Biophysical Characterization of Light-gated Ion Channels Using Planar Automated Patch Clamp

Channelrhodopsins (ChRs) are proteins that guide phototaxis in protists and exhibit light-gated channel conductance when their genes are heterologously expressed in mammalian cells. ChRs are widely used as molecular tools to control neurons and cardiomyocytes with light (optogenetics). Cation- and anion-selective ChRs (CCRs and ACRs, respectively) enable stimulation and inhibition of neuronal activity by depolarization and hyperpolarization of the membrane, respectively. More than 400 natural ChR variants have been identified so far, and high-throughput polynucleotide sequencing projects add many more each year. However, electrophysiological characterization of new ChRs lags behind because it is mostly done by time-consuming manual patch clamp (MPC). Here we report using a high-throughput automated patch clamp (APC) platform, SyncroPatch 384i from Nanion Technologies, for ChR research. We find that this instrument can be used for determination of the light intensity dependence and current-voltage relationships in ChRs and discuss its advantages and limitations

Rational design of a (S)-selective-transaminase for asymmetric synthesis of (1S)-1-(1,1′-biphenyl-2-yl)ethanamine

Amine transaminases offer an environmentally sustainable synthesis route for the production of pure chiral amines. However, their catalytic efficiency toward bulky ketone substrates is greatly limited by steric hindrance and therefore presents a great challenge for industrial synthetic applications. We hereby report an example of rational transaminase enzyme design to help alleviate these challenges. Starting from the Vibrio fluvialis amine transaminase that has no detectable catalytic activity toward the bulky aromatic ketone 2-acetylbiphenyl, we employed a rational design strategy combining in silico and in vitro studies to engineer the transaminase enzyme with a minimal number of mutations, achieving an high catalytic activity and high enantioselectivity. We found that, by introducing two mutations W57G/R415A, detectable enzyme activity was achieved. The rationally designed variant, W57F/R88H/V153S/K163F/I259M/R415A/V422A, showed an improvement in reaction rate by more than 1716-fold toward the bulky ketone under study, producing the corresponding enantiomeric pure (S)-amine (enantiomeric excess (ee) value of >99%)

Molecular details of the unique mechanism of chloride transport by acyanobacterial rhodopsin

Microbial rhodopsins are well known as versatile and ubiquitous light-driven ion transporters and photosensors. While the proton transport mechanism has been studied in great detail, much less is known about various modes of anion transport. Until recently, only two main groups of light-driven anion pumps were known, archaeal halorhodopsins (HRs) and bacterial chloride pumps (known as ClRs or NTQs). Last year, another group of cyanobacterial anion pumps with a very distinct primary structure was reported. Here, we studied the chloride- transporting photocycle of a representative of this new group, Mastigocladopsis repens rhodopsin (MastR), using time-resolved spectroscopy in the infrared and visible ranges and site-directed mutagenesis. We found that, in accordance with its unique amino acid sequence containing many polar residues in the transmembrane region of the protein, its photocycle features a number of unusual molecular events not known for other anion-pumping rhodopsins. It appears that light-driven chloride ion transfers by MastR are coupled with translocation of protons and water molecules as well as perturbation of several polar sidechains. Of particular interest is transient deprotonation of Asp-85, homologous to the cytoplasmic proton donor of light- driven proton pumps (such as Asp-96 of bacteriorhodopsin), which may serve as a regulatory mechanism

Kalium Channelrhodopsins Are Natural Light-gated Potassium Channels That Mediate Optogenetic Inhibition

