Calcium-sensitive synaptotagmin 11-lipid interaction modulates exo-endocytosis

Domain: neuroscience
Published: 2025-12-16
Editorial outcome: major_revision

Abstract
Synaptotagmins (Syts) are the primary Ca2+ sensors for synaptic vesicle exocytosis, while most mammalian Syts are non-Ca2+-affinitive and play critical roles in neurotransmission and synaptic plasticity with unclear mechanisms. Here, we show that high-alkaline non-Ca2+-binding Syt11 exhibits higher affinity for acidic phospholipids and Ca2+-inhibited liposome-binding, thereby competing with the Ca2+-binding Syt1. Physiological levels of Ca2+ eliminate this competition by promoting Ca2+-dependent membrane insertion of Syt1 while suppressing Syt11’s binding through electrostatic shielding of the membrane surface. Site-directed mutagenesis reveals a dual-regional lipid-binding mode (a lysine-rich motif for Ca2+-independent binding and Ca2+-binding loops for Ca2+-facilitation) for Syt1, and a redundant multi-point lipid-binding interface for Syt11. Consistent with the Ca2+-dependent competition, Syt11 inhibits both the early stages of exocytosis and endocytosis in neurons, while the maximal rate of exocytosis remains intact. This Ca2+-sensitivity of Syt11 proposes Syt1-Syt11 inter-switching in membrane-occupancy as a critical step precisely controlling exocytosis and endocytosis during synaptic transmission.

Introduction
Ca2+ is the key messenger triggering neural transmission, yet how Ca2+ signals are faithfully translated into transmission events is not fully understood. Synaptotagmins (Syts) are the primary Ca2+ sensors responsible for triggering synaptic vesicle exocytosis by forming fusion machinery with the soluble N-ethylmaleimide sensitive factor (NSF) attachment protein receptor (SNARE) complex. However, the molecular architecture and assembly mechanisms of this fusion machinery remain largely unknown1. Mammalian Syts constitute a large gene family with 17 members (Syt1–17), each sharing a conserved structural framework: (1) a luminal (or extracellular) domain; (2) a single-helix transmembrane domain (TMD, or membrane-anchoring sites in Syt17); (3) a juxtamembrane linker; and (4 and 5) two tandem C2 domains (C2A and C2B). These C2 domains bind 2–4 Ca2+ ions each through aspartate residues (D) in their loop regions. Notably, only 8 of them (Syt1, 2, 3, 5, 6, 7, 9, and 10) are canonical Ca2+-affinitive Syts (Ca+-Syts), while the remaining 9 isoforms are non-Ca2+-affinitive (Ca−-Syts) due to evolutionary mutations in these key aspartate or related residues, e.g., the aspartate-to-serine substitutions in the C2A domains of Syt4 and Syt112. Despite their lack of Ca2+-affinity, several Ca−-Syts are abundantly expressed in the central nervous system (CNS) and have been implicated in neurodegenerative and psychiatric disorders, e.g., Syt11 has been defined as a risk factor mediating the pathogenesis of Parkinson’s disease (PD) and schizophrenia3–6. However, whether and how these Ca−-Syts directly participate in vesicular exocytosis remain unclear.

Ca+-Syts, despite their distinct binding affinities, serve as the primary Ca2+ sensors initiating vesicular exocytosis and synaptic transmission7–9. They play critical roles in vesicle docking, priming, and fusion pore dynamics during exocytosis10–13, as well as the coupling of endocytosis to exocytosis during synaptic transmission. Our previous work has depicted a bidirectional regulatory role of Syt1 in promoting clathrin-mediated endocytosis (CME) but inhibiting activity-dependent bulk endocytosis during sustained synaptic activity14. Meanwhile, Syt3 has been reported to localize post-synaptically to drive the off-membrane trafficking of α-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid receptor (AMPAR) through Ca2+-dependent endocytosis15. In contrast, Ca−-Syts have been shown to function opposite to Ca+-Syts. Ca−-Syt4 has been shown to inhibit exocytosis and endocytosis16–18. We have also demonstrated that Syt11 exerts inhibitory effects on both CME and bulk endocytosis in dorsal root ganglion, hippocampal, and midbrain dopaminergic neurons4,19. However, the molecular basis and mechanisms underlying the inhibitory roles of Ca−-Syts remain poorly understood. Furthermore, whether and how Ca+-Syts and Ca−-Syts cooperate to regulate exocytosis and endocytosis during synaptic transmission remain virtually unknown.

The molecular mechanism by which Syt proteins regulate exocytosis and endocytosis remains a subject of debate1. In the case of Syt1, crystallographic studies have revealed its multiple distinct interaction interfaces with SNARE complex, including a conserved R-apex (residues R398/R399)20,21, which is essential for Syt1 to initiate Ca2+-dependent exocytosis. Syt1 also adopts a clamping conformation with SNARE to prevent spontaneous membrane fusion before Ca2+ influx22–24. Meanwhile, the interactions of Syt1 with phospholipids and Ca2+ are equally critical for vesicle docking, fusion initiation, and pore dilation25–27. Syt1 is proposed to bind acidic phosphatidylserine (PS) liposomes in a Ca2+-dependent manner28–31, while a conserved K-rich motif (also known as the polybasic patch) enables Ca2+-independent binding to phosphatidylinositol 4,5-bisphosphate (PIP2)32–34. Lipid-binding enhances the Ca2+ affinity of Syt135, forming a Syt-Ca2+-lipid complex that enables a deep penetration of the Ca2+-binding loops into phospholipids bilayer and causes the deforming of the plasma membrane to facilitate fusion36,37. While some hypotheses suggest that Syt-Ca2+-lipid interactions maybe more critical than Syt-SNARE interactions in exocytosis38. According to our model, the Ca2+-dependent membrane remodeling effects of Syt1 also provide a compelling explanation for its bidirectional role in endocytosis14. Therefore, elucidating the molecular basis of Syt-lipid interactions and their Ca2+-sensitivity is essential for understanding the dual roles of Syt proteins in coordinating exocytosis and endocytosis during neurotransmission.

In the present work, by using a combination of biochemical assays, in silico analyses, in vitro liposome experiments, and pHlourin imaging in primary cultured neurons, we show that the C2 domains of Ca+-Syts exhibit distinct dynamics in Ca2+/lipid binding, define the Ca2+-sensitive binding of Ca−-Syts to liposomes, and propose a Ca2+-dependent Ca+-Syt/Ca−-Syt inter-switch working model for the excitation-coupled exocytosis and endocytosis during synaptic transmission.

Results
Non-Ca2+-affinitive Syts display higher alkaline surfaces
The structures of all 34 rat Syt-C2 domains predicted by AlphaFold39,40 were used to estimate their lipid-binding potential. We found that the C2 domains of Ca+-Syts shared a common characteristic of highly basic surfaces, e.g., the lysine (K)-rich motifs in the C2B domains of Syt1 and Syt7, which is critical for establishing strong electrostatic interactions, thereby conferring high affinity for the acidic surface of the plasma membrane. Based on the full-length amino acid sequence similarity of Syts, the Ca−-Syts 12~17 were distinguished from Ca+-Syts, while the Ca−-Syts 4, 8, and 11 clustered with Ca+-Syts 1, 2, 5, and 7 (Fig. 1a), indicating that not all Ca−-Syts are evolved independently with Ca+-Syts. Thus, Syts 4, 8, and 11 are likely to exhibit greater molecular and functional consistencies rather than differentials with Ca+-Syts, except for Ca2+-affinity. Although the C2A domains of Ca+- and Ca−-Syts showed diverse isoelectric points (pIs), most C2B domains had a higher pI (>8.5), suggesting that the C2B domains of Ca−-Syts also exhibit highly alkaline, positively-charged surfaces (Fig. 1b). Consistent with this, the electrostatic potential mapping of C2 domains revealed that most C2B domains of both Ca+- and Ca−-Syts harbored a large region of high alkalinity (Fig. 1c, red region), centered around the K-rich motif. In contrast, such alkaline region was absent in the C2A domains.

The electrostatic profiles of Ca+- and Ca−-Syt C2 domains were largely similar when analyzed from the N- to C-terminus, except for a short fragment around residue-25 in the C2B domains where Ca+-Syts exhibited a negative and Ca−-Syts displayed a positive potential (Fig. 1d). Most C2A domains in both Ca+- and Ca−-Syts showed a negative electrostatic potential, though this potential was higher in Ca−-Syts compared to Ca+-Syts. In contrast, the C2B domains had a positive potential, which was also higher in Ca−-Syts (Fig. 1e). To further quantify these differences, we then analyzed the solvent-accessible surface area (SASA) of highly basic regions (defined as atoms with an electrostatic potential >75 mV). Most C2A domains had a smaller basic SASA than C2B domains, which was higher in Ca−-C2s than that in Ca+-C2s in average (Fig. 1f). Collectively, these findings indicate a high potential of Ca−-Syts for lipid interactions, which is even stronger than that of Ca+-Syts.

Syt11 binds acidic phospholipids with Ca2+-sensitivity
To investigate the phospholipid binding capabilities of Ca+- and Ca−-Syts, we purified Syt1 (a representative Ca+-Syt) and Syt11 (a representative Ca−-Syt) for the in vitro co-liposome precipitation (co-LP) analysis (Fig. 2a). The TMD and most of the juxtamembrane linker region were removed from both proteins to minimize their interference. We used two distinct lipid compositions: (1) the tubulation assay (TA) lipids, which contain a higher concentration of acidic phospholipids (45% PS + 5% PIP2), as used in our previous research14; and (2) the brain extract total (BET) lipids, which contain a lower concentration of acidic phospholipids (~10.6% PS + 1% PIP2). As a Ca2+ sensor, Syt1 exhibited a clear Ca2+-enhancement in binding to BET liposomes. However, when TA lipids were used, Ca2+ did not enhance the binding (Fig. 2b), suggesting that the interaction is predominantly electrostatic in nature and that the higher negative charge density of the TA membrane compensates for the Ca2+-promotion effect on Syt-lipid interactions. In contrast, Syt11, as a Ca−-Syt, bound to both TA and BET liposomes in the absence of Ca2+, and this binding was suppressed by increasing Ca2+ concentrations. The inhibitory effect became apparent at 0.1 mM Ca2+ and a complete blockage occurred at ~5 mM Ca2+. This Ca2+-dependent inhibition was not due to changes in protein stability or ionic strength, as Ca2+ did not alter Syt11’s behavior in the absence of liposomes, and higher concentrations of KCl (up to 100 mM) did not affect the binding as CaCl2 did (Fig. 2c). These findings validate the Ca2+-sensitive nature (facilitation vs inhibition) of Syt1/11–lipid interactions, most probably mediated by direct electrostatic forces.

An intermediate lipid composition (40% PS + 1.5% PIP2) was used in subsequent experiments to compensate the charge effect. Various lipid compositions have been used to study Syt–lipid interactions in vitro over the past 30 years. For instance, the TA lipids, which contain higher levels of PIP2, can attenuate the Ca2+-promotion effect of Syt1–lipid binding (Fig. 2b). In contrast, BET lipids, sourced from Avanti as whole brain lipids from porcine brains, contain ~10.6% PS but lack the specific presentation of the synaptic active zone (AZ). Meanwhile, human brain PS levels vary between 11.4 and 20.5%, depending on individual differences and brain regions41; and PIP2 in whole-cell lipids accounts for ~1%42,43. On the other hand, since both PS and PIP2 are predominantly located in the inner leaflet of the plasma membrane, their concentrations can reach 22.8-41% and ~2%, respectively. Furthermore, PIP2 is enriched in clusters at AZs, with local concentrations reaching up to ~5%44–46 or even higher47. Therefore, to maintain the potential Ca2+-promoting effects, the lipid composition of 40% PS and 1.5% PIP2 are used to approximate physiological relevance. To explore the molecular basis for the Ca2+-sensitivity of Syt-lipid interactions, the evolutionarily-substituted serine residue in the C2A domain of Syt11 was mutated back to aspartate (Syt11-S247D) to restore Ca2+-binding ability. Interestingly, Ca2+ failed to inhibit the liposome binding of the Syt11-S247D mutant, suggesting that the Ca2+-neutralized loop region facilitates lipid binding. Meanwhile, Ca2+ did not show promotion either, likely because the C2B domain of this mutant remains Ca2+-insensitive. On the contrary, the Syt1-D2N (D230/232/363/365 to asparagine) and D2E (D230/232/363/365 to glutamic acid) mutants, in which D residues in the C2AB were mutated to abolish Ca2+-affinity, replicated the Ca2+-inhibition in lipid binding like that observed in Ca−-Syts. Consistently, the Ca−-Syt4 also exhibited Ca2+-suppressed lipid-binding (Fig. 2d). Collectively, these results validate a conserved Ca2+-inhibition mechanism in the binding of Ca−-Syts to acidic phospholipids, and propose both facilitatory and inhibitory roles of Ca2+ in modulating Syt-lipid interactions.

The Ca2+-sensitive Syt1/11 competition for lipid binding
Given the opposing effects of Ca2+ on lipid-binding properties of Ca+- and Ca−-Syts, we next tested whether these proteins compete for lipid binding. Notably, we observed a dose-dependent competition between Syt1 and Syt11 for lipid binding in the absence of Ca2+. However, this competitive effect was abolished under 2 mM Ca2+ conditions, and Syt11 was mostly removed from liposomes by Ca2+ (Fig. 2e, f). This outcome indicates a dual effect of Ca2+ on Syt1 (facilitation) and Syt11 (inhibition). In contrast, the Syt11-S247D mutant restored the competition with Syt1 in liposome-binding to some extent in 2 mM Ca2+ conditions. This might be due to the Ca2+-facilitation of lipid-binding of the S247D mutant, although its C2B domain remains Ca2+-insensitive, which was supported by that the liposome attachment of S247D mutant was much stronger than that of wild type Syt11 (Fig. 2g). These findings also suggest that Ca2+-binding generates additional membrane attachment sites for Ca+-Syts, thereby reinforcing their Ca2+-independent lipid interactions. Thus, we propose that the competitive effect of Syt11 on Syt1-lipid binding can be effectively negated by Ca2+. Consistent with this, 20 μM Ca2+ was sufficient for Syt1 to overcome competition from Syt11 (Fig. 2h). Considering that the activation of voltage-gated calcium channels (VGCCs) induces a transient (lasting ~1–2 ms) local Ca2+ concentrations of ~10–100 μM within nano/microdomains13,48–50, our results reveal a fascinating inter-switch mechanism between Syt1 and Syt11 on membrane occupancy.

