A Gain-of-Function Mutation in Synaptotagmin-1 Reveals a Critical Role of Ca 2+ -Dependent Soluble N -Ethylmaleimide-Sensitive Factor Attachment Protein Receptor Complex Binding in Synaptic Exocytosis

“After SDS-PAGE, synaptotagmin-1 associated with heavy liposome was quantified by immunoblotting using Cl41.”

“…vesicle exocytosis in a reaction that is catalyzed by soluble N-ethylmaleimidesensitive factor attachment protein receptor (SNARE) complex assembly, and is triggered by Ca 2 binding to the two C2 domains of the synaptic vesicle protein synaptotagmin-1 (Südhof, 2004; Brunger, 2005; Sorensen, 2005).”

“…to form complexes with both phospholipids and with SNARE complexes (Bennett et al., 1992; Yoshida et al., 1992; Davletov and Südhof, 1993; Sollner et al., 1993; Chapman and Jahn, 1994; Chapman et al., 1995; Li et al., 1995; Banerjee et al., 1996; Kee and Scheller, 1996; Schiavo et al.,…”

“Moreover, overexpression of many other proteins also alters fusion pore dynamics in transfected cells, suggesting that sheer overexpression of a protein can change the membrane tension and thereby secondarily affect fusion pores (for review, see Jahn et al., 2003).”

“In the synaptotagmin-1 mutant synapses examined here, just as in the synaptotagmin-1-deficient synapses we studied previously (Geppert et al., 1994; Maximov and Süd- hof, 2005), the effect was restricted to fast synchronous release, and no significant effect on asynchronous release during stimulus…”

“In vitro, Ca 2 binding causes synaptotagmin-1 to form complexes with both phospholipids and with SNARE complexes (Bennett et al., 1992; Yoshida et al., 1992; Davletov and Südhof, 1993; Sollner et al., 1993; Chapman and Jahn, 1994; Chapman et al., 1995; Li et al., 1995; Banerjee et al., 1996; Kee…”

“1 (10 l of ascite) attached to protein A- or G-Sepharose beads (10 l; Amersham Biosciences, Piscataway, NJ) in a 1 ml volume of immunoprecipitation buffer (50 mM HEPES-NaOH, pH 6.8, 0.1 M NaCl, 4 mM EGTA, 2 mM MgCl2, 0.5% Triton X-100 in the presence of different concentrations of free Ca 2 ).”

“In vivo, mutations that selectively decrease synaptotagmin-1 phospholipid binding cause a corresponding decrease of release (Fernandez-Chacon et al., 2001; Li et al., 2006), whereas mutations that enhance phospholipid binding produce an increase of release (Rhee et al., 2005).”

“…et al., 1992; Yoshida et al., 1992; Davletov and Südhof, 1993; Sollner et al., 1993; Chapman and Jahn, 1994; Chapman et al., 1995; Li et al., 1995; Banerjee et al., 1996; Kee and Scheller, 1996; Schiavo et al., 1997; Fernandez et al., 2001; Wang et al., 2001; Zhang et al., 2002;…”

“The fact that complexin, a protein that only binds to assembled SNARE complexes (McMahon et al., 1995), is coimmunoprecipitated with the SNARE antibodies in these experiments demonstrates that synaptotagmin-1 binding to SNARE complexes is studied.”

“…with transfected cells and/or recombinant proteins, and often presented contradictory conclusions (Bennett et al., 1992; Yoshida et al., 1992; Davletov and Südhof, 1993; Sollner et al., 1993; Chapman and Jahn, 1994; Chapman et al., 1995; Li et al., 1995; Banerjee et al., 1996; Kee and…”

“Only recent results uncovered the extreme sensitivity of the synaptotagmin/SNARE interaction to ionic strength (Tang et al., 2006), and thus we were not previously able to analyze the mutant mice properly for changes in SNARE binding.”

