Effects of chlorpromazine and phencyclidine on mouse C2 acetylcholine receptor kinetics.

Abstract

SUMMARY1. Patch-clamp techniques were used to record acetylcholine-(ACh) activated single-channel currents in cell-attached membrane patches from myotubes of the mouse cell line, C2.2. The effects of the phenothiazine derivative chlorpromazine (CPZ) and of the hallucinogen phencyclidine (PCP) on ACh-activated single-channel properties were studied under conditions where both compounds are positively charged (pH 7 2).3. The single-channel conductance was unaffected by either CPZ or PCP at concentrations ranging from 10 to 500 nM.4. 10-200 nM-CPZ and PCP led to shortened mean burst times. 5. CPZ and PCP effects on mean burst times were voltage independent and did not vary in a simple linear manner with concentration.6. 10-200 nM-CPZ and PCP did not reduce channel opening frequencies, suggesting that the fraction ofnon-conducting state (occupied, blocked or desensitized) favoured at equilibrium was not significant at these concentrations.7. On the other hand, concentrations of CPZ and PCP higher than 300 nM did lead to depressed channel opening frequencies. In addition, we observed that, at these concentrations, the shortened burst duration reverses to the longer values found at lower effector concentrations.8. The effects of CPZ and PCP on ACh-activated single-channel kinetics are interpreted in terms of current models of ACh-receptor structure and conformational transitions.

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

“…The degree of symmetry of the AChR molecule inferred from morphological and primary structure data, can thus be experimentally tested at this level. Furthermore, electrophysiological (review in [27][28][29]) as well as biochemical (review in [6,30]) evidence supports the view that the high-affinity NCB site is located within the ion channel. Photoactivatable NCBs are thus useful tools for direct identification of potential channel-forming elements within each of the AChR subunits [1,2,12,13].…”

Allosteric binding site in a Cys-loop receptor ligand-binding domain unveiled in the crystal structure of ELIC in complex with chlorpromazine

“…In the present study, we could not observe CPZ binding in the ELIC pore, and this can be likely explained by the unusual and bulky Phe residue at the 16′ pore position in ELIC, which prevents pore access to noncompetitive pore blockers such as memantine (40) and probably also CPZ. Therefore, it is possible that the β8-β9 loop site identified in our study on ELIC corresponds to an "external" binding site for CPZ described in one of the pioneering studies on mouse C2 muscle-type nAChRs and that is distinct from the high-affinity "internal" pore blocker site (66).…”

The noncompetitive blocker [3H]chlorpromazine labels three amino acids of the acetylcholine receptor gamma subunit: implications for the alpha-helical organization of regions MII and for the structure of the ion channel.

“…Indeed, in such a configuration the a carbons of Ser-y257 and Leu-y260 are predicted to lie, respectively, at 1.1 and 1.9 helix turn from the a carbon of Thr-y253, and, as a consequence, the three labeled residues would be aligned on adjacent turns of the a-helix. The distance between the a carbons of Thr-y253 and Leu-y260 would then be 10.7 A, a distance compatible with the dimensions of CPZ deduced from its crystal structure (21 (25), were suggested to sterically block ion flux by plugging the ionic channel. This hypothesis is reinforced by rapid-mixing and photolabeling experiments in the millisecond time range (6) and by the demonstration that channel-permeant cations competitively block binding of ethidium to the high-affinity site for NCBs (9).…”