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• W1967998294 abstract "The calcium (Ca2+) regulation of neurotransmitter release is poorly understood. Here we investigated several aspects of this process in PC12 cells. We first showed that osmotic shock by 1 m sucrose stimulated rapid release of neurotransmitters from intact PC12 cells, indicating that most of the vesicles were docked at the plasma membrane. Second, we further investigated the mechanism of rescue of botulinum neurotoxin E inhibition of release by recombinant SNAP-25 COOH-terminal coil, which is known to be required in the triggering stage. We confirmed here that Ca2+ was required simultaneously with the SNAP-25 peptide, with no significant increase in release if either the peptide or Ca2+ was present during the priming stage as well as the triggering, suggesting that SNARE (solubleN-ethylmaleimide-sensitive fusion protein attachment protein receptor) complex assembly was involved in the final Ca2+-triggered event. Using this rescue system, we also identified a series of acidic surface SNAP-25 residues that rescued better than wild-type when mutated, due to broadened Ca2+sensitivity, suggesting that this charged patch may interact electrostatically with a negative regulator of membrane fusion. Finally, we showed that the previously demonstrated stimulation of exocytosis in this system by calmodulin required calcium binding, since calmodulin mutants defective in Ca2+-binding were not able to enhance release. The calcium (Ca2+) regulation of neurotransmitter release is poorly understood. Here we investigated several aspects of this process in PC12 cells. We first showed that osmotic shock by 1 m sucrose stimulated rapid release of neurotransmitters from intact PC12 cells, indicating that most of the vesicles were docked at the plasma membrane. Second, we further investigated the mechanism of rescue of botulinum neurotoxin E inhibition of release by recombinant SNAP-25 COOH-terminal coil, which is known to be required in the triggering stage. We confirmed here that Ca2+ was required simultaneously with the SNAP-25 peptide, with no significant increase in release if either the peptide or Ca2+ was present during the priming stage as well as the triggering, suggesting that SNARE (solubleN-ethylmaleimide-sensitive fusion protein attachment protein receptor) complex assembly was involved in the final Ca2+-triggered event. Using this rescue system, we also identified a series of acidic surface SNAP-25 residues that rescued better than wild-type when mutated, due to broadened Ca2+sensitivity, suggesting that this charged patch may interact electrostatically with a negative regulator of membrane fusion. Finally, we showed that the previously demonstrated stimulation of exocytosis in this system by calmodulin required calcium binding, since calmodulin mutants defective in Ca2+-binding were not able to enhance release. soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptor vesicle-associated membrane protein bovine serum albumin polyacrylamide gel electrophoresis soluble N-ethylmaleimide attachment protein The secretion of neurotransmitters from neurons and neuroendocrine cells is regulated by Ca2+. Different populations of vesicles within cells exhibit distinct Ca2+ concentration requirements for the stimulation of exocytosis and undergo fusion with varying kinetics (1Kasai H. Trends Neurosci. 1999; 22: 88-93Abstract Full Text Full Text PDF PubMed Scopus (166) Google Scholar). This is partly attributable to the spatial Ca2+ concentration gradients originating from Ca2+ channels (2Neher E. Neuron. 1998; 20: 389-399Abstract Full Text Full Text PDF PubMed Scopus (856) Google Scholar, 3Artalejo C.R. Adams M.E. Fox A.P. Nature. 1994; 367: 72-76Crossref PubMed Scopus (241) Google Scholar) and partly due to heterogeneity in the maturation states of the vesicles themselves (4Voets T. Neher E. Moser T. Neuron. 1999; 23: 607-615Abstract Full Text Full Text PDF PubMed Scopus (210) Google Scholar). Ca2+ not only triggers the final step of transmitter release, but is also involved in many other events important for vesicle recycling, for example, movement of vesicles from the reserve pool to the immediately releasable pool, which may involve Ca2+-dependent cytoskeletal rearrangements (5Neher E. Zucker R.S. Neuron. 