Channelrhodopsin-2, a directly light-gated cation-selective membrane channel.
25 Nov 2003-Proceedings of the National Academy of Sciences of the United States of America (National Academy of Sciences)-Vol. 100, Iss: 24, pp 13940-13945
TL;DR: It is demonstrated by functional expression, both in oocytes of Xenopus laevis and mammalian cells, that ChR2 is a directly light-switched cation-selective ion channel, and may be used to depolarize small or large cells, simply by illumination.
Abstract: 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.
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TL;DR: In this paper, the authors adapted 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.
Abstract: 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.
4,411 citations
TL;DR: A primer on the application of optogenetics in neuroscience is provided, focusing on the single-component tools and highlighting important problems, challenges, and technical considerations.
1,712 citations
TL;DR: The development of currently available single-component optogenetic tools is outlined and the application of various optogenetics tools in diverse model organisms is summarized.
Abstract: Genetically encoded, single-component optogenetic tools have made a significant impact on neuroscience, enabling specific modulation of selected cells within complex neural tissues. As the optogenetic toolbox contents grow and diversify, the opportunities for neuroscience continue to grow. In this review, we outline the development of currently available single-component optogenetic tools and summarize the application of various optogenetic tools in diverse model organisms.
1,658 citations
Cites background from "Channelrhodopsin-2, a directly ligh..."
...The desensitized population can recover in the dark with a characteristic time constant on the order of 5 seconds, giving rise to a similar peak photocurrent if a second light pulse is applied after sufficient time has elapsed (Nagel et al. 2003)....
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...For example, channelrhodopsin-1 (ChR1) (Nagel et al. 2002) and channelrhodopsin-2 (ChR2) (Nagel et al. 2003) from Chlamydomonas reinhardtii are bluelight-activated nonspecific cation channels....
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...In 2005, one of these micro- bial opsins was brought to neuroscience as the first single-component optogenetic tool (Boyden et al. 2005), and the other microbial opsin subfamilies followed close behind....
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...Typically, a transient peak photocurrent, evoked at the onset of light stimulation, decays modestly to a steady-state photocurrent even in the presence of continuous light, owing in part to the desensitization of a certain population of channels (Nagel et al. 2003)....
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TL;DR: An archaeal light-driven chloride pump from Natronomonas pharaonis is identified and developed for temporally precise optical inhibition of neural activity and forms a complete system for multimodal, high-speed, genetically targeted, all-optical interrogation of living neural circuits.
Abstract: Our understanding of the cellular implementation of systems-level neural processes like action, thought and emotion has been limited by the availability of tools to interrogate specific classes of neural cells within intact, living brain tissue. Here we identify and develop an archaeal light-driven chloride pump (NpHR) from Natronomonas pharaonis for temporally precise optical inhibition of neural activity. NpHR allows either knockout of single action potentials, or sustained blockade of spiking. NpHR is compatible with ChR2, the previous optical excitation technology we have described, in that the two opposing probes operate at similar light powers but with well-separated action spectra. NpHR, like ChR2, functions in mammals without exogenous cofactors, and the two probes can be integrated with calcium imaging in mammalian brain tissue for bidirectional optical modulation and readout of neural activity. Likewise, NpHR and ChR2 can be targeted together to Caenorhabditis elegans muscle and cholinergic motor neurons to control locomotion bidirectionally. NpHR and ChR2 form a complete system for multimodal, high-speed, genetically targeted, all-optical interrogation of living neural circuits.
1,581 citations
Cites background from "Channelrhodopsin-2, a directly ligh..."
...Author Information The GenBank accession number is EF474018 for the ‘mammalianized’ NpHR sequence and EF474017 for the ‘mammalianized’ ChR2(1-315) sequence....
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Journal Article•
TL;DR: In this paper, an archaeal light-driven chloride pump (NpHR) was developed for temporally precise optical inhibition of neural activity, allowing either knockout of single action potentials, or sustained blockade of spiking.
Abstract: Our understanding of the cellular implementation of systems-level neural processes like action, thought and emotion has been limited by the availability of tools to interrogate specific classes of neural cells within intact, living brain tissue. Here we identify and develop an archaeal light-driven chloride pump (NpHR) from Natronomonas pharaonis for temporally precise optical inhibition of neural activity. NpHR allows either knockout of single action potentials, or sustained blockade of spiking. NpHR is compatible with ChR2, the previous optical excitation technology we have described, in that the two opposing probes operate at similar light powers but with well-separated action spectra. NpHR, like ChR2, functions in mammals without exogenous cofactors, and the two probes can be integrated with calcium imaging in mammalian brain tissue for bidirectional optical modulation and readout of neural activity. Likewise, NpHR and ChR2 can be targeted together to Caenorhabditis elegans muscle and cholinergic motor neurons to control locomotion bidirectionally. NpHR and ChR2 form a complete system for multimodal, high-speed, genetically targeted, all-optical interrogation of living neural circuits.
1,520 citations
References
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TL;DR: The extracellular patch clamp method, which first allowed the detection of single channel currents in biological membranes, has been further refined to enable higher current resolution, direct membrane patch potential control, and physical isolation of membrane patches.
Abstract: 1. The extracellular patch clamp method, which first allowed the detection of single channel currents in biological membranes, has been further refined to enable higher current resolution, direct membrane patch potential control, and physical isolation of membrane patches. 2. A description of a convenient method for the fabrication of patch recording pipettes is given together with procedures followed to achieve giga-seals i.e. pipette-membrane seals with resistances of 10(9) - 10(11) omega. 3. The basic patch clamp recording circuit, and designs for improved frequency response are described along with the present limitations in recording the currents from single channels. 4. Procedures for preparation and recording from three representative cell types are given. Some properties of single acetylcholine-activated channels in muscle membrane are described to illustrate the improved current and time resolution achieved with giga-seals. 5. A description is given of the various ways that patches of membrane can be physically isolated from cells. This isolation enables the recording of single channel currents with well-defined solutions on both sides of the membrane. Two types of isolated cell-free patch configurations can be formed: an inside-out patch with its cytoplasmic membrane face exposed to the bath solution, and an outside-out patch with its extracellular membrane face exposed to the bath solution. 6. The application of the method for the recording of ionic currents and internal dialysis of small cells is considered. Single channel resolution can be achieved when recording from whole cells, if the cell diameter is small (less than 20 micrometer). 7. The wide range of cell types amenable to giga-seal formation is discussed.
17,136 citations
Book•
16 Jul 2001
TL;DR: The superfamily of voltage-gated channels was studied in this paper, where a classical biophysics of the squid giant axon was discussed. But the superfamily was not considered in this paper.
Abstract: PART I Classical biophysics of the squid giant axon The superfamily of voltage-gated channels Voltage-gated calcium channels Potassium channels and chloride channels Ligand-gated channels of fast chemical synapses Modulation, slow synaptic action, and second messengers Sensory transduction and excitable cells Calcium dynamics, epithelial transport, and intercellular coupling PART II Elementary properties of ions in solution Elementary properties of pores Counting channels Structure of channel proteins Selective permeability: Independence Selective permeability: Saturation and binding Classical mechanisms of block Structure-function studies of permeation and block Gating mechanisms: Kinetic thinking Gating: Voltage sensing and inactivation Modification of gating in voltage-sensitive channels Cell biology and channels Evolution and origins
3,678 citations
01 Jan 1996
TL;DR: The action potential is triggered when the membrane potential, which was at the resting level, depolarizes and reaches the threshold of excitation, which triggers the action potential.
Abstract: Excitability. Excitability of cell membranes is crucial for signaling in many types of cell. Excitation in the physiological sense means that the cell membrane potential undergoes characteristic changes which, in most cases, go in the depolarizing direction. Single depolarization from the resting potential to potentials near 0 mV has generally been called an action potential. A schematic representation of a neuronal action potential is given in Fig. 12.1 A. The action potential is triggered when the membrane potential, which was at the resting level, depolarizes and reaches the threshold of excitation. This depolarization, which triggers the action potential, is generated by depolarizing synaptic currents, or depolarizing current coming from a membrane region that is already excited (propagation of an action potential), or by pacemaker currents mediated by pacemaker channels, or by current injected externally by an electrode. The duration of different types of action potential varies from seconds to less than 1 ms.
3,016 citations
TL;DR: A complete atomic model for bacteriorhodopsin between amino acid residues 8 and 225 has been built and suggests that pK changes in the Schiff base must act as the means by which light energy is converted into proton pumping pressure in the channel.
2,772 citations
TL;DR: It is shown that the purple colour is due to retinal bound to an opsin-like protein, the only protein present in this membrane fragment, which has been isolated in relatively pure form from Halobacterium halobium.
Abstract: HALOPHILIC bacteria require high concentrations of sodium chloride and lower concentrations of KCl and MgCl2 for growth. The cell membrane dissociates into fragments of varying size when the salt is removed1. One characteristic fragment—termed the “purple membrane” because of its characteristic deep purple colour—has been isolated in relatively pure form from Halobacterium halobium2. We can now show that the purple colour is due to retinal bound to an opsin-like protein, the only protein present in this membrane fragment (see also ref. 3).
1,849 citations