Lipid charge shielding by Ca2+ increases surface Zeta potential
Given the well-established Ca2+-insensitivity of Syt4 and Syt11, the universal Ca2+-inhibition effect observed here is unlikely attributable to the protein itself, but rather suggests a lipid-centered mechanism. We proposed that the Coulomb force between Syts and acidic lipids plays a central role in their interactions. The highly concentrated phosphate groups of PIP2 have been shown to facilitate Ca2+ chelation51, which may be responsible for the Ca2+-modulated Syt-membrane interactions. We performed electrophoretic light scattering (ELS) to measure the Zeta potential of 400-nm liposomes in different Ca2+ conditions (Fig. 3a). Strikingly, 0.05 mM CaCl2 was sufficient to increase the Zeta potential of the liposomes, indicating an effective shielding of negative charges of PIP2. This effect is not attributed to altered physical properties of the solution, such as conductivity or viscosity, because 10 mM KCl did not produce similar effect (Fig. 3b–d). These results are consistent with previous reports that high concentrations of Mg2+ inhibit Syt1-mediated liposome fusion38. Collectively, these findings suggest that the shielding of surface negative charges is a key contributor to the Ca2+-dependent inhibition of Syt-liposome interactions.

Double-regional interactions confer Ca2+-sensitive Syt1-membrane associations
The highly basic surface of C2 domains may provide a large interface for association with acidic membrane. The K-rich motif of Syt1 is composed of 4 consecutive lysine (K) residues in both the C2A and C2B domains. However, in the C2B domain, this motif can be extended to an 8-residue fragment containing ~6 lysine/arginine (K/R) residues, forming a conserved feature shared by both Ca+- and Ca−-Syts (Fig. 4a). To examine the role of these motifs in Ca2+-independent lipid binding, we mutated key residues in the K-rich motifs of Syt1-C2A and C2B domains and assessed their binding capabilities using co-LP assays (Fig. 4b). The Syt1-AKA (K189/190/191 to alanine in C2A) showed a minimal reduction in liposome-binding in Ca2+-free conditions, while Syt1-BKA (R322N-K324/325A in C2B) exhibited a dramatic decrease in binding. Furthermore, Syt1-ABKA (AKA+BKA), in which both motifs were mutated, completely lost its ability to bind liposomes. Importantly, Ca2+-dependent enhancement of lipid binding was retained in all mutants (Fig. 4c), suggesting a double-regional interaction (Ca2+-dependent and Ca2+-independent) between Syt1 and liposomes. The Ca2+-independent lipid-binding was mediated primarily by the K-rich motif in the C2B domain, but not the C2A domain, which is consistent with the electrostatic analyses of Ca+-Syts (Fig. 1). On the other hand, the Ca2+-dependent lipid-binding was mediated by Ca2+-binding loops in both C2A and C2B domains, independent of K-rich motifs (Figs. 2d, 4c). Collectively, these findings define both Ca2+-independent (through the K-rich motif in the C2B) and Ca2+-facilitated (Ca2+-binding loops in both C2 domains) Syt1-liposome interactions, highlighting the dual regulatory roles of Syt1 in lipid interactions.

Multi-regional interactions confer redundant binding of Syt11-C2B to the membrane
As for Ca−-Syt11, we found that the C2B domain, rather than the C2A domain, is responsible for liposome-binding and Ca2+-inhibition. No associations between the C2A and C2B domains were observed (Fig. 4d). Competition assay further demonstrated that only Syt11-C2B, but not Syt11-C2A, competes with Syt1 for liposome binding in Ca2+-free conditions (Fig. 4e), underscoring a central role of the C2B domain in Syt11-membrane interactions.

Strikingly, the mutations in the K-rich motif (K340N-R341N-K344/346A) of Syt11-C2B (Syt11-BKA) failed to abolish Ca2+-inhibition of its lipid-binding ability (Fig. 4f, g), suggesting additional binding interfaces in Syt11. Given that loop-1 and loop-3 are responsible for Ca2+-coordination and membrane insertion of the C2 domains in Ca+-Syt1, we next investigated analogous basic residues in the loop regions of Syt11-C2B, despite their lack of Ca2+-binding ability. Systematic deletion of loops-1, 2, and 3 individually or in combination revealed that none of the Syt11-C2B mutants, including Δloop1 (C2B: aa 319-325 to “GS”), Δloop2 (C2B: aa 348-357 to “GSG”), Δloop3 (C2B: aa 382-390 to “GGS”), or Δloop1+3 (Δloop1+Δloop3), showed abolished liposome-binding either (Fig. 4f, g), and all of them retained Ca2+-inhibition (Fig. 4g). These results suggest a redundant multi-regional interaction mode for Syt11-C2B in liposome binding.

To comprehensively define the membrane-binding mode of Syt11, we generated 14 Syt11-C2B mutants by permuting and combining the K-rich motif (Kmotifm) and 3 loop regions (BLp1m, BLp2m, and BLp3m). To preserve chemical properties of the protein, K/R/histidine (H) residues were mutated to N/glutamine (Q) instead of A, D, E, or glycine (G). These mutants were categorized into 3 groups: single-regional, double-regional, and triple-regional mutations. We found that the lipid-binding ability gently decreased but the Ca2+-inhibition remained in single-regional mutants (Fig. 4h). Some double-regional mutants showed a significant decrease in lipid-binding (Fig. 4i), while most triple-regional mutants lost binding capability entirely (Fig. 4j). This defines a redundant multi-regional membrane-binding hierarchy for Syt11: C2B K-rich motif (primary) > C2B loop-1 > C2B loop-3 ≈ C2B loop-2 ≫ C2A (negligible).

Keysites for Syt11-C2B-lipid binding and the mutations examined in this study are summarized in Fig. 4k. Notably, Ca+-Syt1 and Ca−-Syt11 share similar lipid-binding sites, except for a basic “KARH” fragment that confers sub-regional positive potential in loop-1 of Syt11-C2B, whereas the corresponding “EAKN” fragment in Syt1 exhibits an overall negative potential (Fig. 4l). This motif is exactly located in the short fragment representing a major electrostatic potential difference between the C2B domains of Ca+- and Ca−-Syts (Fig. 1d). This suggests a universal yet distinct membrane-binding mode for Ca−-Syts, which may confer higher lipid-binding affinity in the absence of Ca2+, enabling competitive membrane binding. Together with the universal mechanism of Ca2+-inhibition of the K-rich motif, and the Ca2+-facilitation of the loop region in Ca+-Syts, these findings provide a molecular basis for the activity-dependent Ca+/Ca−-Syts inter-switch model mediating precise control of synaptic transmission.

PIP2 docks to specific regions of Syt11-C2B
To investigate the atomic mechanism underlying the interaction between Syt11 and acidic lipids, we applied in silico docking of the headgroup of PIP2 (PInP2) to predict the lipid-binding structure of Syt11-C2B (Fig. 5a). Previous studies have validated that the K-rich motif in the C2 domains of rabphilin and PKC serves as a basic binding pocket for the acidic headgroup of PIP252,53. Consistent with these findings, we found that PInP2 can dock into the same pocket of Syt11-C2B with various conformations, forming tight interactions mediated by electrostatic forces (Fig. 5b). We then generated a Syt11-Kmotifm mutation to neutralize the electric interference of the K-rich motif, and found that PInP2 could still dock to the basic regions of loop-1 and loop-2 in Syt11-C2B through electrostatic interactions (Fig. 5c). However, PInP2 failed to dock automatically to the loop-3 region of Syt11-Kmotifm or Kmotifm BLp2m unless a predefined binding box was used to enforce interaction in this region (Fig. 5d). This suggests a synergistic effect between the K-rich motif and loop-3 in lipid-interactions, which is supported by the observed interaction between the glycerol backbone of PInP2 and K388 in loop-3 (Fig. 5b). Thus, loop-3 may play a more significant role in liposomal or in vivo conditions, where PIP2 is more abundant or clustered. These results define an atomic model of Syt11-lipid interactions, in which PIP2 binds to multiple regions of Syt11-C2B to form a large multi-point interface (Fig. 5e).

Syt11-membrane interaction regulates exocytosis and endocytosis at different stages
We have previously established that Syt11 inhibits both clathrin-mediated endocytosis (CME) and activity-dependent bulk endocytosis during synaptic transmission4,5,14,19. To investigate the role of Syt11-lipid interactions in synaptic exocytosis and endocytosis, we next performed live synaptophysin-pHluorin (Syp-pH) imaging in cultured primary neurons. Cortical neurons from homozygous floxed Syt11-null mice were infected with synapsin-driven Cre-expressing (Syn-Cre) recombinant adeno-associated virus (rAAV) to generate a knockout (KO) genotype, and full-length wild-type (WT) Syt11 or mutants were over-expressed to rescue the phenotypes (Fig. 6a). A sustained 70 mM KCl depolarization was applied to trigger exocytosis and endocytosis of synaptic vesicles, represented by the rising and decay of Syp-pH fluorescence signals (Fig. 6b, c). The differential of each trace (dF/dt) was calculated to assess the rate of exo-endocytosis (Fig. 6d).

In our experiments, fluorescence changes reflect the collective activity of all vesicles per synapse rather than transient dynamics of individual vesicles. While the rising time depends on stimulation duration, the Vmax of the rising phase reflects the maximal exocytosis. To define the early stage of exocytosis, we calculated the rising time from 0.1·Vmax to Vmax as the acceleration time (taccel), during which interference from Ca2+ accumulation and endocytosis is minimized, allowing assessment of exocytosis driven by the fusion machinery. Strikingly, Syt11-KO reduced the taccel of Spy-pH, which was fully rescued by WT-Syt11 but not Syt11-Kmotifm or Syt11-BLp123m mutant (Fig. 6e), suggesting that Syt11 inhibits exocytosis via membrane-binding during the early stage of synaptic transmission. However, the Vmax of the rising phase remained unchanged in Syt11-KO neurons (Fig. 6f), suggesting that the inhibitory role of Syt11 is alleviated or abolished during sustained exocytosis when accumulated Ca2+ weakens Syt11-lipid interactions and promotes Syt1 competition. In the decaying phase, the exponentially fitted τdecay was dramatically reduced by Syt11-KO, which was rescued by WT-Syt11 but not the mutants (Fig. 6g), suggesting that the inhibitory role of Syt11 in endocytosis is also mediated via membrane binding. Therefore, we proposed a hypothetic working model of the molecular processes during Syp-pH imaging (Fig. 6h). The fluorescence change rate (dF/dt) is composed of a positive exocytosis component and a negative endocytosis component. Vesicular exocytosis mainly depends on Ca2+, while endocytosis depends on both Ca2+ and exocytic extent. Syt11 inhibits exocytosis through competitive binding to the plasma membrane at the early stage of Ca2+ rising, but not the high-activity stage following sustained stimulation, when high Ca2+ promotes a probable Syt1-Syt11 displacement to temporarily retire Syt11. The immediate and rapid decay of Syp-pH after 70 mM KCl treatment indicates an immediate and rapid restore of cytoplasmic Ca2+, removing the Ca2+-inhibition in Syt11-lipid binding and thus enabling Syt11 as a brake of endocytosis at this stage.

To investigate the impact of the altered Ca2+-affinity of Syt11-S247D on vesicle recycling, we conducted membrane capacitance (Cm) recordings in dorsal root ganglion (DRG) neurons (Fig. S1a). A 200-ms depolarization stimulus induced a Cm jump and decay, validating that knock-down (KD) of Syt11 increased both the speed and the amount of endocytosis (Fig. S1b, c). Notably, the expression of the Syt11-S247D mutant led to a further increase in endocytic speed, but it did not affect the amount of endocytosis. The Cm decay in Syt11S247D-rescued neurons was initially very rapid, reaching a stationary phase more quickly than in control conditions, and the overshoot phenomenon observed in KD neurons was effectively eliminated (Fig. S1a). This behavior can be rationalized by considering the dual-stage function of Syt11-S247D. In the early phase, when Ca2+ levels are high, Syt11-S247D can sense Ca2+ and accelerate endocytosis similarly to a canonical Ca+-Syt, such as Syt1. However, as Ca2+ levels decline at the later phase, Syt11-S247D reverts to functioning like WT Syt11, thereby rescuing the KD effect. This switch is likely attributed to that the S247D mutant selectively restores the Ca2+ affinity of the C2A domain, while leaving the C2B domain unaffected. Consequently, its overall Ca2+ sensitivity remains lower than that of canonical Ca+-Syts like Syt1, rendering it inactive under low Ca2+ conditions.

Methods
Ethics statement
For all animal studies, the use and care of animals were conducted in accordance with the guidelines and regulations approved by the Animal Care and Use Committee of Xi’an Jiaotong University (NO. 2016-10).

Structural analyses
All structures of C2 domains used in this study were predicted using AlphaFold 239,40. Amino acid sequence similarity analyses of all Syts were performed using Clustal Omega89. The sequences used from humans are (hSyt1-17): P21579, Q8N9I0, Q9BQG1, O00445, Q5T7P8, O43581, Q86SS6, Q6XYQ8, Q9H2B2, Q8NBV8, Q9BT88, Q8IV01, Q7L8C5, Q8NB59, Q9BQS2, Q17RD7, and Q9BSW7. The sequences used from rat are (rSyt1-17): P21707, P29101, P40748, P47861, Q62746, Q62747, Q925C0, O08625, P50232, Q925B4, O08835, P97610, Q925B5, M0R7W7, P59926, A0A0G2K644, and Q62807. The isoelectric points (pI) of C2 domains were calculated using the ExPASy Server90. Electrostatic potential calculations for each C2 domain were performed using Protein-Sol91. Solvent-accessible surface area (SASA) calculations and protein structure mutagenesis were performed using PyMol. In silico docking of the PIP2 headgroup (PInP2, CID 125105) to Syt11-C2B was performed using DockThor92.

Plasmids
For the expression of cytoplasmic tandem C2A-C2B domains of Syt for co-liposome precipitation (co-LP) experiments, the rat Syt1 (rSyt1, aa 139-421) and Syt11 (rSyt11, aa 154-430) sequences were cloned into the pET-His vector. The isolated C2A and C2B domains of rSyt11 were defined as aa 154-282 and aa 290-430, respectively, and cloned into the pET-His and pRSFDuet-sumo vectors. For the Syt11-Syt1 competition assays, the rSyt1-C2AB (aa 96-421) was expressed from the pGEX-KG vector described in our previous research14. All mutants were generated using the QuikChange site-directed mutagenesis kit (Stratagene). The mutated sites are as follows: Syt1-AKA (C2A domain: K189/190/191-to-A), Syt1-BKA (C2B domain: R322-to-N and K324/325-to-A), Syt1-ABKA (combination of AKA and BKA mutations), Syt11-BKA (C2B domain: K340-to-N, R341-to-N, and K344/346-to-A), Syt11-Δloop1 (C2B domain: aa 319-325 replaced with “GS”), Syt11-Δloop3 (C2B domain: aa 382-390 replaced with “GGS”), Syt11-Δloop1+3 (combination of Δloop1 and Δloop3 mutations), Syt11-Δloop2 (C2B domain: aa 348-357 replaced with “GSG”), and Syt11-Kmotifm/BLp1/2/3m (see Fig. 4k for details). For the K-motif of Syt11, 4 of the 6 K/R residues were randomly selected for mutation. The H/K/R residues mutated in loop 1/2/3 were chosen based on their alkaline properties. The primers used to generate these mutants are listed in Table S1. They are available for reuse from the corresponding author upon request.