1993; 10: 21-30Abstract Full Text PDF PubMed Scopus (455) Google Scholar). The norepinephrine-containing dense core vesicles in PC12 cells have been shown to be morphologically docked (6Banerjee 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 (143) Google Scholar) and physically associated with the plasma membrane (7Martin T.F. Kowalchyk J.A. J. Biol. Chem. 1997; 272: 14447-14453Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar) and therefore do not require mobilization steps prior to exocytosis. Osmotic shock by applying high concentrations of sucrose has been used to estimate the size of the readily releasable pool in neurons (8Rosenmund C. Stevens C.F. Neuron. 1996; 16: 1197-1207Abstract Full Text Full Text PDF PubMed Scopus (849) Google Scholar) and is used in this study to confirm that PC12 cell dense core vesicles are docked. In the reconstituted exocytosis system using these cracked PC12 cells, Ca2+ concentration is controlled by buffered solution, and only docked dense core vesicles are assayed, thus the system eliminates Ca2+ concentration dynamics and provides a tool to analyze the late post-docking steps of vesicle fusion. The role of Ca2+ in the triggering of fusion with the plasma membrane of synaptic vesicles in neurons and large dense core vesicles in PC12 cells is likely in regulating the formation of SNARE1 (solubleN-ethylmaleimide-sensitive fusion protein attachment protein receptor) complexes (9Chen 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 (360) Google Scholar). Syntaxin 1a is the t- (for target) or Q-SNARE of the plasma membrane, which together with the peripherally attached t- or Q-SNARE SNAP-25 binds to the vesicle-associated membrane protein VAMP2 (an R-SNARE) to form a trans-SNARE complex (10Söllner T. Whiteheart S.W. Brunner M. Erdjument-Bromage H. Geromanos S. Tempst P. Rothman J.E. Nature. 1993; 362: 318-324Crossref PubMed Scopus (2637) Google Scholar, 11Hanson P.I. Heuser J.E. Jahn R. Curr. Opin. Neurobiol. 1997; 7: 310-315Crossref PubMed Scopus (334) Google Scholar, 12Brunger A.T. Curr. Opin. Neurobiol. 2000; 10: 293-302Crossref PubMed Scopus (82) Google Scholar). Full formation of the SNARE complex bridging the two membranes is believed to force the membranes into close proximity, helping to overcome the energy barrier of membrane fusion (9Chen 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 (360) Google Scholar, 13Weber T. Zemelman B.V. McNew J.A. Westermann B. Gmachl M. Parlati F. Söllner T.H. Rothman J.E. Cell. 1998; 92: 759-772Abstract Full Text Full Text PDF PubMed Scopus (2021) Google Scholar). Current models postulate that full SNARE complex formation is prevented by a Ca2+-sensing protein, which binds to one or more of the SNAREs (or a partial SNARE complex) until it is displaced by the arrival of Ca2+ ions that trigger exocytosis (14Lin R.C. Scheller R.H. Annu. Rev. Cell Dev. Biol. 2000; 16: 19-49Crossref PubMed Scopus (422) Google Scholar). However, the nature of the proposed Ca2+ sensor and its precise binding site are still topics of debate (12Brunger A.T. Curr. Opin. Neurobiol. 2000; 10: 293-302Crossref PubMed Scopus (82) Google Scholar, 15Schiavo G. Gmachl M.J. Stenbeck G. Söllner T.H. Rothman J.E. Nature. 1995; 378: 733-736Crossref PubMed Scopus (158) Google Scholar, 16Gerona 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 (198) Google Scholar, 17Chapman E.R. Hanson P.I. An S. Jahn R. J. Biol. Chem. 1995; 270: 23667-23671Abstract Full Text Full Text PDF PubMed Scopus (323) Google Scholar, 18Peters C. Mayer A. Nature. 1998; 396: 575-580Crossref PubMed Scopus (325) Google Scholar, 19Reim K. Mansour M. Varoqueaux F. McMahon H.T. Südhof T.C. Brose N. Rosenmund C. Cell. 2001; 104: 71-81Abstract Full Text Full Text PDF PubMed Scopus (382) Google Scholar). We previously established a system to study the role of SNAP-25 in large dense core vesicle exocytosis in cracked PC12 cells, in which the SNAP-25 COOH-terminal coil is inactivated by cleavage with botulinum neurotoxin E, and exocytosis of tritiated norepinephrine is rescued by addition of a recombinant SNAP-25 COOH-terminal coil (S25C; Ref. 9Chen 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 (360) Google Scholar). To investigate whether, as in chromaffin cells for example (5Neher E. Zucker R.S. Neuron. 