Dataset Information

Structural foundations of optogenetics: Determinants of channelrhodopsin ion selectivity.

ABSTRACT: The structure-guided design of chloride-conducting channelrhodopsins has illuminated mechanisms underlying ion selectivity of this remarkable family of light-activated ion channels. The first generation of chloride-conducting channelrhodopsins, guided in part by development of a structure-informed electrostatic model for pore selectivity, included both the introduction of amino acids with positively charged side chains into the ion conduction pathway and the removal of residues hypothesized to support negatively charged binding sites for cations. Engineered channels indeed became chloride selective, reversing near -65 mV and enabling a new kind of optogenetic inhibition; however, these first-generation chloride-conducting channels displayed small photocurrents and were not tested for optogenetic inhibition of behavior. Here we report the validation and further development of the channelrhodopsin pore model via crystal structure-guided engineering of next-generation light-activated chloride channels (iC++) and a bistable variant (SwiChR++) with net photocurrents increased more than 15-fold under physiological conditions, reversal potential further decreased by another ? 15 mV, inhibition of spiking faithfully tracking chloride gradients and intrinsic cell properties, strong expression in vivo, and the initial microbial opsin channel-inhibitor-based control of freely moving behavior. We further show that inhibition by light-gated chloride channels is mediated mainly by shunting effects, which exert optogenetic control much more efficiently than the hyperpolarization induced by light-activated chloride pumps. The design and functional features of these next-generation chloride-conducting channelrhodopsins provide both chronic and acute timescale tools for reversible optogenetic inhibition, confirm fundamental predictions of the ion selectivity model, and further elucidate electrostatic and steric structure-function relationships of the light-gated pore.

SUBMITTER: Berndt A

PROVIDER: S-EPMC4743797 | biostudies-literature | 2016 Jan

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Structural foundations of optogenetics: Determinants of channelrhodopsin ion selectivity.

Berndt Andre A Lee Soo Yeun SY Wietek Jonas J Ramakrishnan Charu C Steinberg Elizabeth E EE Rashid Asim J AJ Kim Hoseok H Park Sungmo S Santoro Adam A Frankland Paul W PW Iyer Shrivats M SM Pak Sally S Ährlund-Richter Sofie S Delp Scott L SL Malenka Robert C RC Josselyn Sheena A SA Carlén Marie M Hegemann Peter P Deisseroth Karl K

Proceedings of the National Academy of Sciences of the United States of America 20151222 4

The structure-guided design of chloride-conducting channelrhodopsins has illuminated mechanisms underlying ion selectivity of this remarkable family of light-activated ion channels. The first generation of chloride-conducting channelrhodopsins, guided in part by development of a structure-informed electrostatic model for pore selectivity, included both the introduction of amino acids with positively charged side chains into the ion conduction pathway and the removal of residues hypothesized to s ...[more]

PMID: 26699459

Similar Datasets

Project description:Channelrhodospin-2 (ChR2), a light-sensitive ion channel, and its variants have emerged as new excitatory optogenetic tools not only in neuroscience, but also in other areas, including cardiac electrophysiology. An accurate quantitative model of ChR2 is necessary for in silico prediction of the response to optical stimulation in realistic tissue/organ settings. Such a model can guide the rational design of new ion channel functionality tailored to different cell types/tissues. Focusing on one of the most widely used ChR2 mutants (H134R) with enhanced current, we collected a comprehensive experimental data set of the response of this ion channel to different irradiances and voltages, and used these data to develop a model of ChR2 with empirically-derived voltage- and irradiance- dependence, where parameters were fine-tuned via simulated annealing optimization. This ChR2 model offers: 1) accurate inward rectification in the current-voltage response across irradiances; 2) empirically-derived voltage- and light-dependent kinetics (activation, deactivation and recovery from inactivation); and 3) accurate amplitude and morphology of the response across voltage and irradiance settings. Temperature-scaling factors (Q10) were derived and model kinetics was adjusted to physiological temperatures. Using optical action potential clamp, we experimentally validated model-predicted ChR2 behavior in guinea pig ventricular myocytes. The model was then incorporated in a variety of cardiac myocytes, including human ventricular, atrial and Purkinje cell models. We demonstrate the ability of ChR2 to trigger action potentials in human cardiomyocytes at relatively low light levels, as well as the differential response of these cells to light, with the Purkinje cells being most easily excitable and ventricular cells requiring the highest irradiance at all pulse durations. This new experimentally-validated ChR2 model will facilitate virtual experimentation in neural and cardiac optogenetics at the cell and organ level and provide guidance for the development of in vivo tools.

Project description:Trimeric acid-sensing ion channels (ASICs) contribute to neuronal signaling by converting extracellular acidification into excitatory sodium currents. Previous work with homomeric ASIC1a implicates conserved leucine (L7') and consecutive glycine-alanine-serine (GAS belt) residues near the middle, and conserved negatively charged (E18') residues at the bottom of the pore in ion permeation and/or selectivity. However, a conserved mechanism of ion selectivity throughout the ASIC family has not been established. We therefore explored the molecular determinants of ion selectivity in heteromeric ASIC1a/ASIC2a and homomeric ASIC2a channels using site-directed mutagenesis, electrophysiology, and molecular dynamics free energy simulations. Similar to ASIC1a, E18' residues create an energetic preference for sodium ions at the lower end of the pore in ASIC2a-containing channels. However, and in contrast to ASIC1a homomers, ion permeation through ASIC2a-containing channels is not determined by L7' side chains in the upper part of the channel. This may be, in part, due to ASIC2a-specific negatively charged residues (E59 and E62) that lower the energy of ions in the upper pore, thus making the GAS belt more important for selectivity. This is confirmed by experiments showing that the L7'A mutation has no effect in ASIC2a, in contrast to ASIC1a, where it eliminated selectivity. ASIC2a triple mutants eliminating both L7' and upper charges did not lead to large changes in selectivity, suggesting a different role for L7' in ASIC2a compared with ASIC1a channels. In contrast, we observed measurable changes in ion selectivity in ASIC2a-containing channels with GAS belt mutations. Our results suggest that ion conduction and selectivity in the upper part of the ASIC pore may differ between subtypes, whereas the essential role of E18' in ion selectivity is conserved. Furthermore, we demonstrate that heteromeric channels containing mutations in only one of two ASIC subtypes provide a means of functionally testing mutations that render homomeric channels nonfunctional.

Dataset Information

Structural foundations of optogenetics: Determinants of channelrhodopsin ion selectivity.

Publications

Structural foundations of optogenetics: Determinants of channelrhodopsin ion selectivity.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets