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. 2003 Nov 25;100(24):13940-5.
doi: 10.1073/pnas.1936192100. Epub 2003 Nov 13.

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

Georg Nagel  1 , Tanjef Szellas, Wolfram Huhn, Suneel Kateriya, Nona Adeishvili, Peter Berthold, Doris Ollig, Peter Hegemann, Ernst Bamberg
Affiliations

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

Georg Nagel et al. Proc Natl Acad Sci U S A. .

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 alpha 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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Figures

Fig. 1.
Fig. 1.
Ion dependence of light-activated conductance mediated by ChR2–315. Photocurrents of full-length ChR2–737 were indistinguishable. (a) Scheme of the predicted structure of ChR2–737 and ChR2–315. (b) Two-electrode voltage-clamp records from oocytes, expressing ChR2–315, in Ringer's solution (110 mM NaCl, 5 mM KCl, 2 mM CaCl2, 1 mM MgCl2, pH 7.6). Illumination with blue (450 ± 25 nm) light is indicated by the gray bar. Currents are typical of those in 23 other experiments. (c) Normalized inward photocurrents at –100 mV, pH 7.6, for 115 mM salt solutions of: LiCl, NaCl, KCl, RbCl, CsCl, and NMG-Cl, measured in the same oocyte. Currents are typical of those in four other experiments. (d) Photocurrents at –100 mV, from the same oocyte, in 115 mM NMG-Cl, at pH 9, pH 7.6, or pH 5. Currents are typical of those in three other experiments. (e) Current–voltage relationship of stationary photocurrents for one representative oocyte (from top to bottom): 115 mM NMG-Cl, pH 9; 80 mM MgCl2, pH 9 (current–voltage like for NMG, pH 9); 115 mM LiCl, pH 9; 110 mM NaCl, 5 mM KCl, pH 7.6. Currents are typical of those in three other oocytes. (f) Permability ratios for different monovalent cations, as derived from changes of reversal potentials (31) of photocurrents when replacing Na+ by the cation X+. Solutions used were: 115 mM XCl, 2 mM BaCl2, 1 MgCl2, pH 9. The permeability ratio for H+ was estimated by the Goldmann–Hodgkin–Katz equation (31) from the photocurrent reversal potential for 115 mM NMG-Cl at pH 9, assuming cytoplasmic ≈100 K+ andapHi of 7.3. n = 3. (g) Photocurrent in 80 mM CaCl2, pH9 at –100 mV (Lower and Inset, higher time resolution). Afterward, the oocyte was injected with 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetate (BAPTA) (as K-salt) to a final concentration of 10 mM, and photocurrent was determined again (Upper and Inset). Currents are typical of those in four other experiments. (h) Photocurrents of a HEK293 cell, transiently expressing ChR2–315. Photocurrents were determined at –100, –50, 0, +50, and +100 mV. Pipette solution used was 140 mM NaCl, 5 mM EGTA, 2 mM MgCl2, 10 mM Hepes, pH 7.4. Bath solution used was 140 mM NaCl, 2 mM MgCl2, 1 mM CaCl2, 10 mM Hepes, pH 7.4. Currents are typical of those in more than nine other experiments on HEK293 and more than five in BHK cells.
Fig. 2.
Fig. 2.
Recovery from desensitization of photocurrents is voltage and pHo dependent. Currents are typical of those in three other experiments. (a–c) Two light pulses with a dark phase of 10 s, pHo 7.6, the same oocyte at –120 mV (a), –40 mV (b), and +40 mV (c). (d) Another oocyte at pHo 7.4, +20 mV, 32 s dark between light pulses. (e) The same oocyte, pHo = 5.5. (f) The same oocyte as in d and e, pHo 7.4 during light flash, but pHo = 5.5 for 20 s during the dark phase. Light pulses are indicated by gray bars. (g) Photocurrent of ChR2–315-E123D at –100 mV, pH 7.6. (h) Photocurrent of ChR2–315-E123Q at –100 mV, pH 7.6
Fig. 3.
Fig. 3.