1993; 10: 21-30Abstract Full Text PDF PubMed Scopus (455) Google Scholar, 20Bittner M.A. Holz R.W. J. Biol. Chem. 1992; 267: 16219-16225Abstract Full Text PDF PubMed Google Scholar, 21von Rüden L. Neher E. Science. 1993; 262: 1061-1065Crossref PubMed Scopus (274) Google Scholar), calcium facilitates earlier steps than the final triggering of exocytosis from PC12 cells, we took advantage of the fact that the norepinephrine release assay can be split into two stages: MgATP-dependent priming, followed by Ca2+-dependent triggering (22Hay J.C. Martin T.F. J. Cell Biol. 1992; 119: 139-151Crossref PubMed Scopus (250) Google Scholar). We examine whether the presence of low concentrations of Ca2+ and/or S25C during the priming step influences the kinetics of either rescue or regular exocytosis during subsequent triggering. Prior exposure to calcium did not detectably enhance the rate of release, suggesting that early calcium-dependent maturation steps likely occur prior to priming and confirming that SNARE complex assembly appears to be involved in the final Ca2+-triggered event. We further identify a series of charged surface residues in the COOH-terminal half of S25C, mutation of which results in enhanced rescue of exocytosis compared with wild-type S25C in our rescue assay. These mutants exhibit broadened tolerance to different Ca2+ concentrations, resulting in enhanced release at most of the Ca2+concentrations tested. We speculate that these residues might be involved in the binding of the Ca2+ sensor to the SNARE complexes, which normally suppresses exocytosis at suboptimal Ca2+ concentrations. A candidate calcium sensor for membrane fusion events is calmodulin, which is a ubiquitous calcium mediator in eukaryotic cells. All vertebrates have one identical calmodulin protein, encoded by multiple genes (23Friedberg F. Protein Seq. Data Anal. 1990; 3: 335-337PubMed Google Scholar). There has been substantial evidence implicating calmodulin in various membrane trafficking events (18Peters C. Mayer A. Nature. 1998; 396: 575-580Crossref PubMed Scopus (325) Google Scholar, 24Chamberlain L.H. Roth D. Morgan A. Burgoyne R.D. J. Cell Biol. 1995; 130: 1063-1070Crossref PubMed Scopus (174) Google Scholar, 25Steinhardt R.A. Alderton J.M. Nature. 1982; 295: 154-155Crossref PubMed Scopus (95) Google Scholar, 26Birch K.A. Pober J.S. Zavoico G.B. Means A.R. Ewenstein B.M. J. Cell Biol. 1992; 118: 1501-1510Crossref PubMed Scopus (89) Google Scholar), and while its effector is still unknown, two recent reports proposed VAMP and Rab3 as possible candidates (27Quetglas S. Leveque C. Miquelis R. Sato K. Seagar M. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 9695-9700Crossref PubMed Scopus (86) Google Scholar, 28Coppola T. Perret-Menoud V. Luthi S. Farnsworth C.C. Glomset J.A. Regazzi R. EMBO J. 1999; 18: 5885-5891Crossref PubMed Scopus (83) Google Scholar). Calmodulin binds Ca2+ via a structural motif called the EF-hand. Upon binding Ca2+ at each of its four EF-hands, calmodulin exposes a hydrophobic surface that is thought to be critical for interactions with its effector proteins (29Persechini A. Moncrief N.D. Kretsinger R.H. Trends Neurosci. 1989; 12: 462-467Abstract Full Text PDF PubMed Scopus (292) Google Scholar). Mutation of one of the highly conserved Ca2+-coordinating aspartates to alanine in the EF-hand motif of the essential yeast calmodulin protein dramatically reduces its affinity for Ca2+ (30Geiser J.R. van Tuinen D. Brockerhoff S.E. Neff M.M. Davis T.N. Cell. 1991; 65: 949-959Abstract Full Text PDF PubMed Scopus (253) Google Scholar), but surprisingly has little effect on yeast growth (30Geiser J.R. van Tuinen D. Brockerhoff S.E. Neff M.M. Davis T.N. Cell. 1991; 65: 949-959Abstract Full Text PDF PubMed Scopus (253) Google Scholar), implying that calmodulin has important functions that do not require Ca2+ binding. We have previously identified calmodulin as an active component of the membrane EGTA extract from brain that stimulated exocytosis