Molecular mechanisms and binding site location for the noncompetitive antagonist crystal violet on nicotinic acetylcholine receptors

Hugo R. Arias, Pankaj Bhumireddy, Guillermo Spitzmaul, James R. Trudell, Cecilia Bouzat

Research output: Contribution to journalArticle

32 Citations (Scopus)

Abstract

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 °C); (ii) CrV-induced inhibition of the phencyclidine (PCP) analogue [3H] 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 al-Glu262 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.

Original languageEnglish
Pages (from-to)2014-2026
Number of pages13
JournalBiochemistry
Volume45
Issue number7
DOIs
StatePublished - 21 Feb 2006
Externally publishedYes

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Gentian Violet
Nicotinic Receptors
Binding Sites
Cholinergic Receptors
tenocyclidine
Tetracaine
Torpedo
Phencyclidine
Barbiturates
Fluorescence Spectrometry
Clamping devices
Fluorescence spectroscopy
Molecular Dynamics Simulation
Mouth
Molecular dynamics

Cite this

Arias, Hugo R. ; Bhumireddy, Pankaj ; Spitzmaul, Guillermo ; Trudell, James R. ; Bouzat, Cecilia. / Molecular mechanisms and binding site location for the noncompetitive antagonist crystal violet on nicotinic acetylcholine receptors. In: Biochemistry. 2006 ; Vol. 45, No. 7. pp. 2014-2026.
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abstract = "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 °C); (ii) CrV-induced inhibition of the phencyclidine (PCP) analogue [3H] 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 al-Glu262 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.",
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Molecular mechanisms and binding site location for the noncompetitive antagonist crystal violet on nicotinic acetylcholine receptors. / Arias, Hugo R.; Bhumireddy, Pankaj; Spitzmaul, Guillermo; Trudell, James R.; Bouzat, Cecilia.

In: Biochemistry, Vol. 45, No. 7, 21.02.2006, p. 2014-2026.

Research output: Contribution to journalArticle

TY - JOUR

T1 - Molecular mechanisms and binding site location for the noncompetitive antagonist crystal violet on nicotinic acetylcholine receptors

AU - Arias, Hugo R.

AU - Bhumireddy, Pankaj

AU - Spitzmaul, Guillermo

AU - Trudell, James R.

AU - Bouzat, Cecilia

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N2 - 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 °C); (ii) CrV-induced inhibition of the phencyclidine (PCP) analogue [3H] 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 al-Glu262 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.

AB - 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 °C); (ii) CrV-induced inhibition of the phencyclidine (PCP) analogue [3H] 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 al-Glu262 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.

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DO - 10.1021/bi051752e

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