Aminotriarylmethane dyes are high-affinity noncompetitive antagonists of the nicotinic acetylcholine receptor

A series of aminotriarylmethane dyes were examined for binding to the nicot inic acetylcholine receptor (AChR) from Torpedo californica. Several compou nds were found to bind to the noncompetitive antagonist site of the AChR as demonstrated by inhibition of [H-3]phencyclidine binding; apparent K-D val ues ranged from 50 nM to >100 mu M. One dye with high affinity, crystal vio let, revealed a greater than 200-fold fluorescence enhancement upon binding the AChR. Using fluorescence to measure binding, we determined that one cr ystal violet bound per receptor with a dissociation constant of 100 nM; in the presence of the agonist carbamylcholine this value decreased to 10 nM. The K-D for [H-3]acetylcholine binding likewise was decreased in the presen ce of crystal violet. These results are consistent with preferential bindin g of crystal violet to the desensitized conformation of the AChR. Crystal v iolet binding blocked agonist-induced Na-22 ion efflux from AChR-rich vesic les. It is concluded that crystal violet and other dyes of similar structur e bind to the high-affinity noncompetitive antagonist site of the AChR asso ciated with the channel lumen. Because of their optical properties, crystal violet and several of the other homologous dyes are likely to be useful li gands for further characterization of the AChR channel. Structure-activity comparison of the various dyes suggests the importance of nonquaternary nit rogens in binding the pore. Additional steric bulk on amines or at meta pos itions increase or have neutral effect on affinity, suggesting that steric considerations alone do not limit high affinity for the binding site.