Molecular mechanism of SNARE-induced membrane fusion
At the atomic level, the participation of calcium in SNARE-induced membrane fusion has further been determined by Jena and his research team . Calcium ion is essential to life’s processes, and participates in diverse cellular and physiological functions. Although calcium is present in abundance within cells, it is well sequestered and is available only on demand . Upon certain cellular stimulus, Ca2+ concentration at specific nano environments in a cell becomes elevated by several orders of magnitude within a brief period (2+ is essential for many physiological functions, such as the release of neurotransmitters. Not surprisingly, calcium ion channels have been found in direct association with t-SNARE (SNAP-23), at the base of fusion pores or porosomes (permanent supramolecular structures at the cell plasma membrane [12–19]), where secretory vesicles dock and fuse to release their contents . Calcium ion [Ca2+] exists in its hydrated state within cells . Since hydrated calcium [Ca(H2O)n]2+ with its first hydration shell possess six water molecules, and measure >6 Å, it would be impossible to squeeze between the 2.8–3 Å space established by t-/v-SNAREs, between the apposing vesicle bilayers [8, 11]. The answer is simple, as elucidated by Jena and his research team .
When t- and v-SNARE vesicles are allowed to mix in a calcium-free buffer, prior to the addition of calcium, no fusion occurs. On the contrary, when t and v-SNARE vesicles are allowed to mix in a calcium-buffer, vesicles aggregate and fuse . When NSF-ATP (for SNARE complex disassembly)  is present in the assay buffer containing calcium, a significant inhibition in aggregation and fusion of proteoliposomes is observed. NSF, in the absence of ATP, has no effect. 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+-phosphate bridges, preventing membrane fusion. Similarly, if the restricted area between adjacent bilayers delineated by the circular arrangement of the t-/v-SNARE complex is formed, then hydrated Ca2+ ions are too large to be accommodated between the bilayers, and hence subsequent addition of Ca2+ would have no effect. However, when t-SNARE vesicles interact with v-SNARE vesicles in the presence of Ca2+, the t-/v-SNARE complex formed allow 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 . The calcium bridging of apposing bilayers allows for the release of water from the hydrated Ca2+ ion, leading to bilayers destabilization and membrane fusion. In addition, the binding of calcium to the phosphate head groups of the apposing bilayers, may also displace the loosely coordinated water at the phosphate groups, further contributing to the destabilization of the lipid bilayer, leading to membrane fusion . Following determination of the molecular mechanism of calcium on SNARE-induced membrane fusion by Jena and his research team [7, 8, 11], the important role of calcium in cell secretion was further confirmed by Rothman in collaboration with another group . These discoveries by Jena and Rothman, have finally determined the molecular machinery and mechanism of membrane fusion in cells. The main steps in membrane fusion, porosome opening and cell secretion of SNARE-induced membrane fusion during secretion, are highly controlled and regulated events (Fig. 1): 1) vesicles dock at the base of porosomes by tethering to porosome-associated t-SNAREs by vesicle-associated v-SNARE; 2) the t-/v-SNARE interact in a circular array to form a ring complex,
pulling the opposing bilayers into close proximity (2.8–3 Å) complex; 3) during this process, the hydrated Ca2+ present between the opposing bilayers (i.e., the base of porosome and the secretory vesicle membrane), is able to bridge with the phospholipid head groups, allowing the release of the water shell from the hydrated Ca2+ ion; 4) this leads to the destabilization of the lipid bilayers within the t-/v-SNARE channel complex, leading to lipid mixing and membrane fusion; 5) the porosomes opening then dilates; 6) secretory vesicles swell, generating intravesicular pressure, which drive the expulsion (partially or completely, depending on the intravesicular pressure); 7) following secretion, vesicle reseals and detaches from the membrane (empty or partially empty) due to SNARE complexes isassembly by NSF-ATP.
Finally, after almost a century of studies to understand the molecular mechanism of membrane fusion in cells, the subject has been greatly elucidated. With
further developments of technology and tools, we may one day in the future be able to understand the rearrangement and interactions of individual atoms in real time (
The author thanks Dr. M. Hinescu for critical reading of the manuscript. This work was partially supported by Ministry of Education and Research CEEX 62/2005 Grant.