Synaptotagmins form a hierarchy of exocytotic Ca 2+ sensors with distinct Ca 2+ affinities
2002; Springer Nature; Volume: 21; Issue: 3 Linguagem: Inglês
10.1093/emboj/21.3.270
ISSN1460-2075
AutoresShuzo Sugita, Ok-Ho Shin, Weiping Han, Ye Lao, Thomas C. Südhof,
Tópico(s)Pancreatic function and diabetes
ResumoArticle1 February 2002free access Synaptotagmins form a hierarchy of exocytotic Ca2+ sensors with distinct Ca2+ affinities Shuzo Sugita Shuzo Sugita Present address: Division of Cellular and Molecular Biology, Toronto Western Research Institute, Ontario, Canada Search for more papers by this author Ok-Ho Shin Ok-Ho Shin The Center for Basic Neuroscience, Department of Molecular Genetics, and Howard Hughes Medical Institute, UT Southwestern Medical Center, Dallas, TX, 75390 USA Search for more papers by this author Weiping Han Weiping Han The Center for Basic Neuroscience, Department of Molecular Genetics, and Howard Hughes Medical Institute, UT Southwestern Medical Center, Dallas, TX, 75390 USA Search for more papers by this author Ye Lao Ye Lao The Center for Basic Neuroscience, Department of Molecular Genetics, and Howard Hughes Medical Institute, UT Southwestern Medical Center, Dallas, TX, 75390 USA Search for more papers by this author Thomas C. Südhof Corresponding Author Thomas C. Südhof The Center for Basic Neuroscience, Department of Molecular Genetics, and Howard Hughes Medical Institute, UT Southwestern Medical Center, Dallas, TX, 75390 USA Search for more papers by this author Shuzo Sugita Shuzo Sugita Present address: Division of Cellular and Molecular Biology, Toronto Western Research Institute, Ontario, Canada Search for more papers by this author Ok-Ho Shin Ok-Ho Shin The Center for Basic Neuroscience, Department of Molecular Genetics, and Howard Hughes Medical Institute, UT Southwestern Medical Center, Dallas, TX, 75390 USA Search for more papers by this author Weiping Han Weiping Han The Center for Basic Neuroscience, Department of Molecular Genetics, and Howard Hughes Medical Institute, UT Southwestern Medical Center, Dallas, TX, 75390 USA Search for more papers by this author Ye Lao Ye Lao The Center for Basic Neuroscience, Department of Molecular Genetics, and Howard Hughes Medical Institute, UT Southwestern Medical Center, Dallas, TX, 75390 USA Search for more papers by this author Thomas C. Südhof Corresponding Author Thomas C. Südhof The Center for Basic Neuroscience, Department of Molecular Genetics, and Howard Hughes Medical Institute, UT Southwestern Medical Center, Dallas, TX, 75390 USA Search for more papers by this author Author Information Shuzo Sugita2, Ok-Ho Shin1, Weiping Han1, Ye Lao1 and Thomas C. Südhof 1 1The Center for Basic Neuroscience, Department of Molecular Genetics, and Howard Hughes Medical Institute, UT Southwestern Medical Center, Dallas, TX, 75390 USA 2Present address: Division of Cellular and Molecular Biology, Toronto Western Research Institute, Ontario, Canada *Corresponding author. E-mail: [email protected] The EMBO Journal (2002)21:270-280https://doi.org/10.1093/emboj/21.3.270 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Synaptotagmins constitute a large family of membrane proteins implicated in Ca2+-triggered exocytosis. Structurally similar synaptotagmins are differentially localized either to secretory vesicles or to plasma membranes, suggesting distinct functions. Using measurements of the Ca2+ affinities of synaptotagmin C2-domains in a complex with phospholipids, we now show that different synaptotagmins exhibit distinct Ca2+ affinities, with plasma membrane synaptotagmins binding Ca2+ with a 5- to 10-fold higher affinity than vesicular synaptotagmins. To test whether these differences in Ca2+ affinities are functionally important, we examined the effects of synaptotagmin C2-domains on Ca2+-triggered exocytosis in permeabilized PC12 cells. A precise correlation was observed between the apparent Ca2+ affinities of synaptotagmins in the