©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Distinct Ca and Sr Binding Properties of Synaptotagmins
DEFINITION OF CANDIDATE Ca SENSORS FOR THE FAST AND SLOW COMPONENTS OF NEUROTRANSMITTER RELEASE (*)

(Received for publication, July 6, 1995)

Cai Li Bazbek A. Davletov (§) Thomas C. Südhof (¶)

From the Department of Molecular Genetics and Howard Hughes Medical Institute, The University of Texas Southwestern Medical Center, Dallas, Texas 75235

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Ca-dependent neurotransmitter release consists of at least two components: a major fast component that is insensitive to Sr and a minor slow component that is potentiated by Sr (Goda, Y., and Stevens, C. F.(1994) Proc. Natl. Acad. U. S. A. 91, 12942-12946). These results suggest that at least two Ca sensors act in synaptic vesicle fusion with distinct Ca and Sr binding properties. We have now investigated the relative Ca and Sr binding activities of synaptotagmins to evaluate their potential roles as Ca sensors for the fast and slow components. Our results demonstrate that the first C(2) domains of synaptotagmins I, II, III, V, and VII have very similar Ca requirements for phospholipid binding (range of EC = 2.6 µM to 5.0 µM), but distinct Sr requirements (EC range = 23 µM to 133 µM); synaptotagmins I and II had the lowest Sr affinity, and synaptotagmin III the highest Sr affinity. Purified synaptotagmin I from bovine brain exhibited similar properties as its recombinant first C(2) domain, suggesting that the first C(2) domain fully accounts for its Ca-dependent phospholipid binding properties. Sr was unable to trigger syntaxin binding by synaptotagmin I at all concentrations tested, whereas it was effective for synaptotagmin III. These results suggest that different C(2) domains have distinct Sr binding properties. They support the hypothesis that synaptotagmins localized on the same vesicle perform distinct functions, with synaptotagmins I and II serving as candidate Ca sensors for the fast component in release and synaptotagmin III for the slow component.


INTRODUCTION

Synaptotagmins constitute an abundant family of Ca-binding proteins of synaptic vesicles. Structurally, synaptotagmins are composed of a short intravesicular sequence, a single transmembrane region, and two copies of an cytoplasmic repeat homologous to the C(2) domain of protein kinase C (reviewed in Südhof(1995)). Synaptotagmins are highly conserved in evolution (Perin et al., 1991a; Bommert et al., 1993; Nonet et al., 1993) and expressed in multiple isoforms with at least 9 genes in mammals (Perin et al., 1990; Geppert et al., 1991; Mizuta et al., 1994; Hilbush and Morgan, 1994; Ullrich et al., 1994; Li et al., 1995; Craxton and Goedert, 1995). Ca binds to purified synaptotagmin I at micromolar concentrations and triggers phospholipid binding by synaptotagmin I (Brose et al., 1992). Ca-dependent phospholipid binding by synaptotagmin I is at least partly mediated by its first C(2) domain since the isolated first C(2) domains of synaptotagmin I and other synaptotagmins, when produced as recombinant proteins, also bind phospholipids as a function of Ca (Davletov and Südhof, 1993; Ullrich et al., 1994; Li et al., 1995). Only the C(2) domains of synaptotagmins IV, VI, and VIII were unable to bind phospholipids in a Ca-regulated manner, suggesting that they may not represent Ca binding domains. Ca binding causes a conformational change in synaptotagmin I and its first C(2) domain (Davletov and Südhof, 1994). Crystallization of the first C(2) domain of synaptotagmin I confirmed that it forms a novel Ca binding module which is almost entirely composed of beta sheets and in which Ca is coordinated between apical loops (Sutton et al., 1995).