“…et al., 1993; Chapman and Jahn, 1994; Chapman et al., 1995; Li et al., 1995; Banerjee et al., 1996; Kee and Scheller, 1996; Schiavo et al., 1997; Fernandez et al., 2001; Wang et al., 2001; Zhang et al., 2002; Rickman and Davletov, 2003; Shin et al., 2003; Bai et al., 2004; Rickman et al.,…”

“…and with SNARE complexes (Bennett et al., 1992; Yoshida et al., 1992; Davletov and Südhof, 1993; Sollner et al., 1993; Chapman and Jahn, 1994; Chapman et al., 1995; Li et al., 1995; Banerjee et al., 1996; Kee and Scheller, 1996; Schiavo et al., 1997; Fernandez et al., 2001; Rickman and…”

“With Ca 2 influx triggered by action potentials, this coupling could occur by simultaneous binding of a single synaptotagmin-1 molecule to both SNAREs and phospholipids, or by separate binding of the two synaptotagmin-1 molecules in a constitutive synaptotagmin-1 dimer (Perin et al., 1991) to SNAREs or phospholipids.”

“…Südhof, 1993; Sollner et al., 1993; Chapman and Jahn, 1994; Chapman et al., 1995; Li et al., 1995; Banerjee et al., 1996; Kee and Scheller, 1996; Schiavo et al., 1997; Fernandez et al., 2001; Wang et al., 2001; Zhang et al., 2002; Rickman and Davletov, 2003; Shin et al., 2003; Bai et al.,…”

“(2) The D232N and D238N mutations are very similar [both neutralize a negatively charged amino acid in the second Ca 2 -binding loop (and the third top loop overall) (Ubach et al., 1998) of the C2A domain of synaptotagmin-1], and the amino acids involved are only five residues apart.”

“…and Jahn, 1994; Chapman et al., 1995; Li et al., 1995; Banerjee et al., 1996; Kee and Scheller, 1996; Schiavo et al., 1997; Fernandez et al., 2001; Wang et al., 2001; Zhang et al., 2002; Rickman and Davletov, 2003; Shin et al., 2003; Bai et al., 2004; Rickman et al., 2004; Bowen et al., 2005).”

“In vitro, Ca 2 binding causes synaptotagmin-1 to form complexes with both phospholipids and with SNARE complexes (Bennett et al., 1992; Yoshida et al., 1992; Davletov and Südhof, 1993; Sollner et al., 1993; Chapman and Jahn, 1994; Chapman et al., 1995; Li et al., 1995; Banerjee et al., 1996; Kee…”

“…and Jahn, 1994; Chapman et al., 1995; Li et al., 1995; Banerjee et al., 1996; Kee and Scheller, 1996; Schiavo et al., 1997; Fernandez et al., 2001; Wang et al., 2001; Zhang et al., 2002; Rickman and Davletov, 2003; Shin et al., 2003; Bai et al., 2004; Rickman et al., 2004; Bowen et al., 2005).”

“For the hippocampal neuronal cultures, offspring from D232N/D238N-mutant compound heterozygotes were used.”

“We thank I. Leznicki, J. Mitchell, L. Fan, N. Hamlin, I. Herfort, and A. Roth for excellent technical assistance and Dr. Jose Rizo for insightful discussions.”

“In vitro, Ca 2 binding causes synaptotagmin-1 to form complexes with both phospholipids and with SNARE complexes (Bennett et al., 1992; Yoshida et al., 1992; Davletov and Südhof, 1993; Sollner et al., 1993; Chapman and Jahn, 1994; Chapman et al., 1995; Li et al., 1995; Banerjee et al., 1996; Kee and Scheller, 1996; Sc…”

“In vivo, mutations that selectively decrease synaptotagmin-1 phospholipid binding cause a corresponding decrease of release (Fernandez-Chacon et al., 2001; Li et al., 2006), whereas mutations that enhance phospholipid binding produce an increase of release (Rhee et al., 2005).”

“…et al., 1992; Davletov and Südhof, 1993; Sollner et al., 1993; Chapman and Jahn, 1994; Chapman et al., 1995; Li et al., 1995; Banerjee et al., 1996; Kee and Scheller, 1996; Schiavo et al., 1997; Fernandez et al., 2001; Wang et al., 2001; Zhang et al., 2002; Rickman and Davletov, 2003; Shin et…”

“These data are in agreement with rescue studies in autapses (Stevens and Sullivan, 2003).”

“…et al., 1992; Yoshida et al., 1992; Davletov and Südhof, 1993; Sollner et al., 1993; Chapman and Jahn, 1994; Chapman et al., 1995; Li et al., 1995; Banerjee et al., 1996; Kee and Scheller, 1996; Schiavo et al., 1997; Fernandez et al., 2001; Rickman and Davletov, 2003; Rickman et al., 2004; Bowen…”

“…binding assays were performed using heavy liposomes with a synaptic phospho- lipid composition and phosphatidylinositol phosphate (PIP) and phosphatidylinositol bisphosphate (PIP2) as indicated previously (Deutsch and Kelly, 1981; Benfenati et al., 1989; Rhee et al., 2005; Li et al., 2006).”