presence of phospholipids and their action in PC12 cell exocytosis. This was extended to PC12 cell exocytosis triggered by Sr2+, which was also selectively affected by high-affinity C2-domains of synaptotagmins. Together, our results suggest that Ca2+ triggering of exocytosis involves tandem Ca2+ sensors provided by distinct plasma membrane and vesicular synaptotagmins. According to this hypothesis, plasma membrane synaptotagmins represent high-affinity Ca2+ sensors involved in slow Ca2+-dependent exocytosis, whereas vesicular synaptotagmins function as low-affinity Ca2+ sensors specialized for fast Ca2+-dependent exocytosis. Introduction When an action potential invades a presynaptic nerve terminal, Ca2+ influx triggers neurotransmitter release (Katz, 1969). Ca2+ activates two components of release: a fast synchronous component that accounts for >90% of total release, and a slow asynchronous component that mediates 500 μM Ca2+). The apparent Ca2+ affinity of the synaptotagmin 1 C2-domains is dramatically increased in the presence of phospholipid membranes (∼5–50 μM Ca2+ depending on the lipid composition), presumably because the phospholipid headgroups provide additional coordination sites for the bound Ca2+ ions (Zhang et al., 1998; Fernandez et al., 2001; Fernandez-Chacon et al., 2001). In the ternary C2-domain– Ca2+–phospholipid complex, the C2A-domain probably contains three Ca2+ ions, but the C2B-domain contains only two Ca2+ ions. As a result, the C2B-domain complex is more labile, and Ca2+-dependent phospholipid binding to the synaptotagmin 1 C2B-domain was only detected recently by methods in which binding was studied in solution (Fernandez et al., 2001). The sequences of most C2-domains of other synaptotagmins are very similar to those of synaptotagmin 1, suggesting that most synaptotagmins also form Ca2+-dependent phospholipid complexes via both C2-domains (Li et al., 1995a,b). Ca2+-dependent phospholipid interactions probably constitute an intrinsic component of synaptotagmin function since phospholipid binding is the only confirmed function of C2-domains, as exemplified by enzymes such as phospholipase A2 or protein kinase C in which the C2-domain mediates the Ca2+-dependent recruitment of the enzymes to the membrane (reviewed in Nalefski and Falke, 1996; Newton and Johnson, 1998). Knockout mice revealed that, in hippocampal synapses, synaptotagmin 1 is essential for fast but not for slow Ca2+-stimulated neurotransmitter release, suggesting that synaptotagmin 1 is an essential Ca2+ sensor for fast exocytosis (Geppert et al., 1994). This hypothesis is supported by mutant mice in which a point mutation, R233Q, was introduced into synaptotagmin 1 by homologous recombination (Fernandez-Chacon et al., 2001). The R233Q mutation caused an ∼2-fold reduction in the overall Ca2+ affinity of synaptotagmin 1, and a similar decrease in the apparent Ca2+ affinity of transmitter release, suggesting that the Ca2+ affinity of synaptotagmin 1 determines the Ca2+ affinity of synaptic exocytosis (Fernandez-Chacon et al., 2001). In contrast to synapses, however, deletion of synaptotagmin 1 in neuroendocrine cells had a surprisingly small effect on exocytosis. In chromaffin cells, the synaptotagmin 1 knockout caused only a minor decrease in fast exocytosis (Voets et al., 2001). Similarly, in PC12 cells lacking synaptotagmins 1 and 2, Ca2+ still induced robust secretion (Shoji-Kasai et al., 1992). The lack of a requirement for synaptotagmin 1 for most large dense-core vesicle exocytosis, but its necessity for synaptic vesicle exocytosis, suggested that other Ca2+ sensors may be more important for large dense-core vesicle exocytosis. Indeed, studies in permeabilized PC12 cells demonstrated that the C2A- and C2B-domains of synaptotagmin 7 potently inhibited exocytosis, whereas the C2A- and C2B-domains of synaptotagmin 1 were without significant effect, indicating that synaptotagmin 7 may constitute a Ca2+ sensor for exocytosis