Dataset DOI: 10.5061/dryad.k98sf7mm2

Description of the data and file structure

The CSV files contain multiple tables, each corresponding to one figure of the manuscript and labeled with the respective figure number. Each table provides the underlying data required to reproduce and replot the corresponding figure.

Files and variables

File: Figure_1i.csv

Description: The ratio of maximal ion channel current I(cGMP)/I(cAMP) for different ion channel mutants of the SthK K+-channel is given for different experiments.

Variables

• Figure 1i: Figure identifier
• SthK1.0: I(cGMP)/I(cAMP)
• SthK1.1: I(cGMP)/I(cAMP)
• SthK2.0: I(cGMP)/I(cAMP)
• SthK2.1: I(cGMP)/I(cAMP)

File: Figure_4b.csv

Description: This file gives the diastolic resting membrane potential in between spikes of cardiomyocytes during different phases of an optogenetic experiment activating the RoCK2.1 (Rhodopsin Guanylyl Cyclase coupled to the SthK2.1 K+ channel) inhibitory tool. APs are evoked at 0.25 Hz with a 2 ms pulse of 400 pA. Cell types are grouped into those with full AP inhibition, those with AP shortening, and uninduced control (cells variable Cells AP with inhibition).

Variables

• Figure 4b: Figure identifier
• Diastolic resting membrane potential in mV (after liquid junction potential correction): description of parameter displayed here
• Cells with AP inhibition: Cell identifier and grouping of cells according to phenotype
• before light: Diastolic membrane potential in mV
• action potentials 2-7: Diastolic membrane potential in mV
• action potentials 10-15: Diastolic membrane potential in mV
• action potentials 95-100: Diastolic membrane potential in mV

File: Figure_4c.csv

Description: This file gives the duration of the action potential until 90% of the voltage has been repolarized (APD_90; s) during different phases of an optogenetic experiment activating the RoCK2.1 (Rhodopsin Guanylyl Cyclase coupled to the SthK2.1 K+ channel) inhibitory tool. Cell types are grouped into those with full AP inhibition, those with AP shortening, and uninduced control cells. During full inhibition, there is no AP, therefore there is no data for these conditions (marked n.a.).

Variables

• Figure 4c: Figure identifier
• Action potential duration at 90% repolarization (APD_90; s): description of parameter displayed here
• Cells with AP inhibition: Cell identifier and grouping of cells according to phenotype
• before light: APD_90; s
• during action potentials 2-7 : APD_90; s
• during action potentials 10-15: APD_90; s
• during action potentials 95-100: APD_90; s

File: Figure_4d.csv

Description: This figure gives the maximal change in sarcomere length (µm) for RoCK2.1 (Rhodopsin Guanylyl Cyclase coupled to the SthK2.1 K+ channel) expressing cells before, during and after activation of RoCK2.1 with light. Sometimes during the experiment, cells drift out of focus, then sarcomere length can't be determined (n.d.). If no contraction occurred (due to inhibition), it is indicated (no contraction)

Variables

• Figure 4d: Figure identifier
• Contraction amplitude = maximal change in sarcomere length (µm): description of the parameter studied here
• Time (s): experiment time
• Cell 1:maximal change in sarcomere length (µm)
• Cell 2:maximal change in sarcomere length (µm)
• Cell 3:maximal change in sarcomere length (µm)
• Cell 4:maximal change in sarcomere length (µm)
• Cell 5:maximal change in sarcomere length (µm)
• Cell 6:maximal change in sarcomere length (µm)
• Cell 7:maximal change in sarcomere length (µm)
• Cell 8:maximal change in sarcomere length (µm)
• Cell 9:maximal change in sarcomere length (µm)
• Cell 10:maximal change in sarcomere length (µm)
• Cell 11:maximal change in sarcomere length (µm)
• Mean: Mean value of the individual cells
• Standard derivation: SD
• Number of analyzed data points = number of cells

File: Figure_5_and_S12.csv

Description: This file gives the analysis of movies recording the coiling behavior of zebrafish embryos during an optogenetic experiment activating the RoCK2.1 (Rhodopsin Guanylyl Cyclase coupled to the SthK2.1 K+ channel) inhibitory tool.

