A lever hypothesis for Synaptotagmin-1 action in neurotransmitter release

“…complexes ( 10 ) that consist of four - helix bundles ( 11 , 12 ) and bring the membranes together as they assemble (zipper) from the N - to the C - terminus ( 13 ), likely inducing membrane fusion ( 14 ) by promoting lipid acyl chain encounters at the polar membrane–membrane interface ( 15 ).”

“The SNAP receptors (SNAREs) syntaxin - 1, SNAP - 25, and synaptobrevin form tight complexes ( 10 ) that consist of four - helix bundles ( 11 , 12 ) and bring the membranes together as they assemble (zipper) from the N - to the C - terminus ( 13 ), likely inducing membrane fusion ( 14 ) by promoting…”

“N - ethylmaleimide sensitive factor (NSF) and soluble NSF attachment proteins (SNAPs) disassemble SNARE complexes ( 10 ) to recycle the SNAREs ( 16 ).”

“The function of Syt1 as the Ca 2+ sensor that triggers neurotransmitter release and its essential nature for fast, synchronous release ( 28 , 66 ) are well established, but its mechanism of action has remained enigmatic despite intense research for over three decades.”

“…is what is the role of the Syt1 C 2 A domain, as Ca 2+ binding to this domain contributes to trigger neurotransmitter release even if it is not as critical as Ca 2+ binding to the C 2 B domain ( 28 , 32 , 34 ), and a Syt1 mutant lacking the C 2 A domain cannot rescue Syt1 function ( 71 , 72 ).”

“…complexes ( 10 ) that consist of four - helix bundles ( 11 , 12 ) and bring the membranes together as they assemble (zipper) from the N - to the C - terminus ( 13 ), likely inducing membrane fusion ( 14 ) by promoting lipid acyl chain encounters at the polar membrane–membrane interface ( 15 ).”

“…complex formation by an NSF - SNAP - resistant mechanism ( 3 , 17 ) in which Munc18 - 1 binds first to a closed conformation of syntaxin - 1 ( 18 , 19 ) and later binds to synaptobrevin, forming a template complex ( 20 – 22 ) for SNARE assembly while Munc13 bridges the two membranes ( 4 , 23 )…”

“…SNARE complex formation by an NSF - SNAP - resistant mechanism ( 3 , 17 ) in which Munc18 - 1 binds first to a closed conformation of syntaxin - 1 ( 18 , 19 ) and later binds to synaptobrevin, forming a template complex ( 20 – 22 ) for SNARE assembly while Munc13 bridges the two membranes ( 4 , 23…”

“The cytoplasmic region of Syt1 is formed mostly by two C 2 domains (C 2 A and C 2 B) that bind three and two Ca 2+ ions, respectively, via loops at the tip of β-sandwich structures ( 29 – 31 ).”

“Many studies reported diverse interactions of Syt1 with the SNAREs that were generally enhanced by Ca 2+ [reviewed in ( 44 , 45 )].”

“…of Syt1 that have been proposed to facilitate membrane fusion such as bilayer perturbation ( 35 , 37 , 38 ), induction of membrane curvature ( 39 , 40 ), or membrane bridging ( 42 , 43 ) can cooperate with the SNAREs to induce fast membrane fusion, particularly after molecular dynamics (MD)…”

“C 2 B for simplicity) with SNARE complex four - helix bundle bound to a fragment spanning residues 26 - 83 of Complexin - 1 (referred to as CpxSC) were performed as described in (47).”

“…involves several steps, including vesicle tethering to pre-synaptic active zones, priming of the vesicles to a release - ready state(s), and vesicle fusion with the plasma membrane, which occurs very fast upon Ca 2+ influx into a presynaptic terminal ( 1 ) [less than 60 μ s in fast synapses ( 2 )].”

“Linker zippering led to fast (microsecond scale) fusion in the simulations (15) and is in principle energetically favorable (62).”