Channelrhodopsins are used widely for optical control of neurons, in which they generate photoinduced proton, sodium or chloride influx. Potassium (K+) is central to neuron electrophysiology, yet no natural K+-selective light-gated channel has been identified. Here, we report kalium channelrhodopsins (KCRs) from Hyphochytrium catenoides. Previously known gated potassium channels are mainly ligand- or voltage-gated and share a conserved K+-selectivity filter. KCRs differ in that they are light-gated and have independently evolved an alternative K+ selectivity mechanism. The KCRs are potent, highly selective of K+ over Na+, and open in less than 1 ms following photoactivation. The permeability ratio PK/PNa of 23 makes H. catenoides KCR1 (HcKCR1) a powerful hyperpolarizing tool to suppress excitable cell firing upon illumination, demonstrated here in mouse cortical neurons. HcKCR1 enables optogenetic control of K+ gradients, which is promising for the study and potential treatment of potassium channelopathies such as epilepsy, Parkinson\u27s disease and long-QT syndrome and other cardiac arrhythmias

Structures of Channelrhodopsin Paralogs in Peptidiscs Explain Their Contrasting K+ and Na+ Selectivities

Kalium channelrhodopsin 1 from Hyphochytrium catenoides (HcKCR1) is a light-gated channel used for optogenetic silencing of mammalian neurons. It selects K+ over Na+ in the absence of the canonical tetrameric K+ selectivity filter found universally in voltage- and ligand-gated channels. The genome of H. catenoides also encodes a highly homologous cation channelrhodopsin (HcCCR), a Na+ channel with \u3e100-fold larger Na+ to K+ permeability ratio. Here, we use cryo-electron microscopy to determine atomic structures of these two channels embedded in peptidiscs to elucidate structural foundations of their dramatically different cation selectivity. Together with structure-guided mutagenesis, we show that K+ versus Na+ selectivity is determined at two distinct sites on the putative ion conduction pathway: in a patch of critical residues in the intracellular segment (Leu69/Phe69, Ile73/Ser73 and Asp116) and within a cluster of aromatic residues in the extracellular segment (primarily, Trp102 and Tyr222). The two filters are on the opposite sides of the photoactive site involved in channel gating

Photochemical characterization of a novel fungal rhodopsin from Phaeosphaeria nodorum

Eukaryotic microbial rhodopsins are widespread bacteriorhodopsin-like proteins found in many lower eukaryotic groups including fungi. Many fungi contain multiple rhodopsins, some significantly diverged from the original bacteriorhodopsin template. Although few fungal rhodopsins have been studied biophysically, both fast-cycling light-driven proton pumps and slow-cycling photosensors have been found. The purpose of this study was to characterize photochemically a new subgroup of fungal rhodopsins, the so-called auxiliary group. The study used the two known rhodopsin genes from the fungal wheat pathogen, Phaeosphaeria nodorum. One of the genes is a member of the auxiliary group while the other is highly similar to previously characterized proton-pumping Leptosphaeria rhodopsin. Auxiliary rhodopsin genes from a range of species form a distinct group with a unique primary structure and are located in carotenoid biosynthesis gene cluster. Amino acid conservation pattern suggests that auxiliary rhodopsins retain the transmembrane core of bacteriorhodopsins, including all residues important for proton transport, but have unique polar intramembrane residues. Spectroscopic characterization of the two yeast-expressed Phaeosphaeria rhodopsins showed many similarities: absorption spectra, conformation of the retinal chromophore, fast photocycling, and carboxylic acid protonation changes. It is likely that both Phaeosphaeria rhodopsins are proton-pumping, at least in vitro.We suggest that auxiliary rhodopsins have separated from their ancestors fairly recently and have acquired the ability to interact with as yet unidentified transducers, performing a photosensory function without changing their spectral properties and basic photochemistry

Broadband velocity modulation spectroscopy of HfF^+: towards a measurement of the electron electric dipole moment

Precision spectroscopy of trapped HfF^+ will be used in a search for the permanent electric dipole moment of the electron (eEDM). While this dipole moment has yet to be observed, various extensions to the standard model of particle physics (such as supersymmetry) predict values that are close to the current limit. We present extensive survey spectroscopy of 19 bands covering nearly 5000 cm^(-1) using both frequency-comb and single-frequency laser velocity-modulation spectroscopy. We obtain high-precision rovibrational constants for eight electronic states including those that will be necessary for state preparation and readout in an actual eEDM experiment.Comment: 13 pages, 7 figures, 3 table

