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Interaction of fatty acids and phospholipids with multiple binding sites on the potassium channel KcsA

Interaction of fatty acids and phospholipids with multiple binding sites on the potassium channel KcsA
Interaction of fatty acids and phospholipids with multiple binding sites on the potassium channel KcsA
The majority of lipids that interact with a transmembrane protein act as a solvent for the protein, forming a shell around it; these lipids are called annular or boundary lipids. However, some lipids interact in a more specific way, binding between transmembrane ?-helices or at protein-protein interfaces, and these lipids are referred to as non-annular lipids. The crystal structure of the bacterial potassium channel KcsA shows a non-annular lipid molecule bound at the subunit interfaces in the homotetrameric structure, and binding of this non-annular lipid has been shown to be essential for channel function, making KcsA an ideal candidate for the study of lipid-protein interactions. Recently, a third type of binding site at the hydrophobic inner cavity of the pore of potassium channels has been proposed for fatty acid molecules, where binding was suggested to cause block of ion flux. Fatty acids are known to affect ion channel activity, but it is not yet certain how they act. Here, fluorescence spectroscopy and electron spin resonance (ESR) are combined to analyse the interaction of fatty acids with the annular and non-annular sites, and with the hydrophobic inner cavity of the pore on KcsA.

To study fatty acid binding by fluorescence spectroscopy, KcsA was reconstituted in bilayers of phosphatidylcholine (PC) containing brominated fatty acid. Quenching of the Trp fluorescence of wild type KcsA by brominated fatty acids allows an analysis of their interaction with annular sites on the channel and interaction at non-annular sites was studied using a Trp mutant of KcsA. The results shown that fatty acids can bind with an affinity rather similar to that of PC to both annular and non-annular sites, but uncharged fatty acid analogues show limited binding, emphasising the importance of charged interactions in these systems. In ESR studies KcsA was reconstituted in PC membranes containing a small amount of spin labelled fatty acid. Spin labelled lipids in contact with the protein show a different ESR spectrum from those in the bulk lipid due to the different mobilities of their acyl chains. The ESR spectra show that the spin labelled fatty acid bound to KcsA is strongly immobilised and binds with high affinity. It is proposed that the fatty acid binds to the hydrophobic cavity with a dissociation constant of ca. 0.22 ?M. The studies show that fatty acids can bind to the channel at a variety of sites, suggesting that ion channel function could be modulated directly by interactions with fatty acids.

Other studies here presented focused on the influence of the annular lipids on the aggregation of KcsA. Protein-protein contacts are important for membrane protein association and activity, but little is known about the influence that the lipid bilayer can have on protein-protein association. ESR experiments with spin labelled phospholipids show that at lipid:channel molar ratios of ca. 100:1 or higher, KcsA is solvated by ca. 31 annular phospholipid molecules, as expected from its crystal structure, but that, at lower lipid content, protein-protein contacts become favourable and KcsA aggregates. The ESR data also show that aggregation is reduced in bilayers of anionic phosphatidylglycerol (PG) in comparison to bilayers of zwitterionic PC, as confirmed by quenching experiments where brominated PG was able to quench wild type KcsA more efficiently than brominated PC at the low lipid:channel molar ratios. The results highlight the importance that the lipid bilayer composition can have on membrane protein association.
Bolivar Gonzalez, Juan
ac7fa998-3854-4255-bcab-cf97d1b4475b
Bolivar Gonzalez, Juan
ac7fa998-3854-4255-bcab-cf97d1b4475b
Lee, Anthony
0891914c-e0e2-4ee1-b43e-1b70eb072d8e
East, John
9fe7f794-1d89-4935-9a99-b831d786056e

Bolivar Gonzalez, Juan (2011) Interaction of fatty acids and phospholipids with multiple binding sites on the potassium channel KcsA. University of Southampton, Centre for Biological Sciences, Doctoral Thesis, 253pp.

Record type: Thesis (Doctoral)

Abstract

The majority of lipids that interact with a transmembrane protein act as a solvent for the protein, forming a shell around it; these lipids are called annular or boundary lipids. However, some lipids interact in a more specific way, binding between transmembrane ?-helices or at protein-protein interfaces, and these lipids are referred to as non-annular lipids. The crystal structure of the bacterial potassium channel KcsA shows a non-annular lipid molecule bound at the subunit interfaces in the homotetrameric structure, and binding of this non-annular lipid has been shown to be essential for channel function, making KcsA an ideal candidate for the study of lipid-protein interactions. Recently, a third type of binding site at the hydrophobic inner cavity of the pore of potassium channels has been proposed for fatty acid molecules, where binding was suggested to cause block of ion flux. Fatty acids are known to affect ion channel activity, but it is not yet certain how they act. Here, fluorescence spectroscopy and electron spin resonance (ESR) are combined to analyse the interaction of fatty acids with the annular and non-annular sites, and with the hydrophobic inner cavity of the pore on KcsA.

To study fatty acid binding by fluorescence spectroscopy, KcsA was reconstituted in bilayers of phosphatidylcholine (PC) containing brominated fatty acid. Quenching of the Trp fluorescence of wild type KcsA by brominated fatty acids allows an analysis of their interaction with annular sites on the channel and interaction at non-annular sites was studied using a Trp mutant of KcsA. The results shown that fatty acids can bind with an affinity rather similar to that of PC to both annular and non-annular sites, but uncharged fatty acid analogues show limited binding, emphasising the importance of charged interactions in these systems. In ESR studies KcsA was reconstituted in PC membranes containing a small amount of spin labelled fatty acid. Spin labelled lipids in contact with the protein show a different ESR spectrum from those in the bulk lipid due to the different mobilities of their acyl chains. The ESR spectra show that the spin labelled fatty acid bound to KcsA is strongly immobilised and binds with high affinity. It is proposed that the fatty acid binds to the hydrophobic cavity with a dissociation constant of ca. 0.22 ?M. The studies show that fatty acids can bind to the channel at a variety of sites, suggesting that ion channel function could be modulated directly by interactions with fatty acids.

Other studies here presented focused on the influence of the annular lipids on the aggregation of KcsA. Protein-protein contacts are important for membrane protein association and activity, but little is known about the influence that the lipid bilayer can have on protein-protein association. ESR experiments with spin labelled phospholipids show that at lipid:channel molar ratios of ca. 100:1 or higher, KcsA is solvated by ca. 31 annular phospholipid molecules, as expected from its crystal structure, but that, at lower lipid content, protein-protein contacts become favourable and KcsA aggregates. The ESR data also show that aggregation is reduced in bilayers of anionic phosphatidylglycerol (PG) in comparison to bilayers of zwitterionic PC, as confirmed by quenching experiments where brominated PG was able to quench wild type KcsA more efficiently than brominated PC at the low lipid:channel molar ratios. The results highlight the importance that the lipid bilayer composition can have on membrane protein association.

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Published date: 30 September 2011
Organisations: University of Southampton, Centre for Biological Sciences

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Local EPrints ID: 334068
URI: https://eprints.soton.ac.uk/id/eprint/334068
PURE UUID: 16086fad-8172-42d0-bb87-6a79e409790b

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Date deposited: 03 Apr 2012 15:42
Last modified: 18 Jul 2017 06:12

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