Phillip G. Wood

Ultra light-sensitive and fast neuronal activation with the Ca2+-permeable channelrhodopsin CatCh

The light-gated cation channel channelrhodopsin-2 (ChR2) has rapidly become an important tool in neuroscience, and its use is being considered in therapeutic interventions. Although wild-type and known variant ChR2s are able to drive light-activated spike trains, their use in potential clinical applications is limited by either low light sensitivity or slow channel kinetics. We present a new variant, calcium translocating channelrhodopsin (CatCh), which mediates an accelerated response time and a voltage response that is ∼70-fold more light sensitive than that of wild-type ChR2. CatChs superior properties stem from its enhanced Ca2+ permeability. An increase in [Ca2+]i elevates the internal surface potential, facilitating activation of voltage-gated Na+ channels and indirectly increasing light sensitivity. Repolarization following light-stimulation is markedly accelerated by Ca2+-dependent BK channel activation. Our results demonstrate a previously unknown principle: shifting permeability from monovalent to divalent cations to increase sensitivity without compromising fast kinetics of neuronal activation. This paves the way for clinical use of light-gated channels.

Direct action of genistein on CFTR

Abstract Human cystic fibrosis transmembrane conductance regulator (CFTR) chloride channels were expressed in oocytes from Xenopus laevis after injection of CFTR cRNA and studied with the two-electrode voltage-clamp and the giant patch techniques. The tyrosine kinase inhibitor genistein alone activated a small chloride current in whole oocytes expressing CFTR and substantially increased the chloride current obtained upon stimulation with forskolin and isobutyl methylxanthine (IBMX). In giant excised patches, genistein was unable to open protein-kinase-A-phosphorylated CFTR channels in the absence of ATP, but increased the ATP-induced CFTR channel currents by a factor of 3.8 ± 1.7. This genistein-mediated potentiation in excised patches is independent of protein phosphatase activity, as it is readily reversible, even after complete inhibition of protein kinase A activity. Involvement of protein tyrosine kinases also seems unlikely, because this effect of genistein is not antagonized by high concentrations of the tyrosine phosphatase inhibitor ortho-vanadate. We, therefore, propose a direct interaction of genistein with CFTR, probably at a nucleotide binding site, which leads to a higher open probability.

A gene-fusion strategy for stoichiometric and co-localized expression of light-gated membrane proteins

The precise co-localization and stoichiometric expression of two different light-gated membrane proteins can vastly improve the physiological usefulness of optogenetics for the modulation of cell excitability with light. Here we present a gene-fusion strategy for the stable 1:1 expression of any two microbial rhodopsins in a single polypeptide chain. By joining the excitatory channelrhodopsin-2 with the inhibitory ion pumps halorhodopsin or bacteriorhodopsin, we demonstrate light-regulated quantitative bi-directional control of the membrane potential in HEK293 cells and neurons in vitro. We also present synergistic rhodopsin combinations of channelrhodopsin-2 with Volvox carteri channelrhodopsin-1 or slow channelrhodopsin-2 mutants, to achieve enhanced spectral or kinetic properties, respectively. Finally, we demonstrate the utility of our fusion strategy to determine ion-turnovers of as yet uncharacterized rhodopsins, exemplified for archaerhodopsin and CatCh, or to correct pump cycles, exemplified for halorhodopsin.

The voltage-dependent proton pumping in bacteriorhodopsin is characterized by optoelectric behavior

