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Figure 1. Design of a light-gated Ca2+-permeant cation channel. (A) Photocurrents of Xenopus oocytes expressing the bovine olfactory cyclic nucleotide gated channel (OLF) and the photoactivatable adenylyl cyclase (bPAC) and various fusion constructs. Total cRNA amounts were adjusted to keep the copies of injected OLF constant. Ratios of cRNA mixtures are indicated. Experiments were performed employing blue light illumination (0.2 s, 1 mW/mm2, 473 nm, n = 6). (B) cAMP production of different OLF and bPAC fusion constructs or mixes; blue light (473 nm, 0.3 mW/mm2); n = 3 experiments, each with 6 oocytes; error bars = SD. (C) Schematic of the designed OLF (T537S) and bPAC fusion construct. T, plasma membrane trafficking signal; Ex, ER export signal. (D) Fluorescence picture of Xenopus oocyte expressing OLF-T-YFP-bPAC-Ex. (E) Example photocurrent of OLF-T-YFP-bPAC-Ex and 1:1 mixture of OLF and bPAC. Holding potential −60 mV; illumination with 473 nm blue light, 1 mW/mm2, red dashed arrow indicates 0 nA. (F) Kinetics of photocurrents in oocytes expressing OLF-T-YFP-bPAC-Ex and 1:1 mixture of OLF and bPAC. Light intensity was adjusted to evoke currents of ~0.6 μA. n = 3, error bars = SD. (G) Example OLF-T-YFP-bPAC-Ex photocurrent traces from 1 oocyte induced by 1 s 473 nm light of different intensities, ~15 min recovery time in the dark for each illumination. (H) OLF-T-YFP-bPAC-Ex photocurrents induced by light of different intensities. n = 4, error bars = SD. (I) A current recording trace from an oocyte expressing OLF-T-YFP-bPAC-Ex with 1 s blue light (473 nm, 550 μW/mm2) illumination and switching bath solutions containing different cations (115Na 2Ba2+: 115 mM NaCl, 2 mM BaCl2; 80Ba2+: 80 mM BaCl2; 80Ca2+: 80 mM CaCl2. All buffers contain 1 mM MgCl2, 5 mM HEPES and pH adjusted to 7.6). Orange line indicates basal current, arrows indicate light pulse, ~15 min recovery time in the dark between each illumination.
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Figure 2. Characterization of light-gated OLF channel in hippocampal neurons. (A) Top: Maximum intensity projection of confocal images of hippocampal neurons 6 days after electroporation with DNA encoding OLF-T-YFP-bPAC-Ex (OLF-bP) and mKate2 (excitation 488 nm). Bottom: Single plane of the YFP signal of indicated area in (A) (dark yellow box). (B) Left: Sample photocurrents evoked by a 50 ms light pulse (470 nm) of different intensity in hippocampal neurons expressing mKate2 and OLF-bP. Right: Light intensity-response relationship fitted with a quadratic equation. Photocurrents are normalized to the maximum current recorded from each neuron. n = 6. (C) Left: Whole-cell responses to current injections from −400 to 1,000 pA in an OLF-bP expressing hippocampal neuron. Right: Action potentials of the same cell generated by applying a short 470 nm light flash (50 ms at 10 mW/mm2).
