Portal de Periódicos da CAPES

Modifications in the C Terminus of the Synaptosome-associated Protein of 25 kDa (SNAP-25) and in the Complementary Region of Synaptobrevin Affect the Final Steps of Exocytosis

2002; Elsevier BV; Volume: 277; Issue: 12 Linguagem: Inglês

10.1074/jbc.m110182200

1083-351X

Anabel Gil, Luis M. Gutiérrez, Carmen Carrasco-Serrano, Marı́a Teresa Alonso, Salvador Viniegra, Manuel Criado,

Retinal Development and Disorders

Fusion proteins made of green fluorescent protein coupled to SNAP-25 or synaptobrevin were overexpressed in bovine chromaffin cells in order to study the role of critical protein domains in exocytosis. Point mutations in the C-terminal domain of SNAP-25 (K201E and L203E) produced a marked inhibition of secretion, whereas single (Q174K, Q53K) and double mutants (Q174K/Q53K) of amino acids from the so-called zero layer only produced a moderate alteration in secretion. The importance of the SNAP-25 C-terminal domain in exocytosis was also confirmed by the similar effect on secretion of mutations in analogous residues of synaptobrevin (A82D, L84E). The effects on the initial rate and magnitude of secretion correlated with the alteration of single vesicle fusion kinetics since the amperometric spikes from cells expressing SNAP-25 L203E and K201E and synaptobrevin A82D and L84E mutants had lower amplitudes and larger half-width values than the ones from controls, suggesting slower neurotransmitter release kinetics than that found in cells expressing the wild-type proteins or zero layer mutants of SNAP-25. We conclude that a small domain of the SNAP-25 C terminus and its counterpart in synaptobrevin play an essential role in the final membrane fusion step of exocytosis. Fusion proteins made of green fluorescent protein coupled to SNAP-25 or synaptobrevin were overexpressed in bovine chromaffin cells in order to study the role of critical protein domains in exocytosis. Point mutations in the C-terminal domain of SNAP-25 (K201E and L203E) produced a marked inhibition of secretion, whereas single (Q174K, Q53K) and double mutants (Q174K/Q53K) of amino acids from the so-called zero layer only produced a moderate alteration in secretion. The importance of the SNAP-25 C-terminal domain in exocytosis was also confirmed by the similar effect on secretion of mutations in analogous residues of synaptobrevin (A82D, L84E). The effects on the initial rate and magnitude of secretion correlated with the alteration of single vesicle fusion kinetics since the amperometric spikes from cells expressing SNAP-25 L203E and K201E and synaptobrevin A82D and L84E mutants had lower amplitudes and larger half-width values than the ones from controls, suggesting slower neurotransmitter release kinetics than that found in cells expressing the wild-type proteins or zero layer mutants of SNAP-25. We conclude that a small domain of the SNAP-25 C terminus and its counterpart in synaptobrevin play an essential role in the final membrane fusion step of exocytosis. soluble NSF attachment protein N-ethylmaleimide-sensitive fusion protein soluble NSF attachment protein receptor green fluorescent protein Herpes simplex virus synaptobrevin The SNAP1 (soluble NSF attachment protein) receptor (SNARE) hypothesis (1Rothman J.E. Nature. 1994; 372: 55-63Crossref PubMed Scopus (2011) Google Scholar) has been crucial to our understanding of the molecular machinery responsible for exocytosis. The molecular events taking place during the exocytotic fusion of cellular and vesicular membranes should provide fundamental information about the mechanism of membrane fusion common to different membrane trafficking processes (2Bennett M. Scheller R. Proc. Natl. Acad. Sci. U. S. A. 1993; 0: 2559-2563Crossref Scopus (548) Google Scholar). The assembly of a ternary complex formed by the plasma membrane proteins syntaxin (3Bennett M.K. Calakos N. Scheller R.H. Science. 1992; 257: 255-259Crossref PubMed Scopus (1077) Google Scholar) and SNAP-25 (4Oyler G.A. Higgins G.A. Hart R.A. Battenberg E. Billigsley M. Bloom F.E. Wilson M.C. J. Cell Biol. 1989; 109: 3039-3052Crossref PubMed Scopus (690) Google Scholar) and the vesicle-associated protein synaptobrevin (5Trimble W.S. Cowan D.M. Scheller R.H. Proc. Natl. Acad. Sci. U. S. A. 1988; 5: 4538-4542Crossref Scopus (451) Google Scholar) is considered to be one of the molecular events driving vesicle priming, involving maturation steps needed to promote the apposition and final fusion of membranes (6Südhof T.C. Nature. 