The light-driven proton pump bacteriorhodopsin (bR) was functionally expressed in Xenopus laevis oocytes and in HEK-293 cells. The latter expression system allowed high time resolution of light-induced current signals. A detailed voltage clamp and patch clamp study was performed to investigate the DeltapH versus Deltapsi dependence of the pump current. The following results were obtained. The current voltage behavior of bR is linear in the measurable range between -160 mV and +60 mV. The pH dependence is less than expected from thermodynamic principles, i.e., one DeltapH unit produces a shift of the apparent reversal potential of 34 mV (and not 58 mV). The M(2)-BR decay shows a significant voltage dependence with time constants changing from 20 ms at +60 mV to 80 ms at -160 mV. The linear I-V curve can be reconstructed by this behavior. However, the slope of the decay rate shows a weaker voltage dependence than the stationary photocurrent, indicating that an additional process must be involved in the voltage dependence of the pump. A slowly decaying M intermediate (decay time > 100 ms) could already be detected at zero voltage by electrical and spectroscopic means. In effect, bR shows optoelectric behavior. The long-lived M can be transferred into the active photocycle by depolarizing voltage pulses. This is experimentally demonstrated by a distinct charge displacement. From the results we conclude that the transport cycle of bR branches via a long-lived M(1)* in a voltage-dependent manner into a nontransporting cycle, where the proton release and uptake occur on the extracellular side.

Effect of site-directed mutagenesis of the arginine residues 509 and 748 on mouse band 3 protein-mediated anion transport

Using site-directed mutagenesis, the arginine residues 509 and 748 in mouse band 3 protein were substituted by Lys, Thr, and Cys, or by Lys and Gln, respectively. After expression in Xenopus oocytes of the cRNAs encoding wild type band 3 or any one of the band 3 mutants, chloride equilibrium exchange was measured. When the flux measurements were performed two to three days after microinjection of the cRNAs, in contrast to the wild type, neither one of the mutants was able to accomplish transport, with the possible exception of the mutants R509K and R748K both of which showed some transport activity of doubtful significance. Immunoprecipitates revealed that the Arg 748 mutants were expressed similar to the wild type band 3 while no expression of the Arg 509 mutants could be detected. When the flux measurements were performed only 3 h after microinjection of the cRNAs, transport activity was observed in the oocytes that had received cRNAs encoding wild type band 3. In some oocytes of a population, a very slight transport activity was brought about by cRNA encoding Arg 509 mutants. No transport activity could be detected after injection of the Arg 748 mutant. Immunoprecipitation demonstrated the successful biosynthesis of wild type band 3 and of both the Arg 509 and the Arg 748 mutants. The experiments suggest that mutation of Arg 748 leads to biosynthesis of an inactive form of the band 3 protein, while that of Arg 509 results in expression of an abnormally folded, possibly functionally more or less intact form, which is proteolytically degraded within less than one day.

The spontaneous activation of a potassium channel during the preparation of resealed human erythrocyte ghosts.

Resealed erythrocyte ghosts prepared under conditions which deplete the cell of its endogenous chelators and metabolites are found to be selectively permeable to potassium. The net efflux of potassium is stimulated by low concentrations of external potassium and can be inhibited by oligomycin. The effect is not expressed when resealed ghosts are formed by hemolysis in the presence of chelators or magnesium. The spontaneously activated pathway is actually the calcium-activated potassium channel, first discovered by Gardos in 1958. In the intact cell, the combined actions of the calcium pump and endogenous chelators maintain the calcium concentration below the threshold for activation. Current observations indicate that the channel is spontaneously activated by traces of calcium originating from the cell itself or from the unavoidable background of calcium found in the media. The channel in ghosts depleted of endogenous chelators exhibits its high affinity for calcium. Channel activation occurs during hemolysis and persists throughout subsequent washings.

Functional Maturation of Human Stem Cell-Derived Neurons in Long-Term Cultures

Differentiated neurons can be rapidly acquired, within days, by inducing stem cells to express neurogenic transcription factors. We developed a protocol to maintain long-term cultures of human neurons, called iNGNs, which are obtained by inducing Neurogenin-1 and Neurogenin-2 expression in induced pluripotent stem cells. We followed the functional development of iNGNs over months and they showed many hallmark properties for neuronal maturation, including robust electrical and synaptic activity. Using iNGNs expressing a variant of channelrhodopsin-2, called CatCh, we could control iNGN activity with blue light stimulation. In combination with optogenetic tools, iNGNs offer opportunities for studies that require precise spatial and temporal resolution. iNGNs developed spontaneous network activity, and these networks had excitatory glutamatergic synapses, which we characterized with single-cell synaptic recordings. AMPA glutamatergic receptor activity was especially dominant in postsynaptic recordings, whereas NMDA glutamatergic receptor activity was absent from postsynaptic recordings but present in extrasynaptic recordings. Our results on long-term cultures of iNGNs could help in future studies elucidating mechanisms of human synaptogenesis and neurotransmission, along with the ability to scale-up the size of the cultures.

