Ernst Bamberg

Millisecond-timescale, genetically targeted optical control of neural activity

Temporally precise, noninvasive control of activity in well-defined neuronal populations is a long-sought goal of systems neuroscience. We adapted for this purpose the naturally occurring algal protein Channelrhodopsin-2, a rapidly gated light-sensitive cation channel, by using lentiviral gene delivery in combination with high-speed optical switching to photostimulate mammalian neurons. We demonstrate reliable, millisecond-timescale control of neuronal spiking, as well as control of excitatory and inhibitory synaptic transmission. This technology allows the use of light to alter neural processing at the level of single spikes and synaptic events, yielding a widely applicable tool for neuroscientists and biomedical engineers.

Channelrhodopsin-2, a directly light-gated cation-selective membrane channel

Microbial-type rhodopsins are found in archaea, prokaryotes, and eukaryotes. Some of them represent membrane ion transport proteins such as bacteriorhodopsin, a light-driven proton pump, or channelrhodopsin-1 (ChR1), a recently identified light-gated proton channel from the green alga Chlamydomonas reinhardtii. ChR1 and ChR2, a related microbial-type rhodopsin from C. reinhardtii, were shown to be involved in generation of photocurrents of this green alga. We demonstrate by functional expression, both in oocytes of Xenopus laevis and mammalian cells, that ChR2 is a directly light-switched cation-selective ion channel. This channel opens rapidly after absorption of a photon to generate a large permeability for monovalent and divalent cations. ChR2 desensitizes in continuous light to a smaller steady-state conductance. Recovery from desensitization is accelerated by extracellular H+ and negative membrane potential, whereas closing of the ChR2 ion channel is decelerated by intracellular H+. ChR2 is expressed mainly in C. reinhardtii under low-light conditions, suggesting involvement in photoreception in dark-adapted cells. The predicted seven-transmembrane α helices of ChR2 are characteristic for G protein-coupled receptors but reflect a different motif for a cation-selective ion channel. Finally, we demonstrate that ChR2 may be used to depolarize small or large cells, simply by illumination.

Light Activation of Channelrhodopsin-2 in Excitable Cells of Caenorhabditis elegans Triggers Rapid Behavioral Responses

For studying the function of specific neurons in their native circuitry, it is desired to precisely control their activity. This often requires dissection to allow accurate electrical stimulation or neurotransmitter application , and it is thus inherently difficult in live animals, especially in small model organisms. Here, we employed channelrhodopsin-2 (ChR2), a directly light-gated cation channel from the green alga Chlamydomonas reinhardtii, in excitable cells of the nematode Caenorhabditis elegans, to trigger specific behaviors, simply by illumination. Channelrhodopsins are 7-transmembrane-helix proteins that resemble the light-driven proton pump bacteriorhodopsin , and they also utilize the chromophore all-trans retinal, but to open an intrinsic cation pore. In muscle cells, light-activated ChR2 evoked strong, simultaneous contractions, which were reduced in the background of mutated L-type, voltage-gated Ca2+-channels (VGCCs) and ryanodine receptors (RyRs). Electrophysiological analysis demonstrated rapid inward currents that persisted as long as the illumination. When ChR2 was expressed in mechanosensory neurons, light evoked withdrawal behaviors that are normally elicited by mechanical stimulation. Furthermore, ChR2 enabled activity of these neurons in mutants lacking the MEC-4/MEC-10 mechanosensory ion channel . Thus, specific neurons or muscles expressing ChR2 can be quickly and reversibly activated by light in live and behaving, as well as dissected, animals.

Channel formation kinetics of gramicidin A in lipid bilayer membranes

SummaryPrevious studies have given evidence that the active form of gramicidin A in lipid bilayer membranes is a dimer which acts as an ion channel; it has been further shown that the mean lifetime of the channel strongly depends on the membrane thickness. As the thickness slightly decreases when a voltage is applied to the membrane, the equilibrium between conducting dimers and nonconducting monomers may be displaced by a voltage jump. From the relaxation of the electrical current after the voltage jump, information about the kinetics of channel formation is obtained. For a dioleoyllecithin/n-decane membrane the rate constant of association is found to be 2×1014 cm2 mole−1 sec−1, which is by three orders of magnitude below the limiting value of a diffusion-controlled reaction in a two-dimensional system. The dissociation rate constant is equal to 2 sec−1, a value which is consistent with the channel lifetime as obtained from electrical fluctuation measurements.

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.

Scan speed limit in atomic force microscopy

The scan speed limit of atomic force microscopes has been calculated. It is determined by the spring constant of the cantilever k, its effective mass m, the damping constant D of the cantilever in the surrounding medium and the stiffness of the sample. Techniques to measure k, k/m and D/m are described. In liquids the damping constant and the effective mass of the cantilever increase. A consequence of this is that the transfer function always depends on the scan speed when imaging in liquids. The practical scan speed limit for atomic resolution in vacuum is 0·1 μm/s while in water it increases to about 2 μm/s due to the additional damping of cantilever movements. Sample stiffness or damping of cantilever movements by the sample increase these limits. For soft biological materials imaged in water at a desired resolution of 1 nm the scan speed should not exceed 2 μm/s.

Aspartic acids 96 and 85 play a central role in the function of bacteriorhodopsin as a proton pump.