1995; 75: 645-653Crossref Scopus (1770) Google Scholar). A heptad repeat structural motif typical of coiled-coils forming proteins is present and could be the basis for the formation of the core complex. This was confirmed by the observed increase in α-helix content following assembly of the complex (7Fasshauer D. Otto H. Eliason W.K. Jahn R. Brünger A.T. J. Biol. Chem. 1997; 272: 28036-28041Abstract Full Text Full Text PDF PubMed Scopus (281) Google Scholar, 8Rice L.M. Brennwald P. Brunger A.T. FEBS Lett. 1997; 415: 49-55Crossref PubMed Scopus (60) Google Scholar). In recent years, precise structural studies on the nature of the complex (9Sutton R.B. Fasshauer D. Jahn R. Brunger A.T. Nature. 1998; 395: 347-353Crossref PubMed Scopus (1932) Google Scholar, 10Poirier M.A. Xiao W. Macosko J.C. Chan C. Shin Y.K. Bennett M.K. Nat. Struct. Biol. 1998; 5: 765-769Crossref PubMed Scopus (419) Google Scholar) have shown that single fragments of 60–70 residues of syntaxin and synaptobrevin together with two segments of SNAP-25 form helices in a four-stranded coil. Although most of the interactions between these helices are hydrophobic, there is a polar layer embedded in the middle of this rod-shaped structure formed by three glutamines and one arginine (zero layer), which is believed to be critical for SNARE complex formation. In addition, functional studies using specific neurotoxins (11Montecucco C. Schiavo G. Mol. Microbiol. 1994; 13: 1-8Crossref PubMed Scopus (496) Google Scholar), antibodies against critical domains (12Xu T. Rammer B. Margittal M. Artalejo A. Neher E. Jahn R. Cell. 1999; 99: 713-722Abstract Full Text Full Text PDF PubMed Scopus (255) Google Scholar), peptides imitating regions of SNAREs (13Gutiérrez L.M. Viniegra S. Rueda J. Ferrer-Montiel A.-V. Cànaves J. Montal M. J. Biol. Chem. 1997; 272: 2634-2639Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar), and overexpression of altered forms of these proteins (14Criado M. Gil A. Viniegra S. Gutiérrez L.M. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 7256-7261Crossref PubMed Scopus (83) Google Scholar, 15Wei S. Xu T. Ashery U. Kollewe A. Matti U. Antonin W. Rettig J. Neher E. EMBO J. 2000; 19: 1279-1289Crossref PubMed Google Scholar) have revealed important details about the participation of SNAREs in the exocytotic process. These and other studies suggest that SNARE interaction results in a tighter structure reducing the repulsive energy barrier between vesicular and plasma membranes. Assembly of the complex may proceed from the distal N-terminal domains of SNAREs assembled in parallel and, in “zipper-like” fashion, end with the interaction of C-terminal domains relatively close to the fusion pore. Even though there is not direct evidence for this model, a number of authors suggest that SNAREs assembly is directly linked to membrane fusion (16Weber T. Zemelman B.V. McNew J.A. Westermann B. Gmachl M. Parlati F. Sollner T.H. Rothman J.E. Cell. 1998; 92: 759-772Abstract Full Text Full Text PDF PubMed Scopus (2021) Google Scholar, 17Chen Y.A. Scales S.J. Patel S.J. Doung Y.C. Scheller R.H. Cell. 1999; 97: 165-174Abstract Full Text Full Text PDF PubMed Scopus (360) Google Scholar). We have overexpressed several mutants of SNAP-25 and synaptobrevin critical domains in adrenal chromaffin cells and studied their effect on catecholamine secretion. Our results indicate that specific residues at the SNAP-25 C-terminal domain and the equivalent synaptobrevin domain influence vesicle fusion kinetics, suggesting a role in the very last events of the exocytotic process. By contrast, zero layer amino acid mutants, despite their ability to negatively affect the formation and stability of SNAREs complexes assembled in vitro, did not produce strong alterations. To produce an in-frame fusion of SNAP-25 to the C terminus of GFP, the cDNA corresponding to the SNAP-25a isoform (18Bark I.C. Wilson M.C. Gene. 1994; 139: 291-292Crossref PubMed Scopus (131) Google Scholar) was cloned into the expression vector pEGFP-C3 (CLONTECH, Palo Alto, CA) as previously described (14Criado M. Gil A. Viniegra S. Gutiérrez L.M. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 7256-7261Crossref PubMed Scopus (83) Google Scholar). The generation of the nine amino acids C-terminal deletion (Δ9) and the point mutation L203E has also been described (14Criado M. Gil A. Viniegra S. Gutiérrez L.M. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 7256-7261Crossref PubMed Scopus (83) Google Scholar). The strategy for generating the other C-terminal mutants was based in the presence of a TaiI site at the 5′-end of the region containing the nucleotides to be mutated and a BamHI site at the 3′-end. Complementary oligonucleotides carrying the desired mutations and the mentioned restriction sites were annealed and cloned into the corresponding construct in place of the original sequence. The point mutation Q53K was generated by PCR of a fragment corresponding to the N terminus of SNAP-25 using the GFP-SNAP-25 construct as template, a sense primer (5′-CGGCATGGACGAGCTGTACA-3′) corresponding to the C terminus of GFP and close to the junction between GFP and SNAP-25 and an antisense primer, which included the sequence to be mutated (indicated in lower characters) (5′-CGACACGATCGAGTTGTTCTCCtttTTCAT-CCAACATAACC-3′). The PCR product was digested with XhoI and PvuI and used to replace the equivalent fragment into the GFP-SNAP-25 construct, which had been digested with the same enzymes. The point mutation Q174K was generated by PCR of a fragment corresponding to the C terminus of SNAP-25 with a sense primer containing the sequence to be mutated (5′-CAATGAGATCGATACAaAGAATCGCCAGATCG-3′) and an antisense primer downstream of the C terminus of SNAP-25 (5′-CTACAAATGTGGTATGGCTG-3′). The amplified DNA carried an internal ClaI site, close to the 5′-end, and a BamHI site at the 3′-end. These enzymes were used to substitute the original SNAP-25 sequence by the modified one in the GFP-SNAP-25 construct. The double mutant Q53K/Q174K was obtained by combining the single ones through enzyme restriction and ligation reactions. To produce an in-frame fusion of synaptobrevin to the N terminus of GFP, the coding region corresponding to synaptobrevin II (19Archer B.T. Ozcelik T. Jahn R. Franke U. Südhof T.C. J. Biol. Chem. 1990; 265: 17267-17273Abstract Full Text PDF PubMed Google Scholar) was amplified by PCR with the following primers: 5′-GCCGAATTCCCGCCATGTCGGCTACC-3′ (sense) and 5′-GCCGGATCCGAGC-TGAAGTAAACGATGATG-3′ (antisense). The PCR product was digested with EcoRI and BamHI and cloned into the same sites of the expression vector pEGFP-N1 (CLONTECH, Palo Alto, CA). The strategy for generating the synaptobrevin mutants (A81D, A82D and L84E) was based in the presence of an SpeI site at the 5′-end of the region containing the nucleotides to be mutated and the previously mentionedBamHI site at the 3′-end. Adequate sense primers carrying the desired mutations and the SpeI site as well as the above antisense primer with the BamHI site were used in PCR amplifications. The corresponding products were used to substitute the original synaptobrevin sequence. All the introduced cassettes were sequenced to check that only the desired mutations have been produced. Chromaffin cells were prepared from bovine adrenal glands by collagenase digestion and further separated from debris and erythrocytes by centrifugation on Percoll gradients as described elsewhere (13Gutiérrez L.M. Viniegra S. Rueda J. Ferrer-Montiel A.-V. Cànaves J. Montal M. J. Biol. Chem. 1997; 272: 2634-2639Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar). Cells were maintained in monolayer cultures using Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum, 10 μm cytosine arabinoside, 10 μm5-fluoro-2′-deoxyuridine, 50 IU/ml penicillin, and 50 μg/ml streptomycin. Cells were harvested at a density of 25,000 cells/cm2 in 35-mm Petri dishes. Primary cultures of chromaffin cells were infected with a Herpes Simplex virus (HSV-1) amplicon containing the constructs described above. For this purpose they were transferred from the pEGFP-C3 or pEGFP-N1 vectors to the pHSVpUC vector (20Geller A.I. During M.J. Haycock J.W. Freese A. Neve R. Proc. Natl. Acad. Sci. 1993; 90: 7603-7607Crossref PubMed Scopus (63) Google Scholar). The packaging of the different helper viruses (HSV-1 IE2 deletion mutant 5dl 1.2) was carried out as previously described (21Lim F. Hartley D. Starr P. Lang P. Song S. Yu L. Wang Y. Geller A.I. BioTechniques. 