Probing the Structural Dynamics of the Activation Gate of KcsA Using Homo-FRET Measurements

The allosteric coupling between activation and inactivation processes is a common feature observed in K<sup>+</sup> channels. Particularly, in the prokaryotic KcsA channel the K<sup>+</sup> conduction process is controlled by the inner gate, which is activated by acidic pH, a...

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Autores principales:	Clara Díaz-García, Maria Lourdes Renart, José Antonio Poveda, Ana Marcela Giudici, José M. González-Ros, Manuel Prieto, Ana Coutinho
Formato:	article
Lenguaje:	EN
Publicado:	MDPI AG 2021
Materias:	potassium channels homo-FRET fluorescence spectroscopy anisotropy fluorescent dye allosteric coupling Biology (General) QH301-705.5 Chemistry QD1-999
Acceso en línea:	https://doaj.org/article/68b993ff83ff4f3d9480de1f90762109
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spelling	oai:doaj.org-article:68b993ff83ff4f3d9480de1f907621092021-11-11T17:22:25ZProbing the Structural Dynamics of the Activation Gate of KcsA Using Homo-FRET Measurements10.3390/ijms2221119541422-00671661-6596https://doaj.org/article/68b993ff83ff4f3d9480de1f907621092021-11-01T00:00:00Zhttps://www.mdpi.com/1422-0067/22/21/11954https://doaj.org/toc/1661-6596https://doaj.org/toc/1422-0067The allosteric coupling between activation and inactivation processes is a common feature observed in K<sup>+</sup> channels. Particularly, in the prokaryotic KcsA channel the K<sup>+</sup> conduction process is controlled by the inner gate, which is activated by acidic pH, and by the selectivity filter (SF) or outer gate, which can adopt non-conductive or conductive states. In a previous study, a single tryptophan mutant channel (W67 KcsA) enabled us to investigate the SF dynamics using time-resolved homo-Förster Resonance Energy Transfer (homo-FRET) measurements. Here, the conformational changes of both gates were simultaneously monitored after labelling the G116C position with tetramethylrhodamine (TMR) within a W67 KcsA background. At a high degree of protein labeling, fluorescence anisotropy measurements showed that the pH-induced KcsA gating elicited a variation in the homo-FRET efficiency among the conjugated TMR dyes (TMR homo-FRET), while the conformation of the SF was simultaneously tracked (W67 homo-FRET). The dependence of the activation p<i>K</i><sub>a</sub> of the inner gate with the ion occupancy of the SF unequivocally confirmed the allosteric communication between the two gates of KcsA. This simple TMR homo-FRET based ratiometric assay can be easily extended to study the conformational dynamics associated with the gating of other ion channels and their modulation.Clara Díaz-GarcíaMaria Lourdes RenartJosé Antonio PovedaAna Marcela GiudiciJosé M. González-RosManuel PrietoAna CoutinhoMDPI AGarticlepotassium channelshomo-FRETfluorescence spectroscopyanisotropyfluorescent dyeallosteric couplingBiology (General)QH301-705.5ChemistryQD1-999ENInternational Journal of Molecular Sciences, Vol 22, Iss 11954, p 11954 (2021)
institution	DOAJ
collection	DOAJ
language	EN
topic	potassium channels homo-FRET fluorescence spectroscopy anisotropy fluorescent dye allosteric coupling Biology (General) QH301-705.5 Chemistry QD1-999
spellingShingle	potassium channels homo-FRET fluorescence spectroscopy anisotropy fluorescent dye allosteric coupling Biology (General) QH301-705.5 Chemistry QD1-999 Clara Díaz-García Maria Lourdes Renart José Antonio Poveda Ana Marcela Giudici José M. González-Ros Manuel Prieto Ana Coutinho Probing the Structural Dynamics of the Activation Gate of KcsA Using Homo-FRET Measurements
description	The allosteric coupling between activation and inactivation processes is a common feature observed in K<sup>+</sup> channels. Particularly, in the prokaryotic KcsA channel the K<sup>+</sup> conduction process is controlled by the inner gate, which is activated by acidic pH, and by the selectivity filter (SF) or outer gate, which can adopt non-conductive or conductive states. In a previous study, a single tryptophan mutant channel (W67 KcsA) enabled us to investigate the SF dynamics using time-resolved homo-Förster Resonance Energy Transfer (homo-FRET) measurements. Here, the conformational changes of both gates were simultaneously monitored after labelling the G116C position with tetramethylrhodamine (TMR) within a W67 KcsA background. At a high degree of protein labeling, fluorescence anisotropy measurements showed that the pH-induced KcsA gating elicited a variation in the homo-FRET efficiency among the conjugated TMR dyes (TMR homo-FRET), while the conformation of the SF was simultaneously tracked (W67 homo-FRET). The dependence of the activation p<i>K</i><sub>a</sub> of the inner gate with the ion occupancy of the SF unequivocally confirmed the allosteric communication between the two gates of KcsA. This simple TMR homo-FRET based ratiometric assay can be easily extended to study the conformational dynamics associated with the gating of other ion channels and their modulation.
format	article
author	Clara Díaz-García Maria Lourdes Renart José Antonio Poveda Ana Marcela Giudici José M. González-Ros Manuel Prieto Ana Coutinho
author_facet	Clara Díaz-García Maria Lourdes Renart José Antonio Poveda Ana Marcela Giudici José M. González-Ros Manuel Prieto Ana Coutinho
author_sort	Clara Díaz-García
title	Probing the Structural Dynamics of the Activation Gate of KcsA Using Homo-FRET Measurements
title_short	Probing the Structural Dynamics of the Activation Gate of KcsA Using Homo-FRET Measurements
title_full	Probing the Structural Dynamics of the Activation Gate of KcsA Using Homo-FRET Measurements
title_fullStr	Probing the Structural Dynamics of the Activation Gate of KcsA Using Homo-FRET Measurements
title_full_unstemmed	Probing the Structural Dynamics of the Activation Gate of KcsA Using Homo-FRET Measurements
title_sort	probing the structural dynamics of the activation gate of kcsa using homo-fret measurements
publisher	MDPI AG
publishDate	2021
url	https://doaj.org/article/68b993ff83ff4f3d9480de1f90762109
work_keys_str_mv	AT claradiazgarcia probingthestructuraldynamicsoftheactivationgateofkcsausinghomofretmeasurements AT marialourdesrenart probingthestructuraldynamicsoftheactivationgateofkcsausinghomofretmeasurements AT joseantoniopoveda probingthestructuraldynamicsoftheactivationgateofkcsausinghomofretmeasurements AT anamarcelagiudici probingthestructuraldynamicsoftheactivationgateofkcsausinghomofretmeasurements AT josemgonzalezros probingthestructuraldynamicsoftheactivationgateofkcsausinghomofretmeasurements AT manuelprieto probingthestructuraldynamicsoftheactivationgateofkcsausinghomofretmeasurements AT anacoutinho probingthestructuraldynamicsoftheactivationgateofkcsausinghomofretmeasurements
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Probing the Structural Dynamics of the Activation Gate of KcsA Using Homo-FRET Measurements

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