A biological relay system achieves these feats. Neurotransmission kicks off with an electrical pulse that runs down a nerve cell, or neuron. When that signal reaches the tip, calcium enters the cell. In response, the neuron liberates chemical messengers — neurotransmitters — which travel to the next neuron and thus pass the baton.
In the 1950s, the late Bernard Katz figured out that cells eject neurotransmitters in fixed amounts. Electron-micrographic studies by others illuminated how. Balloon-like containers — vesicles — each hold set quantities of the chemicals. Calcium incites these lipid-bound sacs to fuse with the membrane that encases the cell, and their contents spill out (see Figure).
Neurotransmission occurs astonishingly quickly — fast enough for a person to pull a hand off a hot burner or dodge an attacking mountain lion. Calcium entry can spark the release of neurotransmitter packages in less than a millisecond.
Although this general scheme had emerged by the time Südhof and Scheller began their work, its molecular details eluded scientists. No one knew what drives the vesicles to fuse with the cell membrane or how calcium provokes that event.
Actors at the membrane
In the late 1980s, Scheller and Südhof set out independently to unveil the molecular underpinnings of the process. Reasoning that nerve terminals would hold the reaction’s components, the scientists decided to purify and characterize proteins at that site. Focusing first on vesicles, Scheller, then at Stanford University School of Medicine, discovered what would turn out to be an essential piece of the fusion apparatus: vesicle-associated membrane protein (VAMP), which he obtained from the electric organ of a marine ray. The following year, Südhof, then at the University of Texas Southwestern Medical Center in Dallas, isolated the same protein from rat brain and called it synaptobrevin.
Südhof next purified and studied another vesicle protein, synaptotagmin. When calcium is present, this protein binds to phospholipids, major constituents of membranes. Scheller established that synaptotagmin clutches a brain protein that he named syntaxin. In contrast to vesicle-dwelling synaptotagmin and VAMP/synaptobrevin, syntaxin concentrates in the cell membrane — at spots where neurotransmitters are released from nerve cells.
Independently, James Rothman (Yale University) (Lasker Basic Medical Research Award, 2002) had been exploring how substances are ferried from one place to another inside cells. In that process, too, transport vesicles deliver their contents by merging their membranes with those of the target compartment. Rothman had proposed that one of the proteins necessary for his experimental system and for fusion in live yeast cells, NSF, attaches to the membrane through a second protein and its as-yet-unidentified collaborators. He set out to unearth these hypothetical proteins, which he called SNAREs.
To nab them, Rothman sought proteins in rat brains that adhere to NSF through its partner. Three proteins stuck: VAMP/synaptobrevin, syntaxin, and SNAP-25, a protein reported previously to reside on cell membranes of nerve termini.
Two separate lines of inquiry thus pointed at the same three molecules. The results suggested that they promote neurotransmitter release by fostering fusion, but provided only indirect evidence. Meanwhile, a clear link to their physiological function had materialized from a different direction. Scientists had long known that certain bacterial toxins thwart neurotransmission, and in 1992, Cesare Montecucco (University of Padua) showed that botulinum and tetanus toxins block neurotransmitter release by clipping off a chunk of VAMP/synaptobrevin. Subsequent work by Montecucco, Südhof, and Reinhard Jahn (Max Planck Institute, G ttingen) demonstrated that additional neurotoxins attack syntaxin and SNAP-25.
These observations established that the three proteins are vital for neurotransmission. The mechanism of fusion was murky, however, as was the way in which calcium triggers the process.
The previous work had shown that VAMP/synaptobrevin, syntaxin, and SNAP-25 bind to the NSF conglomeration, but not whether they do so alone or together. In 1993, Scheller, in collaboration with Rothman, found that VAMP/synaptobrevin, syntaxin, and SNAP-25 associate stably with one another to form the so-called ‘SNARE complex’. The team also showed that NSF rips apart this assemblage. Scientists later realized that this phenomenon helps recycle the molecular machinery.
In the meantime, researchers were defining the precise interactions among these proteins and discerning how the associations might instigate fusion (see Figure for current model). Scheller found, for instance, that VAMP/synaptobrevin on vesicles clasps plasma membrane-bound syntaxin, and that SNAP-25 helps them pair up. Work by Scheller, Jahn, John Heuser (Washington University School of Medicine, St. Louis), and others advanced the concept that VAMP/synaptobrevin and syntaxin form coils that wrap around each other along their lengths, thus drawing together the membranes in which they’re embedded. Axel Brunger (Yale University) and Jahn confirmed and extended these ideas when they reported the high-resolution structure of VAMP/synaptobrevin, syntaxin, and SNAP-25 in 1998. The results supported a scenario in which the proteins zipper together, eventually forcing fusion (see Figure).
Control mechanisms calcify
These and other observations were fleshing out the basic mechanism of fusion, but they also highlighted crucial open questions. Uncontrolled, the reaction would result in rampant and constant neurotransmitter release. Although scientists knew that the system does not launch until calcium arrives, the details remained obscure. It was clear, however, that the extremely short time between calcium influx and neurotransmitter discharge would not permit assembly of a multi-protein machine. The device must lie on the brink of its fusion-competent state, waiting for calcium to push it over the edge, presumably by a protein that senses this ion.
Südhof’s original observations on synaptotagmin tantalized him because they indicated that the protein performs calcium-dependent activities. Perhaps, he speculated, calcium prompts it to facilitate fusion. In 1993, he showed that two regions of synaptotagmin bind calcium, and this property allows it to efficiently grasp phospholipids. These results bolstered the protein’s candidacy as the calcium sensor.
In 1994, he generated mice that lack operational synaptotagmin. Although the animals died soon after birth, neurons from their embryos could be studied. Südhof found that their core fusion machinery remained functional, but unable to respond to calcium.