from cracked PC12 cells (31Chen Y.A. Duvvuri V. Schulman H. Scheller R.H. J. Biol. Chem. 1999; 274: 26469-26476Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar). Here we show that mutations in the Ca2+-binding domain of calmodulin prevent its ability to stimulate release, demonstrating that Ca2+ binding is required for calmodulin's function in exocytosis. PC12 cells were maintained, loaded with [3H]norepinephrine, and cracked (mechanically permeabilized) as described previously (22Hay J.C. Martin T.F. J. Cell Biol. 1992; 119: 139-151Crossref PubMed Scopus (250) Google Scholar, 31Chen Y.A. Duvvuri V. Schulman H. Scheller R.H. J. Biol. Chem. 1999; 274: 26469-26476Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar). After being incubated on ice in KGlu buffer (50 mm HEPES, pH 7.2, 105 mm potassium glutamate, 20 mm potassium acetate, 2 mm EGTA) with 0.1% BSA and 9 mmadditional EGTA for ≥1 h, the cracked cells were washed in KGlu + 0.1% BSA buffer three times (800 × g centrifugation for 5 min at 4 °C) before the final stimulation. A typical composite release reaction contains ∼106 cells, 2 mmMgATP, ∼0.7 mg/ml rat brain cytosol, and 1.6 mm total Ca2+ (∼1 µm free Ca2+, measured by fura-2 fluorescence as in Ref. 31Chen Y.A. Duvvuri V. Schulman H. Scheller R.H. J. Biol. Chem. 1999; 274: 26469-26476Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar) in total of 200 µl of KGlu buffer. Release reactions were initiated by warming to 30 °C and terminated by chilling on ice. [3H]norepinephrine secretion was measured by centrifuging the cells for 30 min at 2500 × g at 4 °C and calculating the percentage of total radioactivity in the supernatant after scintillation counting. Results were plotted using Cricket Graph 1.5.2 software (Computer Associates International, Inc.). Where indicated, the composite release reaction was split into two stages: MgATP-dependent priming, followed by Ca2+-dependent triggering. In such cases, cells were first primed by incubation in KGlu/BSA buffer containing 2.6 mm MgATP and ∼0.65 mg/ml rat brain cytosol at 30 °C for 5 or 15 min prior to triggering for different lengths of time by addition of Ca2+ to 1.6 mm total. To investigate the effect of adding S25C or the full complement of Ca2+ during triggering, the indicated concentrations of S25C or Ca2+ were added during a 5-min priming reaction, followed by a time course of triggering with the appropriate amounts of extra S25C, Ca2+, MgATP, and cytosol to compensate for the volume increase. There were no washes between the priming and triggering stages. After EGTA extraction and one wash in KGlu/BSA buffer, the cells were treated with 30 nmrecombinant botulinum neurotoxin E light chain in the presence of 2 mm MgATP and ∼0.35 mg/ml rat brain cytosol for 6–10 min at 30 °C. Cells were then washed three times in KGlu/BSA buffer prior to rescue by addition to tubes containing S25C in addition to cytosol, MgATP, and Ca2+ as in the typical release reactions above. For the dose response curves, the purity and concentration of the S25C mutants (measured using the BCA assay (Pierce)) were verified by SDS-PAGE to ensure equivalent amounts were used in the assays. To determine the Ca2+ sensitivity of the S25C proteins, the cells were stimulated for 30 min at 30 °C in the presence of MgATP, cytosol, 40 µm of each S25C protein, and Ca2+ of different concentrations to achieve final total free concentrations ranging from 0 to 2 µm as indicated. Cracked cells were primed in KGlu buffer containing 0.7–1 mg/ml rat brain cytosol and 2 mm MgATP at 30 °C for 30 min. The primed cells were centrifuged, washed once with KGlu buffer, and distributed into triggering reactions containing ∼1 µm free Ca2+ and 1.5 µm recombinant calmodulin (in the absence of cytosol and MgATP) and stimulated for 6 min at 30 °C. The percentage of [3H]norepinephrine release achieved during S25C rescue or regular release, was calculated and the averages of the indicated number of independent experiments plotted versus time using DeltaGraph® 4.0.5 software. The curves were fitted with exponential curves given by y = a * (1 −e −bx) + c(r 2 is typically 0.99–0.999). The initial rate of release (initial slope of each time