Activation of ChR2 photocurrents in excised inside-out giant membrane patches from Xenopus oocytes. This configuration allows a rapid concentration change (≈100 ms; ref. 43) at the cytoplasmic side by changing the superfusing bath solution. (a) Activation of inward photocurrents by light pulses of 442 nm (gray bars). Pipette solution used was 115 mM NMG-Cl, pH 5; bath (cytoplasmic solution) used was 115 mM NMG-Cl, pH 7. Currents typical of those in nine other experiments are shown. (b) Activation of photocurrents by two laser flashes of 10-ns duration (arrows), 440 nm. Pipette solution used was 115 mM NMG-Cl, pH 5; bath solution used was 115 mM NMG-Cl, pHi 7or 115 mM NMG-Cl, pHi 4, as indicated. Currents typical of those in three other experiments are shown. (c) Higher time resolution of first photocurrent, shown in b, pHi = 7. (d) Dependence of closing rate on pHi (n = 4–7). (e) Simple three-state model of ChR2 photocycle. Light, curved arrow indicates light-activated step from ChR2-ground state (C) to open state (O). Straight arrows indicate dark reactions, to the closed desensitized state (D) and back to the ground state (C). For further details see text. (f) Simulated photocurrent with τDC = 2 s and other time constants as in the model depicted in e. (g) Simulated photocurrent with τDC = 60 ms and other time constants as in e.
Fig. 4.
Fig. 4.
Action spectra of ChR2 and ChR1, antibody staining of C. reinhardtii membrane fractions, and ChR2-induced membrane depolarization. (a) Normalized action spectra for the ChR2-mediated inward current from oocytes at pHo = 7.6 and –100 mV (□, n = 3) and for the high-light saturating photoreceptor current from C. reinhardtii strain CW2 at pH 9 (▪). C. reinhardtii cells were grown at low light (0.5 W·m–2) for 3 days. Photocurrents were recorded directly from the eye spot with NMG-Hepes, 2 mM Ca2+, and 2 mM K+ in the pipette (21). Solid lines show the standard rhodopsin spectrum [Dartnall spectrum (41)], fitted to the data points. Spectra of the ChR1 photocurrent, measured in oocytes (24), and of photoreceptor currents, recorded from C. reinhardtii at pH 7 (21), are plotted for comparison. (b and c) C. reinhardtii cells were grown in darkness (D), low light conditions (LL, 0.5 W·m–2) and high light conditions (HL; 10 W·m–2). Membrane fractions were collected and analyzed by protein immunoblotting. Chop1310–546 antiserum (1:1,000 dilution) was raised against a Chop1–310-546 fragment in rabbits. (b) It identified Chop1 and Chop2. (c) Chop2617–723 antiserum (raised against a Chop2–617-723-fragment) is specific for Chop2. Binding was detected by using an alkaline phosphatase-coupled second antibody. Chop1 and Chop2 fragments expressed in Escherichia coli (lanes 4, 5, 9, and 10) are blotted for comparison. (d and e) Depolarization of ChR2-expressing cells by blue light, as indicated by gray bar. Voltages typical of those in four other experiments are shown. (d)An oocyte, expressing ChR2–315, in Ringer's solution (110 mM NaCl/5 mM KCl/2 mM CaCl2/1 mM MgCl2, pH 7.6). (e) A HEK293 cell, transiently expressing ChR2–315. Pipette solution used was 140 mM KCl, 5 mM EGTA, 2 mM MgCl2, 10 mM Hepes, pH 7.4. Bath solution used was 140 mM NaCl, 2 mM MgCl2, 1 mM CaCl2, 10 mM Hepes, pH 7.4.
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References

    1. Kaupp, U. B. (1995) Curr. Opin. Neurobiol. 5, 434–442. - PubMed
    1. Okada, T., Ernst, O. P., Palczewski, K. & Hofmann, K. P. (2001) Trends Biochem. Sci. 26, 318–324. - PubMed
    1. Sakmar, T. P., Menon, S. T., Marin, E. P. & Awad, E. S. (2002) Annu. Rev. Biophys. Biomol. Struct. 31, 443–484. - PubMed
    1. Zuker, C. S. (1996) Proc. Natl. Acad. Sci. USA 93, 571–576. - PMC - PubMed
    1. Hardie, R. C. & Raghu, P. (2001) Nature 186–193. - PubMed

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