271
Viktor Stein (ed.), Synthetic Protein Switches: Methods and Protocols, Methods in Molecular Biology, vol. 1596,
DOI 10.1007/978-1-4939-6940-1_17, © Springer Science+Business Media LLC 2017
Chapter 17
Yeast-Based Screening System for the Selection
of Functional Light-Driven K
+
Channels
Cristian Cosentino, Laura Alberio, Gerhard Thiel, and Anna Moroni
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
1 Introduction
Potassium (K
+
) channels are membrane proteins ubiquitous in living organisms. They drive the flux of K
+
ions across the ionimpermeant lipid membrane. In higher organisms, they play a
fundamental role in controlling cell excitability and intercellular
communication, particularly between neurons. Potassium channels
are tetrameric proteins in which each monomer contributes to the
formation of a central ion conductive pore surrounded by regulatory or sensor domains. All K
+
channels operate according to a
common principle: upon perception of the stimuli, the peripheral
sensors convey the information to the pore that opens or closes in
response (gating). Given their common modular architecture, K
+
channels tolerate swapping of pore and sensor modules between
members of different families without losing functionality [1, 2].
More surprising is the evidence that exogenous sensors found in
proteins unrelated to the K
+
channel superfamily can be grafted on
the pore to control gating. In the past, we have engineered a
Viktor Stein (ed.), Synthetic Protein Switches: Methods and Protocols, Methods in Molecular Biology, vol. 1596,
DOI 10.1007/978-1-4939-6940-1_17, © Springer Science+Business Media LLC 2017
Chapter 17
Yeast-Based Screening System for the Selection
of Functional Light-Driven K
+
Channels
Cristian Cosentino, Laura Alberio, Gerhard Thiel, and Anna Moroni
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
1 Introduction
Potassium (K
+
) channels are membrane proteins ubiquitous in living organisms. They drive the flux of K
+
ions across the ionimpermeant lipid membrane. In higher organisms, they play a
fundamental role in controlling cell excitability and intercellular
communication, particularly between neurons. Potassium channels
are tetrameric proteins in which each monomer contributes to the
formation of a central ion conductive pore surrounded by regulatory or sensor domains. All K
+
channels operate according to a
common principle: upon perception of the stimuli, the peripheral
sensors convey the information to the pore that opens or closes in
response (gating). Given their common modular architecture, K
+
channels tolerate swapping of pore and sensor modules between
members of different families without losing functionality [1, 2].
More surprising is the evidence that exogenous sensors found in
proteins unrelated to the K
+
channel superfamily can be grafted on
the pore to control gating. In the past, we have engineered a