Electric Organ Acetylcholine Receptor Research Articles

The 9-Arginine Residue of α-Conotoxin GI Is Responsible for Its Selective High Affinity for the αγ Agonist Site on the Electric Organ Acetylcholine Receptor

The two agonist-binding domains of the electric organ nicotinic acetylcholine receptor are located at the alphagamma and alphadelta subunit interfaces. alpha-Conotoxins GI and MI are competitive antagonists of this receptor and, like d-tubocurarine, bind to the alphagamma site with much higher affinity than to the alphadelta site. In the present study, alpha-conotoxin SIA also displayed strong affinity for the alphagamma site but no measurable affinity for the alphadelta site, thus showing even greater site-selectivity. In contrast, alpha-conotoxin SI does not distinguish between the two agonist sites, although its sequence differs from that of GI at only three positions: GI, ECCNPACGRHYSC; SI, ICCNPACGPKYSC. Analogues of SI and GI modified at these three positions were studied to identify the determinants of GI's alphagamma selectivity. Substituting arginine for proline at position 9 produced peptides which displayed "GI-like" selectivity for the alphagamma site. Conversely, substituting proline for arginine at position 9 resulted in "SI-like" nonselective inhibitors. An SI analogue having alanine in place of proline 9 did not distinguish between the two agonist sites and displayed about the same affinity as SI, indicating the importance of the arginyl cation. Interchanging the residues at position 1 or at position 10 influenced the affinity for the receptor but did not measurably change peptide selectivity. Therefore, of the three sequence differences in SI and GI, the variation at position 9, proline and arginine, respectively, is sufficient to account for GI's selective high-affinity binding to the alphagamma site on the electric organ acetylcholine receptor.

Biochemical interactions of carbamates and ecothiophate with the activated conformation of nicotinic acetylcholine receptor.

Purified Torpedo nobiliana electric organ acetylcholine receptor (AChR) was reconstituted into membranes containing natural phospholipids supplemented with cholesterol (25% w/w). The reconstituted system facilitates the study of the effects of drugs on the regulation of the AChR channel complex under both resting and carbachol (carb)-stimulated conditions. Neostigmine (Neo) was the only carbamate to induce activation of [3-H]-phencyclidine ([3-H]-PCP) binding to the channel sites, acting as a weak agonist. The activation of [3-H]-PCP binding is dependent upon the nature of the reconstituted systems, with carb/Neo activation ratios of 8, 3, and 1 for the intact purified AChR vesicles fraction (PVF), the PVF reconstituted in phospholipid/cholesterol (CRPVF), and the PVF reconstituted in phospholipid (RPVF), respectively. The carbamates Neo, physostigmine (Physo), and pyridostigmine (Pyrido) inhibited carb-activated [3-H]-PCP binding with Ki values of 10, 20, and 1,600 microM, respectively. The inhibition was mixed competitive-noncompetitive in nature. The characteristic response of CRPVF to carb-stimulated [22-Na] influx was inhibited by the three carbamates, with IC-50 values of 6, 50, and 1,000 microM for Neo, Physo, and Pyrido, respectively. The quaternary ammonium organophosphate ecothiophate (Eco) inhibited carb-stimulated [22-Na] influx with potency similar to that of Neo. Preincubation of AChR preparation with the carbamates and ecothiophate caused a reduction in the binding of [125-I]-alpha-bungarotoxin ([125-I]-alpha-BGT) with the following decreasing order of potency: Neo less than Physo less than Eco less than Pyrido. Calcium has a direct modulatory role on the time-course inhibition of [125-I]-alpha-BGT binding by these drugs. While we observed a high potency of Neo and Physo in inhibiting [125-I]-alpha-BGT binding, it was undetectable for the carbamate insecticide 2-methyl-2-(methylthio)propionaldehyde-O-(methylcarbamoyl)oxime (aldicarb). These data suggest that the potent anticholinesterase carbamate agents interact differently with the AChR and its ionic channel. Their interactions with the nicotinic AChR channel system can be described as (a) weakly agonist, (b) directly acting on the open conformation of the channel, and (c) blocking the AChR-binding sites.

Characterization of a component in chick ciliary ganglia that cross- reacts with monoclonal antibodies to muscle and electric organ acetylcholine receptor

Chick ciliary ganglion neurons have previously been shown to contain a component that shares an antigenic determinant with the "main immunogenic region" of the alpha-subunit in nicotinic acetylcholine receptor from skeletal muscle and electric organ. Ultrastructural studies of antibody binding in the ganglion have shown that the cross-reacting antigen exposed on the surface of the neurons is located predominantly in synaptic membrane. Here we show that the neuronal antigen can be identified in detergent extracts of ciliary and sympathetic ganglia, but not in extracts of heart, liver, spinal cord, retina, or dorsal root ganglia. In the ciliary ganglion the component is present as an integral membrane constituent, and, when detergent solubilized, it sediments as a 10 S species and binds to concanavalin A. The component is distinct from the alpha-bungarotoxin-binding site on the neurons since toxin-binding sites and antibody-binding sites can be precipitated separately in ganglion extracts. The component reaches peak levels per ganglionic protein between embryonic days 8 and 12. These are some of the properties expected for the nicotinic acetylcholine receptor on ciliary ganglion neurons.

Antigenic modulation and receptor loss in experimental autoimmune myasthenia gravis.

Immunization of groups of rats with 0.1- 100 microgram of acetylcholine receptor (AChR) purified from the electric organ of Torpedo californica resulted in dose-dependent (1) loss of acetylcholine receptor from the rats' muscles, (2) binding of antibodies to many of the receptors remaining in muscle and (3) production of antibodies in serum capable of cross-reacting with receptor solubilized from rat muscle. Addition of antibodies from rats immunized with electric organ acetylcholine receptors to muscle cells in culture caused loss of receptor by accelerating the rate of receptor degradation. Monovalent antibody fragments did not accelerate degradation unless antiantibody was added to cross-link the monovalent antibody fragments bound to receptors. This indicates that cross-linking of receptors by antibody molecules triggers accelerated receptor degradation, leading to receptor loss. The rate of increase in receptor destruction due to antigenic modulation observed in vitro appears sufficient to account for the extent of receptor loss observed in vivo. Endocytosis of antibody cross-linked receptors may be a rate-limiting step common to antigenic modulation in vitro and in vivo.