As briefly elucidated earlier in this article, membrane fusion is mediated via a specialized set of proteins at the secretory vesicle membrane and the cell plasma membrane. Three soluble N-ethylmaleimide-sensitive factor (NSF)-attachment protein receptors (SNAREs) have been implicated in membrane fusion (29). Target membrane proteins, SNAP-25 and syntaxin (t-SNARE), and secretory vesicle-associated membrane protein (v-SNARE), are part of the conserved protein complex involved in fusion of opposing bilayers (29). The molecular mechanism of the involvement of SNAREs to bring about membrane fusion remained unknown until 2002 (9, 17, 18). The structure and arrangement of SNAREs, when associated with lipid bilayers, were first determined using the AFM (9). A bilayer electrophysiological assay allowed measurements of membrane conductance and capacitance before and after t-SNARE- or v-SNARE-reconstitution and following exposure to v-SNARE- or t-SNARE-reconstituted lipid vesicles. Results from these studies demonstrate that t-SNAREs and v-SNARE, when present in opposing bilayers, interact in a circular array, and, in the presence of calcium, form conducting pores (9). The interaction of t-/v-SNARE proteins to form a conducting pore or channel is strictly dependent on the presence of t-SNAREs and v-SNARE in opposing bilayers. Addition of purified recombinant v-SNARE to a t-SNARE-reconstituted lipid membrane increased only the size of the globular t-SNARE oligomer without influencing the electrical properties of the membrane (9). However, when t-SNARE vesicles are added to v-SNARE-reconstituted membrane, SNAREs assemble in a ring pattern (Fig. 6) and a stepwise increase in capacitance, and conductance is observed (9). Thus, t- and v-SNAREs are required to reside in opposing bilayers to allow appropriate t-/v-SNARE interactions leading to membrane fusion only in the presence of calcium (9). Studies using SNARE-reconstituted liposomes and bilayers (17) demonstrate (i) a slow fusion rate ( = 16 min) between t- and v-SNARE-reconstituted liposomes in the absence of Ca2+; and (ii) exposure of t-/v-SNARE liposomes to Ca2+ drives vesicle fusion on a near physiological-relevant time scale ( ~ 10 sec), demonstrating an essential role of Ca2+ in membrane fusion. Because the Ca2+ effect on membrane fusion in SNARE-reconstituted liposomes is downstream of SNAREs, it suggests a regulatory role for Ca2+-binding proteins in membrane fusion in the physiological state (17). It is further demonstrated from these studies that, in the physiological state in cells, both SNAREs and Ca2+ operate as the minimal fusion machinery (17). Native and synthetic vesicles exhibit a significant negative surface charge primarily due to the polar phosphate head groups. These polar head groups produce a repulsive force, preventing aggregation and fusion of apposing vesicles. SNAREs bring opposing bilayers closer, to within a distance of 2–3 Å (Fig. 7), allowing Ca2+ to bridge them (17). The bound Ca2+ then leads to the expulsion of water between the bilayers at the bridging site, allowing lipid mixing and membrane fusion. Hence, SNAREs, besides bringing apposing bilayers closer, dictate the site and size of the fusion area during secretion. The size of the t-/v-SNARE complex forming the pore is dictated by the curvature of the opposing membranes, hence depends on the size of t-/v-SNARE-reconstituted vesicles. The smaller the vesicles, the smaller the pores formed (unpublished observation).
However, at the atomic level, how does Ca2+ bring about membrane fusion? This was resolved in a recent study (18). Calcium ion is essential for life processes and is found in every cell. Ca2+ participates in diverse cellular processes, such as metabolism, secretion, proliferation, muscle contraction, cell adhesion, learning, and memory. Although calcium is abundantly present within the cell, it is well sequestered and is available only on demand. Upon certain cellular stimulus for instance, Ca2+ concentration at specific locations (i.e., nanoenvironment) within the cell is elevated by several orders of magnitude within a brief period (some in of Ca2+ is essential for many physiological processes, such as the release of neurotransmitters or cell signaling. A unique set of chemical and physical properties of the Ca2+ ion make it ideal for performing these biochemical reactions. Calcium ion [Ca2+] exists in its hydrated state within cells. The properties of hydrated calcium have been extensively studied using x-ray diffraction and neutron scattering in combination with molecular dynamics simulations (38–41). The molecular dynamic simulations include three-body corrections compared with ab initio quantum mechanics/molecular mechanics molecular dynamics simulations. First principles molecular dynamics have also been used to investigate the structural, vibrational, and energetic properties of [Ca(H2O)n]2+ clusters and the hydration shell of calcium ion. These studies demonstrate that hydrated calcium [Ca(H2O)n]2+ has more than one shell around the Ca2+, with the first hydration shell around the Ca2+ having six water molecules in an octahedral arrangement (39). In studies using light scattering and x-ray diffraction of SNARE-reconstituted liposomes, it was demonstrated that fusion proceeds only when Ca2+ ions are available between the t- and v-SNARE-apposed bilayers (Fig. 8; Ref. 18).