“The SNAP receptors (SNAREs) syntaxin - 1, SNAP - 25, and synaptobrevin form tight complexes ( 10 ) that consist of four - helix bundles ( 11 , 12 ) and bring the membranes together as they assemble (zipper) from the N - to the C - terminus ( 13 ), likely inducing membrane fusion ( 14 ) by promoting…”

“The cytoplasmic region of Syt1 is formed mostly by two C 2 domains (C 2 A and C 2 B) that bind three and two Ca 2+ ions, respectively, via loops at the tip of β-sandwich structures ( 29 – 31 ).”

“…- SNAP - resistant mechanism ( 3 , 17 ) in which Munc18 - 1 binds first to a closed conformation of syntaxin - 1 ( 18 , 19 ) and later binds to synaptobrevin, forming a template complex ( 20 – 22 ) for SNARE assembly while Munc13 bridges the two membranes ( 4 , 23 ) and opens syntaxin - 1 ( 24 ).”

“Previous studies ( 25 , 50 , 53 ), together with the correlations between our binding data ( 47 ) ( Figs.”

“Many studies reported diverse interactions of Syt1 with the SNAREs that were generally enhanced by Ca 2+ [reviewed in ( 44 , 45 )].”

“…of Syt1 that have been proposed to facilitate membrane fusion such as bilayer perturbation ( 35 , 37 , 38 ), induction of membrane curvature ( 39 , 40 ), or membrane bridging ( 42 , 43 ) can cooperate with the SNAREs to induce fast membrane fusion, particularly after molecular dynamics (MD)…”

“While this model explains a large amount of data and release - of - inhibition models have been popular since 30 years ago ( 56 ), there is evidence that Syt1 is not merely a SNARE inhibitor and exerts an active action to trigger release upon Ca 2+ binding ( 7 , 44 ).”

“Three structures of Syt1–SNARE complexes have been described, each revealing binding through distinct surfaces of the C 2 B domain that are not involved in Ca 2+ binding ( 25 , 48 , 49 ).”

“…to facilitate membrane fusion such as bilayer perturbation ( 35 , 37 , 38 ), induction of membrane curvature ( 39 , 40 ), or membrane bridging ( 42 , 43 ) can cooperate with the SNAREs to induce fast membrane fusion, particularly after molecular dynamics (MD) simulations revealed a natural…”

“Moreover, it is not clear how actions of Syt1 that have been proposed to facilitate membrane fusion such as bilayer perturbation ( 35 , 37 , 38 ), induction of membrane curvature ( 39 , 40 ), or membrane bridging ( 42 , 43 ) can cooperate with the SNAREs to induce fast membrane fusion, particularly…”

“Ca 2+ induces insertion of these loops into membranes ( 35 , 36 ), which was proposed to facilitate membrane fusion by perturbing the bilayers ( 35 , 37 , 38 ) and/or inducing membrane curvature ( 39 , 40 ).”

“…facts that R322E, K325E and K325A, K327A mutations strongly impair neurotransmitter release whereas a K324E, K326E mutation has no significant effect ( 48 , 51 ), and that R322E, K325E disrupts Ca 2+ - dependent binding to PIP 2 - containing nan-odiscs much more strongly than K324E, K326E ( 47 ).”

“…is what is the role of the Syt1 C 2 A domain, as Ca 2+ binding to this domain contributes to trigger neurotransmitter release even if it is not as critical as Ca 2+ binding to the C 2 B domain ( 28 , 32 , 34 ), and a Syt1 mutant lacking the C 2 A domain cannot rescue Syt1 function ( 71 , 72 ).”

“In addition, the Syt1 C 2 B domain binds to phosphatidylinositol 4,5 - bisphosphate (PIP 2 ) through a polybasic region on the side of the β-sandwich ( 41 ) and was proposed to help induce fusion by bridging the two membranes ( 42 , 43 ).”

“4 B ), but such extension is not required for neurotransmitter release ( 70 ) or liposome fusion ( 61 ).”

