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

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.