These biochemical studies demonstrated that synaptotagmin is a major Ca-binding protein of synaptic vesicles, suggesting that it serves as a Ca sensor in the synaptic vesicle cycle. The best characterized Ca action in the nerve terminal is the triggering of neurotransmitter release, although Ca probably plays multiple additional roles in regulating synaptic vesicle traffic. A detailed analysis of the kinetics of neurotransmitter release in cultured hippocampal neurons revealed that at least two components can be distinguished in Ca-dependent release: a major fast component that has a low Ca affinity and cannot be triggered by Sr, and a more minor slow component that is sensitive to Sr and has a higher apparent Ca affinity (Goda and Stevens, 1994). To test the possible function of synaptotagmin I as a Ca sensor, mice homozygous for a synaptotagmin I mutation were analyzed (Geppert et al., 1994). In these mice, a selective impairment of the fast component of Ca-dependent neurotransmitter release but not of the slow component was observed, suggesting that synaptotagmin I is the major Ca sensor for the fast component of release in the synapses that were analyzed in the mutant.

Although the studies on synaptotagmin I-mutant mice demonstrated an essential role for synaptotagmin I in fast Ca-triggered release, the apparent Ca affinity of synaptotagmin I as measured by phospholipid binding (EC = 3-6 µM) was much higher than that of the putative exocytotic Ca sensor (>200 µM; Llinas et al.(1992) and Heidelberger et al. (1994)). This apparent paradox was resolved upon the discovery that synaptotagmins and their first C(2) domains have multiple independent Ca-activated properties (Li et al., 1995). Phospholipid binding triggered at Ca concentrations that are similar for most synaptotagmins, and syntaxin binding at Ca concentrations that differ between synaptotagmins. Phospholipid binding is observed in the low micromolar range of Ca for all C(2) domains that exhibit Ca-dependent phospholipid binding. In contrast, syntaxin binding requires high concentrations of Ca for synaptotagmins I and II (>200 µM) but low Ca concentrations for synaptotagmins III and VII (<10 µM). These studies provided a biochemical correlate for the events during the last stage of synaptic vesicle exocytosis and showed that a single C(2) domain may contain multiple Ca binding sites that have not yet been defined structurally.

Multiple synaptotagmins are co-localized in brain. The synaptotagmin I mutation gave a strong phenotype in spite of the presence of other synaptotagmins; in fact, synaptotagmins I and III are colocalized in the hippocampal neurons that are affected in the knockout (Ullrich et al., 1994). This suggests that different synaptotagmin isoforms perform distinct functions. The Ca binding properties of most synaptotagmins suggest that they also function as Ca sensors similar to synaptotagmin I. The fact that synaptotagmin III has a higher Ca affinity than synaptotagmins I in the syntaxin interaction and that the slow component of neurotransmitter release is unimpaired in the synaptotagmin I mutants raises the possibility that synaptotagmin III could function as a Ca sensor for the slow component of release.

The defining property of the slow component of release is its distinct Sr sensitivity compared to the fast component (Goda and Stevens, 1994). We have now investigated the possibility that the slow component of neurotransmitter release may be mediated by synaptotagmin III by studying the relative Sr sensitivities of different synaptotagmins. Our results demonstrate that although all synaptotagmins exhibit similar Ca affinities for phospholipid binding, they exhibit dramatically different affinities for Sr. Direct comparison of the phospholipid binding properties of recombinant first C(2) domains of synaptotagmin I and of the cytoplasmic domains of purified brain synaptotagmin I demonstrated that they have very similar properties, suggesting that the properties of the first C(2) domain reflect the properties of native synaptotagmin. Together these studies show that results with the recombinant first C(2) domains accurately reflect the activities of the whole protein and that synaptotagmin III has the requisite cation specificity of the sensor for the slow component.


EXPERIMENTAL PROCEDURES

Caand Sr-dependent Phospholipid and Syntaxin Binding Measurements to Recombinant GST-synaptotagmins

