We investigated the molecular mechanisms and the binding site location for the fluorophorcrystal violet (CrV), a noncompetitive antagonist of the nicotinic acetylcholine receptor (AChR). To thisend, radiolabeled competition binding, fluorescence spectroscopy, Schild-type analysis, patch-clamprecordings, and molecular dynamics approaches were used. The results indicate that (i) CrV interactswith the desensitized
Torpedo AChR with higher affinity than with the resting state at several temperatures(5-37
C); (ii) CrV-induced inhibition of the phencyclidine (PCP) analogue [
3H]thienylcyclohexylpiperidinebinding to the desensitized or resting AChR is mediated by a steric mechanism; (iii) tetracaine inhibitsCrV binding to the resting AChR, probably by a steric mechanism; (iv) barbiturates modulate CrV bindingto the resting AChR by an allosteric mechanism; (v) CrV itself induces AChR desensitization; (vi) CrVdecreases the peak of macroscopic currents by acting on the resting AChR but without affecting thedesensitization rate from the open state; and (vii) two tertiary amino groups from CrV may bind to the
1-Glu
262 residues (located at position 20') in the resting state. We conclude that the CrV binding siteoverlaps the PCP locus in the resting and desensitized state. The noncompetitive action of CrV may beexplained by an allosteric mechanism in which the binding of CrV to the extracellular mouth of theresting receptor leads to an inhibition of channel opening. Binding of CrV probably increases desensitizationof the resting channel and stabilizes the desensitized state.