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Conformational Dynamic Studies of Prokaryotic Potassium Channels Explored by Homo-FRET Methodologies

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Potassium Channels

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2796))

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Abstract

Fluorescence techniques have been widely used to shed light over the structure–function relationship of potassium channels for the last 40–50 years. In this chapter, we describe how a Förster resonance energy transfer between identical fluorophores (homo-FRET) approach can be applied to study the gating behavior of the prokaryotic channel KcsA. Two different gates have been described to control the K+ flux across the channel’s pore, the helix-bundle crossing and the selectivity filter, located at the opposite sides of the channel transmembrane section. Both gates can be studied individually or by using a double-reporter system. Due to its homotetrameric structural arrangement, KcsA presents a high degree of symmetry that fulfills the first requisite to calculate intersubunit distances through this technique. The results obtained through this work have helped to uncover the conformational plasticity of the selectivity filter under different experimental conditions and the importance of its allosteric coupling to the opening of the activation (inner) gate. This biophysical approach usually requires low protein concentration and presents high sensitivity and reproducibility, complementing the high-resolution structural information provided by X-ray crystallography, cryo-EM, and NMR studies.

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Acknowledgments

We thank Aleksander Fedorov, Manuel Prieto, and Mário Nuno Berberan-Santos from iBB (Portugal) and Clara Díaz-García, Ana Marcela Giudici, José Manuel González-Ros, and Eva Martínez from IDiBE (Spain) for all the extensive and wise contributions to the work described here. This work was partly supported by grants PGC2018-093505-B-I00 from the Spanish “Ministerio de Ciencia e Innovación”/FEDER, U.E., and national funds from FCT Fundação para a Ciência e a Tecnologia, I.P., under the scope of the projects UIDB/04565/2020 and UIDP/04565/2020 of the Research Unit Institute for Bioengineering and Biosciences (iBB) and the project LA/P/0140/2020 of the Associate Laboratory Institute for Health and Bioeconomy (i4HB).

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Authors and Affiliations

  1. iBB, Institute for Bioengineering and Biosciences, Instituto Superior Técnico, Universidade de Lisboa, Lisbon, Portugal

    Ana Coutinho

  2. Associate Laboratory i4HB, Institute for Health and Bioeconomy at Instituto Superior Técnico, Universidade de Lisboa, Lisbon, Portugal

    Ana Coutinho

  3. Departamento de Química e Bioquímica, Faculdade de Ciências, Universidade de Lisboa, Lisbon, Portugal

    Ana Coutinho

  4. Instituto de Investigación, Desarrollo e Innovación en Biotecnología Sanitaria de Elche, Universidad Miguel Hernández, Elche, Spain

    José Antonio Poveda & María Lourdes Renart

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  1. Ana Coutinho
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  2. José Antonio Poveda
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  3. María Lourdes Renart
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Correspondence to María Lourdes Renart .

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  1. Department of Electrical, Electronic and Information Engineering "Guglielmo Marconi”, University of Bologna, Cesena, Italy

    Simone Furini

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Coutinho, A., Poveda, J.A., Renart, M.L. (2024). Conformational Dynamic Studies of Prokaryotic Potassium Channels Explored by Homo-FRET Methodologies. In: Furini, S. (eds) Potassium Channels. Methods in Molecular Biology, vol 2796. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-3818-7_3

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