Variables

• Figure 5 and S12: Figure identifier
• date: date of the experiment
• condition: Illumination condition (3 conditions: green_1s, green_15s, red_15s)
• video_ID: video identifier (10 videos per condition per experiment)
• fish_ID: fish identifier (40 fish per condition per experiment).
• genotype: genotype identifier, wt (zebrafish larvae not expresssing RoCK), RoCK (zebrafish larvae expressing RoCK)
• coils: frame number in which a coil occurs (max. 4050 frames, 135 seconds per video, 30 fps)
• coil_height: maximum delta pixel intensity at a coil
• raw_data: delta pixel intensity over time within one video

File: Figure_S3a.csv

Description: This file gives the constants K1/2 of halfmaximal activation of different SthK mutant channels for activation with cGMP and cAMP. K1/2 values are determined in inside out membrane patches and superfusing the inside of the patch with various concentrations of cAMP or cGMP. Each dataset is then fitted with a hill equation (I=Imax*Lⁿ/(K1/2ⁿ+Lⁿ) which returns the K1/2 value for the respective cyclic nucleotide.

Variables

• Figure S3a: Figure identifier
• constant of halfmaximal activation: description of parameter displayed here
• cGMP: cGMP or cAMP was used as ligand
• Mutant (+# of experiments): Name of channel mutant that was investigated and how many experiments were analyzed (in brackets)
• Mean: Mean value of the individual K1/2 values
• SD: Standard deviation

File: Figure_S4.csv

Description: Quantification of light- and dark-induced activity of CaRhGC variants. Assays were performed under saturating substrate conditions (2 mM GTP, 10 mM MgCl 2 , pH 7.4) using crude membrane fractions from transfected HEK293T cells. Crude membrane fractions were illuminated for 20 min at 522 nm or kept in the dark. The amount of GTP and cGMP was then determined using HPLC and recording the absorbance of the respective nucleotide peak in the chromatography. The cGMP peaks were integrated (Variables exp1, exp2, exp3, and dark). To normalize according to expression levels, for each preparation, the fluorescence of the YFP reporter was recorded in a fluorescence spectrometer (Variable YFP-Fluorescence). A GTP standard was measured and the resulting mean, SD values were calculated as described in "Processing".

Variables

• Figure S4: Figure identifier
• Reaction Time: experimental conditions
• 20 min: experimental conditions
• RhGC Variant: Which variant was analyzed
• YFP-Fluorescence (CPS x 10⁵): Fluorescence value of the YFP marker attached to RoCK2.1.
• exp2: Result of integration of the cGMP peak
• exp3: Result of integration of the cGMP peak
• dark: Result of integration of the cGMP peak
• Processing: mean(cGMP) x normalization x volume (200 l) / (GTPstandard x time): analysis math
• mean: of the three experiments after processing
• SD: of the three experiments after processing
• compare: Statistics
• p values: Statistics

File: Figure_S6.csv

Description: In-cell measurements of light-dependent CaRhGC activity in HEK293T cells using the cGMP Glo-Sensor 40F (Promega). After a 2-second illumination the luminescence signal is determined for different time points (variables) at varying light intensities (given in mW/cm²). Glosensor only: cells not transfected with CaRhGC. CaRhGC - dark: no illumination.