1996; 20: 460-469Crossref PubMed Scopus (130) Google Scholar). Efficiency of virus infection was determined by fluorescent microscopy, using serial dilutions of purified virus. The dilution producing 15–20% infection efficiency (usually 30–40 μl virus per 35-mm plate containing 1 ml of medium) was chosen in further experiments. GFP fluorescence was observed 1 day after infection and persisted, at least, during the two following days. Amperometric measurements were carried out during this time. To study secretory activity from control non-infected and fluorescence-emitting cells expressing the different constructs, culture media was replaced by Krebs/HEPES basal solution with the following composition (in mm): NaCl 134, KCl 4.7, KH2PO4 1.2, MgCl2 1.2, CaCl2 2.5, Glucose 11, ascorbic acid 0.56, and Hepes 15, and the pH value was adjusted to 7.4 using a NaOH solution. Carbon-fiber electrodes insulated with polypropylene and with 11-μm diameter tips were used to monitor catecholamine release from individual chromaffin granules in cells under superfusion (22Gil A. Viniegra S. Gutiérrez L.M. Eur. J. Neurosci. 1998; 10: 3369-3378Crossref PubMed Scopus (32) Google Scholar). Electrodes were carefully positioned in close apposition to the cell surface using high precision hydraulic micromanipulation and assessing cell membrane deformation with an Axiovert 135 inverted-stage microscope (Zeiss, Oberkochen, Germany) mounting Hoffman optics (Modulation Optics, Greenvale, NY). Electrical connection was accomplished with mercury. An amperometric potential of +650 mVversus an Ag/AgCl bath reference electrode was applied using an Axopatch 200A amplifier (Axon Instruments Inc., Foster City, CA). Current product of the catecholamine oxidation was digitized with an analogical/digital converter (ITC-16, Instrutech Corp., Great Neck, NY) and recorded at 200 μs/point using the program PulseControl (23Herrington J. Bookman R.J. Pulse Control V4.3: IGOR XOPs for Patch Clamp Data Acquisition and Capacitance Measurements. University of Miami Press, Miami, FL1994Google Scholar) running on top of the graphical software Igor Pro (Wavematrics, Lake Oswego, OR) in a PowerMac 7100 computer. Experiments were performed in cells stimulated by superfusion with depolarizing 59 mm high potassium (obtained by replacing isosmotically NaCl by KCl) and applied through a valve-controlled puffer tip commanded by the acquisition software and located near the studied cells. Individual spike characteristics were studied using Igor-Pro macros called “Spike” versus 1.0 (24Segura F. Brioso M.A. Gomez J.F. Machado D. Borges R. J. Neurosci. Methods. 2000; 103: 151-156Crossref PubMed Scopus (73) Google Scholar) supplied by Dr. Ricardo Borges (Unidad de Farmacologı́a, Universidad de la Laguna, Tenerife, Spain), allowing for peak detection, integration, and kinetic parameter calculations. Oxidized current was filtered at a corner frequency of 400 Hz using an 8-poles low-pass Bessel filter and acquired at 0.2 ms/point. Only well defined narrow peaks with amplitude higher than 5 pA and the width at the half-height (half-width) lower than 75 ms were taken to build event histograms. Electrode to electrode variations were alleviated by using the same electrodes for measurements in control (non-infected) and infected cells (electrode tip was frequently cleaned with isobutanol). Controls were taken before and after (post-control) performing measurements in infected cells, and peak analysis included experiments performed in 23–66 cells in each condition. After obtaining the mean spike parameters for each individual cell, they were average for the number (n) of cells analyzed in each experimental condition (25Colliver T.L. Hess E.J. Pothos E.N. Sulzer D. Ewing A.G. J. Neurochem. 