Structure of the high-affinity binding site for noncompetitive blockers of the acetylcholine receptor: [3H]chlorpromazine labels homologous residues in the .beta. and .delta. chains

Abstract

The membrane-bound acetylcholine receptor from Torpedo marmorata was photolabeled by the noncompetitive channel blocker [3H]chlorpromazine under equilibrium conditions in the presence of the agonist carbamoylcholine. The amount of radioactivity incorporated into all subunits was reduced by addition of phencyclidine, a specific ligand for the high-affinity site for noncompetitive blockers. The labeled p chain was purified and digested with trypsin or CNBr, and the resulting fragments were fractionated by high-performance liquid chromatography. Sequence analysis resulted in the identification of Ser-254 and Leu-257 as residues labeled by [3H]chlorpr~mazine in a phencyclidine-sensitive manner. These residues are located in the hydrophobic and potentially transmembrane segment M I1 of the chain, a region homologous to that containing the chlorpromazine-labeled Ser-262 in the 6 chain [Giraudat, J., Dennis, M.These results show that homologous regions of different receptor subunits contribute to the unique high-affinity site for noncompetitive blockers, a finding consistent with the location of this site on the axis of symmetry of the receptor molecule.x e nicotinic acetylcholine receptor (AcChR)' from fish electric organ and vertebrate neuromuscular junction is a ABSTRACT: We have previously described a specific protease in turkey erythrocytes that converts the larger 50-kDa (P50) form of the P,-adrenoceptor to a smaller 40-kDa (P40) form [Jurss, R., Hekman, M., &

The functional architecture of the acetylcholine nicotinic receptor explored by affinity labelling and site-directed mutagenesis

“…The affinity-labelling data obtained with [ 3 H]chlorpromazine (Giraudat et al , 1987(Giraudat et al , 1989Revah et al 1990) and TPMP , thus support the view that: (1) .DSG-EK MTLSISVLLSLTVFLLVIV E..…”

“…5); such disposition supports an organization of Mil into an a-helix, the a-carbons of the labelled amino acids being aligned on the same meridian on adjacent turns of the helix (Giraudat et al 1987;Revah et al 1990). Pedersen & Cohen (19906) have used another noncompetitive blocker, a mustard derivative of meproadifen to affinity label the a-subunit amino acid GIU262, an amino acid belonging to the ' outer ring of negatively charged residues' (Figs 4, 5 and see below) located at the border of the Mil segment in the region linking Mil and M i l l .…”

“…Labelling of #-Ser262 is blocked by phencyclidine and belongs to the hydrophobic segment Mil, thus pointing to Mil as part of (or in the close vicinity to) the high-affinity site for non-competitive blockers within the ion channel . Similar experiments were repeated with the other subunits and sequence analysis resulted in the identification of Ser254 and Leu257 for the /?-subunit (Giraudat et al 1987), Ser248 for the a-subunit (Giraudat et al 1989) and Thr253, Ser257 and Leu26o for the y-subunit (Revah et al 1990) as residues labelled by [ 3 H]chlorpromazine in a phencyclidine sensitive manner. Following the finding of Giraudat et al (1986) with chlorpromazine, Oberthiir et al (1986) using triphenylmethylphosphonium (TPMP) have also identified £-Ser262 as the labelled amino acid.…”

“…In the previous chapter, it was mentioned that a highly conserved Leu ring was labelled by chlorpromazine (Giraudat et al 1987;Revah et al 1990). To unravel its physiological significance, its homologue in the a7-homooligomeric receptor (Leu247) was mutated into a polar residue (e.g.…”

The noncompetitive blocker [3H]chlorpromazine labels segment M2 but not segment M 1 of the nicotinic acetylcholine receptor α‐subunit

“…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.…”