Variables

• Figure S6: Figure identifier
• Time [s]: Experimental conditions
• 0: Luminescence signal for different light intensities at the indicated time
• 52.4: Luminescence signal for different light intensities at the indicated time
• 104.8: Luminescence signal for different light intensities at the indicated time
• 157.3: Luminescence signal for different light intensities at the indicated time
• 209.7: Luminescence signal for different light intensities at the indicated time
• 262.1: Luminescence signal for different light intensities at the indicated time
• 596.3: Luminescence signal for different light intensities at the indicated time
• 648.7: Luminescence signal for different light intensities at the indicated time
• 701.1: Luminescence signal for different light intensities at the indicated time
• 753.5: Luminescence signal for different light intensities at the indicated time
• 806: Luminescence signal for different light intensities at the indicated time
• 858.4: Luminescence signal for different light intensities at the indicated time
• 910.8: Luminescence signal for different light intensities at the indicated time
• 963.2: Luminescence signal for different light intensities at the indicated time
• 1015.6: Luminescence signal for different light intensities at the indicated time
• 1068: Luminescence signal for different light intensities at the indicated time
• 1120.4: Luminescence signal for different light intensities at the indicated time
• 1172.8: Luminescence signal for different light intensities at the indicated time
• 1225.2: Luminescence signal for different light intensities at the indicated time
• 1277.6: Luminescence signal for different light intensities at the indicated time
• 1330.1: Luminescence signal for different light intensities at the indicated time
• 1382.5: Luminescence signal for different light intensities at the indicated time
• 1434.9: Luminescence signal for different light intensities at the indicated time
• 1487.3: Luminescence signal for different light intensities at the indicated time
• 1539.7: Luminescence signal for different light intensities at the indicated time
• 1592.1: Luminescence signal for different light intensities at the indicated time
• 1644.5: Luminescence signal for different light intensities at the indicated time
• 1696.9: Luminescence signal for different light intensities at the indicated time
• 1749.3: Luminescence signal for different light intensities at the indicated time
• 1801.7: Luminescence signal for different light intensities at the indicated time
• 1854.2: Luminescence signal for different light intensities at the indicated time
• 1906.6: Luminescence signal for different light intensities at the indicated time
• 1959: Luminescence signal for different light intensities at the indicated time
• 2011.4: Luminescence signal for different light intensities at the indicated time
• 2063.8: Luminescence signal for different light intensities at the indicated time
• 2116.2: Luminescence signal for different light intensities at the indicated time
• 2168.6: Luminescence signal for different light intensities at the indicated time
• 2221: Luminescence signal for different light intensities at the indicated time
• 2273.4: Luminescence signal for different light intensities at the indicated time
• 2325.9: Luminescence signal for different light intensities at the indicated time
• 2378.3: Luminescence signal for different light intensities at the indicated time
• 2430.7: Luminescence signal for different light intensities at the indicated time
• 2483.1: Luminescence signal for different light intensities at the indicated time
• 2535.5: Luminescence signal for different light intensities at the indicated time
• 2587.9: Luminescence signal for different light intensities at the indicated time
• 2640.4: Luminescence signal for different light intensities at the indicated time
• 2692.8: Luminescence signal for different light intensities at the indicated time

File: Figure_S11b.csv

Description: Quantitative analysis of cell-to-cell variation in eYFP signals. Graph shows average pixel intensity per cell, comparing different time points after adenoviral transduction of the RoCK2.1 tool (N = 2 rabbits, n = 152 cells).

Variables

• Figure S11b: Figure identifier
• cell #: cell identifier
• 48 h after pAdeno-RoCK transduction: Average eYFP fluorescence intensity per pixel
• 72 h after pAdeno-RoCK transduction: Average eYFP fluorescence intensity per pixel
• 96 h 1after pAdeno-RoCK transduction: Average eYFP fluorescence intensity per pixel

File: Figure_S11g.csv

Description: Photocurrents of WiChR (n = 12 cells, mean ± standard deviation) in cardiomyocytes. Photocurrents are given for the individual cells at different holding potentials and the respective mean and SEM is calculated.

Variables

• Figure S11g: Figure identifier
• Holding potential (mV): Voltage Clamp value (mV) at that experiment
• Cell 1: Photocurrents of WiChR
• Cell 2: Photocurrents of WiChR
• Cell 3: Photocurrents of WiChR
• Cell 4: Photocurrents of WiChR
• Cell 5: Photocurrents of WiChR

• Cell 6: Photocurrents of WiChR
• Cell 7: Photocurrents of WiChR
• Cell 8: Photocurrents of WiChR
• Cell 9: Photocurrents of WiChR
• Cell 10: Photocurrents of WiChR
• Cell 11: Photocurrents of WiChR
• Cell 12: Photocurrents of WiChR
• Mean: mean of 12 cells
• SD: Standard deviation of 12 cells

File: Figure_S11i.csv

Description: The file gives the diastolic resting membrane potential of 12 cardiomyocytes during an optogenetic experiment before, during and after activating the WiChR inhibitory tool.