Photocycle and Vectorial Proton Transfer in a Rhodopsin from the Eukaryote Oxyrrhis marina

Retinylidene photoreceptors are ubiquitously present in marine protists as first documented by the identification of green proteorhodopsin (GPR). We present a detailed investigation of a rhodopsin from the protist Oxyrrhis marina (OR1) with respect to its spectroscopic properties and to its vectorial proton transport. Despite its homology to GPR, OR1s features differ markedly in its pH dependence. Protonation of the proton acceptor starts at pH below 4 and is sensitive to the ionic conditions. The mutation of a conserved histidine H62 did not influence the pK(a) value in a similar manner as in other proteorhodopsins where the charged histidine interacts with the proton acceptor forming the so-called His-Asp cluster. Mutational and pH-induced effects were further reflected in the temporal behavior upon light excitation ranging from femtoseconds to seconds. The primary photodynamics exhibits a high sensitivity to the environment of the proton acceptor D100 that are correlated to the different initial states. The mutation of the H62 does not affect photoisomerization at neutral pH. This is in agreement with NMR data indicating the absence of the His-Asp cluster. The subsequent steps in the photocycle revealed protonation reactions at the Schiff base coupled to proton pumping even at low pH. The main electrogenic steps are associated with the reprotonation of the Schiff base and internal proton donor. Hence, OR1 shows a different theme of the His-Asp organization where the low pK(a) of the proton acceptor is not dominated by this interaction, but by other electrostatic factors.

Unmasking of a potassium leak in resealed human red blood cell ghosts

A selective potassium leak is observed in resealed, human red blood cell ghosts when hemolysis is performed with distilled water at pH 6.5, 0 degrees C. The leak, which has a maximum near pH 6.7, is suppressed when either magnesium or a chelating agent is present in the hemolysing medium. The potassium leak has the additional property that it can be suppressed after resealing by washing the ghost membranes in a medium containing a low concentration of ATP or EDTA. The data suggest that through the dilution of endogenous chelating agents at hemolysis a potassium leak may be unmasked.

Functional Green-Tuned Proteorhodopsin from Modern Stromatolites

The sequenced genome of the poly-extremophile Exiguobacterium sp. S17, isolated from modern stromatolites at Laguna Socompa (3,570 m), a High-Altitude Andean Lake (HAAL) in Argentinean Puna revealed a putative proteorhodopsin-encoding gene. The HAAL area is exposed to the highest UV irradiation on Earth, making the microbial community living in the stromatolites test cases for survival strategies under extreme conditions. The heterologous expressed protein E17R from Exiguobacterium (248 amino acids, 85% sequence identity to its ortholog ESR from E. sibiricum) was assembled with retinal displaying an absorbance maximum at 524 nm, which makes it a member of the green-absorbing PR-subfamily. Titration down to low pH values (eventually causing partial protein denaturation) indicated a pK value between two and three. Global fitting of data from laser flash-induced absorption changes gave evidence for an early red-shifted intermediate (its formation being below the experimental resolution) that decayed (τ1 = 3.5 μs) into another red-shifted intermediate. This species decayed in a two-step process (τ2 = 84 μs, τ3 = 11 ms), to which the initial state of E17-PR was reformed with a kinetics of 2 ms. Proton transport capability of the HAAL protein was determined by BLM measurements. Additional blue light irradiation reduced the proton current, clearly identifying a blue light absorbing, M-like intermediate. The apparent absence of this intermediate is explained by closely matching formation and decay kinetics.