The indicated GST-synaptotagmins were produced as described, immobilized on glutathione agarose, and used for divalent cation-dependent phospholipid and syntaxin binding assays essentially as described (Davletov and Südhof, 1993; Ullrich et al., 1994; Li et al., 1995). Liposomes were made from 2.5 mg of phosphatidylcholine and 1.0 mg of phosphatidylserine with trace amounts of [^3H]phosphatidylcholine (Amersham) in a final volume of 20 ml. Free Ca concentrations were set with Ca/EGTA buffers as described (Davletov and Südhof, 1993; Ullrich et al., 1994). All experiments were carried out with MilliQ water that had been treated with Chelex 100 beads (Bio-Rad). For some experiments, Ca concentrations were confirmed using a Ca electrode (Accumet 25, Fisher) and commercial Ca standards. Sr and Ba were titrated in the absence of chelating agents or in the presence of 10 µM EGTA to suppress the background due to contaminating Ca; EGTA does not bind to Ba or Sr at that concentration. For the experiments measuring the ability of Ca, Ba, and Sr to trigger binding of syntaxin to the first C(2) domains of different synaptotagmins, a reaction (100 µl) containing approximately 20 µg of recombinant protein attached to glutathione beads and 150 µg solubilized total rat brain protein were incubated overnight at 4 °C in 10 mM HEPES-NaOH pH 7.4, 0.15 M NaCl, 2 mM MgCl(2), 0.2% Triton X-100, and 0.5 mM EGTA supplemented with either 5 mM EGTA, 3 mM CaCl(2), 3 mM BaCl(2), or 3 mM SrCl(2). Beads were washed 3 times in their incubation buffers and then analyzed by SDS-PAGE (^1)and immunoblotting. For the titration of the Sr concentration dependence of syntaxin binding, the assays were performed similarly except that EGTA and Sr were added at the indicated final concentrations and that the reaction buffer contained 0.4% Triton X-100. All experimental results were confirmed in at least three independent experiments. Curve-fitting and binding constant calculations were performed using GraphPad-InPlot software.

Caand Sr-dependent Phospholipid and Syntaxin Binding Measurements to Purified Bovine Brain Synaptotagmin I

Synaptotagmin I was purified from bovine brain (Hata et al., 1993). Mild proteolysis of purified synaptotagmin I in order to cleave it at a single site close to the membrane region was performed by incubating synaptotagmin I (0.2 g/liter) in 50 mM Tris-HCl, pH 7.7, with 0.5 mg/liter trypsin for 30 min at room temperature. The reaction was stopped by addition of phenylmethylsulfonyl fluoride (50 mg/liter), and the cleaved synaptotagmin I was incubated overnight with monoclonal antibody Cl41.1 that reacts with the first C(2) domain of synaptotagmin I (kind gift of Dr. R. Jahn, Yale University) at 4 °C. The cytoplasmic synaptotagmin fragment was immobilized by the addition of Protein A-Sepharose; uncleaved synaptotagmin I was immobilized similarly. Phospholipid and syntaxin binding assays were carried out as described above and in previous studies (Davletov and Südhof, 1993; Ullrich et al., 1994; Li et al., 1995). Background binding was determined as phospholipid bound in the absence of Ca and as Ca-dependent binding to columns lacking antibody for the Protein A-beads.

Miscellaneous Procedures

SDS-PAGE was performed according to Laemmli(1970); molecular weights of proteins were estimated using prestained SDS-PAGE standards (Bio-Rad). Protein concentrations were estimated with a Coomassie Blue-based assay kit (Bio-Rad) or the BCA kit (Pierce). Immunoblotting with synaptotagmin I and syntaxin I antibodies was performed as described previously (Perin et al., 1991b; Li et al., 1995).


RESULTS

The First C(2)Domains of Different Synaptotagmins Exhibit a Similar CaConcentration Dependence for Phospholipid Binding

Previous studies revealed that the first C(2) domains of synaptotagmins I, II, III, V, and VII bound phospholipids as a function of Ca, whereas those of synaptotagmins IV, VI, and VIII were inactive (Ullrich et al., 1994; Li et al., 1995). To evaluate quantitatively how similar the Ca dependence of different synaptotagmins is, we performed a series of Cadependent phospholipid binding measurements ( Fig. 1and Table 1). All synaptotagmins exhibited very similar Ca affinities with this assay, with the EC values observed ranging from 2.6 µM for synaptotagmin VII to 5.0 µM for synaptotagmin II. Thus, in spite of their considerable sequence divergence, the first C(2) domains of different synaptotagmins have similar Ca affinities for phospholipid binding. This observation was also confirmed with the C(2) domain of protein kinase Cbeta which had a Ca affinity in the same range (data not shown).


