Metadynamics simulations reveal mechanisms of Na+ and Ca2+ transport in two open states of the channelrhodopsin chimera, C1C2.

Cation conducting channelrhodopsins (ChRs) are a popular tool used in optogenetics to control the activity of excitable cells and tissues using light. ChRs with altered ion selectivity are in high demand for use in different cell types and for other specialized applications. However, a detailed mechanism of ion permeation in ChRs is not fully resolved. Here, we use complementary experimental and computational methods to uncover the mechanisms of cation transport and valence selectivity through the channelrhodopsin chimera, C1C2, in the high- and low-conducting open states. Electrophysiology measurements identified a single-residue substitution within the central gate, N297D, that increased Ca2+ permeability vs. Na+ by nearly two-fold at peak current, but less so at stationary current. We then developed molecular models of dimeric wild-type C1C2 and N297D mutant channels in both open states and calculated the PMF profiles for Na+ and Ca2+ permeation through each protein using well-tempered/multiple-walker metadynamics. Results of these studies agree well with experimental measurements and demonstrate that the pore entrance on the extracellular side differs from original predictions and is actually located in a gap between helices I and II. Cation transport occurs via a relay mechanism where cations are passed between flexible carboxylate sidechains lining the full length of the pore by sidechain swinging, like a monkey swinging on vines. In the mutant channel, residue D297 enhances Ca2+ permeability by mediating the handoff between the central and cytosolic binding sites via direct coordination and sidechain swinging. We also found that altered cation binding affinities at both the extracellular entrance and central binding sites underly the distinct transport properties of the low-conducting open state. This work significantly advances our understanding of ion selectivity and permeation in cation channelrhodopsins and provides the insights needed for successful development of new ion-selective optogenetic tools.

The high-light-sensitivity mechanism and optogenetic properties of the bacteriorhodopsin-like channelrhodopsin GtCCR4.

Channelrhodopsins are microbial light-gated ion channels that can control the firing of neurons in response to light. Among several cation channelrhodopsins identified in Guillardia theta (GtCCRs), GtCCR4 has higher light sensitivity than typical channelrhodopsins. Furthermore, GtCCR4 shows superior properties as an optogenetic tool, such as minimal desensitization. Our structural analyses of GtCCR2 and GtCCR4 revealed that GtCCR4 has an outwardly bent transmembrane helix, resembling the conformation of activated G-protein-coupled receptors. Spectroscopic and electrophysiological comparisons suggested that this helix bend in GtCCR4 omits channel recovery time and contributes to high light sensitivity. An electrophysiological comparison of GtCCR4 and the well-characterized optogenetic tool ChRmine demonstrated that GtCCR4 has superior current continuity and action-potential spike generation with less invasiveness in neurons. We also identified highly active mutants of GtCCR4. These results shed light on the diverse structures and dynamics of microbial rhodopsins and demonstrate the strong optogenetic potential of GtCCR4.

Channelrhodopsins with distinct chromophores and binding patterns.

Channelrhodopsins are popular optogenetic tools in neuroscience, but remain poorly understood mechanistically. Here we report the cryo-EM structures of channelrhodopsin-2 (ChR2) from Chlamydomonas reinhardtii and H. catenoides kalium channelrhodopsin (KCR1). We show that ChR2 recruits an endogenous N-retinylidene-PE-like molecule to a previously unidentified lateral retinal binding pocket, exhibiting a reduced light response in HEK293 cells. In contrast, H. catenoides kalium channelrhodopsin (KCR1) binds an endogenous retinal in its canonical retinal binding pocket under identical condition. However, exogenous ATR reduces the photocurrent magnitude of wild type KCR1 and also inhibits its leaky mutant C110T. Our results uncover diverse retinal chromophores with distinct binding patterns for channelrhodopsins in mammalian cells, which may further inspire next generation optogenetics for complex tasks such as cell fate control.

Exploring Protonation State, Ion Binding, and Photoactivated Channel Opening of an Anion Channelrhodopsin by Molecular Simulations.