Variables

• Figure S11i Figure identifier

• Cell #: cell identifier
• before light: diastolic resting membrane potential (in mV)
• during light: diastolic resting membrane potential (in mV)
• 2 s after light: diastolic resting membrane potential (in mV)

File: Figure_S11h.csv

Description: Recovery from inhibition of cardiomyocytes after activation of the WiChR inhibitory tool. Current-Clamp recordings with triggered AP stimulation at the indicated time point after light off.

Variables

• Figure S11h: Figure identifier

• Time (ms): Experiment time (ms)
• delta t=50 ms after light: voltage progression during the attempt to trigger an AP
• delta t=200 ms after light: voltage progression during the attempt to trigger an AP
• delta t=350 ms after light: voltage progression during the attempt to trigger an AP
• 500 ms: voltage progression during the attempt to trigger an AP
• 650 ms: voltage progression during the attempt to trigger an AP
• 800 ms: voltage progression during the attempt to trigger an AP
• 950 ms: voltage progression during the attempt to trigger an AP
• 1100 ms: voltage progression during the attempt to trigger an AP
• 1250 ms: voltage progression during the attempt to trigger an AP
• 1400 ms: voltage progression during the attempt to trigger an AP
• 1550 ms: voltage progression during the attempt to trigger an AP
• 1700 ms: voltage progression during the attempt to trigger an AP
• 1850 ms: voltage progression during the attempt to trigger an AP
• 2000 ms: voltage progression during the attempt to trigger an AP
• 2150 ms: voltage progression during the attempt to trigger an AP
• 2300 ms: voltage progression during the attempt to trigger an AP
• 2450 ms: voltage progression during the attempt to trigger an AP
• 2600 ms: voltage progression during the attempt to trigger an AP
• 2750 ms: voltage progression during the attempt to trigger an AP

File: Figure_S10c-e.csv

Description: Representative traces showing sarcomere length (SL) measurements of

RoCK2.1-expressing CMs. 2/11 CMs showed full inhibition (c), contraction amplitude

was reduced in 7/11 CMs (d), and contraction amplitude was not altered in 2/11 CMs

(e). CM were field stimulated at 0.2 Hz and green light (520 nm, 3 mW mm -2 ) was

applied for 5 s.

Variables

• Figure S10c: Figure identifier
• 0.20818: Time of the experiment (s)
• 1.85: sarcomere length (µm), example of full inhibition
• Figure S10d: Figure identifier
• 0.044055: Time of the experiment (s)
• 1.769: sarcomere length (µm), example for reduced amplitude
• Figure S10e: Figure identifier
• 0.004055: Time of the experiment (s)
• 1.857: sarcomere length (µm), example for no impact

File: Figure_S8.csv

Description: Duration of neuronal silencing of individual cultured hippocampal neurons expressing WiChR in response to 10 ms pulses of 470 nm or 550 nm light.

Variables

• Figure S8: Figure identifier and light stimulation conditions

• Intensities [mW/mm^2^]: Light intensity of the experiment
• cell 1: duration (s) of neuronal silencing
• cell 2: duration (s) of neuronal silencing
• cell 3: duration (s) of neuronal silencing
• cell 4: duration (s) of neuronal silencing
• cell 5: duration (s) of neuronal silencing
• cell 6: duration (s) of neuronal silencing
• cell 7: duration (s) of neuronal silencing
• cell 8: duration (s) of neuronal silencing
• cell 9: duration (s) of neuronal silencing

File: Figure_S10a.csv

Description: Comparison of action potential duration at 30% repolarization (APD30) of cultured control CM to RoCK2.1-expressing CMs before and at different time points following light application (N = 3 rabbits, n = 24 cells). Individual points correspond to the average of six APs. APs were stimulated at 0.25 Hz. Experiments are grouped into those with AP inhibition, those with AP shortening, and untransduced control cells. In cells with full inhibition, there are no APs and no APD_30 values can be determined. Those are marked n.a.

