Presynaptic membrane proteins

The influx of Ca(Il) across the presynaptic membrane is essential for nerve signal transmission involving excitation by acetylcholine (26). Calcium is important in transducing regulatory signals across many membranes and is an important secondary messenger hormone. The increase in intracellular Ca(Il) levels can result from either active transport of Ca(Il) across the membrane via an import channel or by release of Ca(Il) from reticulum stores within the cell. More than 30 different proteins have been linked to regulation by the calcium complex with calmoduhn (27,28). 

G0 was isolated as an other PTx-ribosylated G-protein which co-purifies with G, but which does not inhibit adenylate cyclase. There are two main isoforms (G0l and Go2), with additional splice-variants. G0 is particularly abundant in the nervous system, comprising up to 1% of membrane proteins. Its main function is to reduce the opening probability of those voltage-gated Ca2+ channels (N- and P/Q-type) involved in neurotransmitter release. Hence, it is largely responsible for the widespread auto-inhibition of transmitter secretion by presynaptic receptors and this effect is mediated through released py subunits. 

G -protein-coupled receptors are often located on the presynaptic plasma membrane where they inhibit neurotransmitter release by reducing the opening of Ca2+ channels like inactivation and breakdown of the neurotransmitter by enzymes, this contributes to the neuron s ability to produce a sharply timed signal. An a2 receptor located on the presynaptic membrane of a noradrenaline-containing neuron is called an autoreceptor but, if located on any other type of presynaptic neuronal membrane (e.g., a 5-HT neuron), then it is referred to as a heteroreceptor (Langer, 1997). Autoreceptors are also located on the soma (cell body) and dendrites of the neuron for example, somatodendritic 5-HTia receptors reduce the electrical activity of 5-HT neurons. 

Vesicular proteins and lipids that are destined for the plasma membrane leave the TGN sorting station continuously. Incorporation into the plasma membrane is typically targeted to a particular membrane domain (dendrite, axon, presynaptic, postsynaptic membrane, etc.) but may or may not be triggered by extracellular stimuli. Exocytosis is the eukaryotic cellular process defined as the fusion of the vesicular membrane with the plasma membrane, leading to continuity between the intravesicular space and the extracellular space. Exocytosis carries out two main functions it provides membrane proteins and lipids from the vesicle membrane to the plasma membrane and releases the soluble contents of the lumen (proteins, peptides, etc.) to the extracellular milieu. Historically, exocytosis has been subdivided into constitutive and regulated (Fig. 9-6), where release of classical neurotransmitters at the synaptic terminal is a special case of regulated secretion [54]. 

Specific membrane components must be delivered to their sites of utilization and not left at inappropriate sites [3]. Synaptic vesicles and other materials needed for neurotransmitter release should go to presynaptic terminals because they serve no function in an axon or cell body. The problem is compounded because many presynaptic terminals are not at the end of an axon. Often, numerous terminals occur sequentially along a single axon, making en passant contacts with multiple targets. Thus, synaptic vesicles cannot merely move to the end of axonal MTs. Targeting of synaptic vesicles thus becomes a more complex problem. Similar complexities arise with membrane proteins destined for the axolemma or a nodal membrane. 

All botulin neurotoxins act in a similar way. They only differ in the amino-acid sequence of some protein parts (Prabakaran et al., 2001). Botulism symptoms are provoked both by oral ingestion and parenteral injection. Botulin toxin is not inactivated by enzymes present in the gastrointestinal tracts. Foodborne BoNT penetrates the intestinal barrier, presumably due to transcytosis. It is then transported to neuromuscular junctions within the bloodstream and blocks the secretion of the neurotransmitter acetylcholine. This results in muscle limpness and palsy caused by selective hydrolysis of soluble A-ethylmalemide-sensitive factor activating (SNARE) proteins which participate in fusion of synaptic vesicles with presynaptic plasma membrane. SNARE proteins include vesicle-associated membrane protein (VAMP), synaptobrevin, syntaxin, and synaptosomal associated protein of 25 kDa (SNAP-25). Their degradation is responsible for neuromuscular palsy due to blocks in acetylcholine transmission from synaptic terminals. In humans, palsy caused by BoNT/A lasts four to six months. 

The arrival of the action potential at the presynaptic terminal opens voltage-dependent Ca ion channels in the plasma membrane so that the Ca ions enter the cytosol down their concentration gradient. This results in activation of a Ca -binding cytosolic or a membrane protein. This facilitates movement of the vesicles to the membrane and formation of a fusion pore through which the neurotransmitter is discharged into the synaptic cleft (i.e. exocytosis). This occurs within about 0.1 ms of the arrival of the depolarisation (Figure 14.8). The process of exocytosis lasts for only a short time, since the Csl ion concentration in the cytosol is rapidly lowered due to the ion extrusion from the cell (Appendix 14.3). 

The a.1 receptors are excitatory in their action, while the a2 receptors are inhibitory, these activities being related to the different types of second messengers or ion channels to which they are linked. Thus, a2 receptors hyperpolarize presynaptic membranes by opening potassium ion channels, and thereby reduce noradrenaline release. Conversely, stimulation of ai receptors increases intracellular calcium via the phosphatidyl inositol cycle which causes the release of calcium from its intracellular stores protein kinase C activity is increased as a result of the free calcium, which then brings about further changes in the membrane activity. 

