Mapping the alpha-subunit site photolabeled by the noncompetitive inhibitor [3H]quinacrine azide in the active state of the nicotinic acetylcholine receptor

• Published in: The Journal of biological chemistry 1990-07, Vol.265 (19), p.11017-11029
• Main Authors: DIPAOLA, M, KAO, P. N, KARLIN, A
• Format: Article
• Language: English
• Subjects: Acetylcholine - metabolism; Affinity Labels; Amino Acid Sequence; Amphibian Venoms - pharmacology; Animals; Azides - metabolism; Binding Sites; Biological and medical sciences; Bupivacaine - pharmacology; Cell receptors; Cell structures and functions; Chlorpromazine - pharmacology; Chromatography, High Pressure Liquid; Cyanogen Bromide; Fundamental and applied biological sciences. Psychology; Molecular and cellular biology; Molecular Sequence Data; Molecular Weight; Monoamines receptors (catecholamine, serotonine, histamine, acetylcholine); Nicotinic Antagonists; Peptide Fragments - isolation & purification; Peptide Mapping; Photochemistry; Proadifen - pharmacology; Quinacrine - pharmacology; Receptors, Nicotinic - metabolism; Torpedo
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1016/S0021-9258(19)38551-5

We have characterized the time-resolved labeling of a site on the Torpedo californica electrocyte acetylcholine receptor (ACHR) by the photoreactive noncompetitive inhibitor derivative quinacrine azide (QA). The dependence of [3H]QA labeling on acetylcholine (ACH) concentration and on time is consistent with the preferential labeling by [3H]QA of ACHR in the open state. The ACH-dependent [3H]QA labeling, which was associated predominantly with the alpha-subunit, was blocked by other noncompetitive inhibitors including quinacrine, chlorpromazine, proadifen, histrionicotoxin, and bupivacaine. alpha-Subunit from ACHR labeled with [3H]QA 20 ms after the addition of ACH was cleaved with CNBr, and the fragments were separated by high pressure liquid chromatography. A peptide containing a major site of specific labeling was purified on two different reverse-phase columns. By N-terminal sequencing, amino acid composition, binding to mercurial-agarose, and apparent molecular weight, this [3H]QA-labeled peptide was identified as alpha-208-243, a CNBr fragment containing the putative membrane-spanning helix M1.