Effects of lipids and detergents on the conformation of the nicotinic acetylcholine receptor from Torpedo californica.
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.
McCarthy
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MP
Center for Advanced Biotechnology and Medicine, University of Medicine and Dentistry, New Jersey-Robert Wood Johnson Medical School, Piscataway 08854.
Moore
M A
MA
D000345:Affinity Labels; D000818:Animals; D001389:Azirines / Q000737:chemistry; D002784:Cholesterol / Q000378:metabolism; D002846:Chromatography, Affinity; D003902:Detergents / Q000494:pharmacology*; D004591:Electrophoresis, Polyacrylamide Gel; D008055:Lipids / Q000494:pharmacology*; D010712:Phosphatidic Acids / Q000378:metabolism; D010713:Phosphatidylcholines / Q000378:metabolism; D011487:Protein Conformation; D011978:Receptors, Nicotinic / Q000737:chemistry / Q000187:drug effects*; D014101:Torpedo
D000345:Affinity Labels; D001389:Azirines; D003902:Detergents; D008055:Lipids; D010712:Phosphatidic Acids; D010713:Phosphatidylcholines; D011978:Receptors, Nicotinic; C037657:dioleoylphosphatidic acid; C033174:3-(trifluoromethyl)-3-(3-iodophenyl)diazirine; D002784:Cholesterol; C017251:1,2-oleoylphosphatidylcholine