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Kinetic evaluation of photosensitivity in bi-stable variants of chimeric channelrhodopsins.

Hososhima S, Sakai S, Ishizuka T, Yawo H - PLoS ONE (2015)

Bottom Line: The turning-on rate constant of each photocurrent followed a linear relationship to 0-0.12 mW mm(-2) of blue LED light or to 0-0.33 mW mm(-2) of cyan LED light.Each photocurrent of bi-stable ChR was shut off to the non-conducting state by yellow or orange LED light in a manner dependent on the irradiance.On the other hand, in another group of neurons, the threshold irradiance was not dependent on the irradiation time.

View Article: PubMed Central - PubMed

Affiliation: Department of Developmental Biology and Neuroscience, Tohoku University Graduate School of Life Sciences, Sendai, Japan; Core Research of Evolutional Science & Technology (CREST), Japan Science and Technology Agency (JST), Tokyo, Japan.

ABSTRACT
Channelrhodopsin-1 and 2 (ChR1 and ChR2) form cation channels that are gated by light through an unknown mechanism. We tested the DC-gate hypothesis that C167 and D195 are involved in the stabilization of the cation-permeable state of ChRWR/C1C2 which consists of TM1-5 of ChR1 and TM6-7 of ChR2 and ChRFR which consists of TM1-2 of ChR1 and TM3-7 of ChR2. The cation permeable state of each ChRWR and ChRFR was markedly prolonged in the order of several tens of seconds when either C167 or D195 position was mutated to alanine (A). Therefore, the DC-gate function was conserved among these chimeric ChRs. We next investigated the kinetic properties of the ON/OFF response of these bi-stable ChR mutants as they are important in designing the photostimulation protocols for the optogenetic manipulation of neuronal activities. The turning-on rate constant of each photocurrent followed a linear relationship to 0-0.12 mW mm(-2) of blue LED light or to 0-0.33 mW mm(-2) of cyan LED light. Each photocurrent of bi-stable ChR was shut off to the non-conducting state by yellow or orange LED light in a manner dependent on the irradiance. As the magnitude of the photocurrent was mostly determined by the turning-on rate constant and the irradiation time, the minimal irradiance that effectively evoked an action potential (threshold irradiance) was decreased with time only if the neuron, which expresses bi-stable ChRs, has a certain large membrane time constant (eg. τm > 20 ms). On the other hand, in another group of neurons, the threshold irradiance was not dependent on the irradiation time. Based on these quantitative data, we would propose that these bi-stable ChRs would be most suitable for enhancing the intrinsic activity of excitatory pyramidal neurons at a minimal magnitude of irradiance.

No MeSH data available.


Related in: MedlinePlus

OFF response kinetics.A–C, Sample photocurrent records of ChR2-C128A (A), ChRWR-C167A (B) and ChRFR-C167A (C) opened by blue LED light (0.12 mWmm−2) and closed by yellow LED light (0.058–0.32 mWmm−2). D-F, Photocurrents of each bi-stable ChR opened by cyan LED light (0.33 mWmm−2) and closed by orange LED light (0.10–1.4 mWmm−2). G, Shutting-off rate constant (τOFF−1) of ChR2-C128A as a function of irradiance by yellow LED light (yellow circles) and orange LED light (orange diamonds). Each line was fitted for the least-squares protocol; y = 16x+0.073 (yellow) and y = 4.4x+0.058 (orange). H, Similar relationships in ChRWR-C167A; y = 47x+0.23 (yellow) and y = 9.0x+0.12 (orange). I, Similar relationships in ChRFR-C167A; y = 25x+0.08 (yellow) and y = 5.8x+0.070 (orange).
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pone.0119558.g003: OFF response kinetics.A–C, Sample photocurrent records of ChR2-C128A (A), ChRWR-C167A (B) and ChRFR-C167A (C) opened by blue LED light (0.12 mWmm−2) and closed by yellow LED light (0.058–0.32 mWmm−2). D-F, Photocurrents of each bi-stable ChR opened by cyan LED light (0.33 mWmm−2) and closed by orange LED light (0.10–1.4 mWmm−2). G, Shutting-off rate constant (τOFF−1) of ChR2-C128A as a function of irradiance by yellow LED light (yellow circles) and orange LED light (orange diamonds). Each line was fitted for the least-squares protocol; y = 16x+0.073 (yellow) and y = 4.4x+0.058 (orange). H, Similar relationships in ChRWR-C167A; y = 47x+0.23 (yellow) and y = 9.0x+0.12 (orange). I, Similar relationships in ChRFR-C167A; y = 25x+0.08 (yellow) and y = 5.8x+0.070 (orange).

