Quinacrine and Ethidium Bind to Different Loci on the Torpedo Acetylcholine Receptor

Hugo R. Arias, C. Fernando Valenzuela, David A. Johnson

Research output: Contribution to journalArticle

26 Citations (Scopus)

Abstract

Fluorescence spectroscopy was used to determine whether quinacrine and ethidium, two high-affinity noncompetitive inhibitors of the Torpedo acetylcholine receptor (AcChR), bind to the same loci. The ability of three nitroxide spin-labels, 5-doxylstearate (5-SAL), spin-labeled androstane (ASL), and TEMPO, to quench receptor-bound quinacrine and ethidium fluorescence was measured. When bound to a phencyclidine-displaceable site on the AcChR, quinacrine was 16.9 and 19 times more efficiently quenched than ethidium by the highly lipophilic 5-SAL and ASL, respectively. TEMPO, which has a limited ability to partition into Torpedo plasma membranes (<1%), was only twice as efficient at quenching receptor-bound quinacrine than ethidium fluorescence. The relative sensitivity of quinacrine and ethidium fluorescence to paramagnetic quenching was examined in three solvents, 1-butanol, sodium phosphate buffer, and acetonitrile, with TEMPO as a quencher. The results from the different solvents demonstrate that quinacrine fluorescence is intrinsically 1.4–3.6 times more sensitive than ethidium fluorescence to quenching by nitroxide spin-labels. Examination of the effect of high concentrations of 5-SAL on ethidium and quinacrine dissociation constants showed that quinacrine but not ethidium binding was competitively inhibited. Together, these results indicate that although quinacrine and ethidium bind in a mutually exclusive manner, the two inhibitors interact at different loci on the AcChR. Whereas the ethidium binding site is at a distance from membrane lipids, probably in or near the lumen, the quinacrine binding site appears to be at a lipid–protein interface in the transmembrane domain and at a distance from the lumen.

Original languageEnglish
Pages (from-to)6237-6242
Number of pages6
JournalBiochemistry
Volume32
Issue number24
DOIs
StatePublished - 1 Jan 1993
Externally publishedYes

Fingerprint

Torpedo
Quinacrine
Ethidium
Cholinergic Receptors
Fluorescence
Spin Labels
Quenching
Binding Sites
Phencyclidine
1-Butanol
Fluorescence Spectrometry
Fluorescence spectroscopy
Cell membranes
Membrane Lipids
Buffers
Cell Membrane

Cite this

Arias, Hugo R. ; Valenzuela, C. Fernando ; Johnson, David A. / Quinacrine and Ethidium Bind to Different Loci on the Torpedo Acetylcholine Receptor. In: Biochemistry. 1993 ; Vol. 32, No. 24. pp. 6237-6242.
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Quinacrine and Ethidium Bind to Different Loci on the Torpedo Acetylcholine Receptor. / Arias, Hugo R.; Valenzuela, C. Fernando; Johnson, David A.

In: Biochemistry, Vol. 32, No. 24, 01.01.1993, p. 6237-6242.

Research output: Contribution to journalArticle

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T1 - Quinacrine and Ethidium Bind to Different Loci on the Torpedo Acetylcholine Receptor

AU - Arias, Hugo R.

AU - Valenzuela, C. Fernando

AU - Johnson, David A.

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N2 - Fluorescence spectroscopy was used to determine whether quinacrine and ethidium, two high-affinity noncompetitive inhibitors of the Torpedo acetylcholine receptor (AcChR), bind to the same loci. The ability of three nitroxide spin-labels, 5-doxylstearate (5-SAL), spin-labeled androstane (ASL), and TEMPO, to quench receptor-bound quinacrine and ethidium fluorescence was measured. When bound to a phencyclidine-displaceable site on the AcChR, quinacrine was 16.9 and 19 times more efficiently quenched than ethidium by the highly lipophilic 5-SAL and ASL, respectively. TEMPO, which has a limited ability to partition into Torpedo plasma membranes (<1%), was only twice as efficient at quenching receptor-bound quinacrine than ethidium fluorescence. The relative sensitivity of quinacrine and ethidium fluorescence to paramagnetic quenching was examined in three solvents, 1-butanol, sodium phosphate buffer, and acetonitrile, with TEMPO as a quencher. The results from the different solvents demonstrate that quinacrine fluorescence is intrinsically 1.4–3.6 times more sensitive than ethidium fluorescence to quenching by nitroxide spin-labels. Examination of the effect of high concentrations of 5-SAL on ethidium and quinacrine dissociation constants showed that quinacrine but not ethidium binding was competitively inhibited. Together, these results indicate that although quinacrine and ethidium bind in a mutually exclusive manner, the two inhibitors interact at different loci on the AcChR. Whereas the ethidium binding site is at a distance from membrane lipids, probably in or near the lumen, the quinacrine binding site appears to be at a lipid–protein interface in the transmembrane domain and at a distance from the lumen.

AB - Fluorescence spectroscopy was used to determine whether quinacrine and ethidium, two high-affinity noncompetitive inhibitors of the Torpedo acetylcholine receptor (AcChR), bind to the same loci. The ability of three nitroxide spin-labels, 5-doxylstearate (5-SAL), spin-labeled androstane (ASL), and TEMPO, to quench receptor-bound quinacrine and ethidium fluorescence was measured. When bound to a phencyclidine-displaceable site on the AcChR, quinacrine was 16.9 and 19 times more efficiently quenched than ethidium by the highly lipophilic 5-SAL and ASL, respectively. TEMPO, which has a limited ability to partition into Torpedo plasma membranes (<1%), was only twice as efficient at quenching receptor-bound quinacrine than ethidium fluorescence. The relative sensitivity of quinacrine and ethidium fluorescence to paramagnetic quenching was examined in three solvents, 1-butanol, sodium phosphate buffer, and acetonitrile, with TEMPO as a quencher. The results from the different solvents demonstrate that quinacrine fluorescence is intrinsically 1.4–3.6 times more sensitive than ethidium fluorescence to quenching by nitroxide spin-labels. Examination of the effect of high concentrations of 5-SAL on ethidium and quinacrine dissociation constants showed that quinacrine but not ethidium binding was competitively inhibited. Together, these results indicate that although quinacrine and ethidium bind in a mutually exclusive manner, the two inhibitors interact at different loci on the AcChR. Whereas the ethidium binding site is at a distance from membrane lipids, probably in or near the lumen, the quinacrine binding site appears to be at a lipid–protein interface in the transmembrane domain and at a distance from the lumen.

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