“However, Sr 2 -triggered release is also mostly asynchronous, limiting its use in probing synaptotagmin-1/SNARE interactions in release (Xu-Friedman and Regehr, 2000).”

“(1) Biophysical studies previously demonstrated that the D232N and D238N mutations selectively but differentially alter intrinsic Ca2 binding by the C2A domain but have no significant effect on its atomic structure (Fernandez-Chacon et al. 2002).”

“…binding assays were performed using heavy liposomes with a synaptic phospho- lipid composition and phosphatidylinositol phosphate (PIP) and phosphatidylinositol bisphosphate (PIP2) as indicated previously (Deutsch and Kelly, 1981; Benfenati et al., 1989; Rhee et al., 2005; Li et al., 2006).”

“…and Jahn, 1994; Chapman et al., 1995; Li et al., 1995; Banerjee et al., 1996; Kee and Scheller, 1996; Schiavo et al., 1997; Fernandez et al., 2001; Wang et al., 2001; Zhang et al., 2002; Rickman and Davletov, 2003; Shin et al., 2003; Bai et al., 2004; Rickman et al., 2004; Bowen et al., 2005).”

“…differences between the native and recombinant proteins are probably not attributable to a modification of native synaptotagmin-1, but rather caused by the contamination of recombinant synaptotagmin-1 with bacterial acidic small molecules that is extremely difficult to remove (Ubach et al., 2001).”

“…binding assays were performed using heavy liposomes with a synaptic phospho- lipid composition and phosphatidylinositol phosphate (PIP) and phosphatidylinositol bisphosphate (PIP2) as indicated previously (Deutsch and Kelly, 1981; Benfenati et al., 1989; Rhee et al., 2005; Li et al., 2006).”

“…and Jahn, 1994; Chapman et al., 1995; Li et al., 1995; Banerjee et al., 1996; Kee and Scheller, 1996; Schiavo et al., 1997; Fernandez et al., 2001; Wang et al., 2001; Zhang et al., 2002; Rickman and Davletov, 2003; Shin et al., 2003; Bai et al., 2004; Rickman et al., 2004; Bowen et al., 2005).”

“…and Jahn, 1994; Chapman et al., 1995; Li et al., 1995; Banerjee et al., 1996; Kee and Scheller, 1996; Schiavo et al., 1997; Fernandez et al., 2001; Wang et al., 2001; Zhang et al., 2002; Rickman and Davletov, 2003; Shin et al., 2003; Bai et al., 2004; Rickman et al., 2004; Bowen et al., 2005).”

“…by Syt1 (green; Pang et al., 2006); the R233Q substitution that greatly decreases the apparent Ca2+affinity of Syt1 during phospholipid binding (blue; Fernandez-Chacon et al., 2001); and the D238N substitution that modestly decreases the apparent Ca2+-affinity of Syt1 (red; Pang et al., 2006).”

“Striatal neurons that depend on synaptotagmin-9 for fast release also express synaptotagmin-1 and -2 (Figure 5; Pang et al., 2006b), and deletion of synaptotagmin-1 or -2 in these neurons impairs fast release, consistent with a role in Ca2+ triggering of release (see Figure 7E for synaptotagmin-1;…”

“This behavior was unexpected, because Ca 2+ -dependent binding of the synaptotagmin-1 C 2 domains to liposomes shows a strong preference for PIP 2 because of its higher negative charg”

“However, there are discrepancies in PIP 2 -induced membrane binding between native and recombinant synaptotagmi”

“Ca2+ induces binding of the two Syt1 C2-domains to phospholipid membranes and to assembled SNARE-complexes; both actions contribute to triggering exocytosis (Fernandez-Chacon et al, 2001; Rhee et al., 2005; Pang et al., 2006).”

“, 2007) whereas mutations that enhance SNARE binding facilitate Ca-triggered synaptic vesicle exocytosis in neurons (Pang et al., 2006).”

“Moreover, previous studies revealed that the C2A domain significantly contributes to SNARE binding by Syt1 (12, 17).”

