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docking,which involves SNARE proteins (Fig. 1). A vesicle-associated protein,

synaptobrevin (v-SNARE, VAMP) binds with high affinity to a presynaptic

membrane protein, syntaxin (t-SNARE). Syntaxin is closely associated with voltage-dependent calcium channels ensuring that the release machinery is opti- mally placed to receive the Ca2+ _{signal. Synaptobrevin and syntaxin, together}

Biochemistry of exocytosis Exocytosis from large dense-core vesicles The role of calcium

with a third protein crucial for docking, SNAP-25, are targets for botulinum and tetanus toxins that are powerful inhibitors of neurotransmitter secretion.

After docking comes another calcium-dependent step, priming, in which a number of soluble cytoplasmic proteins form a transient complex with the SNAREs, resulting in partial fusion of vesicle and presynaptic membranes. This step involves the hydrolysis of ATP.

Primed vesicles are poised for exocytosis, requiring only a large pulse of Ca2+

to permit complete fusion of the vesicle and presynaptic membranes and opening of the fusion pore through which exocytosis occurs. A calcium-binding protein located in the vesicle membrane, synaptotagmin, is the Ca2+_{sensor in}

the exocytotic machinery. In the absence of calcium it prevents complete fusion

but when it binds Ca2+ _{it undergoes a conformational change which allows}

fusion to proceed. This final stage is fast. It must occur within 200 µs.

Endocytosis Following exocytosis, synaptic vesicles are recycled within 30–60 s by endo-

cytosis. Firstly, the vesicle membrane acquires a clathrin coat, distorting it so that it invaginates into the terminal. Next a GTP-binding protein, dynamin, forms a collar around the neck of the invagination. Dynamin has an intrinsic GTPase activity which hydrolyzes the bound GTP so triggering the fission of the coated vesicle from the presynaptic membrane. The GTP bound form of dynamin requires calcium, so the same rise in nerve terminal Ca2+_{concentration}

responsible for exocytosis also enables endocytosis. Once free in the terminal the vesicle loses its clathrin coat (Fig. 2).

C5 – Neurotransmitter release 67 Synaptotagmin Vesicle membrane Ca2+_{binding domain} Presynaptic membrane VDCaC Ca2+ Syntaxin SNAP-25 Synaptobrevin

Fig. 1. Proteins involved in the docking of neurotransmitter vesicles. VDCaC, voltage- dependent calcium channel.

GTP Dynamin Clathrin GTP GDP + Pi GDP Vesicle membrane Fusion pore Presynaptic membrane Dynamin Endocytosed vesicle

Fig. 2. Vesicle endocytosis. Reprinted from Revest, P.A. and Longstaff, A. (1998) Molecular Neuroscience. © BIOS Scientific Publishers Ltd, Oxford.

Kiss-and-run cycle The above account describes the Heuser–Reese cycle, based on studies of the neuromuscular junction. A recently documented kiss-and-run cycle also oper- ates at central synapses. Here the vesicle membrane fuses with the presynaptic membrane to open a pore through which transmitter discharges, after which the pore closes and the vesicle disengages. There is no complicated endocytosis. Because this mechanism has a cycle time of only one second it is able to support extended periods of high release with only 35–40 vesicles in the re- cycling pool.

Refilling Vesicles are reloaded with neurotransmitter in the nerve terminals. The vesicles

are acidified by the action of a proton ATPase. The transport of transmitter into vesicles is then driven by secondary active transport with H+_{efflux providing}

the energy (Fig. 3). Vesicle transporters have been identified for a number of transmitters including glutamate, ACh and catecholamines, but not yet for GABA. They are large glycoproteins with 12 transmembrane segments. Surprisingly, they seem unrelated to neurotransmitter transporters located in plasma membranes of neurons or glia. Peptide transmitters, after synthesis on ribosomes in the cell body, are secreted into the lumen of the rough endo- plasmic reticulum (RER), and packaged for export by the Golgi apparatus, from which the loaded vesicles are budded. These are then moved to the terminal by fast axoplasmic transport. This is necessary because nerve terminals are devoid of ribosomes and incapable of protein synthesis.

Autoreceptors Neurotransmitter receptors are not confined to the postsynaptic membrane but

also exist in the presynaptic membrane, where they are termed presynaptic receptors, and over the cell body and dendrites. If these are receptors for the transmitter released by the neuron in which they are located, they are auto- receptors. Autoreceptors, which are invariably metabotropic receptors, have several functions that are normally homeostatic. Those on the presynaptic membrane are involved in regulating neurotransmitter release. Most (but not all) presynaptic autoreceptors decrease the release of neurotransmitter by reducing calcium influx into the presynaptic terminal. This is a negative feedback mechanism either to avoid excessive excitation, or to curtail post- synaptic receptor desensitization, which would reduce the sensitivity of the synapse.

Autoreceptors can also decrease the synthesis of transmitter (e.g. of cate- cholamines and serotonin by their respective neurons) and some dopaminergic neurons have dopamine receptors on their dendrites and cell body which regu- late neuron firing rate.

Vesicle neurotransmitter transporter Neurotransmitter Proton ATPase ATP ADP + Pi H+ H+

Fig. 3. Vesicle refilling. Reprinted from Revest, P.A. and Longstaff, A. (1998) Molecular

Some presynaptic receptors are receptors for transmitters not secreted by the neuron in which they are situated. These heteroceptors also regulate transmitter release. For example, GABABreceptors exist presynaptically at glutamatergic

synapses where they reduce glutamate release. It is assumed that they are acti- vated by GABA that has diffused from neighboring synapses.