New light-driven channels and transporters for optogenetics – NEWOPTOGENETICSTOOLS
Optogenetics is one of the most important technological recent advances in biology. It revolutionized our ability to study the mechanisms of processes in neuronal circuits, promising new approaches to the treatment of different diseases and handicaps. The core tools of optogenetics are light-driven retinal membrane proteins. Unfortunately, few retinal proteins are available for optogenetics. Specifically, these are the nonselective channel rhodopsin ChR2 (discovered by partner MPI) 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.
A crucial aspect for future progress is to identify and/or engineer new light-driven proteins with novel properties, such as channels and transporters with high selectivity and conductivity for a particular ion. The ions Na+, K+, Ca2+, relevant for neuronal function, have not been addressable by microbial rhodopsin pumps so far. In particular, an outward K+ pump would be highly desirable, as K+ is the main ion used for neuronal re- or hyperpolarization.
Our project consists of two complementary parts.
The first part, generation of new light-driven pumps, is based on recent work of members of our consortium. The ground state structure of the first known light-driven Na+ pump, Krokinobacter eikastus rhodopsin 2 (KR2), was solved at high resolution. It revealed the ion-translocation pathway and allowed the authors to engineer a light-driven K+ pump [Gushchin et al, Nat Struct Mol Biol, 2015; European patent application by Gordeliy, Bamberg et al, 2015]. This essential data is only a first step. We plan here to optimize the pumps for optogenetic application, by improving expression in neurons of KR2 and its K+-selective variants. Moreover, we will select new prospective candidates from genome databanks and study new Na+ pumps with properties different from KR2 to identify the better candidates for optogenetic applications. Subsequently, new K+ pumps will be engineered using the selected Na+ pumps and their potential for optogenetics will be assessed in vitro and in vivo. To understand the molecular mechanism of ion pumping, atomic details of the photocycle intermediate states are required. We will solve the structures of the intermediate states of KR2 and the related K+ pump. This structural information will enable rational design of optimal pumps and will be used to elaborate also a light-driven Ca2+ pump.
The 2nd part of the project focuses on channels. 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 customized selective channels. An important goal will be to obtain high-resolution structures of the intermediate states of the cation-selective ChR2.
The consortium of 3 French and 3 German teams includes pioneers of studies of light-driven proteins and founders of optogenetics. Its complementary multidisciplinary expertise spans the full range of available techniques, from the most basic -- retinal protein production, function and structure determination, biophysical characterization, rational protein design – to the most applied -- application of light-driven proteins to neuroscience, in neuronal cell culture and in the nematode C. elegans.
Our ambitions are (1) to establish a high-throughput pipe-line for the studies of light-driven ion-pumps and channels, thus facilitating selection and design of new optogenetic tools, (2) to extend our mechanistic understanding of rhodopsins, and (3) to suggest and implement a set of new light-driven proteins required to substantially advance optogenetics.
Monsieur Michel Vivaudou (Institut de Biologie Structurale)
The author of this summary is the project coordinator, who is responsible for the content of this summary. The ANR declines any responsibility as for its contents.
ICS Institute of Complex Systems, Zelluläre Biophysik (ICS-4), Forschungszentrum Jülich
IBS/Membrane+UJF Institut de Biologie Structurale+Université Joseph Fourier Grenoble 1
Inria - NANO-D Centre de recherche Inria Grenoble Rhône-Alpes - NANO-D
MPI Max Planck Institute (MPI) of Biophysics, Department of Biophysical Chemistry
BMLS Buchmann Institute for Molecular Life Sciences & Institute of Biochemistry, Goethe-University Frankfurt
IBS/Channels Institut de Biologie Structurale
Help of the ANR 508,994 euros
Beginning and duration of the scientific project: February 2016 - 48 Months