“The excessive redundancy of the Syt orthologs is either due to critical nature of Syt in regulated exocytosis (38) or as part of an intricate addressing system that allows for the temporal and spatial fine-tuning of fusion between specific vesicles and specific membrane targets in various cell types (39).”

“This interaction is believed to position SYT on the prefusion SNARE complexes to trigger rapid exocytosis in response to Ca (20, 22).”

“Syt1 is known to bind to assembled SNARE complexes in both a Ca-dependent and a Ca-independent manner [54], and we show here that Syt7 also binds to SNARE complexes (Fig 6), and that Syt1 can promote SNARE complex assembly in the presence of complexin (Figs 7 and 8).”

“…2011), with some binding activity being sensitive to Ca 2 and some being apparently Ca 2 -independent (Sollner et al., 1993; Chapman et al., 1995; Gerona et al., 2000; Zhang et al., 2002; Rickman and Davletov, 2003; Bai et al., 2004b; Rickman et al., 2004b; Pang et al., 2006; Lynch et al., 2008).”

“Particularly relevant is the observation that a C2A Ca2 ligand mutation D232N increases the Ca2 sensitivity of neurotransmitter release and the Ca2 -dependent binding of synaptotagmin 1 to SNAREs (Stevens and Sullivan, 2003; Pang et al., 2006), which is in accord with our conclusion that C2A contains a Ca2 -dependent…”

“In contrast, a D229E substitution in the Drosophila C2A domain reduced vesicle fusion (Striegel et al., 2012), whereas neutralization of D232 (D232N) in mice enhanced synaptic transmission (Fernández-Chacón et al., 2002; Stevens and Sullivan, 2003; Pang et al., 2006a).”

“Calcium binding to syt1 promotes its interaction with the t-SNAREs, syntaxin-1 and SNAP-25, and to phospholipids, thereby facilitating synaptic vesicle and presynaptic membrane fusion (Davis et al., 1999; Bai et al., 2004; Pang et al., 2006).”

“Indeed, C2A D3N mutants that include D4N enhanced synchronous fusion as shown by an increase in the apparent Ca 2 affinity of release (Stevens and Sullivan, 2003; Pang et al., 2006; Yoshihara et al., 2010).”

“A great deal of functional mutagenesis work has been undertaken to elucidate the mechanism by which Syt I triggers exocytosis, and to determine the specific roles of Ca-triggered binding of Syt I to PS and SNAREs (Fernandez-Chacon et al., 2001; Fernandez-Chacon et al., 2002; Mackler et al., 2002; Stevens and Sullivan,…”

“In animals bearing these mutations, presynaptic release probability was changed, indicating that synaptotagmin acts as a Ca2 regulator of synaptic vesicle exocytosis (Fernandez-Chacon et al.,. 2001; Pang et al., 2006).”

“…D166/E170A, D166A, and D51/E52/ E55/D166A on evoked release, and no effect of D186/193A, are complementary to those obtained previously by mutation of syt-1 (Brewer et al., 2015; Zhou et al., 2015) and overall support the physiological relevance of syt-1:SNARE interactions (Pang et al., 2006b).”

“© 2010 by The American Society for Cell Biology 2821 mutations were subsequently reported to have very different effects on lipid and SNARE binding and to alter synaptic transmission quite markedly (Pang et al., 2006).”

“…increased the mEJP frequency at several other synapses, including excitatory and inhibitory synapses between cultured cortical neurons, the calyx of Held, and mature neuromuscular junctions in Drosophila and mice (DiAntonio and Schwarz, 1994; Pang et al., 2006a,c) (but see Marek and Davis, 2002).”

“The latter finding extend quantitatively the results of previous studies showing an association between SYT2 and GABAergic neurons (Pang et al., 2006a; Fox and Sanes, 2007).”

“…literature—to change the intracellular calcium-dependence of exocytosis triggering are mutations in synaptotagmins and SNAP-25 (Sorensen et al., 2003a; Wang et al., 2003; Chieregatti et al., 2004; Rhee et al., 2005; Wang et al., 2005; Nagy et al., 2006; Pang et al., 2006; Sorensen et al., 2006).”

“…studies suggesting an important role for this interaction during fusion (Bennett et al., 1992; Brewer et al., 2015; Chicka et al., 2008; Choi et al., 2010; Li et al., 1995; Littleton et al., 2001; Lynch et al., 2008; Lyubimov et al., 2016; Pang et al., 2006; Schupp et al., 2016; Zhou et al., 2015).”