Protein purification
Constructs were transformed into E. coli BL21(DE3) and cultured in lysogeny broth at 37°C to an optical density of ~1.0. Protein expression was induced with 0.2 mM isopropyl β-d-1-thiogalactopyranoside (IPTG) at 18°C for 10 h. Cells were harvested by centrifugation at 8000×g for 5 min at 4°C and resuspended in lysis buffer (50 mM HEPES pH 7.4, 500 mM NaCl, 20 mM imidazole, 5% glycerol, 0.2% Triton X-100, 5 mM β-mercaptoethanol, and 1 mM PMSF). After sonication softly on ice and centrifugation at 20,000×g for 30 min, the supernatant was filtered through a 0.45 μm pore-size membrane. For pGEX-KG-expressed proteins, the supernatant was further incubated with Glutathione Sepharose beads (GE Healthcare) at 4°C for 2 h. Beads were collected by centrifugation at 400×g for 3 min and washed thoroughly with 10 μg/mL DNase I and 10 μg/mL RNase I in the binding buffer (50 mM HEPES pH 7.4, 100 mM NaCl, and 1 mM β-mercaptoethanol), then eluted with additional 10 mM reduced glutathione (GSH). For the preparation of tag-free Syt1, thrombin digestion was carried out on the beads at room temperature for 30 min, and then the flow-through containing the cleaved protein was collected. For pET-His and pRSFDuet-sumo-expressed proteins, Ni-NTA Sepharose 6FF beads were used for purification. Proteins on column were washed with additional 40 mM imidazole, then eluted with 400 mM imidazole. The purified proteins were checked via Nanodrop for a 280:260 nm absorbance ratio >1.8 and concentrated to ~10 mg/mL.

Liposome preparation
For co-LP assays, phospholipids (Avanti) were mixed in chloroform with indicated compositions and dried under vacuum. Lipids were resuspended in binding buffer to a final concentration of 0.5 mg/mL, vortexed thoroughly for 5 min, and subjected to 5 freeze-thaw cycles to form large multilamellar vesicles. For Zeta potential measurements, liposomes containing either 2% or 10% PIP2 were used to assess their coordination with Ca2+. The liposomes were prepared in ELS buffer (10 mM HEPES pH 7.4, 40 mM NaCl; the lower salt concentration was chosen to facilitate electrophoresis), with 15 freeze-thaw cycles, followed by extrusion through 400-nm polycarbonate filters for another 15 cycles to generate homogenous unilamellar vesicles.

Co-LP assays
Co-LP assays were performed at room temperature (RT) in binding buffer with additional amounts of EDTA, CaCl2, or KCl. Syt proteins (5 μg) were pre-incubated in binding buffer for 10 min, centrifuged to remove unstable precipitates, and incubated with 10 μg liposomes for 30 min. Liposomes were collected by centrifugation at 20,000×g for 20 min, and co-precipitated Syt proteins were analyzed via SDS-PAGE and Coomassie blue staining. Band intensities were quantified using Fiji93. Full gel scans and quantified data were supplied in the Source Data file.

Zeta potential measurements
The surface potential of liposomes was measured via electrophoretic light scattering using a Zetasizer Nano ZSE (Malvern). Liposomes were diluted to 0.1 mg/mL in buffers with indicated concentrations of CaCl2 or KCl, and Zeta potential was calculated using phase analysis light scattering.

Animals and viruses
Floxed Syt11-null (syt11f/f) transgenic mice were obtained from The Jackson Laboratory (strain B6.129-Syt11tm1Sud/J). C57BL/6J (B6) mice were sourced from Charles River Laboratories. The use and care of animals were approved and directed by the Animal Care and Use Committee of Xi’an Jiaotong University. All animals were housed in the animal facility of Xi’an Jiaotong University, maintained under a 12-h light/dark cycle at 22±2°C and 40–60% humidity, and provided with ad libitum access to food and water. The animals were finally euthanized with CO2, followed by cervical dislocation. For each experiment, the postnatal day 0 (P0) to P2 C57 or syt11f/f mice of both sexes (overall number of 20) were used for primary cortical neuron culture. Wistar rats (male, ~60 g, overall number of 6) were used for DRG culture. AAV2/9 used for primary neuron transfection included: AAV2/9-Psyn-Cre produced by Shanghai Heyuan Biotech, AAV2/9-Pcag-DIO-Syp-pHluorin produced by Hanbio Biotechnology, and AAV2/9-Pef1α-DIO-rSyt11-P2A-mCherry and its mutants (Kmotifm, BLp123m) produced by BrainVTA.

Cortical neuron culture and transfection
Primary cortical neurons were isolated and cultured as previously described with few modifications94. Anterior cortex tissues from P0 to P2 mice were dissected and cut into ~1 mm pieces in the Hank’s balanced salt solution (HBSS). The tissue fragments were digested with 0.25% trypsin at 37°C for ~12 min, followed by three rounds of mechanical dissociation using the 1 mL tips on pipettes. Neurons were plated on poly-L-lysine-coated glass coverslips and maintained in DMEM (Gibco) + 10% FBS medium for 4 h, which was then replaced with Neurobasal (Gibco) + 2% B27 + 1% GlutaMAX medium. Five micromolar cytosine arabinoside was added at day 2 in vitro (DIV2) to suppress glia growth. Medium were half-changed every 3–4 days. rAAV transfection was performed at DIV6, and experiments were conducted on mature neurons after DIV14.

DRG neuron culture and Cm recording
The culture of dorsal root ganglion (DRG) neurons and the membrane capacitance (Cm) recordings were performed as previously described (PMID: 26589353). Adult Wistar rats (male, ~60 g) were used to isolate DRGs in ice-cold L15 medium (Gibco). The ganglia were treated with a mixture of trypsin (0.3 mg/ml) and collagenase (1 mg/ml) for 40 min at 37°C. Subsequently, cells were dissociated, collected, and transfected using the NeonTM transfection system (MPK10096, Invitrogen). Cells were then plated on poly-L-lysine-coated coverslips and maintained in Dulbecco’s modified Eagle’s medium (DMEM)-F12 supplemented with 10% FBS (Gibco). For gene silencing experiments, 24 h post-transfection, the culture medium was replaced with Neurobasal-A medium supplemented with 2% B27 (Gibco), 0.5 mM L-glutamine (Gibco), 10 ng/ml nerve growth factor, and 5 μM cytosine arabinoside. Experiments were performed at day 2 in vitro (DIV2).

To silence the expression of Syt11, the nucleotide target sequences GGCTGAGATCACAAATATACG (shSyt11) was synthesized and inserted into the plasmid pRNAT-H1.1-GFP-shSyt11. A random sequence (TTCTCCGAACGTGTCACGT), which was predicted to target no genes in human, rat, and mouse cells, was used as a negative control. For rescue experiments, an RNAi-resistant form of rSyt11 was generated by introducing the following silent mutations: G GCG GAA ATT ACC AAT ATA CG.

Cm recordings were performed under the whole-cell configuration using an EPC10/2 amplifier controlled by Pulse software (HEKA Elektronik). The membrane potential was clamped at −70 mV, and pipette resistance was maintained between 3 and 4 MΩ. The external solution contained (in mM) 150 NaCl, 5 KCl, 2.5 CaCl2, 1 MgCl2, 10 H-HEPES, and 10 D-glucose (pH 7.4). The intracellular pipette solution contained (in mM) 153 CsCl, 1 MgCl2, 10 H-HEPES, and 4 Mg-ATP (pH 7.2). A 200 ms membrane depolarization was used to induce exocytosis and endocytosis. Data were collected with PatchMaster v2x90.2 (HEKA EPC10). Offline data analysis was performed using Igor Pro 6.22 software (Wavemetrics), with capacitance traces low-pass filtered at 30 Hz.

Syp-pH imaging
The real-time synaptic fluorescence imaging was performed using total internal reflection fluorescence (TIRF) microscopy. An inverted microscope equipped with a 100× TIRF objective lens (Nikon ECLIPSE Ti-U; numerical aperture 1.45) was used for image-capturing via an Andor EMCCD camera with NIS-Elements BR software, with an exposure time of 100 ms per frame. Neurons were bathed in resting solution (in mM: 140 NaCl, 2.5 KCl, 2 CaCl2, 2 MgCl2, 10 D-glucose, and 10 HEPES pH 7.4), and high K+ solution (changed to 72.5 mM NaCl, 70 mM KCl) was locally applied to induce neuronal activity. Time-series signals were analyzed using Fiji 1.54f and Graphpad Prism 8.0 for smoothing and differentiation.

