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Fig. 1
Engineering and characterization of NCR1. a The schematic diagram of the NCR1 full-length protein sequence, illustrated by Protter—visualize proteoforms [20]. The seven transmembrane helices are according to the structure predicted by AlphaFold 2 [16]. b Different truncated constructs of NCR1. LR: the Lucy-Rho membrane targeting signal, T: plasma membrane trafficking signal, E: ER export signal peptides, eYFP: yellow fluorescent protein. Please note that NCR1-273 had a further 10 aa truncation at the N-terminal. c Fluorescence pictures and photocurrents of different NCR1 variants. 30 ng cRNA was injected into the Xenopus oocyte for each construct. Photocurrent was measured with extracellular ORi solution (Ori, 110 mM NaCl, 5 mM KCl, 2 mM CaCl2, 1 mM MgCl2, 5 mM HEPES and pH 7.6). The green bar indicated the 0.5 s green light illumination, 532 nm at 0.5 mW/mm2. Holding potential: −40 mV. d Photocurrents of different NCR1 variants under different 532 nm light intensities ranging from 0.03 to 4 mW/mm2 (0.031, 0.062, 0.125, 0.125, 0.25, 0.5, 1, 2, 3, 4 mW/mm2). Other conditions are the same as indicated in c. n = 6, error bars = SEM. e Action spectra of different NCR1 variants. The wavelengths were changed from 420 to 620 nm. Photocurrent was measured with extracellular ORi solution, pH 7.6. Holding potential: −40 mV. n = 6, error bars = SEM
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Fig. 2
Comparing the ion conductance of NCR1 and NCR1-273 2.0. a Representative photocurrent traces and current-potential curves of NCR1 in high Na+ (115 mM NaCl, 2 mM CaCl2, 1 mM MgCl2, 5 mM Hepes, pH = 7.6), high K+ (115 mM KCl, 2 mM CaCl2, 1 mM MgCl2, 5 mM Hepes, pH = 7.6) and low Na+ and K+ (112.7NMG+, 1.15 mM NaCl, 1.15 mM KCl, 2 mM CaCl2, 1 mM MgCl2, 5 mM Hepes, pH = 7.6) buffers. Photocurrents were measured with oocytes upon 0.5 s green light (532 nm, 0.5 mW/mm2) illumination, indicated by the green bars. Holding potentials were changed from −80 to +60 mV. The blue color marked the traces at holding potential of 40 mV, while the red color marked the traces at holding potential of 20 mV. For the current-potential curves, n = 6 experiments, error bars = SEM. b The calculated reversal potentials of different NCR1s in high Na+, high K+ and low Na+ and K+ buffer. The calculated PNa/PK of NCR1-273 2.0 was shown on the right. n = 6. c Representative photocurrent traces and d current-potential curves of NCR1-273 2.0 in 2 mM Ca2+ (115 mM NMG, 2 mM CaCl2, 1 mM MgCl2, 5 mM Hepes, pH =7.6), 80 mM Ca2+ (80 mM CaCl2, 2 mM MgCl2, 5 mM Hepes, pH =7.6) and 80 mM Ba2+ (80 mM BaCl2, 2 mM CaCl2, 5 mM Hepes, pH =7.6) buffers. Photocurrents were measured with oocytes upon 0.5 s green light (532 nm, 0.5 mW/mm2) illumination. Holding potentials were from −160 to −60 mV. For the current-potential curves, n = 6 experiments, error bars = SEM
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Fig. 3
Combination of NCR1 2.0 and GtACR1 for light-controlled water influx into the oocytes. a The schematic diagram shows the water transport through the AQP1, driven by the ion gradients created by the Na+ and Cl+ channels. The aquaporin AQP1 is constitutively active. NCR1 and ACR1 channels are opened by the green light illumination. b Fluorescence pictures of Xenopus oocytes after expressing different proteins. 5 ng AQP1, 20 ng NCR1 2.0, and 5 ng GtACR1 cRNAs were injected into the oocyte singly or as mixtures. Pictures were taken with oocytes in ND96 buffer. c Light-induced oocyte swelling in ND96 buffer after expressing different combinations of channels for 2 days. The oocyte swelling was indicated by the explosion (or rupture) at the red arrow-marked positions. The oocytes were illuminated by 520 nm LEDs with light intensity of 400 μW/mm2. Red arrow: the explosive part in oocytes. 5 ng AQP1, 20 ng NCR1 2.0, and 5 ng GtACR1 cRNAs were injected into the oocyte singly or as mixtures. d Oocyte swelling efficacy after different times of green light illumination. The percentage of ruptured oocytes was recorded every 20 min. The y-axis in the graph represents the percentage of oocytes displaying rupture (due to osmotic swelling). Error bars = SEM, n = 6 groups, each with 10 oocytes. Other conditions are the same as c
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Fig. 4
Improving the expression and photocurrent of KCR1 in Xenopus oocytes. a The schematic diagram and representative photocurrent traces of KCR1 and KCR1 2.0. The green bars indicate the duration of 0.5 s green light illumination, using a 532-nm laser at an intensity of 0.5 mW/mm2. The holding potentials ranged from −60 to +20 mV. The measurements were performed using the high K+ buffer described in Fig. 2a. A 30 ng KCR1 or 30 ng KCR1 2.0 cRNAs were injected for oocyte expression. b Fluorescence images of Xenopus oocytes expressing KCR1 and KCR1 2.0 with 30 ng cRNAs for each. c Photocurrents of KCR1 and KCR1 2.0 in high Na+ and high K+ buffers, and buffer contents were described in Fig 2a. A 0.5-s 532-nm laser flash at 0.5 mW/mm2 was used for illumination. The holding potentials ranged from −100 to +20 mV. n = 6. Error bars = SEM
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Fig. 5
Combine the KCR1 2.0 and GtACR1 for light-controlled water efflux of the oocytes. a The schematic diagram shows the water transport through the AQP1 driven by the ion gradients created by the K+ and Cl+ channels. The aquaporin AQP1 is constitutively active. KCR1 and ACR1 channels are opened by the green light illumination. b Fluorescence pictures of Xenopus oocytes after expressing different proteins. 5 ng AQP1, 20 ng KCR1 2.0, and 5 ng GtACR1 cRNAs were injected into the oocyte singly or as mixtures. Pictures were taken in ND96 buffer. c Light-induced oocyte shrinking in ND96 buffer after expressing different combinations of channels for 2 days. The oocyte shrinking was indicated by the separation of the cytoplasmic membrane and the vitelline membrane at the red arrow-marked positions. The oocytes were illuminated for 20 min by 520-nm LEDs with light intensity of 1 mW/mm2. 5 ng AQP1, 20 ng KCR1 2.0, and 5 ng GtACR1 cRNAs were injected into the oocyte singly or as mixtures. d Oocyte shrinking efficacy after different times of green light illumination. The number of shrunk oocytes was recorded every 20 min. The y-axis in the graph represents the percentage of oocytes displaying clearly the shrunken phenotype. Error bars = SEM, n = 6 groups, each with 10 oocytes. Other conditions are the same as c
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