (38Sollner T. Bennett M.K. Whiteheart S.W. Scheller R.H. Rothman J.E. Cell. 1993; 75: 409-418Abstract Full Text PDF PubMed Scopus (1574) Google Scholar, 39Sollner T. Whiteheart S.W. Brunner M. Erdjument-Bromage H. Geromanos S. Tempst P. Rothman J.E. Nature. 1993; 362: 318-324Crossref PubMed Scopus (2611) Google Scholar) suggests that BoNT/A damage compromises the ability of the complex to facilitate vesicle fusion. Calcium induces a conformational transition of the trimeric SNARE complex from a cis(nonsecretory) to a trans orientation which triggers vesicle fusion and neurotransmitter release (40Chen Y.A. Scales S.J. Patel S.M. Doung Y.C. Scheller R.H. Cell. 1999; 97: 165-174Abstract Full Text Full Text PDF PubMed Scopus (357) Google Scholar, 41Wei S. Xu T. Ashery U. Kollewe A. Matti U. Antonin W. Rettig J. Neher E. EMBO J. 2000; 19: 1279-1289Crossref PubMed Google Scholar). Given these two effects, SNAP-25 cleavage and reduced Ca2+ sensitivity, it is possible that BoNT/A action on SNAP-25 alters other interactions which affect neurotransmitter release. To this end, it was demonstrated that Ca2+ stimulates binding between SNAP-25 and P197 with synaptotagmin (30Gerona R.R. Larsen E.C. Kowalchyk J.A. Martin T.F. J. Biol. Chem. 2000; 275: 6328-6336Abstract Full Text Full Text PDF PubMed Scopus (196) Google Scholar). Hence, there may be multiple roles for SNAP-25 and Ca2+ in the release process (34von Ruden L. Neher E. Science. 1993; 262: 1061-1065Crossref PubMed Scopus (272) Google Scholar, 41Wei S. Xu T. Ashery U. Kollewe A. Matti U. Antonin W. Rettig J. Neher E. EMBO J. 2000; 19: 1279-1289Crossref PubMed Google Scholar, 42Neher E. Zucker R.S. Neuron. 1993; 10: 21-30Abstract Full Text PDF PubMed Scopus (453) Google Scholar, 43Olafsson P. Soares H.D. Herzog K.H. Wang T. Morgan J.I. Lu B. Brain Res. Mol. Brain Res. 1997; 44: 73-82Crossref PubMed Scopus (49) Google Scholar). Calcium ionophores have been used successfully to discriminate between the effects of voltage-gated Ca2+ channels and direct effects 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; 75: 2017-2028Crossref PubMed Scopus (95) Google Scholar, 44Fassio A. Sala R. Bonanno G. Marchi M. Raiteri M. Neuroscience. 1999; 90: 893-902Crossref PubMed Scopus (23) Google Scholar, 45Gil A. Viniegra S. Gutierrez L.M. Eur. J. Neurosci. 1998; 10: 3369-3378Crossref PubMed Scopus (32) Google Scholar). This approach has been extended to the present study to investigate the requirement for intracellular Ca2+ in overcoming the inhibitory action of BoNT/A in mouse spinal cord cultures. The findings demonstrate that a mathematical correlation exists between the generation of P197 by BoNT/A and inhibition of Ca2+-dependent neurotransmitter release, indicating that the amount of P197 attained within neurons will block exocytosis. Monoclonal antibody to SNAP-25 (SMI-81) was obtained from Sternberger Monoclonals, Inc. (Lutherville, MD). Thapsigargin, ionomycin, monoclonal antibody to syntaxin, and alkaline phosphatase-labeled anti-mouse antibody were purchased from Sigma-Aldrich Chemical Co. All electrophoresis reagents were from Bio-Rad. Polyvinylidene difluoride membrane was obtained from Bio-Rad. Preparations of BoNT/A and -E toxin complex were from Wako Chemicals Inc. (Richmond, VA) with reported activities of 2.0 × 107 LD50 and 1.0 × 107LD50 per mg of protein, respectively. BoNT/E (1 mg/ml) was activated (nicked) by incubating for 30 min at 37 °C with 0.3 mg/ml trypsin (type XI, bovine pancreas) in 30 mm HEPES, pH 6.75. Trypsin was subsequently inhibited by addition of 0.5 mg/ml trypsin inhibitor (type I-S, soybean) and incubation for 15 min at 20 °C (16Keller