Directed evolution of potassium channels for the understanding of neuronal activity including mechanisms of sleep and anaesthesia

Abstract

Directed evolution is a technique able to modify functions on existing proteins which won the Noble Prize for Chemistry in 2018. It is based on diversifying the DNA coding sequence of the target protein, expression in a biological platform, and applying a selection pressure for screening. This is repeated for several rounds, each time generating incremental mutations directing the protein towards carrying out the desired function. Directed evolution has led to the production of molecules which have contributed to the understanding of neuronal activity underlying a range of physiological phenomena including loss of consciousness during sleep and anaesthesia. This thesis includes a short review of optogenetic tools developed by the technique including genetically-encoded calcium indicators which fluoresce upon detecting intracellular calcium ion influx and genetically-encoded voltage indicators which fluoresce upon detecting change in membrane potential of neurons. An additional short review covers the handful of metabotropic designer receptors exclusively activated by designer drugs also developed by the same technique. In an endeavour to further contribute to this field, we have conducted preliminary studies on ionotropic channels 5HT3A, and tandem two-pore potassium channels TASK3, TREK1, and TOK1 onto which directed evolution could be applied to add to this molecular toolbox. Parallel preliminary experiments, also preparing for directed evolution, investigate the binding site of halothane anaesthetic on TOK1 in order to better understand the activation mechanism of halothane on tandem two-pore potassium channels. Further work suggests experiments on TOK1 to conduct directed evolution and future perspectives for the developed molecules: a TOK1 receptor hypersensitive to halothane and a TOK1 receptor exclusively activated by Compound 21, a designer drug with no physiological targets.