Synaptic Vesicle Function and Formation

In this final section we consider the regulated secretion of neurotransmitters that is the basis for signaling by many nerve cells. These small, water-soluble molecules (e.g., acetyl-

choline, dopamine) are released at chemical synapses, specialized sites of contact between a signaling neuron and a receiving cell. Generally signals are transmitted in only one direction: an axon terminal from a presynaptic cell releases neurotransmitter molecules that diffuse through a narrow extracellular space (the synaptic cleft) and bind to receptors on a postsynaptic cell (see Figure 7-31). The membrane of the postsynaptic cell, which can be another neuron, a muscle cell, or a gland cell, is located within approximately 50 nm of the presynaptic membrane.

Neurotransmitters are stored in specialized regulated secretory vesicles, known as synaptic vesicles, which are 40-50 nm in diameter. Exocytosis of these vesicles and release of neurotransmitters is initiated when a stimulatory electrical impulse (action potential) travels down the axon of a presynaptic cell to the axon terminal where it triggers opening of voltage-gated Ca2+ channels. The subsequent localized rise in the cytosolic Ca2+ concentration induces some synaptic vesicles to fuse with the plasma membrane, releasing their contents into the synaptic cleft. We described the major events in signal transmission at chemical synapses and the effects of neurotransmitter binding on postsynaptic cells in Chapter 7. Here we focus on the regulated secretion of neurotransmitters and the formation of synaptic vesicles in the context of the basic principles of vesicular trafficking already outlined in this chapter.

Synaptic Vesicles Loaded with Neurotransmitter Are Localized Near the Plasma Membrane

The exocytosis of neurotransmitters from synaptic vesicles involves targeting and fusion events similar to those that lead to release of secreted proteins in the secretory pathway. However, several unique features permit the very rapid release of neurotransmitters in response to arrival of an action potential at the presynaptic axon terminal. For example, in resting neurons some neurotransmitter-filled synaptic vesicles are "docked" at the plasma membrane; others are in reserve in the active zone near the plasma membrane at the synaptic cleft. In addition, the membrane of synaptic vesicles contains a specialized Ca2+-binding protein that senses the rise in cytosolic Ca2+ after arrival of an action potential, triggering rapid fusion of docked vesicles with the presynaptic membrane.

A highly organized arrangement of cytoskeletal fibers in the axon terminal helps localize synaptic vesicles in the active zone (Figure 17-35). The vesicles themselves are linked together by synapsin, a fibrous phosphoprotein associated with the cytosolic surface of all synaptic-vesicle membranes. Filaments of synapsin also radiate from the plasma membrane and bind to vesicle-associated synapsin. These interactions probably keep synaptic vesicles close to the part of the plasma membrane facing the synapse. Indeed, synapsin knockout mice, although viable, are prone to seizures; during repetitive stimulation of many neurons in such mice, the number of synaptic vesicles that fuse with the plasma mem-

▲ EXPERIMENTAL FIGURE 17-35 Fibrous proteins help localize synaptic vesicles to the active zone of axon terminals. In this micrograph of an axon terminal obtained by the rapid-freezing deep-etch technique, synapsin fibers can be seen to interconnect the vesicles and to connect some to the active zone of the plasma membrane. Docked vesicles are ready to be exocytosed. Those toward the center of the terminal are in the process of being filled with neurotransmitter. [From D. M. D. Landis et al., 1988, Neuron 1:201.]

brane is greatly reduced. Thus synapsins are thought to recruit synaptic vesicles to the active zone.

Rab3A, a GTP-binding protein located in the membrane of synaptic vesicles, also is required for targeting of neuro-transmitter-filled vesicles to the active zone of presynaptic cells facing the synaptic cleft. Rab3A knockout mice, like synapsin-deficient mice, exhibit a reduced number of synap-tic vesicles able to fuse with the plasma membrane after repetitive stimulation. The neuron-specific Rab3 is similar in sequence and function to other Rab proteins that participate in docking vesicles on particular target membranes in the secretory pathway.

A Calcium-Binding Protein Regulates Fusion of Synaptic Vesicles with the Plasma Membrane

Fusion of synaptic vesicles with the plasma membrane of axon terminals depends on the same proteins that mediate membrane fusion of other regulated secretory vesicles. The principal v-SNARE in synaptic vesicles (VAMP) tightly binds syntaxin and SNAP-25, the principal t-SNAREs in the plasma membrane of axon terminals, to form four-helix SNARE complexes. After fusion, SNAP proteins and NSF

within the axon terminal promote disassociation of VAMP from t-SNAREs, as in the fusion of secretory vesicles depicted previously (see Figure 17-11).

0 Strong evidence for the role of VAMP in neurotransmitter exocytosis is provided by the mechanism of action of botulinum toxin, a bacterial protein that can cause the paralysis and death characteristic of botulism, a type of food poisoning. The toxin is composed of two polypep-tides: One binds to motor neurons that release acetylcholine at synapses with muscle cells, facilitating entry of the other polypeptide, a protease, into the cytosol of the axon terminal. The only protein this protease cleaves is VAMP. After the bot-ulinum protease enters an axon terminal, synaptic vesicles that are not already docked rapidly lose their ability to fuse with the plasma membrane because cleavage of VAMP prevents assembly of SNARE complexes. The resulting block in acetylcholine release at neuromuscular synapses causes paralysis. However, vesicles that are already docked exhibit remarkable resistance to the toxin indicating that SNARE complexes may already be in a partially assembled, protease-resistant state when vesicles are docked on the presynaptic membrane. I

The signal that triggers exocytosis of docked synaptic vesicles is a rise in the Ca2+ concentration in the cytosol near vesicles from <0.1 ^M, characteristic of resting cells, to 1-100 ^M following arrival of an action potential in stimulated cells. The speed with which synaptic vesicles fuse with the presynaptic membrane after a rise in cytosolic Ca2+ (less than 1 msec) indicates that the fusion machinery is entirely assembled in the resting state and can rapidly undergo a con-formational change leading to exocytosis of neurotransmit-ter. A Ca2+-binding protein called synaptotagmin, located in the membrane of synaptic vesicles, is thought to be a key component of the vesicle fusion machinery that triggers exo-cytosis in response to Ca2+ (Figure 17-36).