in these cells (Sugita et al., 2001). However, since synaptotagmin 7 is also present in synapses, this raises the question of why synaptotagmin 7 can substitute for synaptotagmin 1 in PC12 cells but not in synapses. One possible explanation for this conundrum is that in synapses and endocrine cells, vesicular synaptotagmins may be responsible for fast exocytosis triggered at higher Ca2+ concentrations, and plasma membrane synaptotagmins for slower exocytosis stimulated at lower Ca2+ concentrations. The more severe phenotype of the synaptotagmin 1 knockout at synapses, and the less severe phenotype in endocrine cells, would then be due to the fact that most synaptic exocytosis, but only a small part of endocrine exocytosis, are mediated by the fast component. However, it is unclear whether plasma membrane and vesicular synaptotagmins have intrinsic functional differences as predicted by this hypothesis, and also whether these differences apply to other synaptotagmins, especially synaptotagmin 3, which is co-localized with synaptotagmin 7 on synaptic plasma membranes but whose location in neuroendocrine cells is unclear. In the present study, we set out to address these issues. Our results demonstrate an unsuspected functional specialization of synaptotagmins whereby plasma membrane synaptotagmins exhibit a higher Ca2+ affinity than vesicular synaptotagmins, and even vesicular synaptotagmins are heterogeneous with respect to Ca2+ affinity. These findings indicate that at central synapses, a series of Ca2+ sensors with distinct affinities may operate in triggering fast release. In large dense-core vesicle exocytosis, by contrast, fusion is probably largely driven by high-affinity synaptotagmins that operate more slowly but require lower Ca2+ levels. Results Relative Ca2+ affinities of the synaptotagmin C2A-domains We compared, in the same experiment, the apparent Ca2+ affinities of the C2A-domains of synaptotagmins 1, 2, 3, 5, 7 and 10. These synaptotagmins were chosen because synaptotagmins 3 and 7 are the most abundant synaptotagmins after 1 and 2 (Butz et al., 1999; Sugita et al., 2001), and because synaptotagmins 3, 5, 6 and 10 form a class of closely related synaptotagmins (Fukuda et al., 1999). Synaptotagmin 6 was not studied because we were unable to produce soluble properly folded C2-domains from this isoform. We estimated the apparent Ca2+ affinities of the C2A-domains by Ca2+-dependent phospholipid binding, and compared two independent methods and three different buffers to control for potential artifacts. Ca2+-dependent phospholipid binding assays were chosen because synaptotagmins bind Ca2+ at physiological concentrations only in the presence of phospholipids and because Ca2+-dependent phospholipid binding most likely constitutes part of their physiological function (reviewed in Südhof, 2002). We first measured the apparent Ca2+ affinity of the C2A-domains with a standard resin-based assay in which immobilized glutathione S-transferase (GST) fusion proteins of the synaptotagmin C2A-domains were incubated with radiolabeled liposomes at different Ca2+ concentrations (Davletov and Südhof, 1993). Figure 1 demonstrates that each synaptotagmin is characterized by a distinct Ca2+ affinity, as measured by this assay. The vesicular synaptotagmins 1 and 2 consistently had the lowest Ca2+ affinities [EC50 ≈ 10–20 μM Ca2+ with liposomes composed of 25% phosphatidylserine (PS)/75% phosphatidylcholine (PC); Figure 1A and B]. By contrast, the plasma membrane synaptotagmins 3 and 7 exhibited the highest Ca2+ affinities (EC50 ≈ 1–2 μM Ca2+; Figure 1C and E). Synaptotagmins 5 and 10 (whose localizations are unknown, but which are most closely related to the plasma membrane synaptotagmins 3 and 6) also displayed relatively high Ca2+ affinities (EC50 ≈ 3 μM Ca2+; Figure 1D and F). Moreover, we observed that synaptotagmins 1 and 2 (which are both localized to synaptic vesicles but differentially distributed in