Statistics
All experiments were performed with side-by-side controls and in random order, and were replicated at least three times. No samples that provided successful measurements were excluded from analysis. All the data are presented as the mean±s.e.m. Statistical comparisons were made with the one/two-way ANOVA, two-tailed Mann-Whitney test or paired Student’s t test as indicated. Normality of the data was tested with the Shapiro–Wilk test, and the equality of variance was determined with Lenene’s test. All the statistical analyses were made using GraphPad Prism 8.0 (Graphpad Software, San Diego, CA), and significant differences were accepted at P<0.05. Numbers of cells or biological repeats analyzed are indicated in the figures and figure legends.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "In this manuscript from the Wang group by Wu et al., authors detail the lipid binding activity of synaptotagmin 11 (syt11), a non Ca++ binding syt and the Ca++ binding Syt1 . While the role of Ca++ binding syts has long been implicated in neurotransmission, the function of the non-Ca++ binding syts expressed in the nervous system during neurotransmission has remained more of a mystery. They utilize a combination of biochemical assays, in silico analyses, in vitro liposome experiments, and finally pHlourin imaging of synaptic firing in primary cultured neurons to show that the C2 domains of Ca-binding Syts exhibit distinct dynamics in Ca2+/lipid binding, define the Ca2+-sensitive lipid binding of Ca-nonbinding-Syts to liposomes, and propose a novel Ca2+- switch between calcium binding and non binding syts model for the excitation-coupled exocytosis and endocytosis during synaptic transmission. Broadly the data and results are interesting and novel, and for the most part convincing, and if concerns detailed below are appropriately addressed, will provide a new understanding of diverse synaptotagmins in neuronal activity. There are multiple instances where additional information is needed to provide full understanding and clarity of the experiments performed and rationale behind them. Further, additional information about replicates and statistics are needed, and potentially additional replicates needed if there is insufficient rigor. Comments are organized by Figures below.\nFigure 1: Analysis of surface charges of all syt family members to find electrostatic differences suggest that non-calcium binding syts have a high likelihood for binding lipids, more so than calcium binding syts. For 1A, Please define the color code and tick marks/x axis. For 1B, The alphafold version needs defining in methods, with reference. Also the Syt1 and Syt11 color in PDB do not match the text. Please include the refseq id of sequences used in methods. 1C: Please provide a lookup table for colors. 1D-F: Are there statistics?\nFigure 2: Using liposome binding assays and bacterially expressed syt1 and syt11, with mutants to block or impart calcium sensitivity, authors demonstrate that calcium inhibits syt11 binding to liposomes, but not syt1, unless they switch the calcium binding. Please specify the sequences used for these constructs, and elaborate more upon the different lipid compositions that were chosen and why, for example, how they relate to physiological conditions. I am confused why the compromised composition to compensate for charge changes was used in later panels as opposed to the BET. 2B: how many replicates and is there any significance testing? Include error bar description in legend. Inclusion of paired data points instead of or in addition to mean is preferred for full understanding of the data. 2C: More details are needed for the is the minus liposome condition. Is this just solution? How do levels compare (a percentage of input). There is no description of this in methods. Discuss stats for rest of calc concentrations-since only done on first? 2D: Is there quantification? The syt11247D looks like a solid line. 2E-F: How many times was this experiment performed, there are no error bars on the quantification, and the syt1 does not appear quantifiable in 2F. 2G: is the evidence for calcium switching, yet liposome binding of syt11 is not blocked by calcium, even though competition with syt1 is blocked. This must be addressed to show that there is indeed switching.\nFigure 3: Authors use electrophoretic light scattering to measure the repulsion (charge) of liposomes in the presence and absence of calcium, to determine if the inhibition of Syt binding is due to the change in the surface charge of the membrane in different Ca2+ conditions. 3A: Adding + and – charges to schematic would be helpful to readers unfamiliar with this technique. 3C: are these averages or single measurements? is there significance or no significance? For KCl? There is also no error bars.\nFigure 4: Authors defined the Calcium sensitive and calcium insensitive lipid binding regions of C2 domains of syt1 and syt11, and found both c2a and c2b of syt1 are not calcium sensitive unless lysine residues are mutated, but only c2b of syt 11 is calcium sensitive and only c2b of syt11 competes with syt1 for lipid binding. Authors could better describe why they chose specific lysines for mutation, and should include the sequences used in methods. 4CDEG: How many replicates were performed, this is missing statistics (CD) or stats/quantification (EG). There is a discussion the calcium sensitive lipid binding of of syt11 through loop mutations and permutations, however, the details of the mutations used were not included in the results or methods, making this part quite difficult interpret,. And there is not statistics.\nFigure 5: Silico docking of pip2 to syt11-c2b to predict the lipid binding structure of Syt11-C2B.\nFigure 6: Authors performed synaptophysin-pHluorin (Syp-pH) imaging in cultured primary neurons, which suggest that syt11 via its lipid binding activity, inhibits both exocytosis and endocytosis after K triggering of firing. What happens in this assay if you express the Syt11247D mutant?\nFigure 7: Schematic of Syt11 function in neuronal secretion- Could authors describe how the SNARE complex fits into their model? Is this mechanism going to be specific to the readily releasable pool or also the reserve pool. In 7A, which membrane is depicted? Plasma or vesicular? How does the competition between Syt11 and Syt1 effect the clamping mechanism of Syt1?", "reviewer_2": "The manuscript titled “Calcium-sensitive Synaptotagmin 11-lipid interaction modulates exo-endocytosis” by Wu et al presents a detailed analysis of the functional interplay between calcium-binding (Ca²⁺-responsive, Caᴿ) and non-calcium-binding (Ca²⁺-tolerant, Caᵀ) synaptotagmins (Syts), focusing on Syt1 and Syt11. Syt1 is the well-established Ca²⁺ sensor for rapid synaptic vesicle exocytosis, while Syt11, despite lacking Ca²⁺-binding capability, plays important roles in synaptic transmission and plasticity. The mechanism by which these Syts coordinate during vesicle trafficking has remained unclear. This study reveals that Syt11, although unable to bind Ca²⁺, has strong affinity for acidic phospholipids and competes with Syt1 for membrane binding at rest. Upon physiological Ca²⁺ influx, this balance shifts: Syt1’s membrane insertion is enhanced, while Syt11’s binding is reduced due to electrostatic repulsion. The authors map a multi-site, Ca²⁺-independent lipid-binding interface in Syt11’s C2B domain and contrast it with Syt1’s dual membrane-binding modes - one Ca²⁺-independent and the other Ca²⁺-facilitated.\nThey propose a Ca²⁺-dependent Syt1–Syt11 membrane occupancy switch as a mechanism for fine-tuning synaptic vesicle exocytosis and endocytosis. In this model, Syt11 inhibits early stages of both processes but does not affect peak exocytosis. These findings redefine Ca²⁺-sensitivity as not solely dependent on Ca²⁺ binding, but also on dynamic electrostatic membrane interactions.\nOverall, by redefining Ca²⁺ sensitivity beyond direct ion binding - to include electrostatic membrane modulation - this work establishes a cooperative yet competitive regulatory mechanism between Caᴿ and Caᵀ Syts, deepening our understanding of synaptic precision. It also highlights the potential pathological relevance of Syt11 misregulation in disorders like Parkinson’s disease and schizophrenia. The manuscript is clearly written, and the findings are novel and impactful, with potential long-term significance in neuroscience and neuropsychiatric research.\nBelow are a few suggestions for improving the manuscript:\n1. In Figure 2, the authors used GST-tagged Syt1 in their assays. It is important to note that GST naturally forms dimers, and when fused to a target protein, GST can promote dimerization or even higher-order oligomerization of the fusion construct - regardless of whether the target protein dimerizes in its native form. This potential for oligomerization should be considered, as it may influence the biochemical behavior and interpretation of the assay results. A brief discussion of this limitation would strengthen the interpretation of the data, e.g., \"While GST dimerization could influence Syt1-GST behavior, our controls suggest this effect is negligible under these conditions.\"\n2. In lines 267–270, the authors describe the generation of Δloop1, Δloop2, Δloop3, and Δloop1+3 deletion constructs of the Syt11-C2B domain. It would be helpful to specify the exact amino acid residues removed in each construct to clarify the experimental design and facilitate reproducibility.\n3. Consider to replace the non-standard notations of “Ca ” and “Ca ” with more conventional terms, such as Ca²⁺-responsive (Caᴿ) and Ca²⁺-tolerant (Caᵀ) (or another widely recognized convention), to improve clarity and consistency with the broader literature.\n4. In line 63, revise “Syt 1~17” to “Syt 1–17” for standard notation of numerical ranges.\n5. Similarly, in line 67, consider to change “2~4 Ca2+” to “2-4 Ca2+”.", "reviewer_3": "Wu et al present a novel study of synaptotagmin-11 (Syt11) in which they show (a) this protein’s C2B and C2AB domains can bind membranes with physiological or higher anionic content, (b) this membrane binding is inhibited by calcium concentrations in the millimolar range, (c) in the absence of calcium, Syt11 can compete with Syt1 for binding to limiting PIP2 ligands, and (d) Syt11 knockout, as well as rescue with membrane binding-defective mutants, subtly but significantly alter the kinetics of exocytosis in primary cortical neurons. These findings provide some of the first information about the mechanism of action of this non-calcium-binding, inhibitory synaptotagmin. However, several of the claims made in the manuscript exceed the evidence presented, and the Syt1 results generally reproduce features of this protein that are already well established.\nMajor concerns:\n1. The proposed electrostatic screening mechanism for the high-Ca inhibition of membrane binding is reasonable and I think it’s likely to be correct. However, I don’t see how it’s relevant to the physiological competition with Syt1 that the manuscript reports occurring at orders of magnitude lower calcium. Inhibition of membrane binding at high calcium has been observed for decades – see for example Fig. 1C-F in PMID 11823420 – but calcium levels in cells seldom exceed 0.1 mM and extracellular Ca is only ~1 mM. The authors’ observation that Syt1 outcompetes Syt7 at 20 uM Ca is much more likely to arise from the increased Syt1 membrane affinity at micromolar calcium levels, not from the negligible decrease in Syt11 membrane affinity (see Fig 2C, EDTA vs. 100 uM Ca; statistical significance does not imply functional relevance). So, I question the authors’ assertion (e.g., in lines 37-41 in the Abstract) that there is a direct connection between these two main observations in the manuscript (Syt11 inhibition by 5-10 mM Ca, and Syt1/Syt11 competition over the 0-20 uM Ca range).\n2. The Abstract (lines 36-38) implies that competing with Syt1 for membrane binding in the absence of Ca is a conserved property of Ca(x)Syts. But the manuscript does not show this. There is one result in the paper (Figure 2D, 2nd from top) that shows that 8 mM Ca inhibits Syt4 from binding membranes, but Syt11 is the only protein that the authors demonstrate to compete with Syt1 for membrane binding. To my knowledge, other Ca(x)Syts have not been demonstrated to bind membranes under near-physiological conditions.\n3. While the model of competition between Syt1 and Syt11 for binding to limited PIP2 sites is reasonable, the conditions used for the liposome co-sedimentation measurements differ from physiological in ways that are likely to exaggerate Syt11’s membrane affinity. This is okay for these mechanistic experiments although it dampens excitement about the physiological relevance.\na. The salt concentration is less than physiological (100 mM NaCl). This condition has previously been shown to exaggerate electrostatic interactions of Syt1 in the absence of calcium (PMID 26389740). Is Syt11 liposome binding also observed at 150 mM salt?\nb. While total brain lipid extracts are used for a few experiments (Fig. 2B-C), the rest of the lipid binding experiments (including the only Syt4 data and all of the Syt1 competition experiments) use higher anionic character (40% PS and 1.5% PIP2, according to line 178). The explanation for this composition is “to compensate the charge effect” which doesn’t make sense to me – the charge effect of what? It would make more sense that the purpose is to strengthen Syt11’s membrane affinity.\nc. The Syt1 C2AB used for the competition experiments was purified without a cation exchange step, and apparently without cleaving the GST tag. The former is known to lead to impurities with lower membrane affinity (PMID 23617808), and the latter could lead to dimerization which could have a minor effect on the competition assay. The data in Figure 2B are consistent with well purified Syt1, but the Methods note that this is a different protein than what was used in the competition assay. Binding measurements akin to Fig 2B using the Syt1 GST fusion protein would increase confidence in the competition assay.\n4. Please clarify what lipid composition and buffer/salt composition(s) were used in Figure 3. This is necessary not only for reproducibility but also to interpret the results. If these experiments were done in the absence of salts except calcium, then the micromolar calcium range of the zeta potential shift seems unlikely to be physiologically relevant.\n5. Lines 281-282: What is the rationale for the claim that the K-motif has a greater contribution to membrane affinity than loop 1? From Figure 4H, the mutation of 3 residues in loop 1 (K/H/R) seems to have more inhibitory effect than the mutation of 4 residues in the K-motif (K/R/K/K). I agree with the authors’ main contention that many basic regions on the C2B domain contribute to affinity. However, Syt11 seems to be unique in its extent of positive charge on loop 1, if I am reading Figure 1C correctly. This suggests to me that the competitive effect in the absence of calcium may be unique (or strongest) for Syt11 among the Ca(x)syts.\n6. The paragraphs on p. 15 (lines 356-380) seem to belong in the Discussion rather than the Results. Most particularly:\na. Lines 364-365: the manuscript does not show that Syt11 prefers or stabilizes curved membranes, although this seems like a reasonable thing to speculate based on its distribution of basic lipid binding sites.\nb. Lines 369-370: the manuscript does not show that Syt11 promotes vesicle docking at resting states. This is speculation.\n7. P. 17, line 407 (and also line 290) states that Syt11 has a “higher resting-state membrane affinity” than Syt1, but the data in Figure 2E are not quantitative enough to make this assertion. (If the blue shapes accurately reflect the relative concentrations, then the affinities of the two proteins in EDTA look approximately equal to me.) What are the Syt1 and Syt11 concentrations used in these titrations?\nMinor concerns:\n1. p. 7, line 160 – please provide a reference for the “previous research.”\n2. P. 7, lines 164-166 – the word “compensates” is confusing. Do the authors mean that the electrostatic interaction saturates the Syt1 membrane binding even in the absence of calcium?