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Yeast-Based Screening System for the Selection of Functional Light-Driven K+ Channels

  • Cristian Cosentino
  • Laura Alberio
  • Gerhard Thiel
  • Anna Moroni
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1596)

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.

Key words

Functional complementation Protein evolution Rational and random mutagenesis Light Screening S. cerevisiae Optogenetics Ion channels Potassium (K+Gating 

Notes

Acknowledgments

This work was supported by MAE (Ministero Affari Esteri) grant n. 01467532013-06-27 and ERC 2015 AdG n. 695078 noMAGIC to A.M.

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Copyright information

© Springer Science+Business Media LLC 2017

Authors and Affiliations

  • Cristian Cosentino
    • 1
  • Laura Alberio
    • 1
  • Gerhard Thiel
    • 2
  • Anna Moroni
    • 1
  1. 1.Department of BiosciencesUniversity of Milan and Biophysics Institute, National Research Council (CNR)MilanItaly
  2. 2.Plant Membrane BiophysicsTechnical University DarmstadtDarmstadtGermany

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