IDENTIFYING THE LIPID-PROTEIN INTERFACE OF THE TORPEDO NICOTINIC ACETYLCHOLINE-RECEPTOR - SECONDARY STRUCTURE IMPLICATIONS

To identify amino acid residues of the Torpedo nicotinic acetylcholine receptor (AchR) interacting with membrane lipid, we have used the pho toactivatable, hydrophobic probe -trifluoromethyl-3-(m-[I-125]-iodophe nyl)diazirine ([I-125]TID). The pattern of [I-125]TID incorporation in to the M3 and M4 hydrophobic segments of each subunit was the same bot h in the presence and absence of the agonist carbamoylcholine and in t he presence of an excess of nonradioactive TID, consistent with nonspe cific photoincorporation from the lipid-protein interface. [I-125]TID reacted with five residues in alpha-M4 [Blanton, M. P., and Cohen, J. B. (1992) Biochemistry 31, 3738-3750] but with only two or three resid ues in M4 segments of beta-, gamma-, and delta-subunits. In delta-M3, [I-125]TID reacted with Met-293, Ser-297, Gly-301, Val-304, and Asn-30 5 as well as with Ile-288 preceding M3. Residues at corresponding posi tions were labeled in beta-M3 (Met-285, Ile-289, Phe-293) and in gamma -M3 (Phe-292, Leu-296, Met-299, and Asn-300) as well as gamma-Ile-283. Within alpha-M3, Phe-284 and Ser-287 were labeled. The periodicity of labeled residues provides the first direct evidence that M3 as well a s M4 segments of each subunit are organized as transmembrane alpha-hel ices each with substantial contact with lipid. In addition, in alpha-M 1 [I-125]TID reacted nonspecifically with Cys-222, Leu-223, Phe-227, a nd Leu-228, a pattern of incorporation inconsistent with the labeling pattern expected either for a ''face'' of an alpha-helix or a beta-she et.