Mapping the alpha-subunit site photolabeled by the noncompetitive inhibitor [3H]quinacrine azide in the active state of the nicotinic acetylcholine receptor.
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.
DiPaola
M
M
Department of Biochemistry and Molecular Biophysics, Columbia University, New York, New York 10032.
Kao
P N
PN
Karlin
A
A
D000109:Acetylcholine / Q000378:metabolism; D000345:Affinity Labels; D000595:Amino Acid Sequence; D000664:Amphibian Venoms / Q000494:pharmacology; D000818:Animals; D001386:Azides / Q000378:metabolism*; D001665:Binding Sites; D002045:Bupivacaine / Q000494:pharmacology; D002746:Chlorpromazine / Q000494:pharmacology; D002851:Chromatography, High Pressure Liquid; D003488:Cyanogen Bromide; D008969:Molecular Sequence Data; D008970:Molecular Weight; D018733:Nicotinic Antagonists; D010446:Peptide Fragments / Q000302:isolation & purification; D010449:Peptide Mapping; D010777:Photochemistry; D011335:Proadifen / Q000494:pharmacology; D011796:Quinacrine / Q000494:pharmacology; D011978:Receptors, Nicotinic / Q000378:metabolism*; D014101:Torpedo
D000345:Affinity Labels; D000664:Amphibian Venoms; D001386:Azides; D018733:Nicotinic Antagonists; D010446:Peptide Fragments; D011978:Receptors, Nicotinic; C032798:quinacrine azide; D011335:Proadifen; D011796:Quinacrine; D000109:Acetylcholine; D003488:Cyanogen Bromide; C007948:histrionicotoxin; D002746:Chlorpromazine; D002045:Bupivacaine