“Intriguingly, while an R398Q, R399Q mutation in region II that disrupts C 2 B–SNARE complex binding ( 47 ) strongly impairs neurotransmitter release ( 25 , 53 ), release is also impaired by a E295A, Y338W mutation in region I ( 25 ) that enhances binding ( 47 ).”

“…is what is the role of the Syt1 C 2 A domain, as Ca 2+ binding to this domain contributes to trigger neurotransmitter release even if it is not as critical as Ca 2+ binding to the C 2 B domain ( 28 , 32 , 34 ), and a Syt1 mutant lacking the C 2 A domain cannot rescue Syt1 function ( 71 , 72 ).”

“1 A ) requires a bimane - tryptophane distance below 10 Å ( 58 ).”

“…is what is the role of the Syt1 C 2 A domain, as Ca 2+ binding to this domain contributes to trigger neurotransmitter release even if it is not as critical as Ca 2+ binding to the C 2 B domain ( 28 , 32 , 34 ), and a Syt1 mutant lacking the C 2 A domain cannot rescue Syt1 function ( 71 , 72 ).”

“…- SNAP - resistant mechanism ( 3 , 17 ) in which Munc18 - 1 binds first to a closed conformation of syntaxin - 1 ( 18 , 19 ) and later binds to synaptobrevin, forming a template complex ( 20 – 22 ) for SNARE assembly while Munc13 bridges the two membranes ( 4 , 23 ) and opens syntaxin - 1 ( 24 ).”

“…containing SNARE complex than to plain nanodiscs in the absence of Ca 2+ while the affinity is similar in the presence of Ca 2+ , suggesting that Ca 2+ actually induces dissociation of C 2 AB from membrane - anchored SNARE complexes ( 46 , 47 ) or at least weakens the C 2 AB–SNARE interactions.”

“Moreover, it is not clear how actions of Syt1 that have been proposed to facilitate membrane fusion such as bilayer perturbation ( 35 , 37 , 38 ), induction of membrane curvature ( 39 , 40 ), or membrane bridging ( 42 , 43 ) can cooperate with the SNAREs to induce fast membrane fusion, particularly…”

“…affect the binding mode between the Syt1 C 2 B domain and the SNARE complex in solution ( 47 ), but remodeling of the binding mode might be triggered because Ca 2+ induces a reorientation of the C 2 B domain on the membrane ( 54 , 55 ) that is incompatible with the primary interface ( 47 ).”

“Note also that other interactions of Syt1 have been described, such as binding of the C 2 B domain to the SNARE complex through a so - called tripartite interface ( 49 ) or Ca 2+ - independent oligomerization ( 73 ).”

“This design led to tryptophan - induced quenching of the bimane fluorescence upon binding of Syt1 C 2 B domain to liposome - anchored SC - 59 - bimane in the absence of Ca 2+ due to the predicted proximity of the tryptophan to the bimane in the primary interface (57).”

“Munc18 - 1 and Munc13s organize SNARE complex formation by an NSF - SNAP - resistant mechanism ( 3 , 17 ) in which Munc18 - 1 binds first to a closed conformation of syntaxin - 1 ( 18 , 19 ) and later binds to synaptobrevin, forming a template complex ( 20 – 22 ) for SNARE assembly while Munc13…”

“…is what is the role of the Syt1 C 2 A domain, as Ca 2+ binding to this domain contributes to trigger neurotransmitter release even if it is not as critical as Ca 2+ binding to the C 2 B domain ( 28 , 32 , 34 ), and a Syt1 mutant lacking the C 2 A domain cannot rescue Syt1 function ( 71 , 72 ).”

“…- SNAP - resistant mechanism ( 3 , 17 ) in which Munc18 - 1 binds first to a closed conformation of syntaxin - 1 ( 18 , 19 ) and later binds to synaptobrevin, forming a template complex ( 20 – 22 ) for SNARE assembly while Munc13 bridges the two membranes ( 4 , 23 ) and opens syntaxin - 1 ( 24 ).”