course curve),V 0, is the product of a andb. For the toxin-treated cells, theV 0 value in the absence of S25C (0 µm) represents the background leakage and so was subtracted from the V 0 values of the appropriate S25C curves. PC12 cells were labeled with [3H]norepinephrine as described previously (22Hay J.C. Martin T.F. J. Cell Biol. 1992; 119: 139-151Crossref PubMed Scopus (250) Google Scholar), then washed three times in MBB (124 mm NaCl, 5 mmKCl, 1.5 mm Na2HPO4, 2 mm MgCl2, 6 mm glucose, 25 mm HEPES, pH 7.4). All cells, except for those stimulated with 5 mm Ca2+ and ionomycin, were treated with 50 µm EGTA-AM (a membrane-permeable methyl ester of EGTA) for 20 min at 37 °C to allow chelation of cytosolic Ca2+to ensure there was no free Ca2+ in the cells. Release was initiated by incubation at 30 °C for various amounts of time by either adding 5 mm calcium and 1 µg/ml ionomycin (in the absence of EGTA) or sucrose to the cells in MBB + 2 mmMgATP and terminated by chilling on ice. [3H]Norepinephrine release was quantitated as for the cracked cells (above). S25C (amino acids 142–206 of mouse SNAP-25) wild-type and mutant proteins, S25N (amino acids 1–82 of mouse SNAP-25), rat syntaxin 1a H3 domain (amino acids 191–266), and rat VAMP2 coil domain (amino acids 25–94) were expressed in bacteria with an NH2-terminal glutathione S-transferase tag that was removed by thrombin cleavage after purification on glutathione-agarose beads, as described previously (32Scales S.J. Chen Y.A. Yoo B.Y. Patel S.M. Doung Y.C. Scheller R.H. Neuron. 2000; 26: 457-464Abstract Full Text Full Text PDF PubMed Scopus (170) Google Scholar). The purity of the proteins was confirmed by SDS-PAGE (>95% pure), and the protein concentration was assayed using the BCA kit (Pierce). Proteins used in the cracked cell assay were all dialyzed into KGlu buffer and, if necessary, concentrated on the day of the experiment to prevent precipitation over time, and no aggregation was observed. S25C mutants were constructed by polymerase chain reaction using the QuikChange site-directed mutagenesis protocol (Stratagene) and verified by sequencing. CD analysis of fast protein liquid chromatography gel filtration-purified S25N/S25C/VAMP2/Syntaxin 1a H3 complexes was performed as described previously (9Chen 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 (360) Google Scholar). His-tagged botulinum neurotoxin E light chain was expressed in bacteria and purified on Ni2+ beads, according to procedures optimized by Heiner Niemann, and dialyzed into KGlu buffer for use in the cracked cell assay. The toxin concentration was estimated by SDS-PAGE, using BSA as a standard. Yeast shuttle vector-based plasmids pJ61, pJG62, pJG65, and pJG66, carrying the EF-hand mutant vertebrate calmodulin genes D20A/D93A (CaM-D1,3A), D56A/D129A CaM-D2,4A), D20A/D56A/D93A/D129A (CaM-D1,2,3,4A), and D56A (CaM-D2A), respectively (30Geiser J.R. van Tuinen D. Brockerhoff S.E. Neff M.M. Davis T.N. Cell. 1991; 65: 949-959Abstract Full Text PDF PubMed Scopus (253) Google Scholar), were restriction-digested to generate a unique 367-base pair HincII/HindIII fragment that then replaced the equivalent wild-type fragment of calmodulin in the pCR2 vector (a pET23d-based construct from Novagen; Ref. 33Persechini A. Blumenthal D.K. Jarrett H.W. Klee C.B. Hardy D.O. Kretsinger R.H. J. Biol. Chem. 1989; 264: 8052-8058Abstract Full Text PDF PubMed Google Scholar). The expression constructs thus obtained were confirmed by sequencing. Mutant and wild-type vertebrate calmodulins were expressed in BL21 (DE3) Escherichia coli cells using the pET system (Novagen). Bacteria were resuspended in cold lysis buffer (50 mm Tris, pH 7.5, 500 mm NaCl, 1 mmEDTA) and disrupted by French pressing, followed by DNase I (Sigma) treatment in 10 mm MgCl2 at 25 °C until no longer viscous. All further steps were performed at room temperature. The lysate was cleared by low speed centrifugation, and EGTA was added to 1 mm prior to gravity loading onto a phenyl-Sepharose (Amersham Pharmacia Biotech) column (10-ml column/liter of culture) equilibrated in PS I buffer (10 mm Tris, pH 7.5, 500 mm NaCl, 1 mm EDTA). After two passages through this column, CaCl2 to 10 mm was