A KcsA/MloK1 chimeric ion channel has lipid-dependent ligand-binding energetics.
Détails
ID Serval
serval:BIB_5C3BAF7003A1
Type
Article: article d'un périodique ou d'un magazine.
Collection
Publications
Institution
Titre
A KcsA/MloK1 chimeric ion channel has lipid-dependent ligand-binding energetics.
Périodique
The Journal of biological chemistry
ISSN
1083-351X (Electronic)
ISSN-L
0021-9258
Statut éditorial
Publié
Date de publication
04/04/2014
Peer-reviewed
Oui
Volume
289
Numéro
14
Pages
9535-9546
Langue
anglais
Notes
Publication types: Journal Article ; Research Support, N.I.H., Extramural ; Research Support, Non-U.S. Gov't
Publication Status: ppublish
Publication Status: ppublish
Résumé
Cyclic nucleotide-modulated ion channels play crucial roles in signal transduction in eukaryotes. The molecular mechanism by which ligand binding leads to channel opening remains poorly understood, due in part to the lack of a robust method for preparing sufficient amounts of purified, stable protein required for structural and biochemical characterization. To overcome this limitation, we designed a stable, highly expressed chimeric ion channel consisting of the transmembrane domains of the well characterized potassium channel KcsA and the cyclic nucleotide-binding domains of the prokaryotic cyclic nucleotide-modulated channel MloK1. This chimera demonstrates KcsA-like pH-sensitive activity which is modulated by cAMP, reminiscent of the dual modulation in hyperpolarization-activated and cyclic nucleotide-gated channels that display voltage-dependent activity that is also modulated by cAMP. Using this chimeric construct, we were able to measure for the first time the binding thermodynamics of cAMP to an intact cyclic nucleotide-modulated ion channel using isothermal titration calorimetry. The energetics of ligand binding to channels reconstituted in lipid bilayers are substantially different from those observed in detergent micelles, suggesting that the conformation of the chimera's transmembrane domain is sensitive to its (lipid or lipid-mimetic) environment and that ligand binding induces conformational changes in the transmembrane domain. Nevertheless, because cAMP on its own does not activate these chimeric channels, cAMP binding likely has a smaller energetic contribution to gating than proton binding suggesting that there is only a small difference in cAMP binding energy between the open and closed states of the channel.
Mots-clé
Bacterial Proteins/chemistry, Bacterial Proteins/genetics, Bacterial Proteins/metabolism, Cyclic AMP/chemistry, Cyclic AMP/genetics, Cyclic AMP/metabolism, Hydrogen-Ion Concentration, Ion Channel Gating, Membrane Lipids/chemistry, Membrane Lipids/genetics, Membrane Lipids/metabolism, Mesorhizobium/chemistry, Mesorhizobium/genetics, Mesorhizobium/metabolism, Potassium Channels/chemistry, Potassium Channels/genetics, Potassium Channels/metabolism, Protein Structure, Tertiary, Recombinant Fusion Proteins/genetics, Recombinant Fusion Proteins/metabolism, Electron Microscopy (EM), Energetics, Fluorescence, Gating, Ion Channels, Isothermal Titration Calorimetry, Nanodisc, Single-channel Recording
Pubmed
Web of science
Open Access
Oui
Création de la notice
09/06/2023 15:03
Dernière modification de la notice
20/07/2023 5:57