title: Yeast-Based Screening System for the Selection of Functional Light-Driven K+Channels.
abstract: Ion channels control the electrical properties of cells by opening and closing (gating) in response to a wide palette of environmental and physiological stimuli. Endowing ion channels with the possibility to be gated by remotely applied stimuli, such as light, provides a tool for in vivo control of cellular functions in behaving animals. We have engineered a synthetic light-gated potassium (K+) channel by connecting an exogenous plant photoreceptor LOV2 domain to the K+channel pore Kcv. Here, we describe the experimental strategy that we have used to evolve the properties of the channel toward full control of light on pore gating. Our method combines rational and random mutagenesis of the channel followed by a yeast-based screening system for light-activated K+conductance.
authors.affiliation: Department of Biosciences, University of Milan and Biophysics Institute, National Research Council (CNR), Via Celoria 26, 201333, Milan, Italy.
authors.forename: Cristian
authors.initials: C
authors.lastname: Cosentino
authors.affiliation: Department of Biosciences, University of Milan and Biophysics Institute, National Research Council (CNR), Via Celoria 26, 201333, Milan, Italy.
authors.forename: Laura
authors.initials: L
authors.lastname: Alberio
authors.affiliation: Plant Membrane Biophysics, Technical University Darmstadt, Darmstadt, Germany.
authors.forename: Gerhard
authors.initials: G
authors.lastname: Thiel
authors.affiliation: Department of Biosciences, University of Milan and Biophysics Institute, National Research Council (CNR), Via Celoria 26, 201333, Milan, Italy. anna.moroni@unimi.it.
authors.forename: Anna
authors.initials: A
authors.lastname: Moroni
mesh_terms: D015640:Ion Channel Gating / Q000502:physiology; D017136:Ion Transport / Q000502:physiology; D008027:Light; D011188:Potassium / Q000378:metabolism*; D015221:Potassium Channels / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000378:metabolism*
chemical_list: D015221:Potassium Channels; D011188:Potassium
keywords: Functional complementation; Gating; Ion channels; Light; Optogenetics; Potassium (K+); Protein evolution; Rational and random mutagenesis; S. cerevisiae; Screening