brain; Ullrich et al., 1994; Marqueze et al., 1995) also differ in Ca2+ affinity (Figure 1A and B), with synaptotagmin 2 reproducibly exhibiting an ∼2-fold lower Ca2+ affinity than synaptotagmin 1. The specificity of the phospholipid binding reaction was confirmed by a point mutant in the predicted Ca2+ binding site of the synaptotagmin 3 C2A-domain (D333N), which abolished Ca2+-dependent phospholipid binding (Figure 1C). Figure 1.Phospholipid binding to the C2A-domains of synaptotagmins 1, 2, 3, 5, 7 and 10 studied by Ca2+-dependent GST pull-downs of radiolabeled liposomes. GST fusion proteins containing the indicated C2A-domains (Syt, synaptotagmin) were immobilized on glutathione–agarose and incubated at increasing concentrations of free Ca2+ with radiolabeled liposomes composed of 25% PS/75% PC. Ca2+ concentrations were clamped by Ca2+/EGTA buffers using the standard NaCl-based buffer (Gerber et al., 2001). Agarose beads were washed three times in the respective incubation buffers and bound liposomes were quantified by liquid scintillation counting. Binding was normalized to 100% for the maximal point. Data shown are means ± SEMs from two experiments performed in triplicate. The binding curve for the synaptotagmin 1 C2A-domain in (A) is repeated in open squares (B–F) as an internal reference point to facilitate comparisons. (C) Gray symbols display binding observed for the synaptotagmin 3 C2A-domain (Syt 3-C2A) containing a point mutation in a Ca2+ binding loop (D333N). Download figure Download PowerPoint The distinct Ca2+ affinities of synaptotagmins could have important implications for the Ca2+-dependent regulation of neurotransmitter release. This is illustrated by the R233Q point mutation in synaptotagmin 1, which causes an ∼2-fold decrease in the Ca2+ affinity of synaptotagmin 1 and a similar decrease in the Ca2+ responsiveness of synapses (Fernandez-Chacon et al., 2001), suggesting that the Ca2+ affinity of vesicular synaptotagmins controls synaptic responses. In view of the potential importance of differences in Ca2+ affinity, we sought to confirm the measured apparent Ca2+ affinities of synaptotagmins with an independent assay. For this purpose we incubated soluble C2A-domain–GST fusion proteins with liposomes at different Ca2+ concentrations, isolated the liposomes by centrifugation, and determined the amount of bound synaptotagmin C2A-domains by Coomassie Blue staining of SDS gels. This assay, referred to as the liposome centrifugation assay, utilizes C2A-domains in solution and thus avoids possible immobilization artifacts of the standard resin-based assay. Figure 2 shows that the liposome centrifugation assay produced results similar to the resin-based assay, revealing an ∼2-fold higher Ca2+ affinity of synaptotagmin 1 than of synaptotagmin 2, and an ∼5- to 10-fold higher affinity of the plasma membrane synaptotagmins than of vesicular synaptotagmins. A major difference between the two assays was that in the resin-based assay, a bell-shaped Ca2+ concentration dependence was observed for synaptotagmins 3 and 7 (Figure 1), whereas the liposome centrifugation assay exhibited no decrease in phospholipid binding at higher Ca2+ concentrations (Figure 2). This difference may reflect the more stringent washes used in the resin-based assay, which would also explain the inability of the resin-based assay to detect Ca2+ binding to the C2B-domain (see below; Fernandez et al., 2001). The concurrence of the apparent Ca2+ affinities determined with the two assays confirms that the Ca2+ affinity is not dependent on whether the C2A-domains are immobilized or in solution. Figure 2.Phospholipid binding by the C2A-domains of synaptotagmins 1, 2, 3, 5, 7 and 10 studied by co-sedimentation with liposomes. Soluble purified GST fusion proteins containing the indicated C2A-domains were incubated with liposomes composed of 25% PS/75% PC in the presence of free Ca2+ at the concentrations shown, clamped by Ca2+/EGTA