J.E. Neale E.A. Oyler G. Adler M. FEBS Lett. 1999; 456: 137-142Crossref PubMed Scopus (144) Google Scholar). Toxins were aliquoted and stored at −20 °C; each experiment utilized a new aliquot of toxin to ensure uniform activity. Molar concentrations stated in the text were based upon masses of 500 and 300 kDa for BoNT/A and -E, respectively. Timed pregnant C57BL/6NCR mice were obtained from the Frederick Cancer Research and Development Center, Frederick, MD. Spinal cord cell cultures were prepared as described (46Williamson L.C. Fitzgerald S.C. Neale E.A. J. Neurochem. 1992; 59: 2148-2157Crossref PubMed Scopus (43) Google Scholar, 47Ransom B.R. Neale E. Henkart M. Bullock P.N. Nelson P.G. J. Neurophysiol. 1977; 40: 1132-1150Crossref PubMed Scopus (418) Google Scholar). Briefly, spinal cords were removed from fetal mice at gestation day 13, dissociated with trypsin, and plated on VitrogenTM 100-coated dishes (Collagen Corp., Palo Alto, CA) at a density of 106 cells/dish. Cultures were maintained for 3 weeks at 37 °C in an atmosphere of 90% air, 10% CO2 before addition of toxins. Growth medium consisted of minimum essential medium (formula 82-0234AJ; Life Technologies, Inc., Bethesda, MD) supplemented with 5% heat-inactivated horse serum and a mixture of complex factors (48Fitzgerald S.C. Sharar A. Devellis J. Vernadakis A. Javer B. A Dissection and Tissue Culture Manual of the Nervous System. Alan R. Liss, Inc., New York1989: 219-222Google Scholar). Cultures were incubated with BoNT diluted with growth medium for times indicated in figure legends. BoNT-containing medium was removed by aspiration; cells were rinsed with toxin-free medium and prepared for Western blot analysis or neurotransmitter release. Protein was prepared by dissolving cells in 1% SDS with 1 mm EDTA and 1 mm EGTA. The slurry was transferred to 1.5-ml microcentrifuge tubes, incubated in a 95 °C water bath for 5 min to inactivate proteases, and then stored at −20 °C. Immediately prior to use samples were thawed, mixed with equal volumes of Tris-Tricine sample buffer (Bio-Rad), heated at 95 °C for 4 min, and then separated by SDS-polyacrylamide gel electrophoresis. Equal quantities of protein were loaded onto 16.5% acrylamide gels prepared by the method of Laemmli (49Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (206631) Google Scholar). Proteins were separated using 0.1m Tris-Tricine, pH 8.3 (50Schagger H. von Jagow G. Anal. Biochem. 1987; 166: 368-379Crossref PubMed Scopus (10460) Google Scholar), and then transferred to polyvinylidene difluoride membrane with a buffer containing 192 mm glycine, 25 mm Tris, pH 8.3, and 12% methanol. Membrane development was performed as previously described (16Keller J.E. Neale E.A. Oyler G. Adler M. FEBS Lett. 1999; 456: 137-142Crossref PubMed Scopus (144) Google Scholar). Potassium-stimulated Ca2+-dependent neurotransmitter release was determined as previously described (51Williamson L.C. Halpern J.L. Montecucco C. Brown J.E. Neale E.A. J. Biol. Chem. 1996; 271: 7694-7699Abstract Full Text Full Text PDF PubMed Scopus (184) Google Scholar). Cultures were labeled with [3H]glycine for 0.5 h at 35 °C and then washed with a series of low K+-containing buffers. Unless otherwise indicated, glycine release was stimulated by addition of 56 mm K+ and 2 mm Ca2+ to cultures; stimulation medium was collected after 5 min at 35 °C. Calcium-dependent release was determined by subtracting baseline radioactivity secreted from cultures in the absence of Ca2+, and expressed as a percentage of the total cellular radioactivity. Scanned images of Western blots were produced and edited utilizing NIH Image(National