Variables

• Figure S10a: Figure identifier
• Action potential duration at 30% repolarization (APD_30; s): description of parameter displayed here
• Cells with AP inhibition: Cell identifier and grouping of cells
• before light: APD_30 (s)
• action potentials 2-7 : APD_30 (s)
• action potentials 10-15: APD_30 (s)
• action potentials 95-100: APD_30 (s)

File: Figure_S10b.csv

Description: Maximal change in sarcomere length during contractions, comparing cultured control CMs (N = 2 rabbits, n = 11 cells) to RoCK2.1-expressing CMs (N = 3 rabbits, n = 11 cells) before and at different time points following light application.

Individual points correspond to the average of six APs.

Variables

• Figure S10b: Figure identifier

• Contraction amplitude = maximal change in sarcomere length (µm): Description of parameter displayed here
• All RoCK2.1-expressing cells: cell identifier and grouping of cells
• before light: sarcomere length (µm)
• contractions 2-7: sarcomere length (µm)
• contractions 10-15: sarcomere length (µm)
• contractions 175-180: sarcomere length (µm)

File: Figure_1e.csv

Description: Normalized current amplitudes of the various SthK mutants as a function of cGMP

concentration.

Variables

• Figure 1e: Figure identifier
• SthK 1.0: respective mutant
• cGMP (M)(1): concentration of cGMP (M)
• Mean(1): mean normalized currents
• STD(1): Standard deviation
• SthK 1.1: respective mutant
• cGMP (M)(2): concentration of cGMP (M)
• Mean(2): mean normalized currents
• STD(2): Standard deviation
• SthK 2.0: respective mutant
• cGMP (M)(3): concentration of cGMP (M)
• Mean(3): mean normalized currents
• STD(3): Standard deviation
• SthK 2.1: respective mutant
• cGMP (M)(4): concentration of cGMP (M)
• Mean(4): mean normalized currents
• STD(4): Standard deviation

File: Figure_1f.csv

Description: Normalized current amplitudes of the various SthK mutants as a function of cAMP

concentration.

Variables

• Figure 1f: Figure identifier
• SthK 1.0: respective mutant
• cAMP (M)(1): concentration of cAMP (M)
• Mean(1): mean normalized currents
• STD(1): Standard deviation
• SthK 1.1: respective mutant
• cAMP (M)(2): concentration of cAMP (M)
• Mean(2): mean normalized currents
• STD(2): Standard deviation
• SthK 2.0: respective mutant
• cAMP (M)(3): concentration of cAMP (M)
• Mean(3): mean normalized currents
• STD(3): Standard deviation
• SthK 2.1: respective mutant
• cAMP (M)(4): concentration of cAMP (M)
• Mean(4): mean normalized currents
• STD(4): Standard deviation

File: Figure_1g.csv

Description: cGMP and cAMP dependent open probabilities for SthK1.0 and SthK1.1.

Variables

• Figure 1g: Figure identifier
• SthK 1.0: respective mutant
• cAMP (M)(1): concentration of cAMP (M)
• Mean(1): mean currents normalized to maximal open probability with cAMP
• STD(1): Standard deviation
• cGMP (M)(2): concentration of cGMP (M)
• Mean(2): mean currents normalized to maximal open probability with cGMP
• STD(2): Standard deviation
• SthK 1.1: respective mutant
• cAMP (M)(3): concentration of cAMP (M)
• Mean(3): mean currents normalized to maximal open probability with cAMP
• STD(3): Standard deviation
• cGMP (M)(4): concentration of cGMP (M)
• Mean(4): mean currents normalized to maximal open probability with cGMP
• STD(4): Standard deviation

File: Figure_1h.csv

Description: cGMP and cAMP dependent open probabilities for SthK2.0 and SthK2.1.