Figure 1: Phospholipid binding to the first C(2) domains of synaptotagmins I, II, III, V, and VII as a function of Ca (top panel) or Sr (bottom panel). Recombinant GST-synaptotagmin fusion proteins containing the first C(2) domains of synaptotagmin I, II, III, V, and VII were immobilized on glutathione agarose beads and incubated with radiolabeled phospholipids at the concentrations of divalent cations indicated. Beads were washed, and binding was determined by scintillation counting and normalized to the range of 0% to 100% to make the results with different recombinant proteins comparable.





The SrConcentration Dependence of Phospholipid Binding Differs between the First C(2)Domains of Different Synaptotagmins

Ca and Sr have similar properties, and previous studies demonstrated that high concentrations of Ba and Sr but not Mg trigger phospholipid binding to the first C(2) domain of synaptotagmin I (Davletov and Südhof, 1993). To determine whether the similar Ca affinity of different C(2) domains is accompanied by a similar Sr affinity, we studied the Sr concentration dependence of phospholipid binding. Surprisingly, major differences between different synaptotagmins were noted, with synaptotagmin III, the most sensitive synaptotagmin, exhibiting an EC of 23 µM for Sr whereas synaptotagmins I and II, the presumptive Ca sensors for the fast component, required much higher Sr concentrations (Fig. 1; Table 1). Thus, the first C(2) domains of different synaptotagmins have similar Ca-dependent phospholipid binding activities but distinct Sr-dependent phospholipid binding activities.

Ca- and Sr-dependent Phospholipid Binding by Native Synaptotagmin I Mirrors the Properties of the First C(2)Domain

To determine the Ca- and Sr-dependent phospholipid binding properties of native synaptotagmin I, we purified synaptotagmin I from bovine brain cortex (Hata et al., 1993). The purified protein was immobilized on Protein A-beads using a monoclonal antibody, and Ca- and Sr-dependent binding of radiolabeled phospholipids to the immobilized protein were measured (Fig. 2). A high background of Ca-independent binding was observed with full-length synaptotagmin I, probably due to nonspecific interactions of the phospholipids with the hydrophobic transmembrane domain of synaptotagmin I. To circumvent this problem, we took advantage of the hypersensitive proteolytic site in synaptotagmin I that cleaves it approximately at residue 120 (Perin et al., 1991b). Purified synaptotagmin I was cleaved with low concentrations of trypsin, and its soluble cytoplasmic domain containing both C(2) domains was then immobilized on Protein A using the monoclonal antibody (Fig. 2).


Figure 2: Immobilization of purified full-length synaptotagmin I (lane A) or of the cytoplasmic domains of synaptotagmin I produced by proteolytic cleavage (lane B) on Protein A-Sepharose via a synaptotagmin I-specific monoclonal antibody (Cl41.1). The figure shows a Coomassie Blue-stained SDS gel of the material used for divalent cation-dependent phospholipid binding measurements to brain synaptotagmin I (Fig. 3). Intact synaptotagmin I migrates on the gel as fuzzy monomeric and dimeric bands because it is glycosylated and multimerizes via a sequence close to the transmembrane region (Perin et al., 1991b). Glycosylated sequences and the multimerization domain are not present in the proteolytic cytoplasmic fragment immobilized by the antibody (lane B) which consists of the two C(2) domains and the C terminus. Filled arrows indicated positions of the different forms of synaptotagmin I. Open arrows point to the heavy and light chain of the monoclonal antibody used for the immobilization. Numbers on the left indicate positions of molecular weight markers.




Figure 3: Phospholipid binding to the immobilized cytoplasmic domains of synaptotagmin I from bovine brain as a function of Ca (left panel), Sr (middle panel), and Ba (right panel). In each panel, filled circles demonstrate phospholipid binding obtained in the presence of synaptotagmin I, and open circles background binding obtained in the absence of immobilizing antibody to control for nonspecific cation-dependent phospholipid precipitation. Numbers in the left upper corner of each panel give the binding constants calculated for the experiment shown, half-maximal binding (EC), and apparent Hill coefficients (n). Bars indicate the standard errors of the mean from triplicate determinations. The experiment was repeated three times with similar results.



