Molecular mechanisms and binding site location for the noncompetitive antagonist crystal violet on nicotinic acetylcholine receptors.
We investigated the molecular mechanisms and the binding site location for the fluorophor crystal violet (CrV), a noncompetitive antagonist of the nicotinic acetylcholine receptor (AChR). To this end, radiolabeled competition binding, fluorescence spectroscopy, Schild-type analysis, patch-clamp recordings, and molecular dynamics approaches were used. The results indicate that (i) CrV interacts with the desensitized Torpedo AChR with higher affinity than with the resting state at several temperatures (5-37 degrees C); (ii) CrV-induced inhibition of the phencyclidine (PCP) analogue [(3)H]thienylcyclohexylpiperidine binding to the desensitized or resting AChR is mediated by a steric mechanism; (iii) tetracaine inhibits CrV binding to the resting AChR, probably by a steric mechanism; (iv) barbiturates modulate CrV binding to the resting AChR by an allosteric mechanism; (v) CrV itself induces AChR desensitization; (vi) CrV decreases the peak of macroscopic currents by acting on the resting AChR but without affecting the desensitization rate from the open state; and (vii) two tertiary amino groups from CrV may bind to the alpha1-Glu(262) residues (located at position 20') in the resting state. We conclude that the CrV binding site overlaps the PCP locus in the resting and desensitized state. The noncompetitive action of CrV may be explained by an allosteric mechanism in which the binding of CrV to the extracellular mouth of the resting receptor leads to an inhibition of channel opening. Binding of CrV probably increases desensitization of the resting channel and stabilizes the desensitized state.
Department of Pharmaceutical Sciences, College of Pharmacy, Western University of Health Sciences, Pomona, California 91766-1854, USA. harias@westernu.edu
Hugo R
HR
Arias
Pankaj
P
Bhumireddy
Guillermo
G
Spitzmaul
James R
JR
Trudell
Cecilia
C
Bouzat
D000494:Allosteric Regulation; D000654:Amobarbital / Q000737:chemistry / Q000494:pharmacology; D000818:Animals; D001665:Binding Sites; D005456:Fluorescent Dyes / Q000737:chemistry; D005840:Gentian Violet / Q000737:chemistry* / Q000494:pharmacology; D007473:Ion Channels / Q000187:drug effects; D008958:Models, Molecular; D018722:Nicotinic Agonists / Q000737:chemistry*; D018733:Nicotinic Antagonists / Q000737:chemistry*; D010424:Pentobarbital / Q000494:pharmacology; D010622:Phencyclidine / Q000031:analogs & derivatives / Q000737:chemistry; D011485:Protein Binding / Q000187:drug effects; D011487:Protein Conformation; D000644:Quaternary Ammonium Compounds / Q000737:chemistry; D011978:Receptors, Nicotinic / Q000737:chemistry*; D013696:Temperature; D013748:Tetracaine / Q000737:chemistry; D013876:Thiophenes / Q000737:chemistry; D014101:Torpedo; D014316:Tritium
D005456:Fluorescent Dyes; D007473:Ion Channels; D018722:Nicotinic Agonists; D018733:Nicotinic Antagonists; D000644:Quaternary Ammonium Compounds; D011978:Receptors, Nicotinic; D013876:Thiophenes; D013748:Tetracaine; D014316:Tritium; C012951:DNS-chol; C012058:tenocyclidine; D000654:Amobarbital; D010424:Pentobarbital; D010622:Phencyclidine; D005840:Gentian Violet