Institutes of Health, Bethesda, MD). Where indicated, images were digitally analyzed using IPLab Gel software (Scanalytics, Inc., Fairfax, VA). Nonlinear regression analysis of data was performed with Sigma Plot (SPSS Science, Inc., Chicago, IL). Error bars represent standard deviations of triplicate determinations unless otherwise indicated. BoNT/E treatment of spinal cord cell cultures results in cleavage of SNAP-25 in a concentration-dependent manner (Fig.1 A). Under the conditions of these experiments, the IC50 for BoNT/E cleavage of SNAP-25 is ∼50 pm. Measurement of synaptic function indicates that K+-evoked glycine release is inhibited to the same extent as SNAP-25 cleavage (Fig. 1 B). In control cultures, the extent of glycine release is related to Ca2+concentrations in the external medium. Cultures depolarized in the presence of 0.15, 0.25, 2, and 10 mm Ca2+release 4.3, 15.7, 26.5, and 25.8% of the total cellular radiolabel. Maximal release is achieved with 2 mm Ca2+(Fig. 3). Varying external Ca2+ concentration over this range did not alter significantly the IC50 for the BoNT/E block of glycine release which varies from 49 to 55 pm for the conditions tested (Fig. 1 B).Figure 3Calcium titration and K+-evoked glycine release. Spinal cord cultures were incubated in growth medium containing either BoNT/A or -E at 1 nm for 24 h at 37 °C, after which Ca2+-dependent release of glycine was assayed. Cultures not treated with toxin (●) demonstrated a standard titration curve relative to Ca2+concentration with an EC50 value for Ca2+ of 0.21 ± 0.01 mm and a Hill coefficient of 1.9 ± 0.2. Maximal glycine release for each experiment ranged from 18 to 27% of the total cellular radioactivity and was set to 100%. BoNT/A-treated cultures (○) demonstrated Ca2+ dependence with an EC50 value of 1.0 ± 0.1 mm and maximal release when Ca2+ exceeded 2 mm. The plateau value for this release was 23.2 ± 2.2% of the maximal release elicited from control cultures. BoNT/E-treated cultures (▪) demonstrated no Ca2+ dependent release when measured in the presence of 1–8 mm Ca2+. Results were compiled from five (control) and four (BoNT/A-treated) experiments. Each symbol represents the average of duplicate determinations.View Large Image Figure ViewerDownload (PPT) The model in Fig. 1 C illustrates the effect of BoNT/E on the release process. In this model, SNAP-25 is shown to interact with elements (X) required for neurotransmitter release with an affinity represented by Kd. Potassium depolarization stimulates release in a Ca2+-dependent manner. Release is directly proportional to the amount of SNAP-25 present. As SNAP-25 is cleaved by increasing concentrations of BoNT/E, neurotransmitter release decreases to a similar extent. BoNT/E-cleaved SNAP-25 (P180) has been shown to interact with VAMP and syntaxinin vitro (36Hayashi T. Yamasaki S. Nauenburg S. Binz T. Niemann H. EMBO J. 1995; 14: 2317-2325Crossref PubMed Scopus (227) Google Scholar, 37Pellegrini L.L. O'Connor V. Lottspeich F. Betz H. EMBO J. 1995; 14: 4705-4713Crossref PubMed Scopus (127) Google Scholar). However, our data indicate that in this intact neuronal system, P180 does not interfere with K+-evoked release, possibly because P180 interacts withX very weakly relative to SNAP-25. Furthermore, increasing extracellular Ca2+ above physiological concentrations does not overcome the block (Fig. 3), similar to findings with neuromuscular preparations (14Adler M. Macdonald D.A. Sellin L.C. Parker G.W. Toxicon. 