“Moreover, it is not clear how actions of Syt1 that have been proposed to facilitate membrane fusion such as bilayer perturbation ( 35 , 37 , 38 ), induction of membrane curvature ( 39 , 40 ), or membrane bridging ( 42 , 43 ) can cooperate with the SNAREs to induce fast membrane fusion, particularly…”

“Although we and others have been unable to observe the tripartite interface in solution ( 47 , 74 ) or the oligomerization by cryo - EM ( 7 , 23 , 75 ), the relevance of these interactions and their compatibility with our model need further investigation.”

“These results, together with previous electron paramagnetic resonance (EPR) data ( 54 ), suggest a model whereby Ca 2+ binding causes dissociation of region I but not region II of the primary interface and reorientation of C 2 B on the membrane, which pulls the SNARE complex through ionic…”

“…linker zippering is expected to be hindered by substantial energy barriers because the zippering pulls the hydrophobic TM regions into the polar bilayer interface (63), and the abundant basic and aromatic residues of the jxt linkers have high propensities to interact with the lipids (15, 27).”

“…phenotypes caused by mutations in the primary interface, most notably the findings that neurotransmitter release is impaired by mutations that impair or enhance binding through this interface and is differentially affected by mutations in the SNARE acidic residues (25, 52, 53, 64) ( Discussion ).”

“…linker zippering is expected to be hindered by substantial energy barriers because the zippering pulls the hydrophobic TM regions into the polar bilayer interface (63), and the abundant basic and aromatic residues of the jxt linkers have high propensities to interact with the lipids (15, 27).”

“…complex upon reorientation of C 2 B might increase the angle between the long axis of the four - helix bundle and the plasma membrane, consistent with data showing that the angle between the SNARE complex and a supported lipid bilayer is increased by binding to Ca 2+ - saturated Syt1 C 2 AB ( 69 ).”

“Since Y338 was replaced by aspartate in the mutant that suppressed lethality in the Drosophila screen, and an A402T mutation also suppressed lethality ( 50 ), we titrated a double Y338D/A204T 15 N -”

“The basic steps that lead to exocytosis have been reconstituted with the main components of the neurotransmitter release machinery ( 3 – 6 ) and the functions of these components have been defined ( 7 – 9 ).”

“…in crystal structures of Syt1–SNARE complexes ( 25 , 49 ), but can be attributed to the increase in the positive electrostatic potential of C 2 B caused by the mutation, as overall electrostatics play a key role in binding of the highly basic C 2 B domain to the highly acidic SNARE complex ( 67 ).”

“…suggested that this interface is important for vesicle priming ( 49 , 51 ), which is strongly supported by our electrophysiological studies ( 52 ) and likely arises because Syt1 cooperates with Munc18 - 1 and Munc13 - 1 in mediating Ca 2+ - independent assembly of trans - SNARE complexes (…”

“Because the R322E/K325E mutation increases the stability of the C 2 B domain and slows down H/D exchange (47, 74), some amide groups in the β-strands are not fully protonated after expression in D 2 O and purification in buffers containing H 2 O, resulting in signal loss for the corresponding peaks.”

“…that the pull on the SNARE complex away from the site of fusion caused by the C 2 B domain reorientation facilitates extension of the syntaxin - 1 and synaptobrevin SNARE motif helices into the jxt linkers, which is critical for neurotransmitter release ( 59 , 60 ) and liposome fusion ( 61 ).”

“The “lever hypothesis” offers an explanation: upon calcium binding, the Syt1 C2B domain undergoes a reorientation on the membrane [27].”

“…further reveal that axons of basal forebrain cholinergic neurons projecting to the bone marrow release acetylcholine, which inhibits NLRP3 inflammasome assembly via α 7nAChR, maintaining the dynamic balance between osteogenesis and osteoclastogenesis (Theis et al., 2025; Jaczynska et al., 2025).”