Noncompetitive inhibitor binding sites on the nicotinic acetylchoune receptor

Research output: Contribution to journalArticle

Abstract

The nicotinic acetylcholine receptor (AChR) is the best characterized member of iht ligand-bindmg receptor superfamily Stnce a major focus of current research on the AChR has been to determine the structural details of ligand binding sites we have used quantitative fluorescence spectroscopy to map the binding sties of two high-af'miiy noncompetitive inhibitors (NCis), qumacnne and ethidium The use of fluorescence resonance energy transfer (FRET) has allowed measurement of the distance between the ethidium (donor) binding site and the hydrophobic probe C12-Texas Red as acceptor at the surface membrane (Johnson and Nuss, Biochemistry 33, 9070, 1994. The calculated transverse distance of about 46 Amst'ongs (A) is compalible with the ethidium binding site located well above the transmembrane domain, probably m a nonlummal site Paralelly the FRET measurement between the qumacrme (donor i binding site and the lipophihc acceptors Cl2-eosm and DMOASP-PS evidenced that the qmnacrme locus is at a distance <10 A apart from the iipid membrane, suggesting that quinacnne binds at the hpid-protein interface (Valenzuela et al JBC 267 8238, 1992) The fact that the efficiency of spin-labeled androstane (ASL) and stearate (5 SAL) to quench AChR-bound ethidium fluorescence was about 5 times lower than the efficiency to quench AChR-bound qjinacnne fiuorescence is in agreement with the location proposed above (Anas et al , JBC 268, 6348, 1993; Additionally by using a senes of different positional isomers of 5-SAL the qumacnne bind.ng Me was located about 7 A below the aqueous-hpid interface (Anas et al. Biochemistry 32 6237, 1993) Finally by comparing the accessibiity of 5-SAL and ASL whrch probe nonannular lipid domains and the accessibility of spin-labeled phosphatidyicholme which only bmds to the annular lipid domain, the qutnacrme binding site was located close to a nonannulai lipid domam (Arias. BB Acta, in press. 1997) The current point of view is that the noncompetitive process is determined by the respective binding of NCIs to the channel lumen However the existence of nonlummal NCl binding sites opens the possibility for a regulation of cation permeation by an aNostenc process.

Original languageEnglish
Pages (from-to)A1057
JournalFASEB Journal
Volume11
Issue number9
StatePublished - 1 Dec 1997
Externally publishedYes

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Nicotinic Receptors
Ethidium
Binding Sites
Cholinergic Receptors
Fluorescence Resonance Energy Transfer
Biochemistry
Lipids
Hordeolum
Ligands
Membranes
Stearates
Fluorescence Spectrometry
Fluorescence spectroscopy
Permeation
Isomers
Cations
Fluorescence
Research
Proteins

Cite this

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title = "Noncompetitive inhibitor binding sites on the nicotinic acetylchoune receptor",
abstract = "The nicotinic acetylcholine receptor (AChR) is the best characterized member of iht ligand-bindmg receptor superfamily Stnce a major focus of current research on the AChR has been to determine the structural details of ligand binding sites we have used quantitative fluorescence spectroscopy to map the binding sties of two high-af'miiy noncompetitive inhibitors (NCis), qumacnne and ethidium The use of fluorescence resonance energy transfer (FRET) has allowed measurement of the distance between the ethidium (donor) binding site and the hydrophobic probe C12-Texas Red as acceptor at the surface membrane (Johnson and Nuss, Biochemistry 33, 9070, 1994. The calculated transverse distance of about 46 Amst'ongs (A) is compalible with the ethidium binding site located well above the transmembrane domain, probably m a nonlummal site Paralelly the FRET measurement between the qumacrme (donor i binding site and the lipophihc acceptors Cl2-eosm and DMOASP-PS evidenced that the qmnacrme locus is at a distance <10 A apart from the iipid membrane, suggesting that quinacnne binds at the hpid-protein interface (Valenzuela et al JBC 267 8238, 1992) The fact that the efficiency of spin-labeled androstane (ASL) and stearate (5 SAL) to quench AChR-bound ethidium fluorescence was about 5 times lower than the efficiency to quench AChR-bound qjinacnne fiuorescence is in agreement with the location proposed above (Anas et al , JBC 268, 6348, 1993; Additionally by using a senes of different positional isomers of 5-SAL the qumacnne bind.ng Me was located about 7 A below the aqueous-hpid interface (Anas et al. Biochemistry 32 6237, 1993) Finally by comparing the accessibiity of 5-SAL and ASL whrch probe nonannular lipid domains and the accessibility of spin-labeled phosphatidyicholme which only bmds to the annular lipid domain, the qutnacrme binding site was located close to a nonannulai lipid domam (Arias. BB Acta, in press. 1997) The current point of view is that the noncompetitive process is determined by the respective binding of NCIs to the channel lumen However the existence of nonlummal NCl binding sites opens the possibility for a regulation of cation permeation by an aNostenc process.",
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Noncompetitive inhibitor binding sites on the nicotinic acetylchoune receptor. / Arias, Hugo R.

