Figure 6. Mathematical model of the fusion pore in the presence of Syt1 and SNAREs suggests a mechanical calcium-triggered pore dilation mechanism.

(A) Schematic of model. The membrane free energy has contributions from membrane tension and bending energy. SNARE complexes may be unzippered and free to roam laterally, or zippered and confined to the pore waist. Crowding among zippered SNARE complexes generates entropic forces that tend to enlarge the pore (top view, shown lower right). The Syt1 C2B domain (green ellipsoid) has a SNARE-binding region, a polybasic patch and Ca²⁺-binding loops. (B) Free energy-minimizing fusion pore shapes determined by solving the membrane shape equation in the presence and absence of constraints applied by the SNARE-C2B complex (see Appendix 1). The C2B calcium-binding loops may either be unburied (top panel) or buried (lower panel) in the membrane. In the buried state the SNARE complex tilts upwards, expanding the fusion pore. The membrane shape constraint is evaluated using the SNARE-C2B complex crystal structure in a space filling representation. Both upper and lower panels depict situations in the presence of Ca²⁺. The model predicts the tilted configuration is strongly favored at high ${\displaystyle \left[\mathrm{C}{\mathrm{a}}^{2+}\right]}$ following equilibration, while the untilted configuration is relevant to the kinetics that establish this equilibrium, and to experiments using low ${\displaystyle \left[\mathrm{C}{\mathrm{a}}^{2+}\right]}$. VAMP2, syntaxin, SNAP25 and the C2B domain are shown blue, red, yellow, and green, respectively. The C2B hydrophobic membrane-inserting residues (V304, I367), polybasic patch (K326, K327) and SNARE-binding region (R398, R399) are shown orange, cyan, and purple, respectively. The protein structure was generated with PyMOL (Schrodinger, LLC, 2015) using the SNARE-C2B crystal structure (PDB ID 5ccg) (Zhou et al., 2015). The TMD of the SNARE complex (PDB ID 3hd7) (Stein et al., 2009) was incorporated using UCSF chimera software (Pettersen et al., 2004). (C) Model-predicted free energy and experimental apparent free energy versus pore radius without calcium and in the presence of excess calcium. (D) Model-predicted normalized conductances shown with experimentally measured values for comparison. Experimental data taken from Figure 2B experiments including Ca²⁺ and PI(4,5)P₂. (E) Pore dilation mechanism emerging from the model. Under conditions of low calcium concentration, the C2B domain is unburied, the SNARE complex lies parallel to the membrane and the membrane separation is set by the maximum thickness of the SNARE-C2B complex. At high calcium concentrations, the calcium binding loops penetrate the plasma membrane, rotating the C2B domain and the entire SNARE-C2B complex which exerts force (red arrows) on the upper and lower membranes of the fusion pore in a lever-like action. These forces increase the fusion pore height, which is coupled by membrane energetics to fusion pore dilation.