The decisive element in exocytosis is the interaction between proteins known as SNAREs that are located on the vesicular membrane (v-SNAREs) and on the plasma membrane (t-SNAREs). In the resting state (1), the v-SNARE synaptobrevin is blocked by the vesicular protein synaptotagmin. When an action potential reaches the presynaptic membrane, voltage-gated Ca "" channels open (see p. 348). Ca "" flows in and triggers the machinery by conformational changes in proteins. Contact takes place between synaptobrevin and the t-SNARE synaptotaxin (2). Additional proteins known as SNAPs bind to the SNARE complex and allow fusion between the vesicle and the plasma membrane (3). The process is supported by the hydrolysis of GTP by the auxiliary protein Rab. 

Mechanism of Action An antidepressant that appears to inhibit serotonin and norepinephrine reuptake at CNS neuronal presynaptic membranes is a less potent inhibitor of dopamine reuptake. Therapeutic Effect Relieves depression. Pharmacokinetics Well absorbed from the G1 tract. Protein binding greater than 90%. Extensively metabolized to active metabolites. Excreted primarily in urine and, to a lesser extent, in feces. Half-life 8-17 hr. 

Mechanism of Action Atricyclicantidepressant, antineuralgic, andantineuriticagent that blocks the reuptake of neurotransmitters, such as norepinephrine and serotonin, at presynaptic membranes, increasing their concentration at postsynaptic receptor sites. Therapeutic Effect Relieves depression and controls nocturnal enuresis. Pharmacokinetics Rapidly, well absorbed following PO administration. Protein binding more than 90%. Metabolized in liver, with first-pass effect. Excreted in urine as metabolites. Half-life 6-18 hr. 

Schematic illustration of a generalized cholinergic junction (not to scale). Choline is transported into the presynaptic nerve terminal by a sodium-dependent choline transporter (CHT). This transporter can be inhibited by hemicholinium drugs. In the cytoplasm, acetylcholine is synthesized from choline and acetyl -A (AcCoA) by the enzyme choline acetyltransferase (ChAT). Acetylcholine is then transported into the storage vesicle by a second carrier, the vesicle-associated transporter (VAT), which can be inhibited by vesamicol. Peptides (P), adenosine triphosphate (ATP), and proteoglycan are also stored in the vesicle. Release of transmitter occurs when voltage-sensitive calcium channels in the terminal membrane are opened, allowing an influx of calcium. The resulting increase in intracellular calcium causes fusion of vesicles with the surface membrane and exocytotic expulsion of acetylcholine and cotransmitters into the junctional cleft (see text). This step can he blocked by botulinum toxin. Acetylcholine s action is terminated by metabolism by the enzyme acetylcholinesterase. Receptors on the presynaptic nerve ending modulate transmitter release. SNAPs, synaptosome-associated proteins VAMPs, vesicle-associated membrane proteins.

Neurotransmitters are reabsorbed by the presynaptic neuron that released them through proteins embedded in the presynaptic membrane. A drug that interferes with reuptake causes a buildup of neurotransmitters in the synaptic cleft. 

FIGURE 13-4 Mechanism of action of botulinum toxin at the skeletal neuromuscular junction. At a normal synapse (shown on left], fusion proteins connect acetylcholine (ACh] vesicles with the presynaptic membrane, and ACh is released via exocytosis. Botulinum toxin (represented by BTX on the right] binds to the presynaptic terminal, and enters the terminal where it destroys the fusion proteins so that ACh cannot be released. See text for details. 

Figure 16.3 Neurotransmitter release, (a) Presynaptic nerve terminal containing vesicles and other organelles, (b) Neurotransmitter-containing vesicles are made of lipid bilayers. Associated proteins participate in the release process, (c) The vesicle associates with the presynaptic membrane via protein complexes that mediate release, (d) Release of neurotransmitter into the synapse is by protein-mediated fusion of vesicle and presynaptic membranes.

Release can be blocked by drugs such as guanethidine and bretylium. After release, norepinephrine diffuses out of the cleft or is transported into the cytoplasm of the terminal (uptake 1 [1], blocked by cocaine, tricyclic antidepressants) or into the postjunctional cell (uptake 2 [2]). Regulatory receptors are present on the presynaptic terminal. (SNAPs, synaptosome-associated proteins VAMPs, vesicle-associated membrane proteins.)... 

The SNAREs involved in the fusion of synaptic vesicles and of secretory granules in neuroendocrine cells, referred to as neuronal SNAREs, have been intensely studied and serve as a paradigm for all SNAREs. They include syntaxin 1A and SNAP-25 at the presynaptic membrane and synaptobrevin 2 (also referred to as VAMP 2) at the vesicle membrane. Their importance for synaptic neurotransmission is documented by the fact that the block in neurotransmitter release caused by botulinum and tetanus neurotoxins is due to proteolysis of the neuronal SNAREs (Schiavo et al. 2000). Genetic deletion of these SNAREs confirmed their essential role in the last steps of neurotransmitter release. Intriguingly, analysis of chromaffin cells from KO mice lacking synaptobrevin or SNAP-25 showed that these proteins can be at least partially substituted by SNAP-23 and cellubrevin, respectively (Sorensen et al. 2003 Borisovska et al. 2005), i.e., the corresponding SNAREs involved in constitutive exocytosis. 

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