Receptor, acetylcholine

Acetylcholine is the natural transmitter at several different kinds of synaptic site, e.g. (a) the somatic nerve voluntary muscle junctions, (b) the ganglionic synapses which are nerve — nerve junctions in the autonomic system, (c) such postganglionic nerve-endings as are parasympathetic. In addition it has some transmitting duties in the central nervous system, e.g. between spinal cord root fibres and Renshaw cells. 

Nicotine mimics acetylcholine at sites (a) and (b), whereas muscarine does so at site (c) (see Table 7.1). Hence it is usual to divide acetylcholine receptors into nicotinic and muscarinic receptors, a useful classification first made by Dale (1914). The work of muscarinic and nicotinic receptors is carried out on very different time scales. A single nervous stimulus affects muscarinic receptors for at least 500 ms, a long duration that is preceded by a latency of about 100 ms. In contrast to this, nicotinic receptors at voluntary neuromuscular junctions are stimulated for only 0.2 ms, and even the ganglionic synapses, which are slower, average only 60 ms. Cyclic GMP is thought to be a necessary mediator of muscarinic reponses. One consequence of interest is that smooth muscle reacts far more slowly than voluntary muscle. Heart muscle is distinguished from both by the fact that acetylcholine increases its polarization (and hence slows the heart), whereas it decreases polarization in other types of muscle. 

Because the ACh receptor does not hydrolyse acetylcholine, the esteratic site of acetylcholinesterase (see Fig. 12.3) must be absent. Other fundamental differences in the two sites are indicated by the following (a) dimethylbutyl acetate 12.66) is a good substrate for the enzyme, but barely activates the receptor which requires a basic group for marked activity (b) muscarine is not a substrate for the enzyme and yet it is a powerful agonist for the muscarinic receptor (c) di-isopropyl phosphorofluoridate (isoflurophate) 13.26) binds to the active site of AChase but not to an ACh receptor (d) acetyl-bungarotoxin specifically binds to the ACh nicotinic receptor but not to the enzyme. 

The next few pages will be devoted to the muscarinic receptor followed by several pages on the nicotinic receptor. Which is the easier to study The structural requirements of the nicotinic receptor seem to be less demanding, for 

The muscarinic receptor has also been extracted from heads of the fruitfly. Drosophila (Dudai and Ben-Barak, 1977), whereas the housefly has, in its head, a quite different type of ACh receptor (p. 536). 

Nicotine mimics acetylcholine at sites (a) and (b), whereas muscarine does so at site (c) (see Table 7.1). Hence it is usual to divide acetylcholine receptors into nicotinic and muscarinic receptors, a useful classification first made by Dale (1914). The work of muscarinic and nicotinic receptors 

Of all the synapses which have cholinergic transmission, that at the motor end-plate (at the volimtary neuromuscular junction) has been most studied. Its structure, visible even in the light microscope and sketched in Fig. 7.1, has revealed yet further complexities to the electron microscope, to electro-physiological measurements, to assays of acetylcholine vesicles and of acetylcholinesterase, and to autoradiography. The chemical nature of the receptors in the end-plate is imperfectly known, but a disulphide (S-S) group is essential for its functioning. This follows from the inhibition of the receptor by dithiothreitol (a disulphide reducer) and restoration of sensitivity by 5,5 -dithio-fe 5-2-nitrobenzoic acid (which restores a dithiol to the disulphide state) (Karlin and Bartels, 1966). 

A practical detail, useful in recognizing the receptor, is that the Formosan snake toxin, a-bungarotoxin (acetyl- W) binds only to the acetylcholine (ACh) receptor site (in mouse diaphragm muscle), whereas diwopropyl-phosphorofluoridate ( H) 12,15) binds specifically to the active site of acetylcholinesterase. An equal number (3 x 10 per end-plate) of the receptor and the enzyme sites is found, i.e. one active molecule per 5000 A of the membrane, which is consequently densely occupied by these two proteins. Blockade becomes marked only when 70 per cent of the ACh receptor sites are occupied, and hence there are not many spare receptors at this site (Barnard, Wieckowski, and Chiu, 1971). 