Variables

• Figure 1h: Figure identifier
• SthK 2.0: respective mutant
• cGMP (M)(1): concentration of cGMP (M)
• Mean(1): mean currents normalized to maximal open probability with cGMP
• STD(1): Standard deviation
• cAMP (M)(2): concentration of cAMP (M)
• Mean(2): mean currents normalized to maximal open probability with cAMP
• STD(2): Standard deviation
• SthK 2.1: respective mutant
• cGMP (M)(3): concentration of cGMP (M)
• Mean(3) : mean currents normalized to maximal open probability with cGMP
• STD(3): Standard deviation
• cAMP (M)(4): concentration of cAMP (M)
• Mean(4): mean currents normalized to maximal open probability with cAMP
• STD(4): Standard deviation

File: Figure_2c-e.csv

Description: Operational light sensitivity (d) and (e) current densities at EC 50 for different N-terminal truncations compared to the full-length CaRhGC upon 10 ms 525 nm illumination, all measured in the following configuration eYFP-CaRhGC-T2A-SthK1.0 (Independent two-sample t-test: CaRhGC vs Tr79: p = 0.03; CaRhGC vs Tr100: p = 0.0008; Tr64 vs Tr100: p = 0.03; mean ± SD, n = 4-7).

Variables

• Fig. 2 c-e: Figure identifier and cell identifier
• 10 ms, 525 nm: illumination conditions
• CaRhGC-T2A-SthK1.0 (cap): Cell capacity (pF)
• CaRhGC-T2A-SthK1.0 (Peak): Peak Current (pA)
• Tr64CaRhGC-T2A-SthK1.0 (cap): Cell capacity (pF)
• Tr64CaRhGC-T2A-SthK1.0 (Peak): Peak Current (pA)
• Tr79CaRhGC-T2A-SthK1.0 (cap): Cell capacity (pF)
• Tr79CaRhGC-T2A-SthK1.0 (Peak): Peak Current (pA)
• Tr100CaRhGC-T2A-SthK1.0 (cap): Cell capacity (pF)
• Tr100CaRhGC-T2A-SthK1.0 (Peak): Peak Current (pA)

File: Figure_2f-k.csv

Description:  Current responses to various illumination conditions and analysis of rise and decay times of the currents elicited by a 10 ms 525 nm pulse of 6.4 mW mm^-2.

Variables

• Fig. 2 f-k: Figure identifier
• RoCK1.0 (10 ms 525nm): respective mutant and illumination condition
• Cell Rock1.0: cell identifier
• Capacity(1): Cell capacity (pF)
• Intensity (mW/mm²)(1): illumination intensity (mW/mm²)
• Area (pAs)(1): result of integration of the current peak
• Peak Current (pA)(1): Peak current (pA)
• Rise Time (5%-90%) (s)(1): duration of the current rise (s)
• Decay Time (100%-10%) (s)(1): duration of the current decline (s)
• RoCK2.0 (10 ms, 525nm): respective mutant
• Cell RoCK2.0: cell identifier
• Capacity (pF)(2): Cell capacity (pF)
• Intensity (mW/mm2)(2): illumination intensity (mW/mm²)
• Area (pAs)(2): result of integration of the current peak
• Peak Current (pA)(2): Peak current (pA)
• Rise Time (5%-90%) (s)(2): duration of the current rise (s)
• Decay Time (100%-10%) (s)(2): duration of the current decline (s)
• RoCK1.1 (10 ms, 525nm): respective mutant
• Cell RoCK1.1:cell identifier
• Capacity (pF)(3): Cell capacity (pF)
• Intensity (mW/mm2)(3): illumination intensity (mW/mm²)
• Area (pAs)(3): result of integration of the current peak
• Peak Current (pA)(3): Peak current (pA)
• Rise Time (5%-90%) (s)(3): duration of the current rise (s)
• Decay Time (100%-10%) (s)(3): duration of the current decline (s)
• RoCK2.1 (10 ms, 525nm): respective mutant
• Cell RoCK2.1: cell identifier
• Capacity (pF)(4): Cell capacity (pF)
• Intensity (mW/mm2)(4): illumination intensity (mW/mm²)
• Area (ms pA)(4): result of integration of the current peak
• Peak Current (pA)(4): Peak current (pA)
• Rise Time (5%-90%) (s)(4): duration of the current rise (s)
• Decay Time (100%-10%) (s)(4): duration of the current decline (s)

File: Figure_3a-d.csv

Description: Inhibition of current ramp-driven AP firing. Ramp 0–500 pA. Rheobase changes following RoCK2.1 activation (n = 7 cells). Duration of spike-free intervals after RoCK2.1 activation as a function of light intensity (n = 7 cells).

