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Multiple binding sites for fatty acids on the potassium channel KcsA

Multiple binding sites for fatty acids on the potassium channel KcsA
Multiple binding sites for fatty acids on the potassium channel KcsA
Interactions of fatty acids with the potassium channel KcsA were studied using Trp fluorescence quenching and electron paramagnetic resonance (EPR) techniques. The brominated analogue of oleic acid was shown to bind to annular sites on KcsA and to the nonannular sites at each protein-protein interface in the homotetrameric structure with binding constants relative to dioleoylphosphatidylcholine of 0.67 ± 0.04 and 0.87 ± 0.08, respectively. Mutation of the two Arg residues close to the nonannular binding sites had no effect on fatty acid binding. EPR studies with a spin-labeled analogue of stearic acid detected a high-affinity binding site for the fatty acid with strong immobilization. Fluorescence quenching studies with the spin-labeled analogue showed that the binding site detected in the EPR experiments could not be one of the annular or nonannular binding sites. Instead, it is proposed that the EPR studies detect binding to the central hydrophobic cavity of the channel, with a binding constant in the range of ~0.1-1 ?M.
2889-2898
Bolivar, Juan H.
e3b126b7-e597-4dad-ac73-bda0c0cab016
Smithers, Natalie
63ead01b-6515-4f82-a963-884f572af872
East, J. Malcolm
9fe7f794-1d89-4935-9a99-b831d786056e
Marsh, Derek
e2ab9365-4e03-43f2-8abe-cb03f222e7ae
Lee, Anthony G.
0891914c-e0e2-4ee1-b43e-1b70eb072d8e
Bolivar, Juan H.
e3b126b7-e597-4dad-ac73-bda0c0cab016
Smithers, Natalie
63ead01b-6515-4f82-a963-884f572af872
East, J. Malcolm
9fe7f794-1d89-4935-9a99-b831d786056e
Marsh, Derek
e2ab9365-4e03-43f2-8abe-cb03f222e7ae
Lee, Anthony G.
0891914c-e0e2-4ee1-b43e-1b70eb072d8e

Bolivar, Juan H., Smithers, Natalie, East, J. Malcolm, Marsh, Derek and Lee, Anthony G. (2012) Multiple binding sites for fatty acids on the potassium channel KcsA. Biochemistry, 51 (13), 2889-2898. (doi:10.1021/bi300153v). (PMID:22409348)

Record type: Article

Abstract

Interactions of fatty acids with the potassium channel KcsA were studied using Trp fluorescence quenching and electron paramagnetic resonance (EPR) techniques. The brominated analogue of oleic acid was shown to bind to annular sites on KcsA and to the nonannular sites at each protein-protein interface in the homotetrameric structure with binding constants relative to dioleoylphosphatidylcholine of 0.67 ± 0.04 and 0.87 ± 0.08, respectively. Mutation of the two Arg residues close to the nonannular binding sites had no effect on fatty acid binding. EPR studies with a spin-labeled analogue of stearic acid detected a high-affinity binding site for the fatty acid with strong immobilization. Fluorescence quenching studies with the spin-labeled analogue showed that the binding site detected in the EPR experiments could not be one of the annular or nonannular binding sites. Instead, it is proposed that the EPR studies detect binding to the central hydrophobic cavity of the channel, with a binding constant in the range of ~0.1-1 ?M.

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e-pub ahead of print date: 12 March 2012
Published date: 3 April 2012
Organisations: Centre for Biological Sciences

Identifiers

Local EPrints ID: 342267
URI: http://eprints.soton.ac.uk/id/eprint/342267
PURE UUID: d768cd94-909a-48ce-92b5-b097718f0cb3

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Date deposited: 20 Aug 2012 14:16
Last modified: 14 Mar 2024 11:48

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Contributors

Author: Juan H. Bolivar
Author: Natalie Smithers
Author: J. Malcolm East
Author: Derek Marsh
Author: Anthony G. Lee

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