1-2-oleoylphosphatidylcholine and 3-(trifluoromethyl)-3-(3-iodophenyl)diazirine

Previous studies have shown that the pattern and degree of 3-(trifluoromethyl)-3-(m-[125I]iodophenyl)diazirine ([125I]TID) photoincorporation into the nicotinic acetylcholine receptor (nAChR) can be used as a sensitive measure of nAChR conformation. Upon desensitization by prolonged exposure to agonists, certain drugs and detergents, or reconstitution into desensitizing lipids, the levels of [125I]TID incorporation into the subunits of the nAChR are dramatically reduced. In this study, we characterized the effects of the snake venom proteins alpha-bungarotoxin and alpha-cobrotoxin, as well as the smaller antagonists tubocurarine and gallamine, on [125I]TID incorporation into the subunits of both partially-purified nAChR in native lipids, or affinity-purified nAChR reconstituted into different combinations of lipids. Unlike all other compounds previously tested, alpha-bungarotoxin and alpha-cobrotoxin reproducibly increased the level of [125I]TID incorporation into all four subunits of nAChR reconstituted into dioleoylphosphatidylcholine, dioleoylphosphatidic acid and cholesterol. Gallamine had little or no effect on [125I]TID incorporation at any concentration tested (0.1 microM-5 mM). Tubocurarine had no effect on [125I]TID incorporation at low concentrations, but at higher concentrations reduced the level of [125I]TID labeling. The snake venom proteins may shift the population of nAChR, which exists as a mixture of resting state and desensitized conformations, entirely to the resting state. However, the binding of the snake venom toxins does not appear sufficient to induce the resting state conformation in nAChR which have been desensitized by other means, such as solubilization in desensitizing detergents or reconstitution in densitizing lipids.

Topics: Affinity Labels; Animals; Azirines; Bungarotoxins; Cobra Neurotoxin Proteins; Gallamine Triethiodide; Iodine Radioisotopes; Nicotinic Antagonists; Phosphatidylcholines; Photochemistry; Protein Conformation; Receptors, Nicotinic; Torpedo; Tubocurarine

The hydrophobic, photoreactive probe 3-(trifluoromethyl)-3-(m-[125I]iodophenyl)diazirine ([125I]TID) was used to characterize the effects of lipids and detergents on acetylcholine receptor (AChR) conformation. Affinity purified AChR reconstituted into dioleoylphosphatidylcholine (DOPC), dioleoylphosphatidic acid (DOPA), and cholesterol showed the same pattern of [125I]TID-labeling and demonstrated the same reduction in labeling of all four subunits upon desensitization by the agonist carbamylcholine, as partially purified AChR in native lipids. On the basis of the patterns of [125I]TID incorporation, reconstitution into DOPC/DOPA also appeared to stabilize the resting (functional) conformation of the AChR, while reconstitution in DOPC/cholesterol or DOPC alone largely desensitized the AChR. The effects of lipids on the functional state of the AChR was determined independently by measuring the ability of AChR reconstituted into different lipid combinations to undergo the change in affinity for agonist diagnostic of desensitization. The dramatic reduction in the apparent levels of [125I]TID associated with the subunits of the AChR observed upon agonist-induced desensitization was shown not to be due to a change in affinity for tightly bound lipid. Solubilization of affinity purified AChR reconstituted into DOPC/DOPA/cholesterol by the non-ionic detergents octyl glucoside, Triton X-100, and Tween 20 (final detergent concentration = 1%) was shown to produce the same pattern of [125I]TID-labeling as desensitization by agonist, while solubilization in 1% sodium cholate appeared to stabilize a conformation of the AChR more similar to the resting state.

Topics: Affinity Labels; Animals; Azirines; Cholesterol; Chromatography, Affinity; Detergents; Electrophoresis, Polyacrylamide Gel; Lipids; Phosphatidic Acids; Phosphatidylcholines; Protein Conformation; Receptors, Nicotinic; Torpedo