Mentions: The DC-gate mutant molecules of ChR2, such as ChR2-C128A, were turned on to the ion-conducting state by blue light and shut off to the non-conducting state by light of longer wavelengths [16] (Fig. 3A). Quite similarly, ChRWR-C167A and ChRFR-C167A were turned on by the blue LED light (peak, 460–490 nm) and shut off by the yellow LED light (peak, 587–597 nm) in a manner dependent on the irradiance (Fig. 3B and 3C). They were also turned on by the cyan LED light (peak, 490–520 nm) and shut off by the orange LED light (peak, 612–620 nm) (Fig. 3D-F). For each C167 variant, the shutting-off rate constant (τOFF−1) was linearly dependent on the irradiance of either yellow LED light or orange LED light, as summarized in Fig. 3G (ChR2-C128A: slope, 16 s−1(mWmm−2)−1 for yellow LED light and 4.4 s−1(mWmm−2)−1 for orange LED light), Fig. 3H (ChRWR-C167A: slope, 47 s−1(mWmm−2)−1 for yellow LED light and 9.0 s−1(mWmm−2)−1 for orange LED light) and Fig. 3I (ChRFR-C167A: slope, 25 s−1(mWmm−2)−1 for yellow LED light and 5.8 s−1(mWmm−2)−1 for orange LED light). The ratio of these slopes by the changes of orange/yellow LED light (O/Y ratio) was 0.28 (ChR2-C128A), 0.19 (ChRWR-C167A) and 0.23 (ChRFR-C167A), respectively.


Kinetic evaluation of photosensitivity in bi-stable variants of chimeric channelrhodopsins.

Hososhima S, Sakai S, Ishizuka T, Yawo H - PLoS ONE (2015)

OFF response kinetics.A–C, Sample photocurrent records of ChR2-C128A (A), ChRWR-C167A (B) and ChRFR-C167A (C) opened by blue LED light (0.12 mWmm−2) and closed by yellow LED light (0.058–0.32 mWmm−2). D-F, Photocurrents of each bi-stable ChR opened by cyan LED light (0.33 mWmm−2) and closed by orange LED light (0.10–1.4 mWmm−2). G, Shutting-off rate constant (τOFF−1) of ChR2-C128A as a function of irradiance by yellow LED light (yellow circles) and orange LED light (orange diamonds). Each line was fitted for the least-squares protocol; y = 16x+0.073 (yellow) and y = 4.4x+0.058 (orange). H, Similar relationships in ChRWR-C167A; y = 47x+0.23 (yellow) and y = 9.0x+0.12 (orange). I, Similar relationships in ChRFR-C167A; y = 25x+0.08 (yellow) and y = 5.8x+0.070 (orange).
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Related In: Results  -  Collection