buffers. Liposomes were centrifuged and washed, and bound proteins were estimated by SDS–PAGE. Data shown are Coomassie Blue-stained gels from a single representative experiment repeated multiple times. Numbers on the right indicate approximate Ca2+ concentrations required for half-maximal binding as estimated from multiple experiments. Download figure Download PowerPoint Synaptotagmin C2B-domains exhibit similar differences in Ca2+ affinity Synaptotagmins have two Ca2+ binding domains: the C2A- and C2B-domains. Initially the C2B-domains were thought to be unable to bind to phospholipids as a function of Ca2+ because the resin-based assay did not detect such binding (reviewed in Südhof and Rizo, 1996). Recently, however, less stringent assays revealed that the C2B-domain of synaptotagmin 1 specifically binds to phospholipids in a Ca2+-dependent manner with an apparent Ca2+ affinity that resembles that of the C2A-domain (Fernandez et al., 2001). The C2B-domain probably forms a less tight Ca2+–phospholipid complex than the C2A-domain because the C2B-domain contains only two Ca2+ binding sites as opposed to the three Ca2+ binding sites of the C2A-domain. To test whether the C2B-domains of synaptotagmins 3 and 7 also bind phospholipids in response to Ca2+ and whether the various C2B-domains exhibit similar differences in Ca2+ affinity as the C2A-domains, we examined the C2B-domains using the liposome centrifugation assay (Figure 2). Figure 3 shows that the synaptotagmin 1 and 7 C2B-domains, but not the synaptotagmin 3 C2B-domain, bind phospholipids as a function of Ca2+. The apparent Ca2+ affinity of the synaptotagmin 7 C2B-domain was ∼10-fold higher than that of the synaptotagmin 1 C2B-domain (Figure 3), demonstrating that the C2A- and C2B-domains of these synaptotagmins exhibit the same difference in Ca2+ affinity. The lack of Ca2+-dependent phospholipid binding by the synaptotagmin 3 C2B-domain, as judged by this assay, is somewhat surprising considering its sequence similarity to other C2B-domains. It may be due either to a lack of Ca2+ binding by this domain (as indicated by the crystal structure; Sutton et al., 1999) or to a selective inability to bind phospholipids as a function of Ca2+. Figure 3.Phospholipid binding by the C2B-domains of synaptotagmins 1, 3 and 7 studied by co-sedimentation with liposomes. Purified GST–C2B-domain fusion proteins were incubated in solution with liposomes composed of 25% PS/75% PC in the presence of the indicated concentrations of free Ca2+ and binding was measured as described in Figure 2. Data shown are from a single representative experiment repeated multiple times; numbers on the right indicate approximate Ca2+ concentrations required for half-maximal binding as estimated from multiple experiments. Download figure Download PowerPoint The relative Ca2+ affinities of synaptotagmin C2A-domains are independent of Ca2+ buffers The apparent Ca2+ affinities for synaptotagmin C2A-domains determined above and in previous studies (Fernandez-Chacon et al., 2001) were determined with the use of Ca2+/EGTA buffers, which may introduce systematic errors. Furthermore, although Ca2+ binding assays and secretion measurements in permeabilized PC12 cells both employ Ca2+/EGTA buffers, these buffers have distinct compositions (Gerber et al., 2001; Sugita et al., 2001). To better relate Ca2+-dependent phospholipid binding to Ca2+-induced secretion in PC12 cells and to evaluate the accuracy of the Ca2+/EGTA buffers, we directly compared three different Ca2+ buffers: the traditional NaCl-based Ca2+/EGTA buffer (Gerber et al., 2001); the K-glutamate Ca2+/EGTA buffer containing 2 mM Mg2+, 2 mM ATP and 0.1% bovine serum albumin (BSA), which is used for permeabilized PC12 cell experiments (Sugita et al., 2001); and a NaCl-based buffer, which contained only Ca2+ but no EGTA (see Materials and methods). All three buffer systems generally gave similar results, suggesting that the Ca2+/EGTA buffers are reliable (data not shown). The only major difference noted was that the synaptotagmin 1 C2A-domain displayed a lower apparent Ca2+ affinity in the K-glutamate Ca2+/EGTA buffer than in the traditional Ca2+/EGTA buffer or the Ca2+-only buffer, probably because the ATP in the K-glutamate buffer inhibits phospholipid binding to the C2A-domain of synaptotagmin 1 but not to that of the other synaptotagmins (data not shown). Synaptotagmins in PC12 cells As a first approach to testing the functional consequences of the different Ca2+ affinities of synaptotagmins, we employed neuroendocrine PC12 cells as a model system. These cells were chosen because they have been productively used in studying Ca2+-triggered exocytosis (Ahnert-Hilger et al., 1987; Walent et al., 1992; McFerran et al., 1998; Avery et al., 2000) and are known to express at least synaptotagmins 1, 3 and 7 (Shoji-Kasai et al., 1992; Mizuta et al., 1994; Sugita et al., 2001). Previous studies have shown that synaptotagmin 1 is present on secretory vesicles in neuroendocrine cells, while synaptotagmin 7 is localized to plasma membranes (Perin et al., 1991; Sugita et al., 2001). The localization of synaptotagmin 3, however, is unclear. Some studies in pancreatic β-cells (the only endocrine cell where it has been studied) reported synaptotagmin 3 on secretory vesicles (Mizuta et al., 1997; Brown et al., 2000; Gao et al., 2000), although a more recent study detected it on the plasma membrane (Gut et al., 2001), similar to its localization in brain (Butz et al., 1999). To address this discrepancy with an independent approach, we examined in PC12 cells the localization of transfected synaptotagmin 3–EYFP fusion protein in comparison with other synaptotagmins. This approach was employed because the low abundance of synaptotagmin 3 made detection of the endogenous protein difficult, and because the direct fluorescence of EYFP avoids possible artifacts due to indirect immunofluorescence procedures. Figure 4 shows that synaptotagmin 3 is quantitatively deposited into the plasma membrane, which is labeled with the fluorescent dye FM4-64. The localization of synaptotagmin 3 is identical to that of synaptotagmin 7–EYFP, whereas synaptotagmin 1–EYFP is exclusively present in intracellular vesicles (Figure 4). These findings suggest that synaptotagmins 3 and 7 are general plasma membrane proteins in neurons and endocrine cells. Figure 4.Localization of synaptotagmins 1, 3 and 7 in PC12 cells analyzed with transfected EYFP fusion proteins. PC12 cells were transfected with expression vectors encoding synaptotagmins 1, 3 or 7 as indicated; all synaptotagmins are expressed as C-terminal fusion proteins with EYFP. Unfixed PC12 cells were incubated with the fluorescent dye FM4-64 to stain the plasma membrane and viewed in a confocal microscope. Synaptotagmin 3 is shown in two examples to document reproducibility. Transfected cells are highlighted by a white arrow. The scale bar (2 μm) applies to all panels. Download figure Download PowerPoint Inhibition of Ca2+-dependent norepinephrine secretion from permeabilized PC12 cells by synaptotagmin C2-domains We loaded PC12 cells with radioactive norepinephrine, permeabilized the cells by freeze–thawing and triggered exocytosis by addition of Ca2+ at increasing concentrations. In the absence of protein additions or in the presence of only GST, we observed a bell-shaped Ca2+ response curve for exocytosis from the permeabilized PC12 cells (control in Figure 5A). This curve was similar to the Ca2+-concentration dependence of phospholipid binding by synaptotagmins 3 and 7 as measured by the resin assay (Figure 1; Sugita et al., 2001). Addition of the synaptotagmin 1 GST–C2A-domain fusion protein to the PC12 cells caused a small but significant inhibition of exocytosis at high Ca2+ concentrations (Figure 5A). A similar but lower amount of inhibition was also observed for the C2A-domain of synaptotagmin 2 (Figure 5B), consistent with its lower