A spectroscopic and functional analysis of two point‐mutated bacteriorhodopsins (BRs) from phototrophic negative halobacterial strains is reported. Bacteriorhodopsin from strain 384 contains a glutamic acid instead of an aspartic acid at position 85 and BR from strain 326 contains asparagine instead of aspartic acid at position 96. Compared to wild‐type BR, the M formation in BR Asp85–‐Glu is accwelerated approximately 10‐fold, whereas the M decay in BR Asp96–‐Asn is slowed down approximately 50‐fold at pH6. Purple membrane sheets containing the mutated BRs were oriented and immobilized in polyacrylamide gels or adsorbed to planar lipid films. The measured kinetics of the photocurrents under various conditions agree with the observed photocycle kinetics. The ineffectivity of BR Asp85–‐Glu resides in the dominance of an inactive species absorbing maximally at approximately 610 nm, while BR Asp96–‐Asn is ineffective due to its slow photocycle. These experimental results suggest that aspartic acid 96 plays a crucial role for the reprotonation of the Schiff base. Both residues are essential for an effective proton pump.

Proteorhodopsin is a Light-driven Proton Pump with Variable Vectoriality

Proteorhodopsin, a homologue of archaeal bacteriorhodopsin (BR), belongs to a newly identified family of retinal proteins from marine bacteria, which could play an important role in the energy balance of the biosphere. We cloned the cDNA sequence of proteorhodopsin by chemical gene synthesis, expressed the protein in Escherichia coli cells, purified and reconstituted the protein in its functional active state. The photocycle characteristics were determined by time-resolved absorption and Fourier transform infrared (FT-IR) spectroscopy. The pH-dependence of the absorption spectrum indicates that the pK(a) of the primary acceptor of the Schiff base proton (Asp97) is 7.68. Generally, the photocycle of proteorhodopsin is similar to that of BR, although an L-like photocycle intermediate was not detectable. Whereas at pH>7 an M-like intermediate is formed upon illumination, at pH 5 no M-like intermediate could be detected. As the photocycle kinetics do not change between the acidic and alkaline state of proteorhodopsin, the only difference between these two forms is the protonation status of Asp97. This is corroborated by time-resolved FT-IR spectroscopy, which demonstrates that proton transfer from the retinal Schiff base to Asp97 is observed at alkaline pH, but the other vibrational changes are essentially pH-independent.After reconstitution into proteoliposomes, light-induced proton currents of proteorhodopsin were measured in a compound membrane system where proteoliposomes were adsorbed to planar lipid bilayers. Our results show that proteorhodopsin is a light-driven proton pump with characteristics similar to those of BR at alkaline pH. However, at acidic pH, the direction of proton pumping is inverted. Complementary experiments were carried out on proteorhodopsin expressed heterologously in Xenopus laevis oocytes under voltage clamp conditions. The following results were obtained. (1) At alkaline pH, proteorhodopsin mediates outwardly directed proton pumping like BR. (2) The direction of proton pumping can be inverted, when Asp97 is protonated. (3) The current can be inverted by changes of the polarity of the applied voltage. (4) The light intensity-dependence of the photocurrents leads to the conclusion that the alkaline form of proteorhodopsin shows efficient proton pumping after sequential excitation by two photons.

A Unifying Concept for Ion Translocation by Retinal Proteins

First, halorhodopsin is capable of pumping protons after illumination with greenand blue light in the same direction as chloride. Second, mutated bacteriorhodopsin where the proton acceptor Asp85 and the proton donor Asp96 are replaced by Asn showed proton pump activity after illumination with blue light in the same direction as wildtype after green light illumination. These results can be explained by and are discussed in light of our new hypothesis: structural changes in either molecule lead to a change in ion affinity and accessibility for determining the vectoriality of the transport through the two proteins.

Phloem-localized, proton-coupled sucrose carrier ZmSUT1 mediates sucrose efflux under the control of the sucrose gradient and the proton motive force.

The phloem network is as essential for plants as the vascular system is for humans. This network, assembled by nucleus- and vacuole-free interconnected living cells, represents a long distance transport pathway for nutrients and information. According to the Münch hypothesis, osmolytes such as sucrose generate the hydrostatic pressure that drives nutrient and water flow between the source and the sink phloem (Münch, E. (1930) Die Stoffbewegungen in der Pflanze, Gustav Fischer, Jena, Germany). Although proton-coupled sucrose carriers have been localized to the sieve tube and the companion cell plasma membrane of both source and sink tissues, knowledge of the molecular representatives and the mechanism of the sucrose phloem efflux is still scant. We expressed ZmSUT1, a maize sucrose/proton symporter, in Xenopus oocytes and studied the transport characteristics of the carrier by electrophysiological methods. Using the patch clamp techniques in the giant inside-out patch mode, we altered the chemical and electrochemical gradient across the sucrose carrier and analyzed the currents generated by the proton flux. Thereby we could show that ZmSUT1 is capable of mediating both the sucrose uptake into the phloem in mature leaves (source) as well as the desorption of sugar from the phloem vessels into heterotrophic tissues (sink). As predicted from a perfect molecular machine, the ZmSUT1-mediated sucrose-coupled proton current was reversible and depended on the direction of the sucrose and pH gradient as well as the membrane potential across the transporter.