The isolation and nature of the acetylcholine receptor was described in Section 2.1. Of the various products of this work, Changeux s micro-sacs are particularly welcome, as they enable much experimentation with drugs to be carried out under conditions more versatile than when the receptors are in the synapse. We shall learn much more about the chemical nature of the active site when the receptor is available, quite pure and in greater quantity, for determination of aminoacid sequence and then for X-ray diffraction study. 

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Mode of Motion. Nicotine, anabasine, and imidocloprid affect the ganglia of the insect central nervous system, faciUtating transsynaptic conduction at low concentrations and blocking conduction at higher levels. The extent of ionisation of the nicotinoids plays an important role in both their penetration through the ionic barrier of the nerve sheath to the site of action and in their interaction with the site of action, which is befleved to be the acetylcholine receptor protein. There is a marked similarity in dimensions between acetylcholine and the nicotinium ion. 

Mode of Action. All of the insecticidal carbamates are cholinergic, and poisoned insects and mammals exhibit violent convulsions and other neuromuscular disturbances. The insecticides are strong carbamylating inhibitors of acetylcholinesterase and may also have a direct action on the acetylcholine receptors because of their pronounced stmctural resemblance to acetylcholine. The overall mechanism for carbamate interaction with acetylcholinesterase is analogous to the normal three-step hydrolysis of acetylcholine however, is much slower than with the acetylated enzyme. 

Autoantibodies are directed against nicotinic acetylcholine receptors in myasthenia gravis, resulting in receptor loss, skeletal muscle paralysis, and dysfunction (100). In addition, antibodies directed against voltage-gated Ca " channels produce similar neuromuscular dysfunction of Lambert-Eaton... 

Given the difficulty of obtaining three-dimensional crystals of membrane proteins, it is not surprising that the electron microscope technique is now widely used to study large membrane-bound complexes such as the acetylcholine receptor, rhodopsin, ion pumps, gap junctions, water channels and light-harvesting complexes, which crystallize in two dimensions. 

Brisson, A., Unwin, RN.T. Quaternary structure of the acetylcholine receptor. Nature 315 474-477, 1985. 

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N-Heterocycles as neuronal acetylcholine receptors in drug discovery 97JMC4169. 

Jakubic, J., and El-Fakahany, E. E. (1997). Positive coopera -tivity of acetylcholine and other agonists with allosteric ligands on muscarinic acetylcholine receptors. Mot. Pharmacol. 52 172-177. 

Positive cooperativity of acetylcholine and other agonists with allosteric ligands on muscarinic acetylcholine receptors. Mol. Pharmacol. 52 172—179. 

Paton, W. D. M., and Rang, H. P. (1965). The uptake of atropine and related drags by intestinal smooth muscle of the guinea pig in relation to acetylcholine receptors. Proc. R. Soc. Lond. [Biot.] 163 1-44. 

Jakubic, J., Bacakova, L., Lisd, V., El-Fakahany, E. E., and Tucek, S. (1996). Activation of muscarinic acetylcholine receptors via their allosteric binding sites. Proc. Natl. Acad. Sci. USA 93 8705-8709. 

Antagonists of muscarinic acetylcholine receptors had widely been used since 1860 for the treatment of Parkinson s disease, prior to the discovery of l-DOPA. They block receptors that mediate the response to striatal cholinergic interneurons. The antiparkinsonian effects of drugs like benzatropine, trihexyphenidyl and biper-iden are moderate the resting tremor may sometimes respond in a favorable manner. The adverse effects, e.g., constipation, urinary retention, and mental confusion, may be troublesome, especially in the elderly. 