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Figure 3. Design of a light-gated potassium channel. (A) Schematic of the designed SthK and bPAC fusion construct. (B) Representative Photocurrent traces recorded from Xenopus oocytes injected with 2 ng SthK-T-YFP-bPAC-Ex (SthK-bP) activated by 1 s blue light (473 nm) of different intensities, ~30 min recovery time in the dark for each illumination. (C) Normalized currents against light intensity curve fitted monoexponentially. The half-maximal light intensity value was determined to be 500 μW/mm2; n = 4, error bars = SEM. (D) Representative traces of membrane potential recording while switching bath solutions containing 5, 30, and 110 mM K+ after 5 s blue light (473 nm, 550 μW/mm2) illumination. (E) Representative traces of current recording (holding at −40 mV) while switching bath solutions containing 5, 30 and 110 mM K+ after 5 s blue light (473 nm, 550 μW/mm2) illumination. (F) On and off kinetics of SthK-bP photocurrents in oocytes with 1 s blue light (473 nm, 550 μW/mm2) illumination; for values, n = 4, error bars = SD. (G) Representative photocurrent traces from oocytes co-injected with 1.2 ng SthK-bP and 5 ng BeCyclop cRNA in Xenopus oocytes. Currents were induced by 200 ms blue light (473 nm, 550 μW/mm2) illumination and reduced by 3 s green light (532 nm, 1 mW/mm2) illumination. (H) cAMP and cGMP production of Xenopus oocyte membranes co-expressing SthK-bP and BeCyclop in the dark or light (532 nm, 80 μW/mm2). n = 3, error bars = SD.
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Figure 4. Characterization of a light-gated potassium channel in hippocampal neurons. (A) Maximum intensity projection of confocal images of hippocampal neurons 3 days after electroporation with DNA encoding SthK-bP and mKate2 (left: excitation 559 nm; right: excitation 488 nm). Lower images are close up of the regions indicated by dark boxes. (B) Single plane of the YFP signal of the region indicated in (A) (dark yellow box). (C) Left: Sample photocurrents evoked by a 50 ms light pulse (470 nm) of different intensity in a hippocampal neuron expressing mKate2 and SthK-bP. The holding potential was −70 mV. Right: Light intensity-response relationship fitted with a quadratic equation. Photocurrents are normalized to the maximum current recorded in each neuron. n = 5. (D) Left: Sample traces of photocurrents recorded from a SthK-bP expressing neuron when stimulated with 0.1 mW/mm2, 470 nm light (50 ms) while holding the cell at membrane potentials from −70 mV down to −115 mV. Baselines are aligned. Right: Current-voltage plot. A non-linear fit was applied to determine the K+ equilibrium potential (−100.5 mV). (E) Photocurrent amplitude recorded from neurons expressing mKate2 and SthK-bP when stimulated with 1 mW mm−2 470 nm light. Shown are individual data points, median and 25–75% interquartile range, n = 5; median peak current SthK-bP 1.37 nA. (F) Kinetics of SthK-bP; data obtained from traces when the stimulation intensity was 1 mW mm−2 (470 nm, 50 ms); n = 5. (G) Whole-cell responses to current injections from −400 to 700 pA in SthK-bP expressing hippocampal neuron. (H) Action potentials generated by repeated somatic current injection (1,000 pA, 600 ms, ISI 5 s) were blocked for 3 min by 3 470 nm light flashes at 40 second intervals (50 ms at 1 mW mm−2); same neuron as in (G).
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Figure 5. Functional expression of OLF-bP and SthK-bP in Drosophila larval motoneurons. (A) Expression of OLF-bP and SthK-bP in the larval motoneurons showed different phenotypes upon continuous blue light illumination, Ctrl-S = UAS-SthK-T-YFP-bPAC-Ex/+, Scale bar = 1 mm. (B) Light induced cAMP production in larvae expressing OLF-bP and SthK-bP in motoneurons, blue light (473 nm, 0.3 mW/mm2), n = 3 experiments, each with 6 larvae, error bars = SD. To calculate the final cAMP concentration in larvae, we assumed 1 larva has a volume of 2 μl.
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Figure 6. Expression of OLF-bP and SthK-bP in larval motoneurons. (A) Staining against YFP (green) reveals a clustered distribution of OLF-bP in motoneuron somata (larval VNC, upper panels), while no signal was detected at the NMJ (lower panels). Anti-HRP (horseradish peroxidase; magenta) was employed to stain neuronal membranes. (B) SthK-bP is enriched at cell body membranes in the VNC (upper panels). Similar to OLF-bP, no signal was detected at the NMJ (lower panels). Scale bars: 20 μm.
