Time-resolved photolabeling by quinacrine azide of a noncompetitive inhibitor site of the nicotinic acetylcholine receptor in a transient, agonist-induced state.

233

107

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., &

Section: Discussionsupporting

confidence: 75%

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

Giraudat¹,

Dennis²,

Heidmann³

et al. 1987

233

107

“…As already discussed, our results show directly that procaine-induced channel inactivation occurs only after ACh binding and, presumably, channel opening. Analogous results have been provided by AChR photolabeling experiments using radiolabeled LA's (Oswald et al, 1983;Cox et al, 1985;Heidmann & Changeux, 1984). These agents bind slowly to Torpedo AChR protein in the absence of agonists, but simultaneous addition of labeling agents and agonists greatly enhances labeling rates.…”

Section: Discussionsupporting

confidence: 67%

Procaine rapidly inactivates acetylcholine receptor from Torpedo and competes with agonist for inhibition sites

Forman

Miller²

1989

The relationship between the high-affinity procaine channel inhibition site (apparent dissociation constant Kp congruent to 200 microM) and the agonist self-inhibition site on acetylcholine receptors (AChRs) from Torpedo electroplaque was investigated by using rapid 86Rb+ quenched-flux assays at 4 degrees C in native AChR-rich vesicles on which 50-60% of ACh activation sites were blocked with alpha-bungarotoxin (alpha-BTX). In the presence of channel-activating acetylcholine (ACh) concentrations (10 microM-10 mM) alone, AChR undergoes one phase of inactivation (fast desensitization, rate = kd) in under a second. Addition of procaine produces two-phase inactivation similar to that seen with self-inhibiting (greater than 10 mM) ACh concentrations [Forman & Miller (1988) Biophys. J. 54, 149-158]--rapid inactivation (rate = kr) complete in 30-75 ms is followed by fast desensitization at the same kd observed without procaine. The dependence of kr on [procaine] is consistent with a bimolecular association between procaine and its AChR site with kon = 2.5 X 10(5) M-1 s-1, koff = 36 s-1, and Kp = 145 +/- 36 microM). Inhibition of AChR function by mixtures of procaine (up to 12Kp) plus self-inhibiting concentrations of ACh or suberyldicholine ([SubCh] up to 13 X the 50% self-inhibiting agonist concentration, KB) was studied by reducing the level of alpha-BTX block in vesicles. The apparent KB increased in the presence of procaine, and the apparent KP increased linearly with [SubCh], indicating mutually exclusive actions at a common AChR site. Our data support a mechanism where procaine binds preferentially to the open-channel AChR state, since no procaine-induced inactivation is observed without agonist and kr's dependence on [ACh] in the channel-activating range closely parallels that of 86Rb+ flux response to ACh.

“…Two of the groups (Cox et al, 1984;Heidmann & Changeux, 1984) first interpreted their data in terms of a mechanism in which the inhibitors selectively label the open-channel form of the receptor. Subsequently Cox et al (1985) suggested inhibitor binding to the desensitized state. These conclusions are based on the observations that the labeling is rapid, agonist specific, develops in the high agonist concentration range, and is transient.…”

Section: Discussionmentioning

confidence: 99%

Cocaine, phencyclidine, and procaine inhibition of the acetylcholine receptor: characterization of the binding site by stopped-flow measurements of receptor-controlled ion flux in membrane vesicles

Karpen¹,

Hess²

1986

Noncompetitive inhibition of acetylcholine receptor-controlled ion translocation was studied in membrane vesicles prepared from both Torpedo californica and Electrophorus electricus electroplax. Ion flux was measured in the millisecond time region by using a spectrophotometric stopped-flow method, based on fluorescence quenching of entrapped anthracene-1,5-disulfonic acid by Cs+, and a quench-flow technique using 86Rb+. The rate coefficient of ion flux prior to receptor inactivation (desensitization), JA, was measured at different acetylcholine and inhibitor concentrations, in order to assess which active (nondesensitized) receptor forms bind noncompetitive inhibitors. The degree of inhibition of JA by the inhibitors studied (cocaine, procaine, and phencyclidine) was found to be independent of acetylcholine concentration. The results are consistent with a mechanism in which each compound inhibits by binding to a single site that exists with equal affinity on all active receptor forms. Mechanisms in which the inhibitors bind exclusively to the open-channel form of the receptor are excluded by the data. The same conclusions were reached in cocaine experiments at 0-mV and procaine experiments at -25-mV transmembrane voltage in T. californica vesicles. It had been previously shown that phencyclidine, in addition to decreasing JA (by binding to active receptors), also increases the rate of rapid receptor inactivation (desensitization) and changes the equilibrium between active and inactive receptors (by binding better to inactivated receptor than to active receptor in the closed or open conformations). These effects were not observed with cocaine or procaine. Here it is shown that despite these differential effects on inactivation, cocaine and phencyclidine bind to the same inhibitory site on active receptors (in E. electricus vesicles).(ABSTRACT TRUNCATED AT 250 WORDS)