From the
The release of neurotransmitter via regulated exocytosis is the primary mode of communication in the nervous system. Changes in the efficacy of this process are at the basis of nervous system plasticity. Our understanding of the biochemical events that mediate neurotransmitter secretion has undergone a revolution in the last few years. The initial focus of research, identifying proteins associated with transmitter-containing (synaptic) vesicles, has spawned the current functional analyses of synapse proteins. These analyses have revealed that neurotransmitter release utilizes molecular events common to all membrane trafficking.
Neurotransmitters are released from small (50 nM) vesicles that undergo many rounds of fusion and recycling at presynaptic terminals. Synaptic vesicles appear to be assembled not in the Golgi but rather in presynaptic endosomal compartments(1, 2) . Unlike neuropeptide-containing secretory granules, synaptic vesicles fill with transmitter after they are formed. Filling with neurotransmitter is mediated by specific transport molecules(3) . Loaded vesicles either sequestered in a reserve pool via interaction with cytoskeletal elements (4) or cluster at the presynaptic terminal at specialized regions termed active zones. The fusion of these ``docked'' vesicles with the presynaptic plasma membrane occurs when intracellular calcium concentrations rise during an action potential. Calcium regulates one of the final events in synaptic vesicle fusion because the release of neurotransmitter following calcium elevation occurs in less than 1 ms.
Neurotransmitter release is therefore the sum of many molecular processes. This minireview will concentrate on what has been learned about vesicle targeting and fusion from a combination of yeast genetics and biochemical analyses. How calcium might regulate these events is discussed in light of this common pathway.
Neurosecretion Utilizes Mechanisms Common to All Eukaryotic
Membrane Transport
The discovery that several proteins localized to the synapse are homologs of yeast proteins required for constitutive secretion provided the first solid evidence that neurosecretion is a specialized version of general membrane trafficking(5) . This realization has led to the search for non-synaptic forms of these proteins in animal cells and for mammalian homologs of other secretory yeast proteins(6) . While neurosecretion is distinguished from constitutive secretion by being regulated by calcium, it shares features with all membrane trafficking including the mechanisms that mediate vesicle targeting and docking and the involvement of the actin cytoskeleton and acidic phospholipids.
Figure 1: A series of protein interactions hypothesized to mediate vesicle docking and fusion. The current working model of vesicle docking and fusion proposes that specificity of vesicle docking is provided by the specific interactions of the proteins VAMP, syntaxin, and SNAP-25. See text for a detailed description.
Before vesicles dock,
the v-SNARE VAMP is hypothesized to be associated with the synaptic
vesicle protein synaptophysin. On the plasma membrane, the t-SNARE
syntaxin is bound to a soluble protein called n-Sec1. Syntaxin and
n-Sec1 dissociate by an unknown mechanism (i) allowing the
formation of a protein complex that sediments at 7 S. This complex
includes VAMP, syntaxin, the t-SNARE SNAP-25, and the synaptic vesicle
protein synaptotagmin. Since synaptotagmin is expressed only in neural
and endocrine cells, the 7 S complex may be unique to regulated
secretion. Synaptotagmin dissociates from the 7 S complex (ii)
allowing the addition of the soluble protein -SNAP (soluble NSF
attachment protein). As its name suggests, the binding of
-SNAP
allows the subsequent binding of NSF (N-ethylmaleimide-sensitive fusion protein) and formation of a
20 S complex (iii). NSF is an ATPase, and its activity (iv) results in the disruption of the 20 S complex. The
dissociation of the 20 S particle may initiate membrane fusion via
unknown intermediates as illustrated in step v.
Evidence for this model includes the protein interactions discussed below and the following observations. 1) VAMP and syntaxin each have homologs in yeast (5) and in animal cells(13, 14) . In yeast, unique homologs of these proteins exist at different stages of the secretory pathway where they are required for secretory vesicle docking and/or fusion. 2) VAMP, syntaxin, and SNAP-25 are substrates of the clostridial and tetanus neurotoxins that block neurosecretion, an observation that indicates that these proteins are involved in the exocytosis of synaptic vesicles(15) .