Channelrhodopsins are light-gated ion channels with a retinal chromophore found in microbes and are widely used in optogenetics, a field of neuroscience that utilizes light to regulate neuronal activity. GtACR1, an anion conducting channelrhodopsin derived from Guillardia theta, has attracted attention for its application as a neuronal silencer in optogenetics because of its high conductivity and selectivity. However, atomistic mechanisms of channel photoactivation and ion conduction have not yet been elucidated. In the present study, we investigated the molecular characteristics of GtACR1 and its photoactivation processes by molecular simulations. The QM/MM RWFE-SCF method which combines highly accurate quantum chemistry calculations with long-time molecular dynamics (MD) simulations were used to model protein structures of the wild-type and mutants with different protonation states of key groups and to calculate absorption energies for verification of the models. The QM/MM modeling together with MD simulations of free-energy calculations favors protonation of a key counterion carboxyl group of Asp234 with a strong binding of a chloride ion in the extracellular pocket in the dark state. A channel open state was also successfully modeled by the QM/MM RWFE-SCF free-energy optimizations, providing atomistic insights into the channel activation mechanism.

Unique hydrogen-bonding network in a viral channelrhodopsin.

Channelrhodopsins (CRs) are used as key tools in optogenetics, and novel CRs, either found from nature or engineered by mutation, have greatly contributed to the development of optogenetics. Recently CRs were discovered from viruses, and crystal structure of a viral CR, OLPVR1, reported a very similar water-containing hydrogen-bonding network near the retinal Schiff base to that of a light-driven proton-pump bacteriorhodopsin (BR). In both OLPVR1 and BR, nearly planar pentagonal cluster structures are comprised of five oxygen atoms, three oxygens from water molecules and two oxygens from the Schiff base counterions. The planar pentagonal cluster stabilizes a quadrupole, two positive charges at the Schiff base and an arginine, and two negative charges at the counterions, and thus plays important roles in light-gated channel function of OLPVR1 and light-driven proton pump function of BR. Despite similar pentagonal cluster structures, present FTIR analysis revealed different hydrogen-bonding networks between OLPVR1 and BR. The hydrogen bond between the protonated Schiff base and a water is stronger in OLPVR1 than in BR, and internal water molecules donate hydrogen bonds much weaker in OLPVR1 than in BR. In OLPVR1, the bridged water molecule between the Schiff base and counterions forms hydrogen bonds to D76 and D200 equally, while the hydrogen-bonding interaction is much stronger to D85 than to D212 in BR. The present interpretation is supported by the mutation results, where D76 and D200 equally work as the Schiff base counterions in OLPVR1, but D85 is the primary counterion in BR. This work reports highly sensitive hydrogen-bonding network in the Schiff base region, which would be closely related to each function through light-induced alterations of the network.

Parallel photocycle kinetic model of anion channelrhodopsin GtACR1 function.

The light-gated anion channelrhodopsin GtACR1 is an important optogenetic tool for neuronal silencing. Its photochemistry, including its photointermediates, is poorly understood. The current mechanistic view presumes BR-like kinetics and assigns the open channel to a blue-absorbing L intermediate. Based on time-resolved absorption and electrophysiological data, we recently proposed a red-absorbing spectral form for the open channel state. Here, we report the results of a comprehensive kinetic analysis of the spectroscopic data combined with channel current information. The time evolutions of the spectral forms derived from the spectroscopic data are inconsistent with the single chain mechanism and are analyzed within the concept of parallel photocycles. The spectral forms partitioned into conductive and nonconductive parallel cycles are assigned to intermediate states. Rejecting reversible connections between conductive and nonconductive channel states leads to kinetic schemes with two independent conductive states corresponding to the fast- and slow-decaying current components. The conductive cycle is discussed in terms of a single cycle and two parallel cycles. The reaction mechanisms and reaction rates for the wild-type protein, the A75E, and the low-conductance D234N and S97E protein variants are derived. The parallel cycles of channelrhodopsin kinetics, its relation to BR photocycle, and the role of the M intermediate in channel closure are discussed.

Molecular Mechanisms behind Circular Dichroism Spectral Variations between Channelrhodopsin and Heliorhodopsin Dimers.

Channelrhodopsin (ChR) and heliorhodopsin (HeR) are microbial rhodopsins with similar structures but different circular dichroism (CD) spectra: ChR shows biphasic negative and positive bands, whereas HeR shows a single positive band. We explored the physicochemical factors underlying these differences through computational methods. Using the exciton model based on first-principles computations, we obtained the CD spectra of ChR and HeR. The obtained spectra indicate that the protein dimer structures and the quantum mechanical treatment of the retinal chromophore and its interacting amino acids are crucial for accurately reproducing the experimental spectra. Further calculations revealed that the sign of the excitonic coupling was opposite between the ChR and HeR dimers, which was attributed to the contrasting second term of the orientation factor between the two retinal chromophores. These findings demonstrate that slight variations in the intermolecular orientation of the two chromophores can result in significant differences in the CD spectral shape.