\n3. P. 7, line 177 – I think the authors mean “intermediate” rather than “compromised”\n4. In Figure 1, why the switch from human to rat sequences/structures between 1B and 1C? Because rodent proteins were used in this paper, it would seem more appropriate to include rodent sequence/structure data in the main paper. Any human comparisons could go in a supplement.\n5. Figure 1C: the usual convention is to display positive charges blue and negative charges red. Is there a particular reason to use the opposite scheme here?\n6. Line 184, “consistently” may not be the right word to describe a result that is visible in only one lane of one gel.\n7. Lines 209-211: this sentence is an overstatement of the results to this point, and may be more appropriate for the Discussion.\n8. Lines 217-218: calcium chelation by PIP2 is already known (PMID 19630438)\n9. Line 292: Please provide a reference for the “calcium inhibition of the K-rich motif in all Syts”, which was not shown in this study (this study did not use all Syts).\n10. Line 402: can the authors explain what is meant by “certain dimensions”? This sentence doesn’t make sense to me in light of the authors’ model that Syt1 and Syt11 have opposing effects on exo- and endocytosis."}, "author_response": "Point-by-point response to the reviewers’ comments\nREVIEWER COMMENTS\nReviewer #1 (Remarks to the Author):\nIn this manuscript from the Wang group by Wu et al., authors detail the lipid binding activity of\nsynaptotagmin 11 (syt11), a non Ca++ binding syt and the Ca++ binding Syt1. While the role of\nCa++ binding syts has long been implicated in neurotransmission, the function of the non-Ca++\nbinding syts expressed in the nervous system during neurotransmission has remained more of a\nmystery. They utilize a combination of biochemical assays, in silico analyses, in vitro liposome\nexperiments, and finally pHlourin imaging of synaptic firing in primary cultured neurons to show\nthat the C2 domains of Ca-binding Syts exhibit distinct dynamics in Ca2+/lipid binding, define the\nCa2+-sensitive lipid binding of Ca-nonbinding-Syts to liposomes, and propose a novel Ca2+-\nswitch between calcium binding and non binding syts model for the excitation-coupled exocytosis\nand endocytosis during synaptic transmission. Broadly the data and results are interesting and novel,\nand for the most part convincing, and if concerns detailed below are appropriately addressed, will\nprovide a new understanding of diverse synaptotagmins in neuronal activity. There are multiple\ninstances where additional information is needed to provide full understanding and clarity of the\nexperiments performed and rationale behind them. Further, additional information about replicates\nand statistics are needed, and potentially additional replicates needed if there is insufficient rigor.\nComments are organized by Figures below.\nResponse: Thank you very much for your kind commendation and encouragement on\nthis work. Your insightful comments have been carefully addressed by providing\nadditional information and the conduction of new experiments. These efforts have\nsignificantly enhanced the quality and impact of this work.\nFigure 1: Analysis of surface charges of all syt family members to find electrostatic differences\nsuggest that non-calcium binding syts have a high likelihood for binding lipids, more so than\ncalcium binding syts. For 1A, Please define the color code and tick marks/x axis. For 1B, The\nalphafold version needs defining in methods, with reference. Also the Syt1 and Syt11 color in PDB\ndo not match the text. Please include the refseq id of sequences used in methods. 1C: Please provide\na lookup table for colors. 1D-F: Are there statistics?\nResponse: Thank you very much for your suggestions.\n1) For 1A, Ca-Syts are shown in red, while Ca-Syts are depicted in gray. The tick marks\nof x-axis: the distance values showing the number of substitutions as a proportion of the\nalignment length (excluding gaps), thereby reflecting the extent of genetic change. This\ninformation has now been included in the figure legend.\n2) For 1B, the data illustrates the calculated pI values of each C2 domain derived from\nprotein sequences, but not structural information. The calculation tool used has been\nincluded and cited in the ‘Methods’ section. Structural data from AlphaFold 2 is presented\nin panels 1C-F. We have cited the reference in the first sentence of the ‘Results’ section\nin original manuscript. Following your advice, we have included the alphafold\nversion/reference in the ‘Methods’ as well. In addition, sequence IDs have been included\nin the ‘Methods’ now.\n3) For 1C, color labeling has been included following your advice (Fig. R1A, also see Fig.\n1C in the revised manuscript).\n4) Regarding the statistics for 1D-F, we initially did not calculate P-values because these\npanels display complete data collections of every residue/protein, rather than being based\non random sampling from a group or independent experimental repeats. P-values are\nusually used to assess the probability of differences between groups arising from random\nsampling. Nonetheless, following your advice, we have performed a two-tailed Mann-\nWhitney test for statistics by treating each amino acid residue as a sampling repeat (Fig.\nR1B-C, also see Fig. 1E-F in the revised manuscript).\nFigure R1 | Non-Ca2+-affinitive Syts exhibit high alkalinity. (A) AlphaFold-predicted\nstructures of all rat C2 domains, illustrating surface electrostatic potential. Top left: Syt11-\nC2 domains aligned with a Ca2+-bound Syt1 crystal structure (PDB: 5CCH); top right: Ca-\nSyt C2 domains, with positive (red) and negative (blue) electrostatic potentials; bottom:\nCa-Syt C2 domains, showing extensive alkaline surfaces. (B) Averaged electrostatic\npotential of residues across all C2 domains. (C) Calculated solvent-accessible surface area\n(SASA) of highly basic regions (potential >75 mV) for all C2 domains. Box-and-whisker:\nmedian + min-to-max range; two-tailed Mann-Whitney test, ***P < 0.001, n.s. no significant\ndifference.\nFigure 2: Using liposome binding assays and bacterially expressed syt1 and syt11, with mutants to\nblock or impart calcium sensitivity, authors demonstrate that calcium inhibits syt11 binding to\nliposomes, but not syt1, unless they switch the calcium binding. Please specify the sequences used\nfor these constructs, and elaborate more upon the different lipid compositions that were chosen and\nwhy, for example, how they relate to physiological conditions. I am confused why the compromised\ncomposition to compensate for charge changes was used in later panels as opposed to the BET. 2B:\nhow many replicates and is there any significance testing? Include error bar description in legend.\nInclusion of paired data points instead of or in addition to mean is preferred for full understanding\nof the data. 2C: More details are needed for the is the minus liposome condition. Is this just solution?\nHow do levels compare (a percentage of input). There is no description of this in methods. Discuss\nstats for rest of calc concentrations-since only done on first? 2D: Is there quantification? The\nsyt11247D looks like a solid line. 2E-F: How many times was this experiment performed, there are\nno error bars on the quantification, and the syt1 does not appear quantifiable in 2F. 2G: is the\nevidence for calcium switching, yet liposome binding of syt11 is not blocked by calcium, even though\ncompetition with syt1 is blocked. This must be addressed to show that there is indeed switching.\nResponse: Thank you for your good suggestions.\n1) Constructs sequences. Information on the AA-truncations in His-rSyt1-C2AB,\nHis-rSyt11-C2AB, His-rSyt1-D2N&D2E, and GST-rSyt1-C2AB has been included in the\n“Materials and Methods” section (Plasmids). The specific mutation sites for D2N&D2E\nwere also provided in the “Results” section (Syt11 binds acidic phospholipids with Ca2+-\nsensitivity) in the main text. Following your advice, we have now included the primer\nsequences used for generating these constructs in the Supplementary Information.\n2) Lipid composition. Various lipid compositions have been used to study Syt-lipid\ninteractions in vitro over the past 30 years. The lipid mixture used in the tubulation assay\n(TA), as utilized in our previous work for Syt1 tubulation assays (1), contains higher levels\nof PIP2 that can effectively shield the Ca2+-promotion binding of Syt1 to lipids (Fig 2B).\nIn contrast, BET lipids, sourced from Avanti as whole brain lipids from porcine brains, contain\n~10.6% PS but lack the specific presentation of an active zoon (AZ). Meanwhile, human\nbrain PS levels vary between 11.4% and 20.5%, depending on individual differences and\nbrain regions (2), while PIP2 composition in whole-cell lipids is ~1% (3, 4). On the other\nhand, since both PS and PIP2 are predominantly located in the inner leaflet of the plasma\nmembrane, their concentrations can reach 22.8-41% and ~2%, respectively. Furthermore,\nPIP2 is shown to be enriched in clusters at AZs, with local concentrations reaching up to\n~5% (5-7) or even higher (8). In this study, the ubiquitous Ca2+-independent binding of\nSyt11 to liposomes (both TA and BET) and the elimination of Ca2+-facilitation in Syt1-TA\nliposome binding suggest that Ca2+ enhancement of vesicle docking is region-dependent\nand primarily occurs in none-AZ regions. As suggested, we have included this information\nin the “Results - Syt11 binds acidic phospholipids with Ca2+-sensitivity” section.\n3) For 2B, following your advice, statistical analyses have been included in the revised\nmanuscript. Please kindly note that this panel has been revised according to suggestions\nfrom Reviewers #2 and #3.\n4) For 2C, the “Lipo(-)” condition refers to the presence of proteins in the solution without\nliposomes and serves as a negative control. All other groups are quantified as a percentage\nto this condition (Fig. R2A-B, also see Fig. 2B-C in the revised manuscript). We have now\nincluded the relevant data points and updated the figure legend.\nFigure R2 | Ca2+ inhibits Syt11-acidic membrane interaction and blocks its\ncompetition with Syt1. (A) The soluble tandem C2A-C2B domain of Syt1 binds to\nliposomes, exhibiting a significant Ca2+-promotion effect that is dependent on acidic\nphospholipid levels. (B) Syt11-liposome binding is suppressed at high-Ca2+ concentrations.\nthe “Lipo(-)” condition, containing protein in solution without liposomes, was used as a\nnegative control, and other groups are quantified as a percentage relative to this control.\n(C) Ca2+-inhibition persists in Ca-Syts (Syt4 and Ca-deficient Syt1 mutants) but is absent\nin the Ca2+-affinity-restored Syt11-S247D mutant. (D) Syt11 (0, 0.5, 1, 2, 4, and 8 μg)\ncompetes with Syt1 (5 μg) for liposome-binding in Ca2+-free conditions. GST-Syt1(96-421)\nwas used to distinguish the bands of Syt1 and Syt11. (E) To exclude potential effects of\nGST dimerization on competition experiments, tag-free Syt1(96-421) was generated via\nthrombin digestion and showed a similar competition phenomenon. (F) The Syt11-S247D\nmutant partially regains competitive ability in the presence of Ca2+. (G) 20 μM Ca2+\nabolishes competitive effects (n = 6), suggesting physiological relevance. Data\npresentation: mean + s.e.m., Student’s t-test for (A), one-way ANOVA for (B, C, G), and\ntwo-way ANOVA for (D-F). *P < 0.05, **P < 0.01, ***P < 0.001, n.s. no significant difference.\n5) For 2D, we have replaced the Syt11-S247D data with a clearer, repeated panel featuring\nmore distinct bands. Syt11 and Syt4 were known as their key mutations in the C2A domain\n(D -to-S), which abolish Ca2+-sensitivity. In this study, we reverted the mutation (S -to-\nCa 247\nD) to restore Ca2+-sensitivity in Syt11-C2A. Consequently, the Ca2+-inhibition of Syt11-\nS247D in lipid-binding disappeared. However, as indicated by the “solid line”, Ca2+ did not\nenhance binding, likely because the C2B domain remained Ca2+-insensitive and exhibited\nCa2+-inhibition. Notably, while the C2Bs of Syt11 and Syt4 do not contain coordinating\nmutations like the C2A domain, their structural arrangements still prevent Ca2+-binding.\nFollowing your advice, we have now included quantification and statistical analysis (Fig.\nR2C, also see Fig. 2D in the revised manuscript).\n6) For 2E-F, we have included additional repeats and quantifications for clear bands\nanalysis (Fig. R2D-E, also see Fig. 2E-F in the revised manuscript).\n7) For 2G, following your advice, we have included statistics and observed a gradual\nincrease in Syt1 levels accompanied by a decline in Syt11 levels as Ca2+ concentration\nincreases (Fig. R2G, also see Fig. 2H in the revised manuscript). Syt11 was indeed\nblocked, but only to a slight degree. Similar phenomena were observed in Syts competition\nwithout Ca2+: as Syt11 levels increased in the presence of EDTA, Syt1 levels decreased.\nHowever, when Syt11 levels continued to rise excessively, Syt1 levels stopped decreasing\nand even rebounded slightly (Fig. R3A). These observations suggest that Syt1 and Syt11\nmay exhibit some form of aggregation (whether specific or nonspecific) at high\nconcentrations. However, our in vitro pull-down experiments (Fig. R3B) indicate that they\ndo not interact clearly in solution, suggesting that this association may occur only on a\nmembrane platform. In fact, it has long been established that Syts exist as monomers in\nsolution but can aggregate into polymers on membranes (e.g., forming ring-like structures)\n(9). The potential involvement of Syt11 in Syt1 ring structures is an important aspect of the\nproposed mechanism for their competition for membrane occupancy competition under\nphysiological conditions. However, due to the lack of direct evidence, we have not\ndiscussed this in the manuscript.\nFigure R3 | Syt1 and Syt11 may aggregate on membranes. (A) Soluble Competition\nassay showing that excessive Syt11 causes a rebound in Syt1 levels (indicated by black\narrows). (B) In vitro pull-down assay demonstrating that Syt11 and Syt1 do not show clear\ninteraction in solution. This supports the hypothesis that aggregation or association\nbetween Syt1 and Syt11 may occur specifically on membrane surfaces rather than in free\nsolution.\nFigure 3: Authors use electrophoretic light scattering to measure the repulsion (charge) of\nliposomes in the presence and absence of calcium, to determine if the inhibition of Syt binding is\ndue to the change in the surface charge of the membrane in different Ca2+ conditions. 3A: Adding\n+ and – charges to schematic would be helpful to readers unfamiliar with this technique. 3C: are\nthese averages or single measurements? is there significance or no significance? For KCl? There\nis also no error bars.\nResponse: Thank you for valuable suggestions. 1) For 3A, following your advice, we have\nincorporated “+” and “-” charges now (Fig. R4A, also see Fig. 3A in the revised\nmanuscript). 2) For 3C, the data presented are averages of 3 biological replicates, and\nthe bar graph now includes the significant differences. Although the KCl conditions were\nnot repeated in the original manuscript, they have been added in the revised version. In\naddition, following Reviewer #3’s suggestion, we have included data for another liposome\ncomposition (2% PIP2) (Fig. R4B, also see Fig. 3C in the revised manuscript).\nFigure R4 | Ca2+ shields the electrostatic surface potential of acidic membranes. (A)\nSchematic of the measurement of Zeta potential via electrophoretic light scattering (ELS).\n(B) 50 μM Ca2+ significantly alters the electrostatic states of acidic membranes. Data\npresentation: mean + s.e.m., one-way ANOVA and two-tailed Mann-Whitney test. *P < 0.05,\n**P < 0.01, ***P < 0.001, n.s. no significant difference.\nFigure 4: Authors defined the Calcium sensitive and calcium insensitive lipid binding regions of C2\ndomains of syt1 and syt11, and found both c2a and c2b of syt1 are not calcium sensitive unless lysine\nresidues are mutated, but only c2b of syt 11 is calcium sensitive and only c2b of syt11 competes with\nsyt1 for lipid binding. Authors could better describe why they chose specific lysines for mutation,\nand should include the sequences used in methods. 4CDEG: How many replicates were performed,\nthis is missing statistics (CD) or stats/quantification (EG). There is a discussion the calcium\nsensitive lipid binding of of syt11 through loop mutations and permutations, however, the details of\nthe mutations used were not included in the results or methods, making this part quite difficult\ninterpret,. And there is not statistics.