Several lines of evidence support a role for synaptotag-min as the Ca2+ sensor for exocytosis of neurotransmitters. For instance, mutant embryos of Drosophila and C. elegans that completely lack synaptotagmin fail to hatch and exhibit very reduced, uncoordinated muscle contractions. Larvae with partial loss-of-function mutations of synaptotagmin survive, but their neurons are defective in Ca2+-stimulated vesicle exocytosis. Moreover, in mice, mutations in synapto-tagmin that decrease its affinity for Ca2+ cause a corresponding

▲ FIGURE 17-36 Release of neurotransmitters and the recycling of synaptic vesicles. Step 1: Synaptic vesicles loaded with neurotransmitter (red circles) move to the active zone and then dock at defined sites on the plasma membrane of a presynaptic cell. Synpatotagmin prevents membrane fusion and release of neurotransmitter. Botulinum toxin prevents exocytosis by proteolytically cleaving VAMP the v-SNARE on vesicles. Step 2: In response to a nerve impulse (action potential), voltage-gated Ca2+ channels in the plasma membrane open, allowing an influx of Ca2+ from the extracellular medium. The resulting Ca2+-induced conformational change in synaptotagmin leads to fusion of docked vesicles with the plasma membrane and release of neurotransmitters into the synaptic cleft. Step 3: After clathrin/AP vesicles containing v-SNARE and neurotransmitter transporter proteins bud inward and are pinched off in a dynamin-mediated process, they lose their coat proteins. Dynamin mutations such as shibire in Drosophila block the re-formation of synaptic vesicles, leading to paralysis. Step 4 : The uncoated vesicles import neurotransmitters from the cytosol, generating fully reconstituted synaptic vesicles and completing the cycle. Most synaptic vesicles are formed by endocytic recycling as depicted here. However, endocytic vesicles containing membrane from the axon terminus can fuse with the endosome; budding from this compartment can then form "new" synaptic vesicles. [See K. Takei et al., 1996, J. Cell. Biol. 133:1237; V. Murthy and C. Stevens, 1998, Nature 392:497; and R. Jahn et al., 2003, Cell 112:519.]

increase in the amount of cytosolic Ca2+ needed to trigger rapid exocytosis.

Several hypotheses concerning how synaptotagmin promotes neurotransmitter exocytosis have been proposed, but the precise mechanism of its function is still unresolved. Synaptotagmin is known to bind phospholipids and after undergoing a Ca2 + -induced conformational change, it may promote association of the phospholipids in the vesicle and plasma membranes. Synaptotagmin also binds to SNARE proteins and may catalyze a late stage in assembly of SNARE complexes when bound to Ca2+. Finally, synaptotagmin may also act to inhibit inappropriate exocytosis in resting cells. At the low cytosolic Ca2+ levels found in resting nerve cells, synaptotagmin apparently binds to a complex of the plasmamembrane proteins neurexin and syntaxin. The presence of synaptotagmin blocks binding of other essential fusion proteins to the neurexin-syntaxin complex, thereby preventing vesicle fusion. When synaptotagmin binds Ca2+, it is displaced from the complex, allowing other proteins to bind and thus initiating membrane docking or fusion. Thus synap-totagmin may operate as a "clamp" to prevent fusion from proceeding in the absence of a Ca2+ signal.

Fly Mutants Lacking Dynamin Cannot Recycle Synaptic Vesicles

Synaptic vesicles are formed primarily by endocytic budding from the plasma membrane of axon terminals. Endocytosis usually involves clathrin-coated pits and is quite specific, in that several membrane proteins unique to the synaptic vesicles (e.g., neurotransmitter transporters) are specifically incorporated into the endocytosed vesicles. In this way, synap tic-vesicle membrane proteins can be reused and the recycled vesicles refilled with neurotransmitter (see Figure 17-36).

As in the formation of other clathrin/AP-coated vesicles, pinching off of endocytosed synaptic vesicles requires the GTP-binding protein dynamin (see Figure 17-20). Indeed, analysis of a temperature-sensitive Drosophila mutant called shibire (shi), which encodes the fly dynamin protein, provided early evidence for the role of dynamin in endocytosis. At the permissive temperature of 20°C, the mutant flies are normal, but at the nonpermissive temperature of 30°C, they are paralyzed (shibire, paralyzed in Japanese) because pinching off of clathrin-coated pits in neurons and other cells is blocked. When viewed in the electron microscope, the shi neurons at 30°C show abundant clathrin-coated pits with long necks but few clathrin-coated vesicles. The appearance of nerve terminals in shi mutants at the nonpermissive temperature is similar to that of terminals from normal neurons incubated in the presence of a nonhydrolyzable analog of GTP (see Figure 17-21). Because of their inability to pinch off new synaptic vesicles, the neurons in shi mutants eventually become depleted of synaptic vesicles when flies are shifted to the nonpermissive temperature, leading to a cessation of synaptic signaling and paralysis.

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