In an elegant set of experiments, Südhof ruled out the possibility that loss of synaptotagmin impairs calcium entry into the cell or hinders some other event peripheral to calcium perception. He generated a series of mice, each of which carried a synaptotagmin with altered calcium-binding affinity. Synaptotagmin’s affinity for the ion correlated with the calcium sensitivity of neurotransmission. Increasing calcium avidity, for instance, decreased by approximately the same amount how much calcium was needed for neurotransmitter release. These results confirmed the notion that synaptotagmin functions as the calcium sensor.
Meanwhile, another layer of regulation was surfacing. Südhof had discovered a protein, complexin, that strongly adheres to the VAMP/synaptobrevin, syntaxin, and SNAP-25 bundle. Subsequent work established that it plays an essential role in calcium-regulated neurotransmitter release. Complexin holds the partially zippered SNARE complex in a form that is poised to trigger fusion, yet inactive until the crucial next step: Calcium binds to synaptotagmin and spurs it to displace complexin, which instantly drives the reaction (see Figure).
Communication within the brain influences how we think and who we are. Defects in the process contribute to schizophrenia, depression, bipolar disorder, and many other pathological conditions. Studies of these illnesses have not yet indicted misbehaving components of the fusion complex itself, but scientists are beginning to uncover connections between this equipment and serious diseases. For example, α-synuclein, a protein that has been linked to Parkinson disease and other neurodegenerative disorders, helps assemble the fusion apparatus and protects animals from age-related neurological problems. Flaws in Munc18, another protein that Südhof and Scheller implicated in neurotransmitter release and that is indispensable for it, have been associated with Ohtahara syndrome, a severe epileptic disorder that strikes during infancy and causes seizures and mental retardation.
By systematically exposing and analyzing the proteins involved in neurotransmitter release, Südhof and Scheller have transformed our description of the process from a rough outline to a series of nuanced molecular transactions. Their work has revealed the elaborate orchestrations that lie at the crux of our most simple and sophisticated neurobiological activities.
by Evelyn Strauss
Key publications of Richard H. Scheller
Trimble, W.S., Cowan, D.M., and Scheller, R.H. (1988). VAMP-1: A synaptic vesicle-associated integral membrane protein. Proc. Natl. Acad. Sci. USA. 85, 4538-4542.
Bennett, M.K., Calakos, N., and Scheller, R.H. (1992). Syntaxin: A synaptic protein implicated in docking of synaptic vesicles at presynaptic active zones. Science. 257, 255-259.
Söllner, T., Bennett, M.K., Whiteheart, S.W., Scheller, R.H., and Rothman, J.E. (1993). A protein assembly-disassembly pathway in vitro that may correspond to sequential steps of synaptic vesicle docking, activation, and fusion. Cell. 75, 409–418.
Calakos, N., Bennett, M.K., Peterson, K.E., and Scheller, R.H. (1994). Protein-protein interactions contributing to the specificity of intracellular vesicular trafficking. Science. 263, 1146-1149.
Lin, R.C. and Scheller, R.H. (1997). Structural organization of the synaptic exocytosis core complex. Neuron. 19, 1087-1094.
Chen, Y.A., Scales, S.J., Patel, S.M., Doung, Y.-C., and Scheller, R.H. (1999). SNARE complex formation is triggered by Ca2+ and drives membrane fusion. Cell. 97, 165-174.
Key publications of Thomas C. Südhof
Perin, M.S., Fried, V.A., Mignery, G.A., Jahn, R., and Südhof, T.C. (1990). Phospholipid binding by a synaptic vesicle protein homologous to the regulatory region of protein kinase C. Nature. 345, 260-263.
Brose, N., Petrenko, A.G., Südhof, T.C., and Jahn, R. (1992). Synaptotagmin: A calcium sensor on the synaptic vesicle surface. Science. 256, 1021-1025.
McMahon, H.T., Missler, M., Li, C., and Südhof, T.C. (1995). Complexins: Cytosolic proteins that regulate SNAP receptor function. Cell. 83, 111-119.
Fernández-Chacón, R., Königstorfer, A., Gerber, S.H., Garcia, J., Matos, M.F., Stevens, C.F., Brose, N., Rizo, J. Rosenmund, C., and Südhof, T.C. (2001). Synaptotagmin I functions as a calcium regulator of release probability. Nature. 410, 41-49.
Rhee, J.-S., Li, L., Shin, O.-H., Rah, J.-C., Rizo, J., Südhof, T.C., and Rosenmund, C. (2005). Augmenting neurotransmitter release by enhancing the apparent Ca2+ affinity of synaptotagmin 1. Proc. Natl. Acad. Sci. USA. 102, 18664–18669.
Tang, J., Maximov, A., Shin, O.-H., Dai, H., Rizo, J., and Südhof, T.C. (2006). A complexin/synaptotagmin-1 switch controls fast synaptic vesicle exocytosis. Cell. 126, 1175-1187.
Review articles by Scheller and Südhof on neurotransmitter release
Bennett, M.K. and Scheller, R.H. (1993). The molecular machinery for secretion is conserved from yeast to neurons. Proc. Natl. Acad. Sci. USA. 90: 2559-2563.
Südhof, T.C. (1995). The synaptic vesicle cycle: A cascade of protein-protein interactions. Nature. 375, 645-653.
Lin, R.C. and Scheller, R.H. (2000). Mechanisms of synaptic vesicle exocytosis. Annu. Rev. Cell Dev. Biol. 16, 19-49.
Südhof, T.C. (2012). Calcium control of neurotransmitter release. Cold Spring Harb. Perspect. Biol. 4:a011353, 1-15.