2000; 74: 1086-1097Crossref PubMed Scopus (84) Google Scholar). The analysis of variance test implemented in the program Graphpad Instat was used for statistical analysis comparing SNAP-25 and synaptobrevin control and post-control parameters with the values obtained for the different mutants. All data was expressed as mean ± S.E. from experiments performed in a number (n) of individual cells. Data represent experiments performed with cells from at least three different cultures. Briefly, cells overexpressing GFP-synaptobrevin constructs were fixed using a 4% paraformaldehyde in phosphate-buffered saline solution during 20 min. Then cells were washed with phosphate-buffered saline solution and mounted in 80% glycerol in the same solution. Fluorescence was investigated using a Laser Scanning confocal TCS Leika microscope. Usually, eight confocal layers covering the total cell volume were obtained (1.25-μm thickness (z)), and individual layers or projections were used to study fluorescence distribution. GST fusion proteins encoding SNAP-25, syntaxin, and synaptobrevin were produced by cloning their respective cDNAs in the pGEX-KG vector (26Guan K.L. Dixon J.E. Anal. Biochem. 1991; 192: 262-267Crossref PubMed Scopus (1641) Google Scholar). The different SNAP-25 and synaptobrevin mutants were transferred to pGEX-KG either through the NcoI site present in the initial methionine of SNAP-25 or using an EcoRI site preceding the coding region of synaptobrevin, thus ensuring the proper reading frame in the GST fusions. Recombinant proteins were purified from expressing bacteria (Escherichia coli BL21 strain for SNAP-25 and synaptobrevin and C43 strain for syntaxin) after expression induction with isopropyl-β-d-thiogalactopyranoside during 5 h at 37 °C. The constructs were bound to affinity columns of glutathione-Sepharose 4B (Amersham Biosciences) in overnight incubations, and after extensive washing the proteins were eluted by proteolysis with thrombin, followed by inactivation of this protein with 2 mm phenylmethylsulfonyl fluoride. Formation of ternary complexes by the purified proteins at a 4-μm concentration and room temperature was assessed in buffer: 100 mm NaCl, 0.005% octylglucoside, and 5 mm dithiothreitol in 25 mm HEPES,pH 7.5. After incubation periods comprised between 10 s and 60 min, 10-μl aliquots were removed, and complex formation was stopped by adding an equal volume of SDS-PAGE buffer (0.1% SDS, 10% (v/v), glycerol, 0.01% β-mercaptoethanol, 0.01% bromphenol blue in 0.062m Tris, pH 6.8). Complex thermostability was assayed by forming complexes during 1-h incubations in the conditions described above and heating samples during 3 min at temperatures ranging from 40 to 90 °C after addition of electrophoresis buffer. Complex dissociation was stopped by cooling the samples to 4 °C before complexes were analyzed by SDS-PAGE. Overexpression of native and mutant forms of SNAP-25 in chromaffin cells is a valuable tool when studying its role in neurosecretion (14Criado M. Gil A. Viniegra S. Gutiérrez L.M. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 7256-7261Crossref PubMed Scopus (83) Google Scholar, 15Wei S. Xu T. Ashery U. Kollewe A. Matti U. Antonin W. Rettig J. Neher E. EMBO J. 2000; 19: 1279-1289Crossref PubMed Google Scholar). In initial studies we used calcium phosphate and electroporation transfection methods, resulting in low levels of transfection (less than 1%). We therefore decided to use amplicons, modified HSV (27Spaete R.R. Frenkel N. Cell. 1982; 30: 285-304Abstract Full Text PDF Scopus (356) Google Scholar). Viral infection produced a greater proportion (15–30%) of cells expressing high amounts of GFP-SNAP-25 constructs, as indicated by their brilliant green fluorescence. We used single cell amperometry (11 micron carbon fiber electrodes) to test the ability of these cells to secrete catecholamines in response to a depolarizing stimulus. Upon stimulation, characteristic spikes depicting single fusion events were obtained for a number of individual cells (Fig.1 A). The secretory profile was obtained by the integration and averaging of amperograms from 22 to 60 cells under each experimental condition (Fig. 1 B). HSV-infected cells incorporating the GFP-SNAP-25 construct and cells expressing GFP alone (Fig. 1, A and B) produced about 50% of the secretory activity normally found in uninfected cells (not shown). The close match of GFP and GFP-SNAP-25 secretory rates when normalized, indicates that secretory kinetics were unaffected by expression of the GFP-SNAP-25 construct and that the reduction in the number of released vesicles observed in infected cells was due to the viral infection itself rather than to a possible inhibition of the secretory machinery by overexpression of SNAP-25. Validity of the HSV amplicon system for testing SNAP-25 function was further confirmed by the catecholamine release in cells overexpressing a GFP-SNAP-25 construct incorporating the mutation of leucine 203 to glutamic acid (L203E) that had previously been shown to greatly affect secretion kinetics (14Criado M. Gil A. Viniegra S. Gutiérrez L.M. