IDENTIFYING THE CHOLESTEROL BINDING DOMAIN IN THE NICOTINIC ACETYLCHOLINE-RECEPTOR WITH [I-125] AZIDO-CHOLESTEROL

A novel photoreactive analog of cholesterol, 3 pha-(4-azido-3-[I-125]i odosalicylic)-cholest-5-3n3 ([I-125]azido-cholesterol), was used to la bel both native acetylcholine receptor (AChR)-rich membranes from Torp edo californica and affinity-purified Torpedo AChRs reconstituted into lipid vesicles. In both cases all four AChR subunits incorporated [I- 125]azido-cholesterol on an equal molar basis and neither the pattern nor the extent of labeling was affected by the presence of the agonist carbamylcholine. Labeled regions in each of the AChR subunits were in itially mapped by Staphylococcus aureus V8 protease digestion to large fragments which contain the AChR transmembrane segments. Sites of [I- 125]azido-cholesterol incorporation were further mapped by exhaustive tryptic digestion of the V8 protease subunit fragments alpha V8-20 (al pha Ser-173-Glu-338), alpha V8-10 (alpha Asn-339-Gly-439), and gamma V 8-14 (gamma Leu-373-Pro-489). The digests were separated by reverse-ph ase high-performance liquid chromatography and labeled peptides identi fied by amino-terminal sequence analysis. [I-125]Azido-cholesterol lab eling was localized to peptides that contain almost exclusively the al pha-M4, alpha-M1 and gamma-M4 membrane spanning segments. These result s establish that the binding domain for cholesterol is at the lipid-pr otein interface of the AChR. (C) 1998 Elsevier Science B.V. All rights reserved.