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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

ON response kinetics.A–C, Sample photocurrent records of ChR2-C128A (A), ChRWR-C167A (B) and ChRFR-C167A (C) opened by blue LED light (0.0021–0.12 mWmm−2) and closed by yellow LED light (0.32 mWmm−2). D-F, Photocurrents of each bi-stable ChR opened by cyan LED light (0.014–0.33 mWmm−2) and closed by orange LED light (1.4 mWmm−2). G, Turning-on rate constant (τON−1) of ChR2-C128A as a function of irradiance by blue LED light (blue circles) and cyan LED light (cyan diamonds). Each line was fitted for the least-squares protocol; y = 640x+2.0 (blue) and y = 190x+0.76 (cyan). H, Similar relationships in ChRWR-C167A; y = 530x+1.1 (blue) and y = 390x+1.9 (cyan). I, Similar relationships in ChRFR-C167A; y = 480x+0.35 (blue) and y = 240x+1.1 (cyan).
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pone.0119558.g004: ON response kinetics.A–C, Sample photocurrent records of ChR2-C128A (A), ChRWR-C167A (B) and ChRFR-C167A (C) opened by blue LED light (0.0021–0.12 mWmm−2) and closed by yellow LED light (0.32 mWmm−2). D-F, Photocurrents of each bi-stable ChR opened by cyan LED light (0.014–0.33 mWmm−2) and closed by orange LED light (1.4 mWmm−2). G, Turning-on rate constant (τON−1) of ChR2-C128A as a function of irradiance by blue LED light (blue circles) and cyan LED light (cyan diamonds). Each line was fitted for the least-squares protocol; y = 640x+2.0 (blue) and y = 190x+0.76 (cyan). H, Similar relationships in ChRWR-C167A; y = 530x+1.1 (blue) and y = 390x+1.9 (cyan). I, Similar relationships in ChRFR-C167A; y = 480x+0.35 (blue) and y = 240x+1.1 (cyan).

Mentions: As each light-dependent state transition of ChR is approximated by a single-photon reaction, each transition rate is presumed to be proportional to the irradiance. To test this, the photocurrents were evoked with varying powers of blue LED light (Fig. 4A-C) or cyan LED light (Fig. 4D-F) and the turning-on time constants (τON) were compared. Indeed, the turning-on rate constant (τON−1) of each photocurrent followed a linear relationship in a wide range of irradiance with either blue or cyan LED light, as summarized in Fig. 4G (ChR2-C128A: slope, 636 s−1(mWmm−2)−1 for blue LED light and 193 s−1(mWmm−2)−1 for cyan LED light), Fig. 4H (ChRWR-C167A: slope, 530 s−1(mWmm−2)−1 for blue LED light and 387 s−1(mWmm−2)−1 for cyan LED light) and Fig. 4I (ChRFR-C167A: 480 s−1(mWmm−2)−1 for blue LED light and 237 s−1(mWmm−2)−1 for cyan LED light). When the ratio of these slopes was calculated by the changes of cyan/blue LED light (C/B ratio), it was 0.30 (ChR2-C128A), 0.73 (ChRWR-C167A) and 0.49 (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)

ON response kinetics.A–C, Sample photocurrent records of ChR2-C128A (A), ChRWR-C167A (B) and ChRFR-C167A (C) opened by blue LED light (0.0021–0.12 mWmm−2) and closed by yellow LED light (0.32 mWmm−2). D-F, Photocurrents of each bi-stable ChR opened by cyan LED light (0.014–0.33 mWmm−2) and closed by orange LED light (1.4 mWmm−2). G, Turning-on rate constant (τON−1) of ChR2-C128A as a function of irradiance by blue LED light (blue circles) and cyan LED light (cyan diamonds). Each line was fitted for the least-squares protocol; y = 640x+2.0 (blue) and y = 190x+0.76 (cyan). H, Similar relationships in ChRWR-C167A; y = 530x+1.1 (blue) and y = 390x+1.9 (cyan). I, Similar relationships in ChRFR-C167A; y = 480x+0.35 (blue) and y = 240x+1.1 (cyan).
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Related In: Results  -  Collection

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pone.0119558.g004: ON response kinetics.A–C, Sample photocurrent records of ChR2-C128A (A), ChRWR-C167A (B) and ChRFR-C167A (C) opened by blue LED light (0.0021–0.12 mWmm−2) and closed by yellow LED light (0.32 mWmm−2). D-F, Photocurrents of each bi-stable ChR opened by cyan LED light (0.014–0.33 mWmm−2) and closed by orange LED light (1.4 mWmm−2). G, Turning-on rate constant (τON−1) of ChR2-C128A as a function of irradiance by blue LED light (blue circles) and cyan LED light (cyan diamonds). Each line was fitted for the least-squares protocol; y = 640x+2.0 (blue) and y = 190x+0.76 (cyan). H, Similar relationships in ChRWR-C167A; y = 530x+1.1 (blue) and y = 390x+1.9 (cyan). I, Similar relationships in ChRFR-C167A; y = 480x+0.35 (blue) and y = 240x+1.1 (cyan).
Mentions: As each light-dependent state transition of ChR is approximated by a single-photon reaction, each transition rate is presumed to be proportional to the irradiance. To test this, the photocurrents were evoked with varying powers of blue LED light (Fig. 4A-C) or cyan LED light (Fig. 4D-F) and the turning-on time constants (τON) were compared. Indeed, the turning-on rate constant (τON−1) of each photocurrent followed a linear relationship in a wide range of irradiance with either blue or cyan LED light, as summarized in Fig. 4G (ChR2-C128A: slope, 636 s−1(mWmm−2)−1 for blue LED light and 193 s−1(mWmm−2)−1 for cyan LED light), Fig. 4H (ChRWR-C167A: slope, 530 s−1(mWmm−2)−1 for blue LED light and 387 s−1(mWmm−2)−1 for cyan LED light) and Fig. 4I (ChRFR-C167A: 480 s−1(mWmm−2)−1 for blue LED light and 237 s−1(mWmm−2)−1 for cyan LED light). When the ratio of these slopes was calculated by the changes of cyan/blue LED light (C/B ratio), it was 0.30 (ChR2-C128A), 0.73 (ChRWR-C167A) and 0.49 (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