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
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Acceso en línea:https://doaj.org/article/68b993ff83ff4f3d9480de1f90762109
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Sumario: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.