Membranes acetylcholine-receptor binding

In spite of the uncertainties concerning membrane-acetylcholine receptors some questions concerning them have been answered in recent years. Although experiments comparing effects obtained in whole cells with effects obtained with purified enzymes can be misleading,the postulate that "acetylcholine receptor" and acetylcholinestarase are identical proteins have been disproved fairly conclusively by experiments showing that the relative binding abilities of related compounds are quite different in the two systems. 

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]. 

Radioiodinated derivatives have been prepared to define more closely the target site of a-conotoxins on the acetylcholine receptor (R. Myers, unpublished data). In membrane preparations from Torpedo electroplax, photoactivatable azidosalicylate derivatives of a-conotoxin GIA preferentially label the p and 7 subunits of the acetylcholine receptor. However, when the photoactivatable derivative is cross-linked to detergent solubilized acetylcholine receptor (AChR), only the 7 subunit is labeled. Since snake a-neurotoxins mainly bind to the a subunits of AChR and a-conotoxins compete directly with a-bungarotoxin, the cross-linking results above are both intriguing and problematic. 

Salminen, O., Whiteaker, P., Grady, S.R., Collins, A.C., McIntosh, J.M., Marks, M.J. The subunit composition and pharmacology of alpha-Conotoxin Mil-binding nicotinic acetylcholine receptors studied by a novel membrane-binding assay. Neuropharmacology. 48 696, 2005. 

The basic reactions of thiolsulfonates have been known for sometime (Field et al., 1961, 1964), but more recently, they have been applied to the study of protein interactions by site-directed modification of native cysteines or through modification of cysteines introduced at particular points in proteins by mutagenesis. Such studies have yielded insights into the structure and binding site characteristics of proteins (Kirley, 1989). Pascual et al. (1998) used AEAETS to probe the acetylcholine receptor from the extracellular side of the membrane in order to investigate the molecular accessibility and electrostatic potential within the open and closed channel. 

The acetyl choline receptor is a ligand-gated ion channel that allows cations to flow out of the neuron to initiate an action potential during neurotransmission (Fig. 9-6). When the receptor binds acetylcholine, a conformational change of the receptor opens a membrane channel that conducts ions. 

Dunn SMJ, Blanchard SG, Raftery MA. 1980. Kinetics of carbamoylcholine binding to membrane-bound acetylcholine receptor monitored by fluorescence changes of a covalently bound probe. Biochemistry 19 5645-5652. 

Valenzuela F, Weign P, Yguerabide J, Johnson DA. 1994. Transverse distance beween the membrane and the agonist binding sites on the Torpedo acetylcholine receptor a fluorescence study. Biophys J 66 674-682. 

To illustrate the principles of an equilibrium dialysis experiment, we will describe the binding of [ H] acetylcholine to the nicotinic acetylcholine receptor (nAChR) in native membranes from Torpedo electro-plax. The data obtained from such an experiment are shown in Figure 10-7 where they are compared with data obtained from a centrifugation assay described below (Protocol 4.2). 

The membranes are diluted to the desired final concentration (usually 0.1-0.5 pM in receptor binding sites) in Torpedo Ringers (20 mM Hepes, 250 mM NaCl, 5 mM KCl, 4 mM CaCl2, 2 mM MgCl2, pH 7.4). For measurement of nonspecific binding, an aliquot of these membranes is incubated with an excess of unlabelled acetylcholine (100 pM-1 mM). 

Muscle contraction is triggered by motor neurons that release the neurotransmitter acetylcholine (see p. 352). The transmitter diffuses through the narrow synaptic cleft and binds to nicotinic acetylcholine receptors on the plasma membrane of the muscle cell (the sarcolemma), thereby opening the ion channels integrated into the receptors (see p. 222). This leads to an inflow of Na which triggers an action potential (see p. 350) in the sarcolemma. The action potential propagates from the end plate in all directions and constantly stimulates the muscle fiber. With a delay of a few milliseconds, the contractile mechanism responds to this by contracting the muscle fiber. 

Each neuron usually releases only one type of neurotransmitter. Neurons that release dopamine are referred to as dopaminergic, for example, while those that release acetylcholine are cholinergic, etc. The transmitters that are released diffuse through the synaptic cleft and bind on the other side to receptors on the postsynaptic membrane. These receptors are integral membrane proteins that have binding sites for neurotransmitters on their exterior (see p. 224). 

Many different receptor types are coupled to G proteins, including receptors for norepinephrine and epinephrine (a- and p-adrenoceptors), 5-hydroxytrypta-mine (serotonin or 5-HT receptors), and muscarinic acetylcholine receptors. Figure 2.1 presents the structure of one of these, the uz-adrenoceptor from the human kidney. All members of this family of G protein-coupled receptors are characterized by having seven membrane-enclosed domains plus extracellular and intracellular loops. The specific binding sites for agonists occur at the extracellular surface, while the interaction with G proteins occurs with the intracellular portions of the receptor. The general term for any chain of events initiated by receptor activation is signal transduction. 

Fig. 16.13. Pore structure of the acetylcholine receptor, based on electron microscopy studies. a) Electron density map of the acetylcholine receptor of the postsynaptic membrane of the electric organ of the ray Torpedo californicus, based on electron microscopy studies. The receptor has a long funnel-like structure in the extracellular region, which narrows at the center of the pore. A smaller funnel structure is observed in the cytoplasmic region of the receptor. Another protein is situated on the cytoplasmic side. The long arrow indicates the direction of ion passage and the small arrow shows the postulated binding site for acetylcholine, b) Schematic representation of the acetylcholine receptor with the M2 hehx as the central block in the ion channel. According to Unwin, (1993).

The adult nicotinic acetylcholine receptor (nAChR) is an intrinsic membrane protein with five distinct subunits (a2B T). A Cartoon of the one of five subunits of the AChR in the end plate surface of adult mammalian muscle. Each subunit contains four helical domains labeled Ml to M4. The 2 domains line the channel pore. B Cartoon of the full nAChR. The N termini of two subunits cooperate to form two distinct binding pockets for acetylcholine (ACh). These pockets occur at the (X-B and the 3-Ct subunit interfaces. 

Gated ion channels of the plasma membrane that open and close (hence the term gating ) in response to the binding of chemical ligands or changes in transmembrane potential. These are the simplest signal transducers. The acetylcholine receptor ion channel is an example of this mechanism (Section 12.2). 

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