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 7256-7261Crossref PubMed Scopus (83) Google Scholar). The expression of this construct partially abrogated the overall extent of the secretory response (Fig. 1 A shows a 70% reduction in the sustained response compared with the control GFP-SNAP-25 construct), and initial rate of release (Fig.1 B, about 0.2 vesicles/s, 32% of the rate found for the GFP-SNAP-25 construct). These results are consistent with those obtained by conventional transfection methods (14Criado M. Gil A. Viniegra S. Gutiérrez L.M. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 7256-7261Crossref PubMed Scopus (83) Google Scholar). Similar results (Fig. 1 B) were observed using a construct lacking the last nine C-terminal residues of this SNARE (GFP-SNAP-25 Δ9) and therefore equivalent to the proteolyzed form produced by the action of botulinum toxin A (28Blasi J. Chapman E.R. Link E. Binz T. De Yamasaki S. Camilli P. Südhof T.C. Niemann H. Jahn R. Nature. 1993; 365: 160-163Crossref PubMed Scopus (1050) Google Scholar, 29Schiavo 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 (379) Google Scholar). These results indicated that the amino acid L203, which is absent in this construct as well as in the SNAP-25 polypeptide cleaved by botulinum neurotoxin A, is critical for exocytosis. The amplicon system was further used to examine the secretory response of cells expressing SNAP-25 proteins mutated at amino acids located in critical areas. It has been suggested that amino acid residues in the zero layer (Gln-53 and Gln-174 of SNAP-25) are critical for SNARE complex formation (9Sutton R.B. Fasshauer D. Jahn R. Brunger A.T. Nature. 1998; 395: 347-353Crossref PubMed Scopus (1932) Google Scholar, 30Fasshauer D. Sutton R.B. Brunger A.T. Jahn R. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 15781-15786Crossref PubMed Scopus (756) Google Scholar). The single mutation in Q174K showed a relatively small effect on secretion when compared with the L203E mutant (both magnitude and initial rate of catecholamine release were reduced 35% in comparison to control values, see Fig. 1, Aand B). A slight effect was also observed with the Q53K mutation (also present in the zero layer but forming part of the SNAP-25 N-terminal domain), which released 15+2 vesicles (n = 20) during prolonged depolarization (52% inhibition, data not shown) Double mutations in the previously mentioned glutamines (Q53K/Q174K) produced lower inhibition (50% in extent and initial rate of secretion, Fig. 1 B) than that obtained with L203E. Although these data imply a role for the zero layer in secretion, it appears to be less important than the part played by Leu-203 at the SNAP-25 C terminus. To assess the importance of regions flanking Leu-203 in molecular events leading to membrane fusion we studied the possible role of residues at the SNAP-25 C-terminal domain in this process. To this end we mutated Ala-199 and Met-202, which have been, respectively, assigned to the seventh and eighth hydrophobic layers of the four-helix bundle in the synaptic fusion complex (30Fasshauer D. Sutton R.B. Brunger A.T. Jahn R. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 15781-15786Crossref PubMed Scopus (756) Google Scholar), as well as Lys-201, which is located in between these two. The secretory behavior of cells expressing mutants A199E and M202E was slightly affected with only the initial rate of vesicle release being slower in the M202E mutant (Fig.2). Mutant K201E had a greater inhibitory effect on the total number of vesicles secreted during cell depolarization, and the initial rate of vesicle release was also retarded (Fig. 2). These results suggest that hydrophobic amino acids in the C-terminal domain of SNAP-25, such as Leu-203 and to a lesser extent Met-202, play a relevant role in membrane fusion events leading to exocytosis and that electrostatic interactions