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Mechanism of ligand-gated potassium efflux in bacterial pathogens.

ABSTRACT: Gram negative pathogens are protected against toxic electrophilic compounds by glutathione-gated potassium efflux systems (Kef) that modulate cytoplasmic pH. We have elucidated the mechanism of gating through structural and functional analysis of Escherichia coli KefC. The revealed mechanism can explain how subtle chemical differences in glutathione derivatives can produce opposite effects on channel function. Kef channels are regulated by potassium transport and NAD-binding (KTN) domains that sense both reduced glutathione, which inhibits Kef activity, and glutathione adducts that form during electrophile detoxification and activate Kef. We find that reduced glutathione stabilizes an interdomain association between two KTN folds, whereas large adducts sterically disrupt this interaction. F441 is identified as the pivotal residue discriminating between reduced glutathione and its conjugates. We demonstrate a major structural change on the binding of an activating ligand to a KTN-domain protein. Analysis of the regulatory interactions suggests strategies to disrupt pathogen potassium and pH homeostasis.

SUBMITTER: Roosild TP

PROVIDER: S-EPMC2993342 | biostudies-literature | 2010 Nov

REPOSITORIES: biostudies-literature

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Mechanism of ligand-gated potassium efflux in bacterial pathogens.

Roosild Tarmo P TP Castronovo Samantha S Healy Jess J Miller Samantha S Pliotas Christos C Rasmussen Tim T Bartlett Wendy W Conway Stuart J SJ Booth Ian R IR

Proceedings of the National Academy of Sciences of the United States of America 20101101 46

Gram negative pathogens are protected against toxic electrophilic compounds by glutathione-gated potassium efflux systems (Kef) that modulate cytoplasmic pH. We have elucidated the mechanism of gating through structural and functional analysis of Escherichia coli KefC. The revealed mechanism can explain how subtle chemical differences in glutathione derivatives can produce opposite effects on channel function. Kef channels are regulated by potassium transport and NAD-binding (KTN) domains that s ...[more]

PMID: 21041667

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Project description:Voltage-gated ion channels play a central role in the generation of action potentials in the nervous system. They are selective for one type of ion - sodium, calcium, or potassium. Voltage-gated ion channels are composed of a central pore that allows ions to pass through the membrane and four peripheral voltage sensing domains that respond to changes in the membrane potential. Upon depolarization, voltage sensors in voltage-gated potassium channels (Kv) undergo conformational changes driven by positive charges in the S4 segment and aided by pairwise electrostatic interactions with the surrounding voltage sensor. Structure-function relations of Kv channels have been investigated in detail, and the resulting models on the movement of the voltage sensors now converge to a consensus; the S4 segment undergoes a combined movement of rotation, tilt, and vertical displacement in order to bring 3-4e(+) each through the electric field focused in this region. Nevertheless, the mechanism by which the voltage sensor movement leads to pore opening, the electromechanical coupling, is still not fully understood. Thus, recently, electromechanical coupling in different Kv channels has been investigated with a multitude of techniques including electrophysiology, 3D crystal structures, fluorescence spectroscopy, and molecular dynamics simulations. Evidently, the S4-S5 linker, the covalent link between the voltage sensor and pore, plays a crucial role. The linker transfers the energy from the voltage sensor movement to the pore domain via an interaction with the S6 C-termini, which are pulled open during gating. In addition, other contact regions have been proposed. This review aims to provide (i) an in-depth comparison of the molecular mechanisms of electromechanical coupling in different Kv channels; (ii) insight as to how the voltage sensor and pore domain influence one another; and (iii) theoretical predictions on the movement of the cytosolic face of the Kv channels during gating.

Project description:Kv7 (KCNQ) voltage-gated potassium channels are critical regulators of neuronal excitability and are candidate targets for development of antiseizure medications. Drug discovery efforts have identified small molecules that modulate channel function and reveal mechanistic insights into Kv7 channel physiological roles. While Kv7 channel activators have therapeutic benefits, inhibitors are useful for understanding channel function and mechanistic validation of candidate drugs. In this study, we reveal the mechanism of a Kv7.2/Kv7.3 inhibitor, ML252. We used docking and electrophysiology to identify critical residues involved in ML252 sensitivity. Most notably, Kv7.2[W236F] or Kv7.3[W265F] mutations strongly attenuate ML252 sensitivity. This tryptophan residue in the pore is also required for sensitivity to certain activators, including retigabine and ML213. We used automated planar patch clamp electrophysiology to assess competitive interactions between ML252 and different Kv7 activator subtypes. A pore-targeted activator (ML213) weakens the inhibitory effects of ML252, whereas a distinct activator subtype (ICA-069673) that targets the voltage sensor does not prevent ML252 inhibition. Using transgenic zebrafish larvae expressing an optical reporter (CaMPARI) to measure neural activity in-vivo, we demonstrate that Kv7 inhibition by ML252 increases neuronal excitability. Consistent with in-vitro data, ML213 suppresses ML252 induced neuronal activity, while the voltage-sensor targeted activator ICA-069673 does not prevent ML252 actions. In summary, this study establishes a binding site and mechanism of action of ML252, classifying this poorly understood drug as a pore-targeted Kv7 channel inhibitor that binds to the same tryptophan residue as commonly used pore-targeted Kv7 activators. ML213 and ML252 likely have overlapping sites of interaction in the pore Kv7.2 and Kv7.3 channels, resulting in competitive interactions. In contrast, the VSD-targeted activator ICA-069673 does not prevent channel inhibition by ML252.

Dataset Information

Mechanism of ligand-gated potassium efflux in bacterial pathogens.

Publications

Mechanism of ligand-gated potassium efflux in bacterial pathogens.

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