1996; 34: 237-249Crossref PubMed Scopus (43) Google Scholar). Unlike BoNT/E, dose-response data with BoNT/A produce nonoverlapping curves for SNAP-25 proteolysis and blockade of neurotransmitter release (Fig.2 B). SNAP-25 cleavage occurs with an approximate IC50 of 3 pm and glycine release is inhibited with IC50 values decreasing from 0.3 to 0.1 pm as Ca2+ concentrations decrease from 2 to 0.15 mm in the release medium (Fig. 2 B). Increasing Ca2+ to 8 or 10 mm yielded results identical to that obtained with 2 mm Ca2+ (data not shown). At Ca2+ concentrations lower than 2 mm, a greater degree of inhibition occurs relative to SNAP-25 cleavage (Fig. 2 B). At all Ca2+concentrations tested, the slopes for neurotransmitter release were the same. Comparing these slopes with the slope for SNAP-25 yields a ratio of ∼2:1. Fig. 2 C depicts possible interactions between SNAP-25 and P197 and their combined effects on the release process. In this model P197 binds to elements X much the same as SNAP-25. Because elevated Ca2+ has been shown previously to reverse 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), PC12 cells (29Banerjee A. Kowalchyk J.A. DasGupta B.R. Martin T.F.J. J. Biol. Chem. 1996; 271: 20227-20230Abstract Full Text Full Text PDF PubMed Scopus (140) Google Scholar), and neuromuscular preparations (52Molgo J. Siegel L.S. Tabti N. Thesleff S. J. Physiol. 1989; 411: 195-205Crossref PubMed Scopus (47) Google Scholar), we hypothesize that neurotransmitter release through the P197-X pathway would require Ca2+ in excess of that achieved during normal neuronal stimulation. Elevated Ca2+ above normal levels is depicted with the term Ca2+′. This model allows us to predict the relationship between SNAP-25 cleavage and block of transmitter release. Assuming (a) that P197 interacts with elements X with the same or nearly the same affinity as SNAP-25, and (b) that at any given dose of BoNT/A, the molar sum of the remaining SNAP-25 and P197 will equal the amount of SNAP-25 in the absence of toxin, the profile in Fig. 2 D is generated. According to this model, the ratio of the slope for neurotransmitter release relative to SNAP-25 is exactly 2:1. The data for neurotransmitter release measured with 2 mm Ca2+ (Fig. 2 B) overlap the predicted result. Because at lower Ca2+ concentrations, BoNT/A-exposed cultures show a greater block in transmitter release (Fig. 2), we examined the effects of extracellular Ca2+concentration on glycine release following exposure to a single, high dose of BoNT/A or -E. Both BoNTs at 1 nm cleave all detectable SNAP-25 after 24 h (Figs. 1 A and 2A). [3H]Glycine release from control cultures (no toxin exposure) is Ca2+ dependent with an EC50 value for Ca2+ of 0.21 mm and a Hill coefficient of about 2 (Fig. 3). Glycine release from BoNT/A-treated cultures is similarly Ca2+ dependent with an EC50 value of 1.0 ± 0.1 mm and a plateau value that is 23% of control release (Fig. 3). Maximum release for both control and BoNT/A-treated cultures is attained when Ca2+ exceeds 2 mm(Fig. 3) and the slope of release is unaffected by toxin with an estimated Hill coefficient of 2.2 ± 0.3 (53Trudeau L.E. Fang Y. Haydon P.G. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 7163-7168Crossref PubMed Scopus (73) Google Scholar, 54Charlton M.P. Smith S.J. Zucker R.S. J. Physiol. 