Phospholipid binding to the immobilized cytoplasmic fragment of native synaptotagmin I was measured as a function of the Ca, Sr, and Ba concentrations (Fig. 3). Native synaptotagmin I containing both C(2) domains had an apparent affinity for Ca-dependent phospholipid binding that was virtually identical with that observed for the GST-C(2) domain fusion protein (EC = 5.4 µM), and its Hill coefficient was also very similar. Thus, the Ca/phospholipid binding properties of the single first C(2) domain of synaptotagmin I accurately reflect the properties of the cytoplasmic domain of the native protein. Both Sr and Ba were found to mediate phospholipid binding to native synaptotagmin I but required much higher divalent cation concentrations than Ca (Fig. 3). The Sr concentration dependence observed was similar to that for the recombinant first C(2) domain ( Fig. 1and Fig. 3). Furthermore, in studies measuring the Ca dependence of syntaxin binding of native synaptotagmin I, a Ca dependence was found that was indistinguishable from that of the first C(2) domain (data not shown; Li et al., 1995). Together these data suggest that the cation-activated phospholipid binding properties of synaptotagmin can be completely accounted for by the activities of its first C(2) domain.

SrActivates Syntaxin Binding by Synaptotagmin III but Not Other Synaptotagmins

Ca triggers the binding of the first C(2) domains of synaptotagmins to syntaxins (Li et al., 1995). The Ca affinity of synaptotagmins I and II for syntaxin binding is much lower than that of synaptotagmins III and VII, supporting the hypothesis that synaptotagmins I and II are the major low affinity Ca sensor for the fast component of release (Geppert et al., 1994). To test if the differences between synaptotagmins in Ca- and Sr-dependent phospholipid binding translate into differences in the ability of these cations to mediate syntaxin binding, we tested the ability of various divalent cations to activate binding of syntaxin I in total rat brain homogenates to the immobilized first C(2) domains of Ca-dependent synaptotagmins (Fig. 4). Surprisingly, Sr or Ba activated only syntaxin binding by synaptotagmin III but not by any other synaptotagmin in spite of the effectiveness of Ca for all synaptotagmins. Sr triggered binding of syntaxin to synaptotagmin III at micromolar concentrations, whereas no binding of synaptotagmin I to syntaxin I was observed even at 5 mM Sr (Fig. 5). Thus, Sr has a very selective effect on synaptotagmin III as compared to other synaptotagmins. The inability of Sr to mediate syntaxin binding to synaptotagmin I agrees well with its inability to trigger the fast component of release (Goda and Stevens, 1994).


Figure 4: Ability of Ca, Sr, and Ba to trigger syntaxin binding to the first C(2) domains of synaptotagmins. Purified GST-synaptotagmin fusion proteins containing the first C(2) domains of the indicated synaptotagmins were immobilized on glutathione agarose beads and incubated with solubilized brain homogenates in the presence of the indicated divalent cations as described under ``Experimental Procedures.'' Beads were washed in the same buffers, and syntaxin bound was determined by immunoblotting.




Figure 5: Concentration dependence of Sr-activated syntaxin binding to the first C(2) domains of synaptotagmins I and III. Binding measurements to the immobilized GST-synaptotagmin fusion proteins were carried out in the presence of the indicated Sr concentrations as described in Fig. 4. Syntaxin binding was measured by SDS-PAGE and immunoblotting for syntaxin I.




DISCUSSION

Previous studies demonstrated that synaptotagmin I has the Ca binding characteristics of the Ca sensor for fast Ca-triggered synaptic vesicle exocytosis (Brose et al., 1992; Davletov and Südhof, 1993, 1994; Chapman and Jahn, 1994; Li et al., 1995), and that, in its absence, fast Ca-dependent release in hippocampal synapses is impaired (Geppert et al., 1994). These studies suggested that synaptotagmin I serves as the major Ca sensor in the very last step of synaptic vesicle exocytosis in the hippocampus but posed puzzling new questions. First, why are other synaptotagmins that are colocalized with synaptotagmin I, especially synaptotagmin III, not redundant with synaptotagmin I? Second, what is the nature of the slow component of Ca-dependent release that is unchanged in the synaptotagmin I mutants? Third, how does synaptotagmin I act in release? Although we are unable to answer these questions definitively at present, results from the current study support a model whereby in hippocampal synapses synaptotagmins I and III serve as distinct Ca sensors for the fast and slow components, respectively, of Ca-dependent neurotransmitter release.