\nResponse: We appreciate your helpful suggestions.\n1) Mutation sites. Since the K-motif is a well-established membrane-binding region of Syt1,\nthe 4 K/R residues mutated within this motif were randomly selected from the 6 K/R\nresidues present. The H/K/R residues mutated in loop1/2/3 were chosen based on their\nalkaline properties. We have now included a brief introduction to all mutations in the\n“Materials and Methods – Plasmids” section.\n2) For 4C-E, we added statistical data from 3-4 repeats (Fig. R5A-C, also see Fig. 4C-E\nin the revised manuscript).\n3) For 4G, since these experiments were preliminary screenings that yielded negative\nresults, we initially did not repeat them. Instead, we provided quantifications and statistical\nanalysis for the more comprehensive screenings presented in Fig. 4H-J, which\nencompassed the data relevant to Fig. 4G. Following your advice, we have now included\nrepeated experiments along with their quantifications (Fig. R5D, also see Fig. 4G in the\nrevised manuscript).\nFigure R5 | Syt11 employs a multi-regional redundant membrane-binding mode. (A)\nMutations in the K-rich motif of Syt1-C2B dramatically reduce liposome affinity, while the\nC2A domain plays a minor role, indicating that Syt1 primarily relies on a single region for\nmembrane binding mode in Ca2+-free conditions. (B) The C2B domain, but not the C2A\ndomain, of Syt11 contributes to liposome binding. (C) The C2B domain, but not the C2A\ndomain, of Syt11 contributes to competition with Syt1 for liposome binding. (D) Mutations\nin the K-rich motif of Syt11-C2B or deletions of loops 1, 2, 3, or 1+3 do not block liposome\nbinding, suggesting redundant interactions. Data presentation: mean + s.e.m. Student’s t-\ntest for (A), two-way ANOVA for (B, C), and one-way ANOVA for (D). *P < 0.05, **P < 0.01,\nn.s. no significant difference.\nFigure 5: Silico docking of pip2 to syt11-c2b to predict the lipid binding structure of Syt11-C2B.\nResponse: Thank you.\nFigure 6: Authors performed synaptophysin-pHluorin (Syp-pH) imaging in cultured primary\nneurons, which suggest that syt11 via its lipid binding activity, inhibits both exocytosis and\nendocytosis after K triggering of firing. What happens in this assay if you express the Syt11247D\nmutant?\nResponse: Thank you for this helpful suggestion. The topic of Ca-Syt11’s role in\nexocytosis and endocytosis is indeed intriguing. However, conducting the proposed\nexperiment would be highly time-consuming, as it would require a new AAV preparation,\nlong-term primary neuron culturing, and multiple repeats. Instead, we have utilized\nmembrane capacitance (Cm) recordings on DRG neurons transfected with plasmids (Fig.\nR6, also see Fig. S1 in the revised manuscript). Fig. R6A presents Cm traces with a 200\nms depolarization stimulus. Statistics of first 5 seconds of endocytosis revealed that knock-\ndown of Syt11 increased the decay speed (Fig. R6A) and amount (Fig. R6C). Interestingly,\nexpression of the Syt11-S247D mutant further accelerated the decay speed (Fig. R6B) but\ndid not affect the amount (Fig. R6C). As shown in Fig. 6A, the decay of the S247D-rescue\nwas very rapid initially but quickly reached a stationary phase, eliminating the overshoot\nphenomenon observed in the KD group. This observation is reasonable because, in the\nearly stage, Syt11-S247D can sense Ca2+ and accelerate endocytosis like a Ca-Syt\n(such as Syt1). However, as Ca2+ levels decrease in later stage, Syt11-S247D reverts to\nfunctioning like WT Syt11 to rescue the KD effect. This is likely because the S247D\nmutation only restores the Ca2+ affinity of the C2A domain but not the C2B domain.\nConsequently, its overall Ca2+ sensitivity is weaker than that of Ca-Syts such as Syt1,\nand it cannot be activated in low Ca2+ conditions.\nFigure R6 | Syt11-S247D promotes early-stage endocytosis. (A) Representative and\naveraged Cm traces following a 200 ms depolarization stimulation in DRG neurons from\nWT, Syt11-KD, and Syt11-S247D-rescue mice. The initial 5 s of endocytosis were analyzed,\nrevealing that Syt11 KD increased the decay speed (B) and amount (C). In contrast, rescue\nwith Syt11-S247D further accelerated the decay speed (B) but did not affect the amount\n(C). The Cm decay in the S247D group quickly reached a stationary phase, eliminating the\novershoot phenomenon observed in the KD group (A). Data presentation: mean + s.e.m.,\none-way ANOVA and two-tailed Mann-Whitney test, *P < 0.05, **P < 0.01, ***P < 0.001,\nn.s. no significant difference.\nFigure 7: Schematic of Syt11 function in neuronal secretion- Could authors describe how the SNARE\ncomplex fits into their model? Is this mechanism going to be specific to the readily releasable pool\nor also the reserve pool. In 7A, which membrane is depicted? Plasma or vesicular? How does the\ncompetition between Syt11 and Syt1 effect the clamping mechanism of Syt1?\nResponse: Thank you for your insightful suggestions.\n1) SNARE. Previous research has established the SNARE-binding capability of all Syts,\nbut only Ca-Syts exhibit Ca2+-promotion effects (13). Therefore, Syt11 is expected to\nfunction similarly to Syt1 (as a clamp) before high levels of Ca2+-influx remove it from the\nmembrane. However, a definitive schematic of how Syt-SNARE interactions drive Ca2+-\ntriggered fusion remains elusive due to ongoing debates about the underlying molecular\nmechanism (14). Various models have been proposed, such as the unclamping of\ncomplexin (Cpx) or self-unclamping of syt, membrane bridging, regulation of vesicle-\nplasma membrane distance, and membrane curvature, among others, with detailed\nmechanisms still unknown. Recent studies have also proposed a “lever” model for Syt1\n(15-17). Nonetheless, all these models highlighted the critical role of diverse membrane-\nbinding modes of Syt. Given the complexity and ongoing debates, we have cautiously\nadded a model of SNAREs in the “Fusion” step of Fig. 7B, illustrating the potential distinct\nbehaviors of Syt11 (continued clamping) and Syt1 (unclamping) on SNARE in response to\nmoderate Ca2+ levels (Fig. R7, also see Fig. 7B in the revised manuscript).\nFigure R7 | Working model of Syt11 function in neural secretion. Synaptically, Syt11 exerts\nan inhibitory role in exocytosis and endocytosis through membrane interactions. This role\nis allocated via a Syt1-Syt11 inter-switch under high-activity, high-Ca2+ conditions (e.g.,\ncontinuous neuronal excitation).\n2) RRP & RP. Syt11 has been shown to regulate both clathrin-mediated endocytosis (CME)\nand activity-dependent bulk endocytosis (10). Although vesicle pool dynamics were not\nmeasured in this manuscript, our unpublished work on Syt11 (titled ‘Syt11 mediates double\nbrakes of synaptic transmission’) revealed dysregulation of the RP (Fig. R8B), while the\nRRP remained unaffected (Fig. R8A).\n3) For 7A, this working model of Syt-liposome interaction is raised from biochemical\nassays mimicking the lipid composition of the plasma membrane.\n4) Clamping. We proposed that both Syt-1/SNAREs and Syt-11/SNAREs contribute to the\ndocking of synaptic vesicles at the resting state, during which they clamp fusion machinery\nwith similar mechanisms. Upon week depolarization (e.g., subthreshold membrane\ndemoralization, TRP channel activation-mediated local Ca2+ influx), micromolar Ca2+\ntriggers the unclamping of Syt-1 but not Syt-11, while more than 3 SNAREs can initiate\nfusion (18). However, under high neural activity, accumulated Ca2+ induces a Syt11-to-Syt1\nswitch, which facilitates vesicular fusion by recruiting more Syt1 to the fusion site.\nWe thank you very much for your helpful and valuable comments.\nReviewer #2 (Remarks to the Author):\nReview comments:\nThe manuscript titled “Calcium-sensitive Synaptotagmin 11-lipid interaction modulates exo-\nendocytosis” by Wu et al presents a detailed analysis of the functional interplay between calcium-\nbinding (Ca²⁺-responsive, Caᴿ) and non-calcium-binding (Ca²⁺-tolerant, Caᵀ) synaptotagmins\n(Syts), focusing on Syt1 and Syt11. Syt1 is the well-established Ca²⁺ sensor for rapid synaptic vesicle\nexocytosis, while Syt11, despite lacking Ca²⁺-binding capability, plays important roles in synaptic\ntransmission and plasticity. The mechanism by which these Syts coordinate during vesicle\ntrafficking has remained unclear.\nThis study reveals that Syt11, although unable to bind Ca²⁺, has strong affinity for acidic\nphospholipids and competes with Syt1 for membrane binding at rest. Upon physiological Ca²⁺ influx,\nthis balance shifts: Syt1’s membrane insertion is enhanced, while Syt11’s binding is reduced due to\nelectrostatic repulsion. The authors map a multi-site, Ca²⁺-independent lipid-binding interface in\nSyt11’s C2B domain and contrast it with Syt1’s dual membrane-binding modes - one Ca²⁺-\nindependent and the other Ca²⁺-facilitated.\nThey propose a Ca²⁺-dependent Syt1–Syt11 membrane occupancy switch as a mechanism for fine-tuning synaptic vesicle\nexocytosis and endocytosis. In this model, Syt11 inhibits early stages of both processes but does not affect peak exocytosis. These findings redefine Ca²⁺-sensitivity as not solely dependent on Ca²⁺ binding, but also on dynamic electrostatic membrane interactions.\nOverall, by redefining Ca²⁺ sensitivity beyond direct ion binding - to include electrostatic membrane\nmodulation - this work establishes a cooperative yet competitive regulatory mechanism between\nCaᴿ and Caᵀ Syts, deepening our understanding of synaptic precision. It also highlights the potential\npathological relevance of Syt11 misregulation in disorders like Parkinson’s disease and\nschizophrenia. The manuscript is clearly written, and the findings are novel and impactful, with\npotential long-term significance in neuroscience and neuropsychiatric research.\nBelow are a few suggestions for improving the manuscript:\nResponse: Thank you very much for your commendation and encouragement on this work.\nYour insightful comments have been carefully addressed, which are very helpful to improve\nthe quality and impact of this work.\n1. In Figure 2, the authors used GST-tagged Syt1 in their assays. It is important to note that GST\nnaturally forms dimers, and when fused to a target protein, GST can promote dimerization or even\nhigher-order oligomerization of the fusion construct - regardless of whether the target protein\ndimerizes in its native form. This potential for oligomerization should be considered, as it may\ninfluence the biochemical behavior and interpretation of the assay results. A brief discussion of this\nlimitation would strengthen the interpretation of the data, e.g., \"While GST dimerization could\ninfluence Syt1-GST behavior, our controls suggest this effect is negligible under these conditions.\"\nResponse: Thank you for your constructive suggestions. The dimerization of GST is\nindeed a notable factor. To address this, we have included a comparison of GST-Syt1 and\nHis-Syt1 (Fig. R9A, also see Fig. 2B in the revised manuscript). Additionally, we have\nincorporated the competition between tag-free Syt1 (96-421, generated by thrombin\ncleavage of GST-Syt1) and Syt11 to ensure data consistency (Fig. R9B, also see Fig. 2F\nin the revised manuscript). Following your advice, we have also included the suggested\nstatement in last paragraph of “Results – The Ca2+-sensitive Syt1/11 competition for\nlipid binding”.\nFigure R9 | GST does not affect Syt1 behavior in co-LP assay. (A) The soluble tandem\nC2A-C2B domain of Syt1 binds to liposomes, exhibiting a significant Ca2+-promotion effect\nthat is dependent on acidic phospholipid levels. Both GST- and His- tagged Syt1 show\nsimilar binding phenomena. (B) Tag-free Syt1(96-421), generated via Thrombin digestion,\ndemonstrates a similar competition phenomenon as GST-Syt1. Data presentation: mean\n+ s.e.m., Student’s t-test for (A), two-way ANOVA for (B). *P < 0.05, **P < 0.01, ***P <\n0.001, n.s. no significant difference.\n2. In lines 267–270, the authors describe the generation of Δloop1, Δloop2, Δloop3, and Δloop1+3\ndeletion constructs of the Syt11-C2B domain. It would be helpful to specify the exact amino acid\nresidues removed in each construct to clarify the experimental design and facilitate reproducibility.\nResponse: Thank you for your valuable suggestions. Following your advice, we have\nincluded the information in the “Results” and the “Materials and Methods” sections.\n3. Consider to replace the non-standard notations of “Ca” and “Ca” with more conventional\nterms, such as Ca²⁺-responsive (Caᴿ) and Ca²⁺-tolerant (Caᵀ) (or another widely recognized\nconvention), to improve clarity and consistency with the broader literature.\nResponse: Thank you for your valuable suggestions. To ensure clarity and alignment with\nestablished knowledge, we used full terms such as “Ca2+-infinitive” and “non-Ca2+-binding”\nIn the abstract and upon their first appearance in the text. However, these terms are quite\nlengthy and not intuitive for repeated use in the main text and figures. Therefore, we opted\nto use the concise abbreviations “Ca” and “Ca” to enhance readability and conciseness.\nMoreover, a key concept of this study is that Ca-Syts (non-Ca2+-binding Syts) also exhibit\nCa2+-sensitivity. Thus, the terms “responsive” and “tolerant” do not accurately reflect this\nassertion. The conventional terms “Ca2+-binding” and “non-Ca2+-binding” are acceptable\nand could serve as alternative phrasing.\n4. In line 63, revise “Syt 1~17” to “Syt 1–17” for standard notation of numerical ranges.\nResponse: Thank you for pointing it out. We have corrected it now.\n5. Similarly, in line 67, consider to change “2~4 Ca2+” to “2-4 Ca2+”.\nResponse: Following your advice, we have corrected it now.\nWe thank you very much for your helpful and valuable comments.\nReviewer #3 (Remarks to the Author):\nWu et al present a novel study of synaptotagmin-11 (Syt11) in which they show (a) this protein’s\nC2B and C2AB domains can bind membranes with physiological or higher anionic content, (b) this\nmembrane binding is inhibited by calcium concentrations in the millimolar range, (c) in the absence\nof calcium, Syt11 can compete with Syt1 for binding to limiting PIP2 ligands, and (d) Syt11 knockout,\nas well as rescue with membrane binding-defective mutants, subtly but significantly alter the kinetics\nof exocytosis in primary cortical neurons. These findings provide some of the first information about\nthe mechanism of action of this non-calcium-binding, inhibitory synaptotagmin. However, several\nof the claims made in the manuscript exceed the evidence presented, and the Syt1 results generally\nreproduce features of this protein that are already well established.\nResponse: Thank you very much for your helpful and valuable comments. Following your\ncomments below, we have conducted new experiments to support the claims made in this\nstudy. Regarding Syt1, we agree with you that many of its characteristics described in this\nmanuscript are not novel. However, Syt1 serves as an ideal positive control for\ncharacterizing the Ca2+-dependent lipid biding of the non-Ca2+-affinitive Syt11. Moreover,\nthe data obtained for Syt1 are crucial for clarifying the kinetic Syt1-Syt11 inter-switching in\nmembrane-occupancy during synaptic transmission.\nMajor concerns:\n1. The proposed electrostatic screening mechanism for the high-Ca inhibition of membrane binding\nis reasonable and I think it’s likely to be correct. However, I don’t see how it’s relevant to the\nphysiological competition with Syt1 that the manuscript reports occurring at orders of magnitude\nlower calcium. Inhibition of membrane binding at high calcium has been observed for decades –\nsee for example Fig. 1C-F in PMID 11823420 – but calcium levels in cells seldom exceed 0.1 mM\nand extracellular Ca is only ~1 mM. The authors’ observation that Syt1 outcompetes Syt7 at 20 uM\nCa is much more likely to arise from the increased Syt1 membrane affinity at micromolar calcium\nlevels, not from the negligible decrease in Syt11 membrane affinity (see Fig 2C, EDTA vs. 100 uM\nCa; statistical significance does not imply functional relevance). So, I question the authors’\nassertion (e.g., in lines 37-41 in the Abstract) that there is a direct connection between these two\nmain observations in the manuscript (Syt11 inhibition by 5-10 mM Ca, and Syt1/Syt11 competition\nover the 0-20 uM Ca range).\nResponse: Thank you for good comments.\n1) PMID 11823420. In this study, Sugita et al described the Ca2+ concentration dependency\nof liposome binding for various Syt-C2 domains. The Ca2+ inhibition was observed in Fig.\n1C-F, where GST-tagged Syts were used in pull-down assays to test the membrane affinity\nof Ca-Syts. However, in the subsequent co-sedimentation assays (Figs. 2-3), this\nphenomenon was not as evident. Following your advice, we have now cited this paper\n(2002) in the Discussion section.\n2) Ca2+ concentrations. This is a constructive and valid concern regarding our work. While\nhigh Ca2+ levels are indeed required to fully inhibit Syt11, we believe our conclusions are\nwell-founded for the following reasons: (a) Synaptic cleft Ca²⁺ concentration: It is true\nthat the Ca2+ concentration in CSF is ~1 mM, but the precise concentration in the synaptic\ncleft remains undetermined due to the complex pathways involving CSF, the Glymphatic\nsystem, interstitial fluid, extracellular matrix, astrocytes, and synapses (20). Astrocytes\nlikely play a key role in regulating extracellular Ca2+ levels. Additionally, there has been\nconcern that a 1.2 mM [Ca2+] may be insufficient to support long-term potentiation (LTP)\ne\n(21). (b) Local Ca²⁺ concentration: We agree with you that global/average [Ca2+] levels\ni\ntypically do not exceed 100 μM. However, local Ca2+ concentrations near voltage-gated\ncalcium channels (VGCCs) can be higher. These channels directly interact with Syt and\nSNARE proteins, positioning them close to the fusion site and enabling the formation of\nmicro- and nano-domains of Ca2+ (22). For instance, a 120 μs membrane depolarization\ncan induce a local [Ca2+] of >150 μM in neuromuscular junction synapses (23). Similarly,\ni\ngiant squid synapses generate Ca2+ microdomains of 200-300 μM (24), and local [Ca2+] in\ni\nanuran hair cell synapses have been measured between 10 μM and 1 mM (25). Collectively,\nthese findings suggest that local Ca2+ concentrations can be sufficiently high to mediate\nkinetic competition between Syt1 and Syt11 for membrane occupancy. (c) Saturation and\nphysiological relevance: Saturation is a carefully regulated mechanism used by living\nsystems to ensure sustainability. For example, although the half-saturation of synaptic\nexocytosis is achieved at a Ca2+ concentration as high as 194 μM (26), 10-20 μM is\nsufficient for transmission under normal conditions. In our study, 5 mM Ca2+ is required for\nfull-inhibition of Syt11-lipid binding, but significant inhibition is observed at 100-200 μM\n(though to a less extent). These effects may play a role in physiological environment.\nFollowing your advice, we have added a detailed discussion on this topic in the\n“Discussion – Efficiency of Ca2+ inhibition” section.\n3) Syt1’s role in Ca2+–sensitive competition. This is also a primary concern for us. Syt1\nis well-known for its highly Ca2+–sensitive membrane binding, which exceeds that of Syt11.\nTo assess the relative contributions of Syt11 and Syt1, we initially planned to use Ca2+-\nbinding deficient Syt1 mutants in Ca2+-sensitive competition assays. However, Syt1-D2N\nand Syt1-D2E, while losing Ca2+-promoted binding, exhibited Ca2+-inhibition in lipid binding\nsimilar to Syt11 (Fig. 2D). This overlap complicates the interpretation and makes it\nchallenging to draw clear conclusions. Nevertheless, we propose that the Ca2+-promoted\nbinding of Syt1 and the Ca2+-inhibited binding of Syt11 work synergistically to drive Ca2+-\nsensitive competition. Even if Syt11’s contribution is relatively smaller, the observed Ca2+-\nsensitive competition supports our model of Syt1-Syt11 inter-switching. To provide clarity,\nwe have incorporated related discussions in the “Discussion – Efficiency of Ca2+\ninhibition” section.\n2. The Abstract (lines 36-38) implies that competing with Syt1 for membrane binding in the absence\nof Ca is a conserved property of Ca(x)Syts. But the manuscript does not show this. There is one\nresult in the paper (Figure 2D, 2nd from top) that shows that 8 mM Ca inhibits Syt4 from binding\nmembranes, but Syt11 is the only protein that the authors demonstrate to compete with Syt1 for\nmembrane binding. To my knowledge, other Ca(x)Syts have not been demonstrated to bind\nmembranes under near-physiological conditions.\nResponse: Thank you for pointing this out. Following your advice, we have revised the\nabstract to specify that this binding property applies specifically to Syt11. We extended this\ngeneralization to other Ca-Syts based on the analyses presented in Fig. 1, which\nrevealed that Ca-Syts exhibit higher alkalinity on average compared to Ca-Syts,\nindicating a strong binding affinity of Ca-Syts to acidic membranes. To test this\nhypothesis, we used Syt4 and Syt11 as representative members of Ca-Syts. Our findings\nshow that they share similar lipid-binding properties, in which the lipid interaction is\nmediated by electrostatic forces and the Ca2+-inhibition is mediated via membrane-\nshielding (Figs.2-3). The mechanisms are broadly applicable.\n3. While the model of competition between Syt1 and Syt11 for binding to limited PIP2 sites is\nreasonable, the conditions used for the liposome co-sedimentation measurements differ from\nphysiological in ways that are likely to exaggerate Syt11’s membrane affinity. This is okay for these\nmechanistic experiments although it dampens excitement about the physiological relevance.\na. The salt concentration is less than physiological (100 mM NaCl). This condition has previously\nbeen shown to exaggerate electrostatic interactions of Syt1 in the absence of calcium (PMID\n26389740). Is Syt11 liposome binding also observed at 150 mM salt?\nResponse: Thank you for raising this point. We agree with you that a 100 mM salt buffer\nis not physiologically relevant. We conducted this in vitro assay following the methods of\nearlier studies (27). However, it is worth noting that Syt11-lipid binding remains persistent\neven in conditions of 100 mM NaCl plus 50/100 mM KCl (Fig 2C, resulting in a total salt\nconcentration of 150-200 mM).\nb. While total brain lipid extracts are used for a few experiments (Fig. 2B-C), the rest of the lipid\nbinding experiments (including the only Syt4 data and all of the Syt1 competition experiments) use\nhigher anionic character (40% PS and 1.5% PIP2, according to line 178). The explanation for this\ncomposition is “to compensate the charge effect” which doesn’t make sense to me – the charge\neffect of what? It would make more sense that the purpose is to strengthen Syt11’s membrane affinity.\nResponse: Thank you for your instructive suggestions.\n1) Liposome composition. Various lipid compositions have been used to study Syt-lipid\ninteractions in vitro over the past 30 years. The BET (brain extract total lipid mixture from\nAvanti) is derived from porcine brain and contains ~10.6% PS, but it lacks specificity for\nthe synaptic active zone. In human brains, PS levels vary between 11.4% and 20.5%,\ndepending on the individual and brain regions (2). Meanwhile, PIP2 accounts for ~1% of\nwhole-cell lipids (3, 4). Notably, both PS and PIP2 are predominantly located in the inner\nleaflet of the plasma membrane, with concentrations of 22.8-41% and 1-2%, respectively.\nFurthermore, PIP2 is enriched in clusters or lipid rafts at the active zoon, with local\nconcentrations reaching up to ~5% (5-7). Given this complexity, it is challenging to perfectly\nreplicate the synaptic active zone lipid environment. Therefore, the conditions used in this\nwork (40% PS and 1.5% PIP2) are intended to approximate physiological relevance.\n2) Compensate for charge effect. The charge effect was evident in Fig. 2B, where high\nPIP2 (5%) blocked Ca2+-promoted Syt1 binding, whereas BET did not. Therefore, we\nreduced the PIP2 concentration to preserve the Ca2+-promoting effects of Syt1 and Syt11\nin subsequent experiments. While we agree with you that this adjustment enhances the\nmembrane affinity of Syt11 within the physiological PS range of 22.8-41%, our primary goal\nwas to maintain an environment relevant to physiological conditions that sustain the Ca2+-\npromoting effects. Following your advice, we have added a brief explanation of this rational\nto the section “Results – Syt11 binds acidic phospholipids with Ca2+-sensitivity”.\nc. The Syt1 C2AB used for the competition experiments was purified without a cation exchange step,\nand apparently without cleaving the GST tag. The former is known to lead to impurities with lower\nmembrane affinity (PMID 23617808), and the latter could lead to dimerization which could have a\nminor effect on the competition assay. The data in Figure 2B are consistent with well purified Syt1,\nbut the Methods note that this is a different protein than what was used in the competition assay.\nBinding measurements akin to Fig 2B using the Syt1 GST fusion protein would increase confidence\nin the competition assay.\nResponse: Thank you for your helpful suggestions.\n1) Cation exchange. We did not employ cation exchange chromatography primarily\nbecause our institute lacks an FPLC system. Given the large number of mutant proteins\nwe needed to purify, relying on equipment at other institutions would have been impractical.\nInstead, we carefully purified the proteins using DNase/RNase digestion and verified their\npurity using OD280/260 measurements to minimize impurities. The membrane affinities of\nthe purified proteins were found to be comparable. As you noted, “the data in Fig. 2B are\nconsistent with well-purified Syt1”. To further ensure comparability, we have included data\nfor both GST-, His-, and tag-free Syt1, confirming that they exhibit comparable qualities in\nour assays (Fig. 2B, E, F).\n2) GST fusion. We used GST-Syt1 to achieve clear separation of Syt1 and Syt11 bands\non gels, as their C2 domains have similar molecular weights. While we recognize that GST\ndimerization could potentially be a concern, it is worth noting that Syt1 is known to form\nlarge clusters or rings on membranes, suggesting that GST dimerization may not\nsignificantly affect the results. Consistent with this, GST-Syt1 and His-Syt1 exhibited similar\nlipid-binding properties (Fig. R10A, also see Fig. 2B in the revised manuscript). Following\nyour advice, we conducted additional competition experiments using tag-free Syt1 (amino\nacids 96-421, generated by thrombin cleavage of GST-Syt1). Both tag-free Syt1 and GST-\nSyt1 showed similar competitive behaviors with Syt11 (Fig. R10B, also see Fig. 2F in the\nrevised manuscript), confirming the Ca2+-dependent removal of Syt11 competition with\nSyt1 during membrane-interaction.\nFigure R10 | GST does not affect Syt1 behavior in co-LP assay. (A) The soluble tandem\nC2A-C2B domain of Syt1 binds to liposomes, exhibiting a significant Ca2+-promotion effect\nthat is dependent on the levels of acidic phospholipid. Both GST- and His-tagged Syt1\nshowed similar binding properties. (B) The tag-free Syt1 (amino acids 96-421), generated\nby thrombin digestion, exhibied a similar competition behavior as GST-Syt1. Data\npresentation: mean + s.e.m., Student’s t-test for (A), two-way ANOVA for (B). *P < 0.05,\n**P < 0.01, ***P < 0.001, n.s. no significant difference.\n4. Please clarify what lipid composition and buffer/salt composition(s) were used in Figure 3. This\nis necessary not only for reproducibility but also to interpret the results. If these experiments were\ndone in the absence of salts except calcium, then the micromolar calcium range of the zeta potential\nshift seems unlikely to be physiologically relevant.\nResponse: Thank you for your helpful suggestions. The liposome compositions and the\nbuffer used in the ELS assay are shown in the “Materials and Methods – Liposome\npreparation” section. Initially, we used liposomes containing 10% PIP2 to test its\ncoordination with Ca2+. Following your advice, we have now conducted new experiments\nusing liposomes with 2% PIP2 to better emphasize its physiological relevance (Fig. R11,\nalso see Fig. 3C in the revised manuscript. For more details on lipid composition, please\nrefer to our response to your comment #3b. Given that the principle of ELS is based on\nelectrophoresis, it is crucial to maintain the solution conductance below 50 mM. Since a\nhigher salt concentration could lead to excessive heat generation, significant liposome\naggregation, and potential damage to the electrodes, we opted for a 40 mM NaCl buffer\nsolution in this work.\nFigure R11 | Ca2+ shields the electrostatic surface potential of acidic membranes. 50\nμM Ca2+ significantly alters the electrostatic states of liposome membrane containing 2%\nand 100% PIP2. Data presentation: mean + s.e.m., one-way ANOVA and two-tailed Mann-\nWhitney test. *P < 0.05, n.s. no significant difference.\n5. Lines 281-282: What is the rationale for the claim that the K-motif has a greater contribution to\nmembrane affinity than loop 1? From Figure 4H, the mutation of 3 residues in loop 1 (K/H/R) seems\nto have more inhibitory effect than the mutation of 4 residues in the K-motif (K/R/K/K). I agree with\nthe authors’ main contention that many basic regions on the C2B domain contribute to affinity.\nHowever, Syt11 seems to be unique in its extent of positive charge on loop 1, if I am reading Figure\n1C correctly. This suggests to me that the competitive effect in the absence of calcium may be unique\n(or strongest) for Syt11 among the Ca(x)syts.\nResponse: Thank you for this insightful comment.\n1) K-motif vs Loop1. The interaction between the K-motif and Loop1 is somewhat\nredundant, which explains why single or double regional mutations did not result in\nsignificant differences. The Loop1 appeared slightly more effective than K-motif in Fig\nm m\n4H, however, the most dramatic difference was observed between Kmotif Lp2 3 (where\nm m m\nbinding was eliminated) and Lp1 2 3 (where considerable binding remained). This\nm m m\ndemonstrates the stronger affinity of the K-motif compared to Loop1. Our summary of “K-\nmotif>lp1>lp2/3” was derived from comprehensive comparisons among all 14 mutants.\n2) Universal Loop1. The Fig 1C primarily displays the K-motifs, rather than the Loop1\nregions (which are on the opposite side and out of view). The citation in the main text refers\nto Fig. 1D (the right panel showing the potential of C2Bs). Here, the red curves illustrate\nthe electrostatic potential statistics for the 9 Ca-Syts, with two vertical dashed lines\nmarking the Loop1 region. The average value (red curve) and the 95% confidence interval\n(red shaded area) indicate that most Ca-Syts exhibit a positive potential (above 0 mV) in\ntheir Loop1 regions. In contrast, most Ca-Syts display a negative potential (black curve\nand gray shaded area, below 0 mV) in their Loop1 regions. Thus, we propose that this\nfeature (opposite to that of Ca-Syts-) is universal among Ca-Syts.\n6. The paragraphs on p. 15 (lines 356-380) seem to belong in the Discussion rather than the Results.\nMost particularly:\nResponse: Thank you for your valuable suggestions. Following your advice, we have\nrevised the content and moved these sentences to the Discussion section.\na. Lines 364-365: the manuscript does not show that Syt11 prefers or stabilizes curved membranes,\nalthough this seems like a reasonable thing to speculate based on its distribution of basic lipid\nbinding sites.\nResponse: Thank you for bringing this to our attention. We have rephrased it to a\nhypothetic expression in the revised manuscript.\nb. Lines 369-370: the manuscript does not show that Syt11 promotes vesicle docking at resting states.\nThis is speculation.\nResponse: Thank you for pointing it out. We proposed this hypothesis based on Syt11’s\nstrong membrane affinity and our previous findings, which demonstrate that Syt11 KO\nresults in a reduced Cm jump following a 200 ms depolarization pulse stimulation in DRG\nneurons We have now rephrased it to present it as a hypothetic description.\n7. P. 17, line 407 (and also line 290) states that Syt11 has a “higher resting-state membrane affinity”\nthan Syt1, but the data in Figure 2E are not quantitative enough to make this assertion. (If the blue\nshapes accurately reflect the relative concentrations, then the affinities of the two proteins in EDTA\nlook approximately equal to me.) What are the Syt1 and Syt11 concentrations used in these titrations?