The noncompetitive blocker [3H]chlorpromazine labels three amino acids of the acetylcholine receptor y subunit: Implications for the a-helical organization of regions MII and for the structure of the ion channel (channel blockers/ligand-gated ion channels/allosteric proteins) FREDERIC REVAH*, JEAN-LUC GALZI*, JfR6ME GIRAUDAT*, PIERRE-YVES HAUMONTt, FLORENCE 8254), -248 of the a chain (Ser-a248), and leucine -257 of the 8 chain (Leu-,8257) (10)(11)(12), which all belong to the second hydrophobic segment of each subunit, MII. Studies using another photolabile NCB, [3H]triphenylmethylphosphonium, have suggested that this compound labels the same serine residues as [3H]CPZ on the a, f3, and 8 subunits (13,14). Finally, site-directed mutagenesis (15)(16)(17)32) has brought complementary information and additional evidence in favor of the notion that segment MII and neighboring amino acids contribute to the regulation of ion transport through the channel.In the present paper, we demonstrate that on the y subunit of T. marmorata AcChoR, three residues belonging to the MII segment are labeled by [3H]CPZ in a PCP-sensitive manner under equilibrium conditions. The implications of these results in terms of a plausible structural model of the AcChoR ion channel are discussed. MATERIALS AND METHODS 4675The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

The membrane bound acetylcholine receptor from Torpedo marmorata was photolabeled by the noncompetitive channel blocker [~H]chlorpromazine under equilibrium conditions in the presence of the agonist carbamoylcholine. The radioactivity incorporated into the AChR subunits was reduced by addition of phencyclidine, a specific ligand for the high-affinity site for noncompetitive blockers. The ~t-subunit was purified and digested with trypsin and/or CNBr and the resulting fragments fractionated by HPLC. Sequence analysis resulted in the identification of Ser-248 as a major residue labeled by pH]chlorpromazine in a phencyclidine-sensitive manner. This residue is located in the hydrophobic and putative transmembrane segment M2 of the ~t-subunit, a region homologous to that containing the chlorpromazine-labeled Ser-262 in the Nchain [1] and the Ser-254 and Leu-257 in the//-chain [2]. Extended sequence analysis of the hydrophobic segment M 1 further showed that no labeling.occurred in this region.

The amino acid sequence of the 297-residuc fragmcnt /I has now bccn cstablishcd. Thc fragmcnt was cleaved with cyanogen bromide; the three peptides thus obtained were submitted to digestions with Staphylococcus nureus V8 protease, chymotrypsin and trypsin, sometimes after succinylation. The complete fragment was also submitted to tryptic cleavage after citraconylation. Peptides were separated by thin-layer finger-printing or high-pressure liquid chromatography. They were mostly sequenced in a liquid-phase sequenator.The 51 1-residue amino acid sequence of the mature protein is thus completely established. Secondary structure predictions indicate an alternation of helical and extended structure, with a higher percentage of the former. Comparisons with other flavoproteins do not detect any significant sequence similarity.