The major defining characteristics of the slow component of neurotransmitter release is that it is triggered by Sr instead of Ca whereas the fast component is not (Goda and Stevens, 1994). In the current study we provide evidence that synaptotagmins exhibit distinct Sr binding activities. We show that the first C(2) domains of all Ca-dependent synaptotagmins are activated to bind phospholipids at similar Ca concentrations but that they differ markedly in their response to Sr. Synaptotagmins I and II required high Sr concentrations for binding, whereas synaptotagmin III bound phospholipids at relatively low Sr concentrations. More decisively, synaptotagmin III was the only synaptotagmin tested in which Sr can substitute for Ca in activating syntaxin binding, with a concentration dependence that is similar to that of phospholipid binding.

Together with the previous demonstration that the apparent Ca affinity of synaptotagmin III is much higher than that of synaptotagmin I as measured by syntaxin binding (Li et al., 1995), these data show that synaptotagmin III has the requisite Ca binding characteristics and specificity of the Ca sensor for the slow component of neurotransmitter release. Furthermore, the new data add to the complexity of C(2) domains by demonstrating that in spite of the similarity in the Ca dependence of phospholipid binding between different C(2) domains, these differ dramatically in their Sr-activated properties. In addition, our results indicate that the currently known properties of synaptotagmins are insufficient to explain the functions of these proteins as Ca sensors in release. Besides the puzzle of why a synapse should have separable fast and slow release components of Ca-dependent release, the fact remains that synaptotagmin III cannot substitute for synaptotagmin I, even though synaptotagmin III requires lower Ca concentrations than I. Thus, the syntaxin interaction of synaptotagmins in itself is not sufficient to explain their in vivo functions. Clearly, synaptotagmins I and III must not only have distinct functions in the nerve terminal but also different Ca-dependent targets that have not yet been identified.

Almost all Ca-dependent activities of synaptotagmins were determined using recombinant proteins. In these studies, only the first C(2) domain shows Ca-dependent activities, and, at least for the currently known Ca-activated targets of synaptotagmins, phospholipids, and syntaxins, the second C(2) domain is not regulated by Ca. Using recombinant proteins, it was suggested that the second C(2) domain binds phospholipids independent of Ca and that it acts synergistically with the first C(2) domain (Damer and Creutz, 1994). To gain a better understanding of how the activities of recombinant single or double C(2) domains relate to those of native synaptotagmins and to determine the behavior of the native protein, we purified synaptotagmin I from brain and measured its Ca-, Sr-, and Ba-dependent phospholipid binding activities. These experiments showed that the native protein and the isolated recombinant first C(2) domain have very similar Ca-dependent properties, indicating that the properties of the first C(2) domain fully account for the known Ca-dependent properties of native synaptotagmin.

Native synaptotagmin showed little Ca-independent phospholipid binding properties or synergistic effect of two versus one C(2) domain. Thus, in the native protein, the second C(2) domain does not appear to contribute to Ca-dependent or Ca-independent phospholipid binding. However, the second C(2) domains of synaptotagmins bind AP-2 (Zhang et al., 1994; Li et al., 1995) and polyinositol phosphates (Fukuda et al., 1994) in a manner that does not require Ca, although this does not necessarily mean that the second C(2) domain is not a Ca binding domain. Future studies will have to determine whether the second C(2) domains of synaptotagmins are Ca binding domains for unknown targets, and whether they functionally cooperate with the first C(2) domains or if the two C(2) domains serve completely separate functions (for example in exo- and endocytosis). Regardless of these limitations in our understanding, synaptotagmins have already now emerged as a focus in Ca-regulated membrane traffic with complex Ca-dependent properties that differ between isoforms.


FOOTNOTES

*
This study was supported by the Perot Family Foundation. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Present address: Dept. of Biochemistry, Imperial College of Science, Technology and Medicine, London SW7 2AY, UK.

To whom correspondence should be addressed.

(^1)
The abbreviations used are: PAGE, polyacrylamide gel electrophoresis; GST, glutathione S-transferase.


ACKNOWLEDGEMENTS

We would like to thank I. Leznicki and A. Roth for excellent technical support and Dr. Nils Brose for his help with the Ca/Sr-dependent syntaxin binding measurements.


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