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pone.0119558.g003: OFF response kinetics.A–C, Sample photocurrent records of ChR2-C128A (A), ChRWR-C167A (B) and ChRFR-C167A (C) opened by blue LED light (0.12 mWmm−2) and closed by yellow LED light (0.058–0.32 mWmm−2). D-F, Photocurrents of each bi-stable ChR opened by cyan LED light (0.33 mWmm−2) and closed by orange LED light (0.10–1.4 mWmm−2). G, Shutting-off rate constant (τOFF−1) of ChR2-C128A as a function of irradiance by yellow LED light (yellow circles) and orange LED light (orange diamonds). Each line was fitted for the least-squares protocol; y = 16x+0.073 (yellow) and y = 4.4x+0.058 (orange). H, Similar relationships in ChRWR-C167A; y = 47x+0.23 (yellow) and y = 9.0x+0.12 (orange). I, Similar relationships in ChRFR-C167A; y = 25x+0.08 (yellow) and y = 5.8x+0.070 (orange).
Mentions: The DC-gate mutant molecules of ChR2, such as ChR2-C128A, were turned on to the ion-conducting state by blue light and shut off to the non-conducting state by light of longer wavelengths [16] (Fig. 3A). Quite similarly, ChRWR-C167A and ChRFR-C167A were turned on by the blue LED light (peak, 460–490 nm) and shut off by the yellow LED light (peak, 587–597 nm) in a manner dependent on the irradiance (Fig. 3B and 3C). They were also turned on by the cyan LED light (peak, 490–520 nm) and shut off by the orange LED light (peak, 612–620 nm) (Fig. 3D-F). For each C167 variant, the shutting-off rate constant (τOFF−1) was linearly dependent on the irradiance of either yellow LED light or orange LED light, as summarized in Fig. 3G (ChR2-C128A: slope, 16 s−1(mWmm−2)−1 for yellow LED light and 4.4 s−1(mWmm−2)−1 for orange LED light), Fig. 3H (ChRWR-C167A: slope, 47 s−1(mWmm−2)−1 for yellow LED light and 9.0 s−1(mWmm−2)−1 for orange LED light) and Fig. 3I (ChRFR-C167A: slope, 25 s−1(mWmm−2)−1 for yellow LED light and 5.8 s−1(mWmm−2)−1 for orange LED light). The ratio of these slopes by the changes of orange/yellow LED light (O/Y ratio) was 0.28 (ChR2-C128A), 0.19 (ChRWR-C167A) and 0.23 (ChRFR-C167A), respectively.

Bottom Line: The turning-on rate constant of each photocurrent followed a linear relationship to 0-0.12 mW mm(-2) of blue LED light or to 0-0.33 mW mm(-2) of cyan LED light.Each photocurrent of bi-stable ChR was shut off to the non-conducting state by yellow or orange LED light in a manner dependent on the irradiance.On the other hand, in another group of neurons, the threshold irradiance was not dependent on the irradiation time.

View Article: PubMed Central - PubMed

Affiliation: Department of Developmental Biology and Neuroscience, Tohoku University Graduate School of Life Sciences, Sendai, Japan; Core Research of Evolutional Science & Technology (CREST), Japan Science and Technology Agency (JST), Tokyo, Japan.

ABSTRACT
Channelrhodopsin-1 and 2 (ChR1 and ChR2) form cation channels that are gated by light through an unknown mechanism. We tested the DC-gate hypothesis that C167 and D195 are involved in the stabilization of the cation-permeable state of ChRWR/C1C2 which consists of TM1-5 of ChR1 and TM6-7 of ChR2 and ChRFR which consists of TM1-2 of ChR1 and TM3-7 of ChR2. The cation permeable state of each ChRWR and ChRFR was markedly prolonged in the order of several tens of seconds when either C167 or D195 position was mutated to alanine (A). Therefore, the DC-gate function was conserved among these chimeric ChRs. We next investigated the kinetic properties of the ON/OFF response of these bi-stable ChR mutants as they are important in designing the photostimulation protocols for the optogenetic manipulation of neuronal activities. The turning-on rate constant of each photocurrent followed a linear relationship to 0-0.12 mW mm(-2) of blue LED light or to 0-0.33 mW mm(-2) of cyan LED light. Each photocurrent of bi-stable ChR was shut off to the non-conducting state by yellow or orange LED light in a manner dependent on the irradiance. As the magnitude of the photocurrent was mostly determined by the turning-on rate constant and the irradiation time, the minimal irradiance that effectively evoked an action potential (threshold irradiance) was decreased with time only if the neuron, which expresses bi-stable ChRs, has a certain large membrane time constant (eg. τm > 20 ms). On the other hand, in another group of neurons, the threshold irradiance was not dependent on the irradiation time. Based on these quantitative data, we would propose that these bi-stable ChRs would be most suitable for enhancing the intrinsic activity of excitatory pyramidal neurons at a minimal magnitude of irradiance.

No MeSH data available.


Related in: MedlinePlus