The high-affinity quinacrine binding site is located at a non-annular lipid domain of the nicotinic acetylcholine receptor

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Abstract

This work deals with the localization of the high-affinity non-competitive quinacrine binding site on the muscle-type nicotinic acetylcholine receptor (AChR). Specifically, quantitative steady-state fluorescence spectroscopy Is used to determine whether quinacrine binds to a site located at either the annular or the non-annular lipid domain. For this purpose, we measure the ability of spin-labelled phosphatidylcholine (SL-PC) to quench AChR-bound quinacrine, AChR-bound ethidium and membrane-partitioned 7-(9-anthroyloxy)stearate (7-AS) fluorescence. Additionally, we compare the accessibility of SL-PC which is considered to bind only to the annular lipid domain of the AChR with the accessibility of two non-annular domain-sensing lipids such as 5-doxylstearate (5-SAL) and spin-labelled androstane (ASL). Initial experiments using 7-AS established the experimental conditions for maximum SL-PC membrane partitioning. The non-specific quenching elicited by increasing turbidity of the sample after addition of SL-PC is corrected by means of parallel experiments with unlabelled egg yolk phosphatidylcholine. After correction, the SL-PC quenching experiments show the following order in quenching efficiency: 7-AS > quinacrine >> ethidium. The relative intrinsic sensitivity of quinacrine to TEMPO paramagnetic quenching in acetonitrile is considered to be approximately two times higher than that for 7-AS. Thus, SL-PC was found to be more accessible (about 5-fold) to the membrane-partitioned 7-AS than to the quinacrine locus. In addition, SL-PC was virtually not accessible to the high-affinity non-luminal binding site for ethidium. The relative capacity of SL-PC, 5-SAL, and ASL to quench AChR-bound quinacrine fluorescence indicated that the spin-labelled lipid accessibility to the quinacrine binding site follows the order: 5-SAL > ASL >> SL-PC. Examination of the effect of high concentrations of 5-SAL, of its unlabelled parent stearate, and of SL-PC on ethidium and quinacrine binding showed that: (a) both fatty acids displace quinacrine, but not ethidium, from its high-affinity binding site, however (b) 5-SAL was found to be more effective than stearate to displace quinacrine from its locus, whereas (c) SL-PC competes neither for the ethidium locus nor for the quinacrine binding site. The results suggest that the high-affinity binding site for quinacrine is located at a non-annular lipid domain of the AChR. This particular area has been considered to be located at the intramolecular interfaces of the five AChR subunits and/or at the interstices of the transmembrane domains.

Original languageEnglish
Pages (from-to)9-22
Number of pages14
JournalBiochimica et Biophysica Acta - Lipids and Lipid Metabolism
Volume1347
Issue number1
DOIs
StatePublished - 12 Jul 1997
Externally publishedYes

Fingerprint

Quinacrine
Nicotinic Receptors
Phosphatidylcholines
Binding Sites
Stearates
Lipids
Ethidium
Cholinergic Receptors
Quenching
Membranes
Fluorescence
Egg Yolk
Experiments
Fluorescence Spectrometry
Fluorescence spectroscopy
Turbidity
Muscle

Keywords

  • Annular lipid domain
  • Fluorescence-quenching spectroscopy
  • High-affinity quinacrine binding site
  • Nicotinic acetylcholine receptor, muscle-type
  • Non-annular lipid domain
  • Non-competitive inhibitor

