PHOTOAFFINITY-LABELING OF THE TORPEDO-CALIFORNICA NICOTINIC ACETYLCHOLINE-RECEPTOR WITH AN ARYL AZIDE DERIVATIVE OF PHOSPHATIDYLSERINE

A photoactivatable analogue of phosphatidylserine, 125I-labeled 4-azidosalicylic acid-phosphatidylserine (125I ASA-PS), was used to label both native acetylcholine receptor (AchR)-rich membranes from Torpedo californica and AchR membranes affinity purified from Torpedo reconstituted into asolectin (a crude soybean lipid extract) vesicles. The radioiodinated arylazido group attaches directly to the phospholipid head group and thus probes for regions of the AchR structure in contact with the negatively charged head group of phosphatidylserine. All four subunits of the AchR incorporated the label, with the α subunit incorporating approximately twice as much as each of the other subunits on a per mole basis. The regions of the AchR α subunit that incorporated 125I ASA-PS were mapped by Staphylococcus aureus V8 protease digestion. The majority of label incorporated into fragments representing a more complete digestion of the α subunit was localized to 11.7- and 10.1-kDa V8 cleavage fragments, both beginning at Asn-339 and of sufficient length to contain the hydrophobic region M4. An 18.7-kDa fragment beginning at Ser-173 and of sufficient length to contain the hydrophobic regions M1, M2, and M3 was also significantly labeled. In contrast, V8 cleavage fragments representing roughly a third of the amino-terminal portion of the α subunit incorporated little or no detectable amount of probe. © 1990, American Chemical Society. All rights reserved.