To monitor interaction(s) between Ca2+ ions and phosphate on the lipid membrane head groups, an x-ray diffraction method was used (17). This experimental approach for monitoring interbilayers contacts essentially requires the presence of (i) highly concentrated lipid suspensions (10 mM and above) favoring a multitude of intervesicular contacts; and (ii) a fully hydrated liposomes, where vesicles have full freedom to interact with each other in solution, hence establishing a confined hydrated area between adjacent bilayers. This small fluid space could arise from interbilayer hydrogen-bond formation through water molecules (42) and additional bridging forces contributed by trans-SNARE complex formation (9, 17). If these two conditions are met, then liposomes diffract as shown (Fig. 8). Mixing of t- and v-SNARE liposomes in the absence of Ca2+ leads to a diffuse and asymmetric diffractogram (depicted by the gray trace; Fig. 7), a typical characteristic of short-range ordering in a liquid system. In contrast, mixing the t-SNARE and v-SNARE liposomes in the presence of Ca2+ leads to a more structured diffractogram (depicted by the black trace; Fig. 7) with an approximately 12% increase in x-ray scattering intensity, pointing to an increase in the number of contacts between apposing bilayers established presumably by calcium-PO bridges, as previously suggested (43). The ordering effect of Ca2+ on interbilayer contacts observed in x-ray studies (18) is in good agreement with recent light microscopy, AFM, and spectroscopic studies suggesting close apposition of PO lipid head groups in the presence of Ca2+ ions followed by formation of Ca2+-PO bridges between adjacent bilayers (17, 44). An x-ray study shows that the effect of Ca2+ on bilayer orientation and interbilayer contacts is most prominent in the area of 3 Å, with additional appearance of a new peak (shoulder) at 2.8 Å (depicted by the arrow; Fig. 7), both of which are within the ionic radius of Ca2+ (18). These studies suggest that the ionic radius of Ca2+ may play an important role in membrane fusion. But there remained a major spatial problem, which was recently resolved (18). As discussed earlier, calcium ions [Ca2+] exist in their hydrated state within cells. Hydrated calcium [Ca(H2O)n]2+ has more than one shell around the Ca2+, with the first hydration shell having six water molecules in an octahedral arrangement (38), measuring ~6 Å (Fig. 8). Studies reveal that for hydrated Ca2+ ion, depending on its coordination number, the nearest average neighbor Ca2+-O and Ca2+-H distances are at r ~ 2.54 Å and r ~ 3.2 Å, respectively, in the first hydration shell. How then would a hydrated calcium ion measuring ~6 Å fit between the 2.8–3 Å space established by t-/v-SNAREs, between the apposing bilayers? One possibility would be that calcium has to be present in the buffer solution when t-SNARE vesicles and v-SNARE vesicles meet. If t- and v-SNARE vesicles are allowed to mix in a calcium-free buffer, no fusion should occur. This was tested in a published study (18). Light-scattering experiments (Fig. 8) were performed on t-SNARE- and v-SNARE-reconstituted phospholipids vesicles in the presence and absence of calcium and in the presence of NSF+ATP. NSF or N-ethylmaleimide-sensitive factor is an ATPase that is known to disassemble the t-/v-SNARE complex. Using the light-scattering measurements, aggregation and membrane fusion of lipid vesicles can be monitored on the second time scale (17, 45). The initial rapid increase in intensity of light scattering was initiated by the addition of t- and v-SNARE vesicles into the cuvette, followed by a slow decay of light scattering (Fig. 8), representing interactions between vesicles in solution. These studies show that, if t-SNARE vesicles and v-SNARE vesicles are allowed to interact before calcium addition (depicted by arrow; Fig. 8), no significant change in light scattering is observed (there is no significant decrease in scattering, attributed to little fusion between the vesicle suspension). On the contrary, when calcium is present in the buffer solution before addition of the t-SNARE and v-SNARE vesicles, there is a marked drop in light scattering, as a result of vesicle aggregation and fusion (Fig. 8). However, in the presence of NSF-ATP in the assay buffer containing calcium, a significant inhibition in aggregation and fusion of proteoliposomes is observed (Fig. 8). NSF, in the absence of ATP, has no effect on the light-scattering properties of the vesicle mixture. These results demonstrate that NSF-ATP disassembles the SNARE complex, thereby reducing the number of interacting vesicles in solution. In addition, disassembly of trans-SNARE complex will then leave apposed bilayers widely separated, out of reach for the formation of Ca2+-PO bridges (Fig. 8). Similarly, if the restricted area between adjacent bilayers deliniated by the circular arrangement of the t-/v-SNARE complex (9) is preformed, then hydrated Ca2+ ions are too large (Fig. 8) to be accommodated between bilayers and, hence, subsequent addition of Ca2+ would have no effect (Fig. 8). However, when t-SNARE vesicles interact with v-SNARE vesicles in the presence of Ca2+, the t-/v-SNARE complex formed allows formation of calcium-phosphate bridges between opposing bilayers, leading to the expulsion of water around the Ca2+ ion to enable lipid mixing and membrane fusion (Fig. 8). Thus, x-ray and light-scattering studies (18) demonstrate that calcium bridging of the apposing bilayers is required to enable membrane fusion. This calcium bridging of apposing bilayers leads to the release or expulsion of water from the hydrated Ca2+ ion, leading to bilayer destabilization and membrane fusion. It could also be argued that the binding of calcium to the phosphate head groups of the apposing bilayers may displace the loosely coordinated water at the PO groups, further adding to the destabilization of the lipid bilayer, leading to membrane fusion.