Vesicular Exocytosis

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Once vesicles detach from the cytoskeleton they are free to participate in the release process but our understanding of precisely how this is brought about is still sketchy, despite the wealth of information which has accumulated over recent years. What is clear is that it involves a complex cascade of regulatory processes focusing on proteins bound to vesicle membranes, the axolemma and some cytoplasmic factors (see Calakos

Figure 4.11 Dephosphorylated synapsin, associated with SSVs, is thought to form a heteromeric complex with CAM kinase II (also partially embedded in the vesicular membrane) and actin filaments. An increase in intracellular Ca2+ triggers phosphorylation of synapsin I which dissociates from the vesicular membrane. This frees the vesicles from the fibrin microfilaments and makes them available for transmitter release at the active zone of the nerve terminal

Figure 4.11 Dephosphorylated synapsin, associated with SSVs, is thought to form a heteromeric complex with CAM kinase II (also partially embedded in the vesicular membrane) and actin filaments. An increase in intracellular Ca2+ triggers phosphorylation of synapsin I which dissociates from the vesicular membrane. This frees the vesicles from the fibrin microfilaments and makes them available for transmitter release at the active zone of the nerve terminal and Scheller 1996). The following sections will deal with those factors about which most is known and which are thought to have a prominent role in exocytosis. The extent to which this scheme explains release from large dense-cored vesicles is unclear, not least because these vesicles are not found near the active zone.

DOCKING AND FUSION

Because exocytosis is so rapid, it is believed that Ca2+ must trigger release from vesicles which are already docked at the active zone. The processes leading to docking and fusion of the vesicle with the axolemma membrane are thought to involve the formation of a complex between soluble proteins (in the neuronal cytoplasm) and those bound to vesicular or axolemma membranes. Much of this evidence is based on studies of a wide range of secretory systems (including those in yeast cells) but which are thought to be conserved in mammalian neurons.

From evidence collected to date, a scheme has emerged, known as the SNARE hypothesis (see Sollner and Rothman 1994) (Fig. 4.12). The soluble proteins referred to above include N-ethylmaleimide sensitive factor ('NSF', an ATPase) and SNAPs which comprise a family of 'soluble NSF attachment proteins'. Evidence, largely derived from studies of the Golgi apparatus, suggests that SNAPs have a general role in proteinprotein interactions underlying membrane fusion. Proteins thought to act as SNAP receptors ('SNARES') are found both in the axolemma (known as 'target SNARES' or tSNARES) and vesicles (vSNARES). The vesicle protein, synaptobrevin (also known as 'vesicle associated membrane protein' or 'VAMP') is thought to act as a vSNARE and couples with the tSNARE proteins: syntaxin and SNAP-25 (synaptosomal associated protein: 25KDa). It is envisaged that this complex of the two SNARES enables sequential binding of the soluble SNAPs and NSF. Subsequent hydrolysis of ATP by NSF enables dissociation of the complex and fusion of the membrane so that the vesicle contents can be discharged into the synapse.

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Figure 4.12 Hypothetical model of the action of SNAP receptors (SNARES) during vesicle docking, activation and fusion. It is thought that synaptic vesicle docking to the presynaptic plasma membrane requires the removal of a regulatory cytosolic protein, n-sec-1, from a VAMP (synaptobrevin) binding site on syntaxin. This results in the dissociation of synaptotagmin from the SNARE complex and binding of SNAPs and NSF, thus initiating fusion. The ATP hydrolysed by NSF results in disassembly of the SNARE complex. How the interconversion of these complexes occurs and which components trigger these processes is poorly understood. Proteins such as rab 3A, Ca2+ binding proteins and Ca2+ channels are likely to be involved. (From Sollner and Rothman 1994, page 346 with permission from Elsevier Science)

Figure 4.12 Hypothetical model of the action of SNAP receptors (SNARES) during vesicle docking, activation and fusion. It is thought that synaptic vesicle docking to the presynaptic plasma membrane requires the removal of a regulatory cytosolic protein, n-sec-1, from a VAMP (synaptobrevin) binding site on syntaxin. This results in the dissociation of synaptotagmin from the SNARE complex and binding of SNAPs and NSF, thus initiating fusion. The ATP hydrolysed by NSF results in disassembly of the SNARE complex. How the interconversion of these complexes occurs and which components trigger these processes is poorly understood. Proteins such as rab 3A, Ca2+ binding proteins and Ca2+ channels are likely to be involved. (From Sollner and Rothman 1994, page 346 with permission from Elsevier Science)

Much evidence supports a role for these proteins in exocytosis. For instance, injection of recombinant SNAP into the squid giant axon increases vesicular exocytosis. Also, membrane SNAP-25 and syntaxin are both targets for botulinum toxin while the vesicule protein, synaptobrevin, is a target for tetanus and botulinum toxins; both these toxins are well known for disrupting transmitter release.

How all these processes are influenced by Ca2+ is uncertain but another vesicle membrane-bound protein, synaptotagmin, is widely believed to effect this regulatory role (Littleton and Bellen 1995). Synaptotagmin has a single membrane-spanning domain with the NH2-tail penetrating the vesicle and the COOH-tail extending into the cytoplasm. This tail binds Ca2+ and could enable synaptotagmin to act as a Ca2+-sensor but, although it is found in adrenergic and sensory neurons, it appears to be absent from motor neurons.

Another protein, synaptophysin (p38), is the most abundant of the vesicle proteins and is found in the membranes of both SSVs and LDCVs. Its transmembrane structure resembles that of connexins which form gap junctions and has provoked the theory that neuronal excitation might cause synaptophysin to act as a fusion pore. There is no doubt that many other factors are involved in regulating the docking-fusion-extrusion process, including the Rab family of GTP-binding proteins and the Rab3 effectors, Rabphilin and Rim. For a detailed review of the role of all these factors in the exo-cytotic cycle, see Benfanati, Onofri and Giovedi 1999.

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