added to the flow-through, before loading onto a second phenyl-Sepharose (Amersham Pharmacia Biotech) column (30-ml column/liter of culture) equilibrated in PS II buffer (10 mm Tris, pH 7.5, 500 mm NaCl, 10 mm CaCl2). The flow-through was passed through the column a second time, the column was washed with 10 volumes of PS II, and then calmodulin was eluted in 3 volumes of PS III buffer (10 mm Tris, pH 7.5, 50 mm NaCl, 50 mm EGTA) with 3-ml fractions being collected. Fractions containing calmodulin were pooled and desalted on a HiTrap desalting column (Amersham Pharmacia Biotech) equilibrated in KGlu buffer. The quadruple mutant calmodulin, CaM(D1,2,3,4A), was purified using a different protocol, as it does not bind phenyl-Sepharose in a Ca2+-dependent manner. The bacterial lysate was heated to 70 °C, chilled on ice, and centrifuged at 100,000 × g for 1 h at 4 °C. The supernatant was gradually brought to a final concentration of 1.5m (NH4)2SO4 at room temperature, and the precipitate was removed by centrifugation. The supernatant was brought to a saturating final concentration (∼5m) of (NH4)2SO4; the precipitate was pelleted, resuspended in 20 mm Tris, pH 7.5, 1 mm EGTA, and dialyzed overnight at 4 °C in the same buffer. After clearing with a low speed spin, the dialysate was loaded onto a Mono Q 5/5 fast protein liquid chromatography column (Amersham Pharmacia Biotech) and eluted with a linear gradient from 0 to 1 m KCl over 85 ml at 1 ml/min. CaM(D1,2,3,4A) eluted at ∼140 mm KCl, and fractions were pooled and dialyzed overnight at 4 °C in KGlu buffer. Protein concentrations of the calmodulins were determined by the Bradford protein assay (Bio-Rad). In neurons, the application of 0.5 osmsucrose induces the fusion of docked synaptic vesicles with the presynaptic membrane and has been used as a tool to estimate the size of the readily releasable pool (8Rosenmund C. Stevens C.F. Neuron. 1996; 16: 1197-1207Abstract Full Text Full Text PDF PubMed Scopus (849) Google Scholar, 34Lonart G. Janz R. Johnson K.M. Südhof T.C. Neuron. 1998; 21: 1141-1150Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar). To investigate whether the fusion of norepinephrine-containing dense core vesicles of PC12 cells can similarly be stimulated, we added various concentrations of sucrose to PC12 cells. As expected, sucrose did not stimulate fusion in cracked PC12 cells (data not shown), as the membrane needs to be intact to be affected by hypertonic stimuli. With intact PC12 cells, while 0.1 osm sucrose had little effect (data not shown), higher concentrations did elicit release (Fig.1, bars 8–16). At 1 osm sucrose, release was maximal (∼47%) within 1 min (bar 10), since longer incubation times (up to 5 min;bars 13 and 16) did not result in further secretion, while 0.5 osm sucrose resulted in slower, less extensive release (bars 8, 11, and14). By comparison, intact cells stimulated with the Ca2+-ionophore ionomycin in the presence of high external [Ca2+] secreted slightly more slowly, perhaps due to delayed [Ca2+]i rise, but released slightly more over a 5-min time period, and by 10 min, over 60% of the total [3H]norepinephrine was secreted. In the absence of external Ca2+, ionomycin also elicited a small amount of release, likely due to release of Ca2+ from internal stores (bars 2–4). The fact that sucrose induced only slightly less norepinephrine release than ionomycin suggests that most of the dense core vesicles in intact PC12 cells are docked at the plasma membrane, constituting a readily releasable pool. In the cracked cells, the small percentage of the nondocked vesicle pool is likely to be further depleted due to cracking and washing, thus the exocytosis signal obtained in the cracked cell system should almost exclusively be from docked vesicles, as suggested previously (6Banerjee 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 (143) Google Scholar, 7Martin T.F. Kowalchyk J.A. J. Biol. Chem. 1997; 272: 14447-14453Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar). The higher apparent release in the cracked cell system (e.g. Fig. 4 and Ref. 35Chen Y.A. Scales S.J. Scheller R.H. Neuron. 