\nResponse: Thank you for your helpful comments. In Fig. 2E, the protein quantities used\nwere 0, 0.5, 1, 2, 4, and 8 μg of Syt11, which competed with 5 μg of Syt1, revealing that\nthe affinity of Syt11 is slightly higher than that of Syt1. The blue shapes in the figure indicate\nthe progressively increased amount of Syt11 used in this assay, rather than providing exact\nquantification. Following your advice, we have included this information in the figure\nlegend. Consistent with these findings, electrostatic potential analyses revealed that Ca-\nC2Bs exhibit higher alkalinity than Ca-C2Bs (Fig 1). This difference is particularly evident\nwhen comparing Syt11 with Syt1. In absence of Ca2+, Syt11 possesses more and larger\ninteraction interfaces than Syt1 (Fig 4C-K, Syt1: only K-motif; Syt11: K-motif + loop1/2/3).\nMinor concerns:\n1. p. 7, line 160 – please provide a reference for the “previous research.”\nResponse: Thank you for pointing it out. We have added the citation now.\n2. P. 7, lines 164-166 – the word “compensates” is confusing. Do the authors mean that the\nelectrostatic interaction saturates the Syt1 membrane binding even in the absence of calcium?\nResponse: Thank you for pointing it out. Yes, that is what we intended to convey, though\nwe were unable to identify a more suitable term at the time.\n3. P. 7, line 177 – I think the authors mean “intermediate” rather than “compromised”\nResponse: Thank you for pointing it out. We have corrected it now.\n4. In Figure 1, why the switch from human to rat sequences/structures between 1B and 1C? Because\nrodent proteins were used in this paper, it would seem more appropriate to include rodent\nsequence/structure data in the main paper. Any human comparisons could go in a supplement.\nResponse: Thank you for your valuable suggestions. We have now included the rat\nsequences in our analysis (Fig. R12A-B, also see Fig. 1A-B in the revised manuscript).\nThe protein sequences of Syt1 (with 98% identity) and Syt11 (with 95% identity) are highly\nconserved between humans and rats, suggesting a conserved role of Syt1-Syt11\ninteractions in mediating the tight coupling of exocytosis and endocytosis across species.\nFigure R12 | Non-Ca2+-affinitive Syts exhibit high alkalinity. (A) Phylogenetic tree of\namino acid sequence similarities among all human and rat Syts, showing that Ca-Syts 4,\n8, and 11 are clustered within Ca-Syts. (B) Calculated isoelectric points (pIs) of all rat\nSyt-C2 domains, highlighting the high pI values of most C2B domains. (C) AlphaFold-\npredicted structures of all rat C2 domains, illustrating surface electrostatic potential.\nTop left: Syt11-C2 domains aligned with a Ca2+-bound Syt1 crystal structure (PDB: 5CCH);\ntop right: Ca-Syt C2 domains with positive (blue) and negative (red) electrostatic potentials;\nbottom: Ca-Syt C2 domains showing extensive alkaline surfaces.\n5. Figure 1C: the usual convention is to display positive charges blue and negative charges red. Is\nthere a particular reason to use the opposite scheme here?\nResponse: Thank you for raising this issue. Interestingly, as is commonly understood in\nvarious scientific and engineering fields, including other areas of biology, the color red in\nheatmaps typically signifies positive values, high values, high activity, or hot-spots. For\ninstance, in physics, high or positive electric potential (or point charges) are usually\ndepicted in red (28). Similarly, in chemistry, most papers use red to present “high values”\nof potential in compounds (29). However, a few studies use red to denote regions with\nconcentrated (“large amounts” of) electrons (negative potential) in their compounds (30).\nIn structural biology, the use of red for oxygen atoms and blue for nitrogen atoms stems\nfrom a classical display mode. This convention may data back to the chemist August\nHoffman in 1865: oxygen is assigned red because it is essential for combustion, while\nnitrogen is given blue as it is the most abundant element in Earth’s atmosphere and the\nsky appears blue. In biochemistry, oxygen is often associated with carboxyl groups and\nnegative charges, while nitrogen is linked to amino groups and positive charges. As a result,\nthe usual convention is to display positive charges in blue and negative charges in red for\nprotein surface.\nBesides electrostatic potential, red is commonly used to present high/large/positive\nvalues across various contexts, such as temperature, concentration, height, enhancement,\ncorrelation coefficient, etc. Given this widespread usage, we prefer our original coloring\nscheme for the following reasons: (1) While it may appear unconventional to structural\nbiologists, it aligns with a much broader scientific consensus. (2) The conventional color\nmode may cause confusion between the representation of atom identity and its\nelectrostatic potential. These two systems are not entirely consistent because the nitrogen\nin Q/N does not provide positive potential, and the oxygen in S/T/Y does not provide\nnegative potential. Therefore, a different coloring system may be needed in the future to\ndistinguish them (e.g., the reversed rainbow we were using). (3) Particularly, this study\nhighlights the positive potential of Syts for mediating membrane-binding activity. Here, a\npositive value indicates high activity or a hot area, and the red color aligns more effectively\nwith this indication. Nonetheless, we have also prepared a figure using the conventional\ncoloring mode for your reference (Fig. R12C). We are open to using this alternative version\nif you prefer it.\n6. Line 184, “consistently” may not be the right word to describe a result that is visible in only one\nlane of one gel.\nResponse: Thank you for pointing it out. We have now included the statistical analysis\nfor the repeat experiments (Fig. R13, also see Fig. 2D in the revised manuscript).\nFigure R13 | Ca2+-inhibition persists in Ca-Syts (e.g., Syt4 and Ca-deficient Syt1\nmutants), but it is absent in the Ca2+-affinity-restored Syt11-S247D mutant. Data\npresentation: mean + s.e.m., one-way ANOVA and two-tailed Mann-Whitney test. *P < 0.05,\n**P < 0.01, n.s. no significant difference.\n7. Lines 209-211: this sentence is an overstatement of the results to this point, and may be more\nappropriate for the Discussion.\nResponse: Thank you for pointing it out. Following your advice, we have removed it.\n8. Lines 217-218: calcium chelation by PIP2 is already known (PMID 19630438)\nResponse: Thank you for raising this point. We initially discussed and cited an early paper\n(1988) on Ca2+/Mg2+-PIP2 interactions in our original manuscript. Following your advice,\nwe have now revised the text flow and added additional citations, including the work you\nmentioned here, in the section “Discussion - Ca2+-affinitive phospholipid membrane”.\n9. Line 292: Please provide a reference for the “calcium inhibition of the K-rich motif in all Syts”,\nwhich was not shown in this study (this study did not use all Syts).\nResponse: Thank you for your valuable comments. We have revised the wording to avoid\nany potential overstatement. This section was part of the summary and inference in the\n“Discussion”, and it was derived from the mechanisms uncovered in this study. The high\nmembrane affinity of Syts stems from their high alkalinity, a trait common to most Syt-C2B\ndomains. Meanwhile, Ca2+-inhibition arises from PIP2 chelation, which is universal to all\nSyts. Given the universality of both mechanisms, we propose that this phenomenon is likely\nwidespread. However, further validations will be needed in future studies.\n10. Line 402: can the authors explain what is meant by “certain dimensions”? This sentence doesn’t\nmake sense to me in light of the authors’ model that Syt1 and Syt11 have opposing effects on exo-\nand endocytosis.\nResponse: Thank you for your helpful comment. We have changed the term “dimensions”\nto “aspects”. This point is addressed in the section “Discussion - Non-Ca2+-affinitive but\nhighly alkaline”, immediately following the analysis of Syt alkalinity presented in Fig. 1.\nSyt11 was grouped with Ca-Syts, implying that their features and functional mechanisms\nshould be similar in “certain aspects”. We then sought to identify those aspects and defined\nmembrane binding as the key features they shared. The opposing effects of Ca2+ on Syt1-\nliposome and Syt11-liposome interactions were further findings that derived from this\nprimary aspect of membrane binding.\nWe thank you very much for your helpful and valuable comments.\nReferences cited in point-by-point responses:\n1. Y. Chen et al., Synaptotagmin-1 is a bidirectional Ca2+ sensor for neuronal\nendocytosis. Proceedings of the National Academy of Sciences 119, e2111051119\n(2022).\n2. L. Svennerholm, Distribution and fatty acid composition of phosphoglycerides\nin normal human brain. J Lipid Res 9, 570-579 (1968).\n3. O. Cremona et al., Essential role of phosphoinositide metabolism in synaptic\nvesicle recycling. Cell 99, 179-188 (1999).\n4. M. R. Wenk et al., Phosphoinositide profiling in complex lipid mixtures using\nelectrospray ionization mass spectrometry. Nature Biotechnology 21, 813-817\n(2003).\n5. K. Aoyagi et al., The activation of exocytotic sites by the formation of\nphosphatidylinositol 4,5-bisphosphate microdomains at syntaxin clusters. J\nBiol Chem 280, 17346-17352 (2005).\n6. I. Milosevic et al., Plasmalemmal phosphatidylinositol-4,5-bisphosphate level\nregulates the releasable vesicle pool size in chromaffin cells. J Neurosci\n25, 2557-2565 (2005).\n7. D. J. James, C. Khodthong, J. A. Kowalchyk, T. F. J. Martin,\nPhosphatidylinositol 4,5-bisphosphate regulates SNARE-dependent membrane\nfusion. Journal of Cell Biology 182, 355-366 (2008).\n8. G. van den Bogaart et al., Membrane protein sequestering by ionic protein–\nlipid interactions. Nature 479, 552-555 (2011).\n9. M. N. Zanetti et al., Ring-like oligomers of Synaptotagmins and related C2\ndomain proteins. eLife 5, e17262 (2016).\n10. C. Wang et al., Synaptotagmin‐11 inhibits clathrin‐mediated and bulk\nendocytosis. EMBO reports 17, 47-63-63 (2016).\n11. M. R. Bowers, N. E. Reist, Synaptotagmin: Mechanisms of an electrostatic\nswitch. Neuroscience Letters 722, 134834 (2020).\n12. E. Hui, C. P. Johnson, J. Yao, F. M. Dunning, E. R. Chapman, Synaptotagmin-\nmediated bending of the target membrane is a critical step in Ca(2+)-regulated\nfusion. Cell 138, 709-721 (2009).\n13. A. Bhalla, M. C. Chicka, E. R. Chapman, Analysis of the Synaptotagmin Family\nduring Reconstituted Membrane Fusion: UNCOVERING A CLASS OF INHIBITORY\nISOFORMS*. Journal of Biological Chemistry 283, 21799-21807 (2008).\n14. Y. Park, J.-K. Ryu, Models of synaptotagmin-1 to trigger Ca2+-dependent\nvesicle fusion. FEBS Letters 592, 3480-3492 (2018).\n15. E. Toulmé, A. Salazar Lázaro, T. Trimbuch, J. Rizo, C. Rosenmund,\nNeurotransmitter release is triggered by a calcium-induced rearrangement in\nthe Synaptotagmin-1/SNARE complex primary interface. Proceedings of the\nNational Academy of Sciences 121, e2409636121 (2024).\n16. K. Jaczynska et al., A lever hypothesis for Synaptotagmin-1 action in\nneurotransmitter release. Proc Natl Acad Sci U S A 122, e2417941121 (2025).\n17. J. Rizo, K. Jaczynska, C. Rosenmund, Evaluation of synaptotagmin-1 action\nmodels by all-atom molecular dynamics simulations. FEBS Open Bio 15, 699-713\n(2025).\n18. R. Mohrmann, H. de Wit, M. Verhage, E. Neher, J. B. Sørensen, Fast Vesicle\nFusion in Living Cells Requires at Least Three SNARE Complexes. Science 330,\n502-505 (2010).\n19. K. Carvalho, L. Ramos, C. Roy, C. Picart, Giant unilamellar vesicles\ncontaining phosphatidylinositol(4,5)bisphosphate: characterization and\nfunctionality. Biophys J 95, 4348-4360 (2008).\n20. J. P. Lopes, R. A. Cunha, What is the extracellular calcium concentration\nwithin brain synapses? J Neurochem 149, 435-437 (2019).\n21. M. Forsberg et al., Ionized calcium in human cerebrospinal fluid and its\ninfluence on intrinsic and synaptic excitability of hippocampal pyramidal\nneurons in the rat. J Neurochem 149, 452-470 (2019).\n22. E. M. Adler, G. J. Augustine, S. N. Duffy, M. P. Charlton,\nAlien intracellular\ncalcium chelators attenuate neurotransmitter release at the squid giant\nsynapse. J Neurosci 11, 1496-1507 (1991).\n23. B. Yazejian, X. P. Sun, A. D. Grinnell, Tracking presynaptic Ca2+ dynamics\nduring neurotransmitter release with Ca2+-activated K+ channels. Nat Neurosci\n3, 566-571 (2000).\n24. R. Llinás, M. Sugimori, R. B. Silver, Microdomains of high calcium\nconcentration in a presynaptic terminal. Science 256, 677-679 (1992).\n25. W. M. Roberts, R. A. Jacobs, A. J. Hudspeth, Colocalization of ion channels\ninvolved in frequency selectivity and synaptic transmission at presynaptic\nactive zones of hair cells. J Neurosci 10, 3664-3684 (1990).\n26. R. Heidelberger, C. Heinemann, E. Neher, G. Matthews, Calcium dependence of\nthe rate of exocytosis in a synaptic terminal. Nature 371, 513-515 (1994).\n27. S. Sugita, O. H. Shin, W. Han, Y. Lao, T. C. Südhof, Synaptotagmins form a\nhierarchy of exocytotic Ca2+ sensors with distinct Ca2+ affinities. EMBO\nJournal 21, 270-280 (2002).\n28. H. A. Radi, J. O. Rasmussen, in Principles of Physics: For Scientists and\nEngineers, H. A. Radi, J. O. Rasmussen, Eds. (Springer Nature Switzerland,\nCham, 2024), pp. 661-697.\n29. C. H. Suresh, G. S. Remya, P. K. Anjalikrishna, Molecular electrostatic\npotential analysis: A powerful tool to interpret and predict chemical\nreactivity. WIREs Computational Molecular Science 12, e1601 (2022).\n30. S. R. Hinze et al., Textbook Treatments of Electrostatic Potential Maps in\nGeneral and Organic Chemistry. Journal of Chemical Education 90, 1275-1281\n(2013).\nPoint-by-point response to the reviewers’ comments\nWe sincerely thank the reviewers for their positive evaluations and constructive\nfeedback.\nReviewer #1 (Remarks to the Author):\nThe Authors have addressed my concerns with the manuscript, as well as in my opinion\nthose of other reviewers. Congratulations on an exciting discovery and robust\nmanuscript. Thank you for adding data, clarifications, and statistical analysis.\nResponse: Thank you very much for your valuable suggestions, which greatly improve\nthe quality of our work.\nReviewer #2 (Remarks to the Author):\nAll points raised by me have been adequately addressed. I have no further comments\nor suggestions.\nResponse: We really appreciate your insightful suggestions and encouragement.\nReviewer #3 (Remarks to the Author):\nThe manuscript is much improved, and it represents a significant advance in the field\nof synaptic transmission and exocytosis. I recommend publication.\nResponse: We truly appreciate your insightful comments throughout the review process,\nwhich greatly improved the quality of our work."}, {"round": 2, "reviewer_comments": {"reviewer_1": "The Authors have addressed my concerns with the manuscript, as well as in my opinion those of other reviewers. Congratulations on an exciting discovery and robust manuscript. Thank you for adding data, clarifications, and statistical analysis.", "reviewer_2": "All points raised by me have been adequately addressed. I have no further comments or suggestions.", "reviewer_3": "The manuscript is much improved, and it represents a significant advance in the field of synaptic transmission and exocytosis. I recommend publication."}, "author_response": "Point-by-point response to the reviewers’ comments\nWe sincerely thank the reviewers for their positive evaluations and constructive\nfeedback.\nReviewer #1 (Remarks to the Author):\nThe Authors have addressed my concerns with the manuscript, as well as in my opinion\nthose of other reviewers. Congratulations on an exciting discovery and robust\nmanuscript. Thank you for adding data, clarifications, and statistical analysis.\nResponse: Thank you very much for your valuable suggestions, which greatly improve\nthe quality of our work.\nReviewer #2 (Remarks to the Author):\nAll points raised by me have been adequately addressed. I have no further comments\nor suggestions.\nResponse: We really appreciate your insightful suggestions and encouragement.\nReviewer #3 (Remarks to the Author):\nThe manuscript is much improved, and it represents a significant advance in the field\nof synaptic transmission and exocytosis. I recommend publication.\nResponse: We truly appreciate your insightful comments throughout the review process,\nwhich greatly improved the quality of our work."}]

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