“Complexin is a small soluble protein that controls (activates or suppresses) the trigger-release and spontaneous release (Fernández-Chacón et al., 2001; Pang et al., 2006; Mohrmann et al., 2015; Yu et al., 2018).”

“These mutations included: i) a D232N substitution in the C2A domain (referred to as DN mutation) that abolishes Ca2+ binding to two of its three Ca2+-binding sites (10) and was described to enhance neurotransmitter release as well as SNARE complex binding (53); ii) an R398Q,R399Q mutation at the bottom of the C2B doma…”

“For example, neutralization of acidic residues in the C2A domain of syt1 abolish the Ca2+-dependent lipid-binding activity of the isolated domain in vitro but either had no effect or enhanced the apparent Ca2+ affinity of the full-length protein during synaptic transmission in neurons (Fernandez-Chacon et al., 2002; R…”

“This observation contrasts with the increase in mini frequency observed at Synaptotagmin mutant larval NMJs in Drosophila and in most mouse cultures, changes that have been attributed to loss of a fusion clamp normally provided by Synaptotagmin [20,21,22].”

“…clearly disrupt function ( Nishiki and Augustine, 2004), while similar mutations in C2A have been reported to either have no effect (Fernández-Chacón et al., 2002), to be gain of function (Stevens and Sullivan, 2003; Pang et al., 2006b), or to cause partial loss of function (Shin et al., 2009).”

“…bind Ca 2 ions and contain many positively and negatively charged amino acids for potential electrostatic interactions with t-SNAREs, PI(4,5)P2, and other interactors (Fernandez et al., 2001; Pang et al., 2006; Chapman, 2008; Pang and Südhof, 2010; Jahn and Fasshauer, 2012; Park et al., 2012).”

“Furthermore, point mutations in Syt1 that result in either reduction of Ca2+ affinity or a decrease in phospholipid binding also selectively suppress the phasic component of evoked release (Pang et al., 2006; Fleidervish et al., 2010).”

“Furthermore, Ca2+ elevation could enhance vesicle recruitment via Ca2+-dependent synaptotagmin binding to the SNARE complex and phospholipids (Pang et al., 2006; Li et al., 2006) and, thus, partially overcome the RP deficiency in Rab3a(−) synapses.”

“promotes additional local lipid rearrangements via C2A domain–lipid interactions that culminate in rapid and efficient calcium-triggered synchronous vesicle fusion (Bhalla et al. 2006; Dai et al. 2007; Martens et al. 2007; Pang et al. 2006; Rettig and Neher 2002).”

“Synaptotagmins interact with SNARE complexes and phospholipids in a Ca 2+ -dependent manner, which in turns triggers synaptic fusion[61,65].”

“Polyclonal rabbit antibody against synaptotagmin-1 (V216, Perin et al., 1990; Pang et al., 2006; Bacaj et al., 2015).”

“Strong support for this conclusion has come from recent studies showing that changing the apparent Ca2+ affinity of the synaptotagmin/ phospholipid complex in vivo changes the Ca2+ sensitivity of release (Fernandez-Chacon et al., 2001;Pang et al., 2006).”

“Alternatively, the energy barrier reduction and increased spontaneous release rate in the D232N mutant do not come from electrostatic effects, but from increased Cadependent SNARE binding [37] at basal Ca levels.”

“Dans les études ultérieures, nous avons étendu cette analyse et introduit chez des souris knock out d’autres mutations ponctuelles, y compris une mutation (D232N) qui augmentait l’interaction Ca2+ dépendante de Syt1 avec les protéines SNARE (figure 7E ; Pang et al., 2006a).”

“Interestingly, synaptotagmin C2domains also binds to SNARE proteins in a manner controlled by its co-factor complexin [121, 123].”

“Complexin is a small soluble protein that controls (activates or suppresses) the trigger-release and spontaneous release (Fernández-Chacón et al., 2001; Pang et al., 2006; Mohrmann et al., 2015; Yu et al., 2018).”

“A number of studies have illustrated that Ca2+-triggered membrane fusion requires Ca2+dependent binding of synaptotagmins to SNARE proteins and membrane phospholipids (Zhang et al., 2002; Bhalla et al., 2006; Pang et al., 2006).”