1982; 323: 173-193Crossref PubMed Scopus (190) Google Scholar). Glycine release from BoNT/E-treated cultures is not detected at any concentration of Ca2+ tested (Fig. 3). This indicates that the rise of intracellular Ca2+ resulting from Ca2+-channel activation can restore some function to BoNT/A- but not to BoNT/E-treated neurons, consistent with results from Figs. 1 and 2. To examine the effects of increased intracellular Ca2+ above levels attained from Ca2+-channel activation, glycine release was evoked by 56 mmK+ in the presence and absence of 2 mmCa2+ and 5 μm ionomycin, a Ca2+-specific ionophore. Ionomycin partially restores glycine release after exposure to BoNT/A (Fig.4). Treatment with BoNT/A reduces glycine release to ∼23% of control release. Addition of ionomycin in combination with Ca2+ reduces the effect of BoNT/A, raising glycine release to 60% of control values. To determine the source of Ca2+ that contributes to this reversal, glycine release was assayed with ionomycin either in the absence of extracellular Ca2+, or after having treated cultures with thapsigargin to deplete intracellular Ca2+ stores (55Brayden D.J. Hanley M.R. Thastrup O. Cuthbert A.W. Br. J. Pharmacol. 1989; 98: 809-816Crossref PubMed Scopus (37) Google Scholar, 56Takemura H. Hughes A.R. Thastrup O. Putney Jr., J.W. J. Biol. Chem. 1989; 264: 12266-12271Abstract Full Text PDF PubMed Google Scholar). The absence of any release with zero external Ca2+ with ionomycin and the failure of thapsigargin to affect the ionomycin-dependent release provide evidence that internal Ca2+ stores do not contribute significantly to ionomycin's reversal of the BoNT/A-induced block. To expand upon this finding, cultures were treated with 1 nm BoNT/A or BoNT/E for 24 h and neurotransmitter release was measured in the presence of 10 μm ionomycin in 56 mm K+ with various external Ca2+ concentrations. Ionomycin produces a significant reversal of the BoNT/A block at all Ca2+ concentrations tested with complete recovery occurring near 2 mmCa2+. Unlike K+/Ca2+ stimulation alone, stimulation in the presence of ionomycin partially alleviates BoNT/E-induced block with maximum glycine release at 53% of control cultures (Fig. 5). Within these experimental conditions, BoNT/A- and/E-treated neurons are about 4- and 7-fold less sensitive to Ca2+, respectively, than untreated neurons. Ionomycin has two measurable effects on reversing BoNT/E action: neurons release more glycine and demonstrate higher sensitivity to Ca2+. In the case of BoNT/A, ionomycin stimulates release of glycine to control values. However, ionomycin does not alter the EC50 value for Ca2+ which is 0.8 ± 0.1 mm (Fig. 5) compared with 1.0 ± 0.1 mm obtained for the partial release achieved in the absence of ionomycin (Fig. 3). Ionomycin at the tested concentration does not affect cultures that were not treated with toxin; neither the Ca2+ sensitivity nor the extent of glycine release was altered (Fig. 5). In this study, we examined the relationship between blockade of neurotransmitter release and cleavage of SNAP-25 caused by BoNT/A and -E to aid in understanding how BoNT/A seems to derive greater potency than BoNT/E (14Adler M. Macdonald D.A. Sellin L.C. Parker G.W. Toxicon. 1996; 34: 237-249Crossref PubMed Scopus (43) Google Scholar, 22Molgo 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, 32Molgo J. Thesleff S. Brain Res. 1984; 297: 309-316Crossref PubMed Scopus (39) Google Scholar). Both BoNTs induce a concentration-dependent block in synaptic vesicle exocytosis and proteolysis of SNAP-25. However, it appears likely that in addition to disabling SNAP-25, BoNT/A action has a secondary inhibitory effect. The nonoverlapping pattern of SNAP-25 cleavage and block of K+-evoked transmitter release in this latter case points to the formation of an inhibitory complex containing a cleavage fragment produced by BoNT/A. Most simply, BoNT/A action on SNAP-25 produces a dual effect: a reduction in functional SNAP-25 and production of a fragment that antagonizes synaptic vesicle fusion. The identity of this fragment could be either the 197-amino acid fragment, P197, or the
Referência(s)