Small GTP-binding proteins are also associated with transport vesicles in animal cells(33) . As in yeast, each species of transport vesicle contains a specific isoform. Rab3, the isoform specific to synaptic vesicles, has been hypothesized to play a role identical to that of Sec4p in yeast. However, mutations that produce a dominant negative phenotype in yeast do not disrupt secretion in animal cells(34) , and targeted disruption of the rab3 gene in mice produces only a mild phenotype(35) . These results may indicate that Rab3 performs a redundant function in neurons. However, they may also indicate either that Rab3 is not the mammalian functional homolog of Sec4p and Ypt1p or that its function is less important in the synapse than in constitutive secretion.
There are several ways acidic
phospholipids could play important roles in membrane transport. Acidic
phospholipids may act as fusogens(41, 42) . This
hypothesis is based on the observation that an increase in the acidic
phospholipid content of synthetic liposomes increases their
fusability(43) . Alternatively, acidic phospholipids may
regulate the interaction of cytoskeletal proteins with vesicles. PI and
its higher phosphorylated forms, PIP and PIP, regulate the
activity of many actin-associated proteins(44) . Therefore,
changes in the concentrations of these lipids in either the vesicle or
acceptor membrane are predicted to alter the surrounding actin
cytoskeleton. This potential link between membrane composition and
actin regulatory proteins is especially appealing in light of
observations that changes in the actin cytoskeleton accompany
secretion.
What is the role of the cytoskeleton in effecting secretion? One clue has come from the identification of a protein required for vesicle docking that has homology to several actin-binding proteins. This protein, termed TAP (transcytosis-associated protein) (48) or p115 (78) was identified through the characterization of soluble proteins required for transcytotic transport in epithelial cells. TAP is required, along with NSF, for the docking and fusion of transport vesicles with the plasma membrane. TAP appears to act prior to NSF to mediate vesicle docking at the acceptor membrane since vesicles incubated with TAP in the absence of ATP will dock, but will not fuse with, the plasma membrane(48) . TAP is a homolog of the yeast USO1 gene product. Mutations in the USO1 gene disrupt ER to Golgi transport indicating that TAP and Uso1p may perform a similar function required at all stages of the secretory pathway(49) . Given its homology to actin-associated proteins and its role in vesicle docking and association with transport vesicles(50) , TAP may mediate cytoskeletal assisted docking of vesicles as illustrated in Fig. 2A. In this model, TAP links transport vesicles to actin filaments and presents the vesicle to the acceptor membrane. TAP may act alone or in conjunction with another myosin-like protein(s). Membrane docking and fusion are then achieved via specific v- and t-SNARE interaction.
Figure 2: Cytoskeleton-associated protein may act at both vesicle docking and fusion. A, TAP (p115) is required for docking of transport vesicles with the plasma membrane. TAP is associated with transport vesicles and contains a region homologous to actin-binding proteins. TAP may therefore facilitate the formation of the 20 S particle by positioning the vesicle over the t-SNARE complex. B, CAPS may resolve docked hemi-fused vesicles toward fusion. This hypothesis predicts that CAPS function would be mediated by a non-calcium-regulated homolog in constitutive secretion. CAPS may also act prior to the formation of the 20 S particle in a fashion similar to that hypothesized for TAP.
Calcium Regulation Is Superimposed on the Machinery
of Membrane Trafficking
While it has become apparent that neurotransmitter secretion
utilizes mechanisms common to all membrane transport, it is
distinguished by its regulation by calcium. Calcium-regulated secretion
is also a feature of endocrine and exocrine cells. Two to three
calcium-mediated processes have been revealed by physiological analyses
of regulated secretion in endocrine
cells(51, 52, 53) . These processes, which
differ in their rates and calcium affinity, have been hypothesized to
reflect calcium-dependent vesicle priming and fusion (52) .