apparent Ca2+ affinity (Figures 1 and 2). The C2A-domain of synaptotagmin 3, however, almost abolished Ca2+-triggered exocytosis at all Ca2+ concentrations (Figure 5C). This inhibition is similar to the effect we previously observed with the C2A-domain of synaptotagmin 7 (Sugita et al., 2001). Inhibition by the C2A-domain of synaptotagmin 3 required Ca2+ binding to the C2A-domain because the Ca2+ binding site mutant of the C2A-domain (D333N), which is unable to mediate Ca2+-dependent phospholipid binding (Figure 1C), had no effect on exocytosis (Figure 5D). Finally, the C2A-domains of synaptotagmins 5 and 10 had an intermediate effect and significantly inhibited Ca2+-triggered exocytosis only at higher Ca2+ concentrations (Figure 5E and F). Figure 5.Inhibition of Ca2+-triggered exocytosis in permeabilized PC12 cells by the C2A-domains of synaptotagmins 1, 2, 3, 5 and 10. PC12 cells were loaded with 3H-labeled norepinephrine, permeabilized and pre-incubated with the indicated purified GST fusion proteins of wild-type synaptotagmin C2A-domains (A–C, E and F) or mutant synaptotagmin 3 C2A-domain containing a single amino acid substitution in the Ca2+ binding site (D333N) (D). Exocytosis was triggered by addition of Ca2+ clamped at the concentrations shown with Ca2+/EGTA buffers. Norepinephrine release was normalized to 100% for the maximal release observed under control conditions run in parallel for each experiment with GST (gray symbols). Synaptotagmin C2A-domains and GST were added at 6 and 9 μM, respectively. Data are means ± SEMs from three experiments performed in duplicate and repeated multiple times. Note that the control traces in each graph (amount of secretion in the same assays observed with addition of 9 μM GST alone) differ slightly between graphs because each assay was performed with its own separate controls. Download figure Download PowerPoint The PC12 cell experiments suggest that the Ca2+ sensor responsible for triggering PC12 cell exocytosis is activated by low micromolar Ca2+ concentrations, which precisely match the apparent Ca2+ affinities of synaptotagmins 3 and 7, and is inhibited by the C2A-domains from these synaptotagmins. The Ca2+ binding measurements described above showed that the C2B-domains of synaptotagmins 1 and 7 exhibited similar differences in Ca2+ affinities, raising the question of whether this also applies to the PC12 cell inhibitions. To test this, we measured the effects of the synaptotagmin 1, 3 and 7 C2B-domains on PC12 cell exocytosis. Figure 6 shows that the synaptotagmin 1 and 3 C2B-domains had little effect on Ca2+-triggered exocytosis, whereas the synaptotagmin 7 C2B-domain effectively inhibited exocytosis at all Ca2+ concentrations. These results again agree well with the Ca2+-dependent phospholipid binding measurements (Figure 3), indicating that there is an overall correlation for synaptotagmins in their apparent Ca2+ affinities as measured by Ca2+-dependent phospholipid binding and their effects in PC12 cells. Figure 6.Inhibition of Ca2+-triggered exocytosis in permeabilized PC12 cells by the C2B-domains of synaptotagmins 1 (A), 3 (B) and 7 (C). PC12 cells were loaded with 3H-labeled norepinephrine, permeabilized, pre-incubated with the indicated purified GST fusion proteins of synaptotagmin C2B-domains or GST alone, and stimulated for exocytosis as described in Figure 5. Data are means ± SEMs from two experiments performed in duplicate. Download figure Download PowerPoint We had previously found that the synaptotagmin 7 C2A-domain inhibits Ca2+-triggered exocytosis at all Ca2+ concentrations except very low ones (Sugita et al., 2001), and a similar trend was observed for the synaptotagmin 3 C2A-domain (Figure 5C). Interestingly, the synaptotagmin 7 C2B-domain exhibited the opposite behavior, in that inhibition of exocytosis was most effective at low Ca2+ concentrations (Figure 6C), suggesting that exoc
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