Cite this

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title = "The high-affinity quinacrine binding site is located at a non-annular lipid domain of the nicotinic acetylcholine receptor",
abstract = "This work deals with the localization of the high-affinity non-competitive quinacrine binding site on the muscle-type nicotinic acetylcholine receptor (AChR). Specifically, quantitative steady-state fluorescence spectroscopy Is used to determine whether quinacrine binds to a site located at either the annular or the non-annular lipid domain. For this purpose, we measure the ability of spin-labelled phosphatidylcholine (SL-PC) to quench AChR-bound quinacrine, AChR-bound ethidium and membrane-partitioned 7-(9-anthroyloxy)stearate (7-AS) fluorescence. Additionally, we compare the accessibility of SL-PC which is considered to bind only to the annular lipid domain of the AChR with the accessibility of two non-annular domain-sensing lipids such as 5-doxylstearate (5-SAL) and spin-labelled androstane (ASL). Initial experiments using 7-AS established the experimental conditions for maximum SL-PC membrane partitioning. The non-specific quenching elicited by increasing turbidity of the sample after addition of SL-PC is corrected by means of parallel experiments with unlabelled egg yolk phosphatidylcholine. After correction, the SL-PC quenching experiments show the following order in quenching efficiency: 7-AS > quinacrine >> ethidium. The relative intrinsic sensitivity of quinacrine to TEMPO paramagnetic quenching in acetonitrile is considered to be approximately two times higher than that for 7-AS. Thus, SL-PC was found to be more accessible (about 5-fold) to the membrane-partitioned 7-AS than to the quinacrine locus. In addition, SL-PC was virtually not accessible to the high-affinity non-luminal binding site for ethidium. The relative capacity of SL-PC, 5-SAL, and ASL to quench AChR-bound quinacrine fluorescence indicated that the spin-labelled lipid accessibility to the quinacrine binding site follows the order: 5-SAL > ASL >> SL-PC. Examination of the effect of high concentrations of 5-SAL, of its unlabelled parent stearate, and of SL-PC on ethidium and quinacrine binding showed that: (a) both fatty acids displace quinacrine, but not ethidium, from its high-affinity binding site, however (b) 5-SAL was found to be more effective than stearate to displace quinacrine from its locus, whereas (c) SL-PC competes neither for the ethidium locus nor for the quinacrine binding site. The results suggest that the high-affinity binding site for quinacrine is located at a non-annular lipid domain of the AChR. This particular area has been considered to be located at the intramolecular interfaces of the five AChR subunits and/or at the interstices of the transmembrane domains.",
keywords = "Annular lipid domain, Fluorescence-quenching spectroscopy, High-affinity quinacrine binding site, Nicotinic acetylcholine receptor, muscle-type, Non-annular lipid domain, Non-competitive inhibitor",
author = "Arias, {Hugo Rub{\'e}n}",
year = "1997",
month = "7",
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doi = "10.1016/S0005-2760(97)00045-3",
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pages = "9--22",
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TY - JOUR

T1 - The high-affinity quinacrine binding site is located at a non-annular lipid domain of the nicotinic acetylcholine receptor

AU - Arias, Hugo Rubén

PY - 1997/7/12

Y1 - 1997/7/12

N2 - This work deals with the localization of the high-affinity non-competitive quinacrine binding site on the muscle-type nicotinic acetylcholine receptor (AChR). Specifically, quantitative steady-state fluorescence spectroscopy Is used to determine whether quinacrine binds to a site located at either the annular or the non-annular lipid domain. For this purpose, we measure the ability of spin-labelled phosphatidylcholine (SL-PC) to quench AChR-bound quinacrine, AChR-bound ethidium and membrane-partitioned 7-(9-anthroyloxy)stearate (7-AS) fluorescence. Additionally, we compare the accessibility of SL-PC which is considered to bind only to the annular lipid domain of the AChR with the accessibility of two non-annular domain-sensing lipids such as 5-doxylstearate (5-SAL) and spin-labelled androstane (ASL). Initial experiments using 7-AS established the experimental conditions for maximum SL-PC membrane partitioning. The non-specific quenching elicited by increasing turbidity of the sample after addition of SL-PC is corrected by means of parallel experiments with unlabelled egg yolk phosphatidylcholine. After correction, the SL-PC quenching experiments show the following order in quenching efficiency: 7-AS > quinacrine >> ethidium. The relative intrinsic sensitivity of quinacrine to TEMPO paramagnetic quenching in acetonitrile is considered to be approximately two times higher than that for 7-AS. Thus, SL-PC was found to be more accessible (about 5-fold) to the membrane-partitioned 7-AS than to the quinacrine locus. In addition, SL-PC was virtually not accessible to the high-affinity non-luminal binding site for ethidium. The relative capacity of SL-PC, 5-SAL, and ASL to quench AChR-bound quinacrine fluorescence indicated that the spin-labelled lipid accessibility to the quinacrine binding site follows the order: 5-SAL > ASL >> SL-PC. Examination of the effect of high concentrations of 5-SAL, of its unlabelled parent stearate, and of SL-PC on ethidium and quinacrine binding showed that: (a) both fatty acids displace quinacrine, but not ethidium, from its high-affinity binding site, however (b) 5-SAL was found to be more effective than stearate to displace quinacrine from its locus, whereas (c) SL-PC competes neither for the ethidium locus nor for the quinacrine binding site. The results suggest that the high-affinity binding site for quinacrine is located at a non-annular lipid domain of the AChR. This particular area has been considered to be located at the intramolecular interfaces of the five AChR subunits and/or at the interstices of the transmembrane domains.