In: FASEB Journal, Vol. 11, No. 9, 01.12.1997, p. A1057.

Research output: Contribution to journalArticle

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N2 - The nicotinic acetylcholine receptor (AChR) is the best characterized member of iht ligand-bindmg receptor superfamily Stnce a major focus of current research on the AChR has been to determine the structural details of ligand binding sites we have used quantitative fluorescence spectroscopy to map the binding sties of two high-af'miiy noncompetitive inhibitors (NCis), qumacnne and ethidium The use of fluorescence resonance energy transfer (FRET) has allowed measurement of the distance between the ethidium (donor) binding site and the hydrophobic probe C12-Texas Red as acceptor at the surface membrane (Johnson and Nuss, Biochemistry 33, 9070, 1994. The calculated transverse distance of about 46 Amst'ongs (A) is compalible with the ethidium binding site located well above the transmembrane domain, probably m a nonlummal site Paralelly the FRET measurement between the qumacrme (donor i binding site and the lipophihc acceptors Cl2-eosm and DMOASP-PS evidenced that the qmnacrme locus is at a distance <10 A apart from the iipid membrane, suggesting that quinacnne binds at the hpid-protein interface (Valenzuela et al JBC 267 8238, 1992) The fact that the efficiency of spin-labeled androstane (ASL) and stearate (5 SAL) to quench AChR-bound ethidium fluorescence was about 5 times lower than the efficiency to quench AChR-bound qjinacnne fiuorescence is in agreement with the location proposed above (Anas et al , JBC 268, 6348, 1993; Additionally by using a senes of different positional isomers of 5-SAL the qumacnne bind.ng Me was located about 7 A below the aqueous-hpid interface (Anas et al. Biochemistry 32 6237, 1993) Finally by comparing the accessibiity of 5-SAL and ASL whrch probe nonannular lipid domains and the accessibility of spin-labeled phosphatidyicholme which only bmds to the annular lipid domain, the qutnacrme binding site was located close to a nonannulai lipid domam (Arias. BB Acta, in press. 1997) The current point of view is that the noncompetitive process is determined by the respective binding of NCIs to the channel lumen However the existence of nonlummal NCl binding sites opens the possibility for a regulation of cation permeation by an aNostenc process.

AB - The nicotinic acetylcholine receptor (AChR) is the best characterized member of iht ligand-bindmg receptor superfamily Stnce a major focus of current research on the AChR has been to determine the structural details of ligand binding sites we have used quantitative fluorescence spectroscopy to map the binding sties of two high-af'miiy noncompetitive inhibitors (NCis), qumacnne and ethidium The use of fluorescence resonance energy transfer (FRET) has allowed measurement of the distance between the ethidium (donor) binding site and the hydrophobic probe C12-Texas Red as acceptor at the surface membrane (Johnson and Nuss, Biochemistry 33, 9070, 1994. The calculated transverse distance of about 46 Amst'ongs (A) is compalible with the ethidium binding site located well above the transmembrane domain, probably m a nonlummal site Paralelly the FRET measurement between the qumacrme (donor i binding site and the lipophihc acceptors Cl2-eosm and DMOASP-PS evidenced that the qmnacrme locus is at a distance <10 A apart from the iipid membrane, suggesting that quinacnne binds at the hpid-protein interface (Valenzuela et al JBC 267 8238, 1992) The fact that the efficiency of spin-labeled androstane (ASL) and stearate (5 SAL) to quench AChR-bound ethidium fluorescence was about 5 times lower than the efficiency to quench AChR-bound qjinacnne fiuorescence is in agreement with the location proposed above (Anas et al , JBC 268, 6348, 1993; Additionally by using a senes of different positional isomers of 5-SAL the qumacnne bind.ng Me was located about 7 A below the aqueous-hpid interface (Anas et al. Biochemistry 32 6237, 1993) Finally by comparing the accessibiity of 5-SAL and ASL whrch probe nonannular lipid domains and the accessibility of spin-labeled phosphatidyicholme which only bmds to the annular lipid domain, the qutnacrme binding site was located close to a nonannulai lipid domam (Arias. BB Acta, in press. 1997) The current point of view is that the noncompetitive process is determined by the respective binding of NCIs to the channel lumen However the existence of nonlummal NCl binding sites opens the possibility for a regulation of cation permeation by an aNostenc process.

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