Membrane protein acetylcholine receptor

FIGURE 6.10 The neuromuscular junction. The region of contact between one nerve and another nerve, or between a nerve and a muscle cell, is called a s)maptic cleft. The secretory vesicles are represented by circles in which acetylcholine is represented by dots. The nerve impulse provokes the entry of calcium ions (not shown) from the extracellular fluid into the nerve cell. Calcium ions act as a signal that stimulates the fusion of vesicles with the plcisma membrane, releasing acetylcholine into the extracellular fluid. Acetylcholine binds to membrane-bound proteins (acetylcholine receptors) on the plasma membrane of the muscle cell, resulting in stimulation of the muscle cell. Acetylcholinesterase of the neuromuscular junction catalyzes the destruction of acetylcholine in the moments after transmission of the nerve impulse. The enzyme is extracellular and is bound to proteoglycan, a molecule of extracellular matrix. 

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. 

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. 

Lipid-protein interactions are of major importance in the structural and dynamic properties of biological membranes. Fluorescent probes can provide much information on these interactions. For example, van Paridon et al.a) used a synthetic derivative of phosphatidylinositol (PI) with a ris-parinaric acid (see formula in Figure 8.4) covalently linked on the sn-2 position for probing phospholipid vesicles and biological membranes. The emission anisotropy decays of this 2-parinaroyl-phosphatidylinositol (PPI) probe incorporated into vesicles consisting of phosphatidylcholine (PC) (with a fraction of 5 mol % of PI) and into acetylcholine receptor rich membranes from Torpedo marmorata are shown in Figure B8.3.1. 

R. J. Bloch, M. Velez, J. Krikorian, and D. Axelrod, Microfilaments and actin-associated proteins at sites of membrane-substrate attachment within acetylcholine receptor clusters, Exp. Cell Res. 182, 583-596 (1989). 

A series of microcentrifuge tubes is set up, containing the membranes (usually 0.1-0.5 pM in receptor binding sites approximately 0.05-0.25 mg protein/ml) in Torpedo membrane protein and [ H]acetyl-choline concentrations ranging from 0 to 2.0 pM. Nonspecific binding is determined in the presence of excess unlabelled acetylcholine (100 pM-1 mM). 

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. 

J. P. Changeux (1995). The acetylcholine receptor A model for allosteric membrane proteins. Biochem. Soc. Trans. 23 195. 

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).

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.

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. 

Barrantes, F.J. Braceras, A. Caldironi, H.A. Mieskes, G. Moser, H. Toren, E.C. Roque, M.E. Walliman, T. Zechel, A. Isolation and characterization of acetylcholine receptor membrane-associated (nonreceptor v2-protein) and soluble electrocyte creatine kinases. J. Biol. Chem., 260, 3024-3034 (1985)... 

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