Vesicle fusion requires higher calcium concentrations (K = 27 µM) than priming, which can be mediated
by less than 1 µM calcium. In neurons, the initiation of
fusion begins approximately 10 times faster than in endocrine cells and
requires even higher calcium concentrations (>100 µM calcium)(54) . Therefore, the calcium regulation of neural
secretion may utilize the high and medium affinity calcium regulators
present in endocrine cells (requiring submicromolar to micromolar
calcium) as well as a low affinity regulator (requiring millimolar
calcium).
Disruption of the synaptotagmin gene in Caenorhabditiselegans and Drosophila produces animals that are severely impaired but maintain limited levels of synaptic transmission, indicating that synaptotagmin is not absolutely required for neurotransmission(62, 63) . Likewise, disruption of the synaptotagmin I gene in mice produces animals that die within 2 days but which are morphologically indistinguishable from normal mice at birth(64) . Analyses of synaptic transmission in synaptotagmin-deficient animals have produced somewhat disparate results. In Drosophila, stimulus evoked neurotransmission at the neuromuscular junction is severely impaired in synaptotagmin null mutants(65, 66, 67) . However, spontaneous transmission at this synapse is increased severalfold. This increased spontaneous release would be predicted to deplete the vesicle pool and therefore diminish evoked responses. These results support the hypothesis that synaptotagmin acts as an inhibitor of constitutive secretion. The absence of synaptotagmin I also produces markedly attenuated synaptic transmission in mice. However, unlike Drosophila synaptotagmin null mutants, cultured hippocampal neurons from synaptotagmin-deficient mice do not display an increase in spontaneous transmitter release. The evoked transmission that remains appears to lack the large, early component while retaining a late, smaller component. These observations suggest that synaptotagmin could mediate the rapid, initial events of transmitter release and argue against it serving as a negative regulator of transmission(64) .
It is unlikely that synaptotagmin performs a different function in mammalian neurons than it does in the neurons of flies. Therefore, it is difficult to rationalize the differences in spontaneous transmitter release between synaptotagmin-deficient flies and mice. These observed differences could reflect inherent differences in the preparations studied (embryonic neuromuscular junction in flies versus cultured central nervous system neurons from mice). Each system may highlight different aspects of synaptotagmin functioning. Synaptotagmin may in fact perform several functions. It is associated with the clathrin adaptor protein AP2(69) , an interaction that implicates synaptotagmin in endocytosis. In addition, quantitative electron microscopic analysis of synapses indicates that synaptotagmin null flies have fewer synaptic vesicles immediately adjacent to the plasma membrane suggesting that synaptotagmin also plays a role in membrane docking(68) . Two observations suggest that synaptotagmin may not be the low affinity calcium regulator of fast neurotransmitter release. First, its affinity for calcium is in the micromolar range(58) , which is significantly higher than the millimolar calcium concentrations required to induce fast transmission(54) . Second, synaptotagmin is present in endocrine cells, which do not exhibit the fast transmission of neurons.
Is there an additional calcium regulator present in neurons? Given the speed of the initial events of neurotransmitter release, it is possible that a protein does not mediate this low affinity calcium-regulated step. Rapid fusion may instead be mediated by the presence of acidic phospholipids, which are fusogenic in the presence of millimolar calcium concentrations(43) . This hypothesis proposes that, in addition to the other calcium-regulated events, neurons, by positioning vesicles near calcium channels, utilize the low calcium affinity fusogenic properties of acidic phospholipids to produce the fast component of neural secretion.
The progress that has resulted from the convergence of biochemistry with yeast genetics has accelerated the pace at which the molecular events of membrane transport are being elucidated. Future research will focus not only on testing the proposed sequence of protein-protein interactions but also on identifying how calcium regulation is imposed on this system. As our understanding of the basic mechanisms of neurosecretion increases, attention will undoubtedly shift to how the molecules of release are modified to produce changes in synaptic efficacy.