New Light-Driven Channels and Transporters for Optogenetics

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Optogenetics is revolutionizing our ability to study signal processing in neuronal circuits and promises new approaches to the treatment of different diseases and handicaps. The core tools of optogenetics are light-driven retinal membrane proteins. Unfortunately, there are at present only few retinal proteins available for optogenetics, the non-selective channel rhodopsin ChR2 used to depolarize and thus activate neurons, the Cl- pump halorhodopsin NphR, and the H+ pump archaerhodopsin Arch3, both of which are used to hyperpolarize and thus silence neurons. Identification and/or engineering light-driven proteins with novel properties, such as channels and transporters with high selectivity and conductivity for a particular ion will be crucial for future progress. Our project is divided in two complementary parts addressing light-regulated pumps (1) and light-regulated channels (2).(1) We will identify/generate new light-driven pumps, based on recent work of members of our consortium. We recently solved the ground state structure of the first known light-driven Na+ pump, Krokinobacter eikastus rhodopsin 2 (KR2), at high resolution. The identification of the ion-translocation pathway allowed engineering a light-driven K+ pump. We will optimize expression of KR2 and the engineered K+ pump in neurons for widespread optogenetic use. Moreover, we will select new prospective candidates from genome databanks to obtain Na+ pumps with novel properties and engineer additional K+ pumps by modifying these selected Na+ pumps. To understand the molecular mechanism of ion pumping, we will solve the structures of intermediate states of KR2 and the related K+ pump. This structural information will be used for improved transport by Na+ and K+ pumps and furthermore for engineering light-driven Ca2+ pumps. To assess the potential for optogenetics, we will express the novel pumps in multiple expressions systems, ranging from mammalian cell lines to C. elegans. (2) Previous attempts to modify the cationic selectivity of ChR2 have met limited success. One reason is that the molecular mechanism of ion permeation is insufficiently clear. We will combine structural biology and computational biology to describe the permeation and selectivity process, and to obtain clues toward the design of light-activated ion channels with improved selectivity and conduction properties. The consortium of three French and three German teams includes pioneers of the studies of light-driven proteins and founders of optogenetics. Its complementary multidisciplinary expertise spans the full range of available techniques, from retinal protein production, function and structure determination, biophysical characterization, rational protein design to the application of light-driven proteins to neuroscience, in neuronal cell culture and in the nematode C. elegans. Our ambition is to suggest and implement a set of new light-driven proteins required to substantially advance optogenetics.