Curare is a generic term for various South American arrow poisons. Curare has been used for centuries by the Indians along the Amazon and Orinoco rivers for immobilizing and paralyzing wild animals used for food. Preparations of curare are derived from Strychnos species, which contain quaternary neuromuscular alkaloids like tubocurarine. Tubocurarine is a potent antagonist at the nicotinic acetylcholine receptor. 

Muscarinic acetylcholine receptors (mAChRs) form a class of cell surface receptors that are activated upon binding of the neurotransmitter, acetylcholine. Structurally and functionally, mAChRs are prototypical members of the superfamily of G protein-coupled receptors. Following acetylcholine binding, the activated mAChRs interact with distinct classes of heterotrimeric G proteins resulting in the activation or inhibition of distinct downstream signaling cascades. 

Wess J (1996) Molecular biology of muscarinic acetylcholine receptors. CritRev Neurobiol 10 69-99... 

Caulfield MP, Birdsall NJM (1998) International Union of Pharmacology. XVII. Classification of muscarinic acetylcholine receptors. Pharmacol Rev 50 279-290... 

Wess J (2004) Muscarinic acetylcholine receptor knockout mice novel phenotypes and clinical implications. Annu Rev Pharmacol Toxicol 44 423-450... 

Lanzafame A A, Christopoulos A, Mitchelson F (2003) Cellular signaling mechanisms for muscarinic acetylcholine receptors. Recept Chann 9 241-260... 

Nicotinic receptors (nicotinic acetylcholine receptors, nACHR) exist not only in the membrane of vertebrate skeletal muscle at the synapse between nerve and muscle (muscle-type nAChR) but also at various synapses throughout the brain, mainly at presynaptic positions (neuronal-type nAChR). Whereas the muscle-type nAChR is precisely composed of two a 1-subunits, one (3 -subunit, one y -subunit and one y -subunit (adult)... 

Dajas-Bailador F, Wonnacott S (2004) Nicotinic acetylcholine receptors and the regulation of neuronal signalling. Trends Pharmacol Sci 25 317-324... 

Gotti C, Zoli M, Clementi F (2006) Brain nicotinic acetylcholine receptors native subtypes and their relevance. Trends Pharmacol Sci 27 482-491... 

Jensen AA, Frolund B, Liljefors T et al (2005) Neuronal nicotinic acetylcholine receptors structural revelations, target identifications, and therapeutic inspirations. J Med Chem 48 4705—4745... 

Karlin A (2002) Emerging structure of the nicotinic acetylcholine receptors. Nat Rev Neurosci 3 102-114... 

Unwin N (2003) Structure and action of the nicotinic acetylcholine receptor explored by electron microscopy. FEES Lett 555 91-95... 

The open channel has in most cases a selective permeability, allowing a restricted class of ions to flow,for example Na+, K+, Ca++ or Cl- and, accordingly, these channels are called Na+-channels, K+-channels, Ca -channels and Cr-channels. In contrast, cation-permeable channels with little selectivity reject all anions but discriminate little among small cations. Little is known about the structures and functions of these non-selective cation channels [1], and so far only one of them, the nicotinic acetylcholine receptor (nAChR, see Nicotinic Receptors), has been characterized in depth [2, 3]. The nAChR is a ligand-gated channel (see below) that does not select well among cations the channel is even permeable to choline, glycine ethylester and tris buffer cations. A number of other plasma... 

Non-selective Cation Channels. Figure 1 The nicotinic acetylcholine receptor (nAChR) is localized within the cell membrane above the cell membrane is the synaptic cleft, below the cytoplasm. Drawing of the closed (left) and open (right) nAChR showing acetylcholine (ACh) binding and cation movement. Dimensions of the receptor were taken from references [2, 3]. 

Miyazawa A, Fujiyoshi Y, Stowell M et al (1999) Nicotinic acetylcholine receptor at 4.6 A resolution transverse tunnels in the channel wall. J Mol Biol 288 765-786... 

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