through residue Lys-201 could also be involved in this process. Current structural data suggest that several hydrophobic synaptobrevin amino acids such as Ala-81, Ala-82, and Leu-84 could interact with Leu-203 of SNAP-25. We therefore decided to mutate these synaptobrevin residues in GFP constructs linked to the synaptobrevin II C-terminal domain, which were also overexpressed following amplicon infection of bovine chromaffin cells (Fig.3). Constructs with wild-type synaptobrevin (Fig. 3 A) as well as the A81D (Fig.3 B), A82D (Fig. 3 C), and L84E (Fig.3 D) mutants, were expressed at similar levels in a punctate pattern suggesting their location in chromaffin vesicles. The secretory activity of cells expressing these constructs was studied using depolarizing stimuli. Cells expressing wild-type synaptobrevin coupled to GFP (SV WT) had an exocytotic behavior resembling that observed in cells expressing GFP and the GFP construct coupled to wild-type SNAP-25 (Fig. 4) in that neither the kinetics nor the level of secretion obtained after 1 min of continuous depolarization was modified. Expression of mutant A81D had a relatively low impact on the release of catecholamines, which showed a 35% inhibition in both overall extent and initial rate of release (Fig. 4). Mutants A82D and L84E had a more pronounced effect on secretory behavior, reaching levels close to 70% inhibition (Fig. 4) similar to that observed in the mutant L203E of SNAP-25. These data strengthen the notion that these synaptobrevin and SNAP-25 hydrophobic residues have a marked and perhaps related influence on secretory behavior.Figure 4Depolarization-evoked secretion in chromaffin cells overexpressing different GFP-synaptobrevin constructs.Secretion was monitored by amperometry as described in previous figures. After building of event cumulative responses for individual cells, the average secretion was obtained from control GFP-synaptobrevin wild-type; SV WT, (n = 42 cells), SV A81D (n = 26), SV A82D(n = 27), and SV L84E (n = 26).View Large Image Figure ViewerDownload Hi-res image Download (PPT) The study of individual fusion kinetics could be potentially useful in elucidating the mechanisms behind the differential effects of SNAP-25 and synaptobrevin mutants. This is possible by analyzing the shape of single amperometric events (31Schroeder T.J. Borges R. Finnegan J.M. Pihel K. Amatore C. Wightman R.M. Biophys. J. 1996; 70: 1061-1068Abstract Full Text PDF PubMed Scopus (143) Google Scholar) occurring in the proximity of the carbon fiber electrode. A semi-automatic analysis was performed using software (24Segura F. Brioso M.A. Gomez J.F. Machado D. Borges R. J. Neurosci. Methods. 2000; 103: 151-156Crossref PubMed Scopus (73) Google Scholar) that measured spike amplitude, half-width, and event charge and selecting well separated spikes with amplitudes over 5 pA and half-width values lower than 75 ms. The collected data were binned to generate distribution histograms for means obtained from individual cell analysis in which parameters were obtained for each individual cell and later averaged, similar to the ones shown for the entire event population in Fig. 5 and summarized in Table I. Non infected cells and cells expressing GFP-SNAP-25 or GFP-synaptobrevin were characterized by very similar distributions, showing means taken from averaged individual cell amplitudes of 70, 59, and 50 pA, respectively (Table I). Amplitudes were also similar in cells expressing single (Q174K, Fig.5 D and Table I; Q53K, Table I) and double (Q174K/Q53K, TableI) mutants of the zero layer as well as A199E and M202E mutants of the SNAP-25 C terminus (Table I). However, the mean amplitudes obtained with L203E (Fig. 5 G and Table I) and K201E mutants (Table I) were clearly reduced to almost half of the control value. Moreover, amplitudes over 75 pA, which were relatively abundant in control cells were less frequent in these cases. These results are in good agreement with those reported for C-terminal deletions in transfected chromaffin cells (14Criado M. Gil A. Viniegra S. Gutiérrez L.M. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 7256-7261Crossref PubMed Scopus (83) Google Scholar). On the other hand, the alteration seen in mean amplitude was not associated with a change in the charge released per eve