Recent observation of short range nucleon correlations in nuclei and their implications for the structure of nuclei and neutron stars

Novel processes probing the decay of nucleus after removal of a nucleon with momentum larger than Fermi momentum by hard probes finally proved unambiguously the evidence for long sought presence of short-range correlations (SRCs) in nuclei. In combination with the analysis of large $Q^2$, A(e,e')X processes at $x>1$ they allow us to conclude that (i) practically all nucleons with momenta $\ge$ 300 MeV/c belong to SRCs, consisting mostly of two nucleons, ii) probability of such SRCs in medium and heavy nuclei is $\sim 25$, iii) a fast removal of such nucleon practically always leads to emission of correlated nucleon with approximately opposite momentum, iv) proton removal from two-nucleon SRCs in 90% of cases is accompanied by a removal of a neutron and only in 10% by a removal of another proton. We explain that observed absolute probabilities and the isospin structure of two nucleon SRCs confirm the important role that tensor forces play in internucleon interactions. We find also that the presence of SRCs requires modifications of the Landau Fermi liquid approach to highly asymmetric nuclear matter and leads to a significantly faster cooling of cold neutron stars with neutrino cooling operational even for $N_p/N_n \le 0.1$. The effect is even stronger for the hyperon stars. Theoretical challenges raised by the discovered dominance of nucleon degrees of freedom in SRCs and important role of the spontaneously broken chiral symmetry in quantum chromodynamics (QCD) in resolving them are considered. We also outline directions for future theoretical and experimental studies of the physics relevant for SRCs.Comment: 74 pages. Review article, updated version to be published in International Journal of Modern Physics

Hadronic properties of the S_{11}(1535) studied by electroproduction off the deuteron

Properties of excited baryonic states are investigated in the context of electroproduction of baryon resonances off the deuteron. In particular, the hadronic radii and the compositeness of baryon resonances are studied for kinematic situations in which their hadronic reinteraction is the dominant contribution. Specifically, we study the reaction $d(e,e'S_{11})N$ at $Q^2\ge 1 GeV^2$ for kinematics in which the produced hadronic state reinteracts predominantly with the spectator nucleon. A comparison of constituent quark model and effective chiral Lagrangian calculations of the $S_{11}$ shows substantial sensitivity to the structure of the produced resonance.Comment: 24 pages, 5 figure

Regulation of Progranulin Expression in Human Microglia and Proteolysis of Progranulin by Matrix Metalloproteinase-12 (MMP-12)

Background: The essential role of progranulin (PGRN) as a neurotrophic factor has been demonstrated by the discovery that haploinsufficiency due to GRN gene mutations causes frontotemporal lobar dementia. In addition to neurons, microglia in vivo express PGRN, but little is known about the regulation of PGRN expression by microglia. Goal: In the current study, we examined the regulation of expression and function of PGRN, its proteolytic enzyme macrophage elastase (MMP-12), as well as the inhibitor of PGRN proteolysis, secretory leukocyte protease inhibitor (SLPI), in human CNS cells. Methods: Cultures of primary human microglia and astrocytes were stimulated with the TLR ligands (LPS or poly IC), Th1 cytokines (IL-1/IFNc), or Th2 cytokines (IL-4, IL-13). Results were analyzed by Q-PCR, immunoblotting or ELISA. The roles of MMP-12 and SLPI in PGRN cleavage were also examined. Results: Unstimulated microglia produced nanogram levels of PGRN, and PGRN release from microglia was suppressed by the TLR ligands or IL-1/IFNc, but increased by IL-4 or IL-13. Unexpectedly, while astrocytes stimulated with proinflammatory factors released large amounts of SLPI, none were detected in microglial cultures. We also identified MMP-12 as a PGRN proteolytic enzyme, and SLPI as an inhibitor of MMP-12-induced PGRN proteolysis. Experiments employing PGRN siRNA demonstrated that microglial PGRN was involved in the cytokine and chemokine production following TLR3/4 activation