AB - This work deals with the localization of the high-affinity non-competitive quinacrine binding site on the muscle-type nicotinic acetylcholine receptor (AChR). Specifically, quantitative steady-state fluorescence spectroscopy Is used to determine whether quinacrine binds to a site located at either the annular or the non-annular lipid domain. For this purpose, we measure the ability of spin-labelled phosphatidylcholine (SL-PC) to quench AChR-bound quinacrine, AChR-bound ethidium and membrane-partitioned 7-(9-anthroyloxy)stearate (7-AS) fluorescence. Additionally, we compare the accessibility of SL-PC which is considered to bind only to the annular lipid domain of the AChR with the accessibility of two non-annular domain-sensing lipids such as 5-doxylstearate (5-SAL) and spin-labelled androstane (ASL). Initial experiments using 7-AS established the experimental conditions for maximum SL-PC membrane partitioning. The non-specific quenching elicited by increasing turbidity of the sample after addition of SL-PC is corrected by means of parallel experiments with unlabelled egg yolk phosphatidylcholine. After correction, the SL-PC quenching experiments show the following order in quenching efficiency: 7-AS > quinacrine >> ethidium. The relative intrinsic sensitivity of quinacrine to TEMPO paramagnetic quenching in acetonitrile is considered to be approximately two times higher than that for 7-AS. Thus, SL-PC was found to be more accessible (about 5-fold) to the membrane-partitioned 7-AS than to the quinacrine locus. In addition, SL-PC was virtually not accessible to the high-affinity non-luminal binding site for ethidium. The relative capacity of SL-PC, 5-SAL, and ASL to quench AChR-bound quinacrine fluorescence indicated that the spin-labelled lipid accessibility to the quinacrine binding site follows the order: 5-SAL > ASL >> SL-PC. Examination of the effect of high concentrations of 5-SAL, of its unlabelled parent stearate, and of SL-PC on ethidium and quinacrine binding showed that: (a) both fatty acids displace quinacrine, but not ethidium, from its high-affinity binding site, however (b) 5-SAL was found to be more effective than stearate to displace quinacrine from its locus, whereas (c) SL-PC competes neither for the ethidium locus nor for the quinacrine binding site. The results suggest that the high-affinity binding site for quinacrine is located at a non-annular lipid domain of the AChR. This particular area has been considered to be located at the intramolecular interfaces of the five AChR subunits and/or at the interstices of the transmembrane domains.

KW - Annular lipid domain

KW - Fluorescence-quenching spectroscopy

KW - High-affinity quinacrine binding site

KW - Nicotinic acetylcholine receptor, muscle-type

KW - Non-annular lipid domain

KW - Non-competitive inhibitor

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U2 - 10.1016/S0005-2760(97)00045-3

DO - 10.1016/S0005-2760(97)00045-3

M3 - Article

C2 - 9233683

AN - SCOPUS:0031565660

VL - 1347

SP - 9

EP - 22

JO - Biochimica et Biophysica Acta (BBA)/Lipids and Lipid Metabolism

JF - Biochimica et Biophysica Acta (BBA)/Lipids and Lipid Metabolism

SN - 0005-2760

IS - 1

ER -