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Figure 7. Optogenetic control of Drosophila larval motility. (A) Velocity of 3rd instar Drosophila larvae expressing OLF-bP in motoneurons under red and blue light. (B) Box plot of velocity data from OLF-bP expressing larvae and controls under red light and blue light conditions, Ctrl-G = +/OK-Gal4; Ctrl-O = UAS- OLF-T-YFP-bPAC-Ex/+. (C) Velocity of 3rd instar Drosophila larvae expressing SthK-bP in motoneurons under red and blue light. (D) Box plot of velocity data from Sthk-bP expressing larvae and controls under red light and blue light conditions. (E) Velocity of different Drosophila control larvae under red and blue light. For box plot graph, box line represents median, box edges represent 25 and 75 percentiles, whiskers represent 1 and 99 percentiles. (F) Velocity of Drosophila larvae expressing OLF-T-YFP-Ex (OLF) in motoneurons under control conditions (red) followed by 30 s of blue light illumination. (G) Velocity of Drosophila larvae expressing CD8-YFP-bPAC (CD8-bP) in motoneurons under control conditions followed by 30 s of blue light illumination. For A-G, n = 20 for each genotype; error bars = SEM. Red light (620 nm, 0.1 μW/cm2), blue light (470 nm, 1.6 mW/cm2). (H) Light to dark velocity ratios of different genotypes. n = 19, error bars = SD. (I) Light induced cAMP production of larvae expressing CD8-bP in motoneurons, blue light (473 nm, 0.3 mW/mm2). n = 3 experiments, each with six larvae, error bars = SD. To calculate the final cAMP concentration in larvae, we assumed that one larva has a volume of 2 μl.
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Figure 8. Optogenetic control of Drosophila larval motility with short illumination. (A) Velocity changes of 3rd instar Drosophila larvae expressing OLF-bP in motoneurons with 1, 2, and 5 s blue light illumination. (B) Velocity changes of 3rd instar Drosophila larvae expressing SthK-bP in motoneurons with 0.2, 0.5, and 1 s blue light illumination. (C–E) Velocity changes of 3 control lines with 5 s blue light illumination. For all, n = 20 for each genotype [except for (C), here n = 16]; error bars (gray) = SEM. Control red light conditions (620 nm, 0.1 μW/cm2), blue light (470 nm 25 μW/cm2).
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Figure 9. Optogenetic control of Drosophila larval body length with long illumination. (A) Body length of 3rd instar Drosophila larvae expressing OLF-bP in motoneurons under red and blue light. (B) Box plot of body length data from OLF-bP expressing larvae and controls under red light and blue light conditions. (C) Body length of 3rd instar Drosophila larvae expressing SthK-bP in motoneurons under red and blue light. (D) Box plot of body length data from Sthk-bP expressing larvae and controls under red light and blue light conditions. For box plot graph (B,D), box line represents median, box edges represent 25 and 75 percentiles, whiskers represent 1 and 99 percentiles. (E) Body length of different Drosophila control larvae under red and blue light. Body length changes of 3rd instar Drosophila larvae expressing OLF (F) and CD8-bP (G) in motoneurons under control conditions followed by 30 s of blue light illumination. For A–G, n = 20 for each genotype; error bars = SEM. Red light (620 nm, 0.1 μW/cm2), blue light (470 nm, 1.6 mW/cm2). (H) Light to dark body length ratios of different genotypes. n = 19, error bars = SD.
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Figure 10. Optogenetic control of Drosophila larval body length with short illumination. Body length changes of 3rd instar Drosophila larvae expressing OLF-bP (A) and SthK-bP (B) in motoneurons with 0.2, 0.5, and 1 s blue light illumination. n = 20 for each genotype; error bars (gray) = SEM. Control conditions (red light 620 nm, 0.1 μW/cm2), blue light (470 nm, 25 μW/cm2).
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