Control of polymers' amorphous-crystalline transition enables miniaturization and multifunctional integration for hydrogel bioelectronics.

Soft bioelectronic devices exhibit motion-adaptive properties for neural interfaces to investigate complex neural circuits. Here, we develop a fabrication approach through the control of metamorphic polymers' amorphous-crystalline transition to miniaturize and integrate multiple components into hydrogel bioelectronics. We attain an about 80% diameter reduction in chemically cross-linked polyvinyl alcohol hydrogel fibers in a fully hydrated state. This strategy allows regulation of hydrogel properties, including refractive index (1.37-1.40 at 480 nm), light transmission (>96%), stretchability (139-169%), bending stiffness (4.6 ± 1.4 N/m), and elastic modulus (2.8-9.3 MPa). To exploit the applications, we apply step-index hydrogel optical probes in the mouse ventral tegmental area, coupled with fiber photometry recordings and social behavioral assays. Additionally, we fabricate carbon nanotubes-PVA hydrogel microelectrodes by incorporating conductive nanomaterials in hydrogel for spontaneous neural activities recording. We enable simultaneous optogenetic stimulation and electrophysiological recordings of light-triggered neural activities in Channelrhodopsin-2 transgenic mice.

Channel Gating in Kalium Channelrhodopsin Slow Mutants.

Kalium channelrhodopsin 1 from Hyphochytrium catenoides (HcKCR1) is the first discovered natural light-gated ion channel that shows higher selectivity to K+ than to Na+ and therefore is used to silence neurons with light (optogenetics). Replacement of the conserved cysteine residue in the transmembrane helix 3 (Cys110) with alanine or threonine results in a >1,000-fold decrease in the channel closing rate. The phenotype of the corresponding mutants in channelrhodopsin 2 is attributed to breaking of a specific interhelical hydrogen bond (the "DC gate"). Unlike CrChR2 and other ChRs with long distance "DC gates", the HcKCR1 structure does not reveal any hydrogen bonding partners to Cys110, indicating that the mutant phenotype is likely caused by disruption of direct interaction between this residue and the chromophore. In HcKCR1_C110A, fast photochemical conversions corresponding to channel gating were followed by dramatically slower absorption changes. Full recovery of the unphotolyzed state in HcKCR1_C110A was extremely slow with two time constants 5.2 and 70 min. Analysis of the light-minus-dark difference spectra during these slow processes revealed accumulation of at least four spectrally distinct blue light-absorbing photocycle intermediates, L, M1 and M2, and a UV light-absorbing form, typical of bacteriorhodopsin-like channelrhodopsins from cryptophytes. Our results contribute to better understanding of the mechanistic links between the chromophore photochemistry and channel conductance, and provide the basis for using HcKCR1_C110A as an optogenetic tool.

Structural basis for ion selectivity in potassium-selective channelrhodopsins.

KCR channelrhodopsins (K+-selective light-gated ion channels) have received attention as potential inhibitory optogenetic tools but more broadly pose a fundamental mystery regarding how their K+ selectivity is achieved. Here, we present 2.5-2.7 Å cryo-electron microscopy structures of HcKCR1 and HcKCR2 and of a structure-guided mutant with enhanced K+ selectivity. Structural, electrophysiological, computational, spectroscopic, and biochemical analyses reveal a distinctive mechanism for K+ selectivity; rather than forming the symmetrical filter of canonical K+ channels achieving both selectivity and dehydration, instead, three extracellular-vestibule residues within each monomer form a flexible asymmetric selectivity gate, while a distinct dehydration pathway extends intracellularly. Structural comparisons reveal a retinal-binding pocket that induces retinal rotation (accounting for HcKCR1/HcKCR2 spectral differences), and design of corresponding KCR variants with increased K+ selectivity (KALI-1/KALI-2) provides key advantages for optogenetic inhibition in vitro and in vivo. Thus, discovery of a mechanism for ion-channel K+ selectivity also provides a framework for next-generation optogenetics.