(143e) Engineering Light-Activated Proteins to Control Biological Processes in the Brain

In nature, diverse organisms ranging from bacteria to plants have acquired the ability to use light as a ubiquitous energy and information source to survive and adapt to its environment.  The main mechanism for sensing light and transducing it into physiological functions is carried out by various classes of light-activated proteins that are often modular and reversible in their ability to regulate downstream effectors.  Taking advantage of these facts, engineered light-activated proteins are emerging as powerful tools to control biological processes with high spatial resolution and temporal accuracy.  However, engineering light-activated proteins to improve existing tools and create novel functionality remains a challenge, especially for multifaceted molecules such as channelrhodopsins, light-activated cation channels which show complex kinetic, spectral, and ion conduction properties.  In this talk, we present an automated microscopy method capable of physiological screening through libraries of mutant channelrhodopsins, optimizing them for multiple properties in parallel.  We use it to find mutations in channelrhodopsin-2 (ChR2) that sculpt its ion selectivity, a physiologically important property that has been hard to engineer, while maintaining high capacity to mediate light-driven control of membrane potential.  We also show that combining multiple mutations discovered through such a screen can result in additive effects on ion selectivity.  Using this methodology and screening principles, we find a mutant with order-of-magnitude improved selectivity for sodium over both calcium and proton ions, which displays photocurrents larger than that of the original wild-type ChR2.  Such tools with high ion selectivity may find use in scientific and prototype medical applications, and the approach utilized may be useful for engineering other difficult properties of light-activated proteins, as well as for creating novel tools.