Structure of the high-affinity binding site for noncompetitive blockers of the acetylcholine receptor: serine-262 of the delta subunit is labeled by [3H]chlorpromazine.

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

“…The present data on the cr-subunit pointing at Ser-248 as a major residue labeled by [3H]CPZ thus bring additional support to the contribution of segment M2 to the structure of the ion channel [1,2,12,33].…”

“…The HPLC system used was as described [1]. Peptides were chromatographed on a Nucleosil C4 (1000 .~-7/~m) reversephase column (4.6 x 250 mm) (Soci&6 Fran~aise Chromato Colonne), or on a/~Bondapack C18 (10/zm) reverse-phase column (3.9 × 300 mm) (Waters).…”

“…More recent works from the same groups have shown that clusters of charged residues neighbouring both ends of segment M2 of the a-, fl-, 7"-and d-subunits are major determinants of the rate of ion transport [33]. Furthermore, mutation of the serines homologous to ~-Ser-262 [1] into alanines on several subunits of the AChR from mouse BC3Ht cells [34] caused a decrease in outward K ÷ currents and reduced residence time of the NCB QX222 thought to bind within the open channel [28]. Also, synthetic peptides corresponding to segment M2, but not to segment M1 of ~-subunit from Torpedo AChR, form discrete ion channels in artificial bilayers [35].…”

“…The noncompetitive blockers (NCBs) are compounds that block the permeability response by interfering directly and/or indirectly with the functioning of the ion channel [5]. Under equilibrium conditions, NCBs reversibly interact with a few categories of belong to the hydrophobic segment M2 of the fland 8-chains (residues ~?-Ser-254 and fl-Leu-257, 8-Ser-262) [1,2]. Also, 8-Ser-262 [13] and homologous residues of the or-and g-chains [12] are labeled by the NCB [3H]triphenylmethylphosphonium.…”

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

“…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 another report, Hucho et al (1986) have obtained fragmentary evidence that also points to Ser248 and Ser254 as the amino acids labelled by TPMP on the a-and /^-subunits, respectively.…”