2001; 30: 161-170Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar) is likely due to the removal of the cytosolic pool of norepinephrine that is not taken up into vesicles during loading (the results are expressed as a percentage of the total [3H]norepinephrine).Figure 4Several S25C surface mutations enhance rescue. A, typical dose response curves of botulinum neurotoxin E rescue by S25C bearing single mutations to alanine at the indicated positions. Cracked, toxin-treated PC12 cells were stimulated with cytosol, MgATP, and 1 µm Ca2+ in the presence of the indicated amounts of the mutant or wild-type S25C proteins, and [3H]norepinephrine release was quantitated as described under “Experimental Procedures.” With the exception of R176A, all the mutations resulted in enhanced rescue relative to wild-type S25C. The mean and S.E. of three independent experiments (after normalization for variations in background release) is shown.B, as in A except multiple surface mutations in S25C were analyzed. The double (D193A/D186A), triple (D193A/D186A/D179A), and quadruple mutants (D193A/D186A/D179A/D172A) formed from combinations of the single mutants shown in Aresulted in increased exocytosis compared with wild-type S25C (the highest rescuing single mutant from A, D179A, is shown again for comparison). The mean and S.E. of three to five independent experiments is plotted.View Large Image Figure ViewerDownload Hi-res image Download (PPT) In adrenal chromaffin cells, Ca2+ concentrations lower than those required to trigger fusion can assist cytoskeletal rearrangements and other upstream events in the maturation of vesicles into the readily releasable pool (5Neher E. Zucker R.S. Neuron. 1993; 10: 21-30Abstract Full Text PDF PubMed Scopus (455) Google Scholar, 20Bittner M.A. Holz R.W. J. Biol. Chem. 1992; 267: 16219-16225Abstract Full Text PDF PubMed Google Scholar, 21von Rüden L. Neher E. Science. 1993; 262: 1061-1065Crossref PubMed Scopus (274) Google Scholar). We therefore took advantage of the fact that exocytosis can be resolved into a MgATP-dependent, Ca2+-independent priming stage and a subsequent Ca2+-dependent (MgATP-independent) triggering of release (22Hay J.C. Martin T.F. J. Cell Biol. 1992; 119: 139-151Crossref PubMed Scopus (250) Google Scholar) to examine whether calcium is required at early, as well as late, steps in PC12 cell exocytosis. We investigated whether the rate of S25C-dependent rescue or regular norepinephrine release during triggering would increase if low concentrations of Ca2+ (<∼100 nm, i.e. less than the ∼1 µm normally added in triggering) were present during the priming step. Triggering was then initiated by addition of the remaining complement of Ca2+ (to ∼1 µmfinal) and S25C in the case of the toxin-treated cells, and the time course of release was followed. We were unable to resolve any differences in the initial rates of fusion following the different priming protocols (data not shown), suggesting that unlike in chromaffin cells, PC12 cell dense core vesicles have probably bypassed early Ca2+-dependent steps of maturation. We reported previously that S25C is required in the triggering but not in the priming step of rescue (9Chen 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 (360) Google Scholar), leading us to conclude that Ca2+ triggers the final step of SNARE complex formation. This means that both Ca2+ and S25C are required to form fully zippered SNARE complexes and drive fusion. We wondered, however, whether S25C's presence in the priming reaction might affect the triggering reaction, for example its release kinetics, that we did not closely examine in the previous study. Thus, we conducted experiments to compare the time course of rescue after priming the cells with two different concentrations (one low and one high) of S25C in the absence of Ca2+ to that of cells primed with Ca2+ in the absence of S25C (Fig.2). The initial rate of release was calculated" @default.
• W1967998294 created "2016-06-24" @default.
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• W1967998294 date "2001-07-01" @default.
• W1967998294 modified "2023-10-14" @default.
• W1967998294 title "Calcium Regulation of Exocytosis in PC12 Cells" @default.
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