Variables

• Figure 3a-d: Figure identifier
• Rheobase: Description of parameter described here and Rheobase conditions
• Time (s): Starting time of the depolarizing ramp.
• cell 1: injected current (pA) at which an AP was elicited, if no spike was elicited, it is indicated
• cell 2: injected current (pA) at which an AP was elicited, if no spike was elicited, it is indicated
• cell 3: injected current (pA) at which an AP was elicited, if no spike was elicited, it is indicated
• cell 4: injected current (pA) at which an AP was elicited, if no spike was elicited, it is indicated
• cell 5: injected current (pA) at which an AP was elicited, if no spike was elicited, it is indicated
• cell 6: injected current (pA) at which an AP was elicited, if no spike was elicited, it is indicated
• cell 7: injected current (pA) at which an AP was elicited, if no spike was elicited, it is indicated
• Rheobase shift:
• Cell 1: difference (in pA) of rheobase currents with respect to value at time = 2s
• Cell 2: difference (in pA) of rheobase currents with respect to value at time = 2s
• Cell 3: difference (in pA) of rheobase currents with respect to value at time = 2s
• Cell 4: difference (in pA) of rheobase currents with respect to value at time = 2s
• Cell 5: difference (in pA) of rheobase currents with respect to value at time = 2s
• Cell 6: difference (in pA) of rheobase currents with respect to value at time = 2s
• Cell 7: difference (in pA) of rheobase currents with respect to value at time = 2s
• Silent Interval: description of parameter displayed here
• Intensity: illumination conditions (mW/mm²)
• cell1: Spike-free duration after RoCK inhibition
• cell2: Spike-free duration after RoCK inhibition
• cell3: Spike-free duration after RoCK inhibition
• cell4: Spike-free duration after RoCK inhibition
• cell5: Spike-free duration after RoCK inhibition
• cell6: Spike-free duration after RoCK inhibition
• cell7: Spike-free duration after RoCK inhibition
• cell8: Spike-free duration after RoCK inhibition
• cell9: Spike-free duration after RoCK inhibition
• cell10: Spike-free duration after RoCK inhibition
• cell11: Spike-free duration after RoCK inhibition
• cell12: Spike-free duration after RoCK inhibition
• cell13: Spike-free duration after RoCK inhibition
• cell14: Spike-free duration after RoCK inhibition
• cell15: Spike-free duration after RoCK inhibition
• cell16: Spike-free duration after RoCK inhibition
• cell17: Spike-free duration after RoCK inhibition

File: Figure_3e-i.csv

Description: Normalized number of APs of granule cells before, during, and after a 100 ms light pulse at 561 nm to activate RoCK2.1. Peak currents during Voltage-Clamp recordings, and voltage dependence of light-evoked currents

Variables

• Figure 3e-i: Figure identifier
• Relative number of action potentials: Description of parameter described here
• cell1: Relative and absolut number of action potentials
• cell2: Relative and absolut number of action potentials
• cell3: Relative and absolut number of action potentials
• cell4: Relative and absolut number of action potentials
• cell5: Relative and absolut number of action potentials
• cell6: Relative and absolut number of action potentials
• mean: mean of relative number of action potentials
• S.E.: Standard Error
• trace: identifier for characteristic traces in the figure
• Voltage Clamp current (pA): Description of parameter described here
• Vhold: Holding voltage (mV)
• Mean: Mean current value (pA) of all six cells recorded
• sem: Standard error
• cell 1: Mean peak current value (pA) of five subsequent traces for the respective cell and individual currents for each trace below.
• cell 2: Mean peak current value (pA) of five subsequent traces for the respective cell and individual currents for each trace below.
• cell 3: Mean peak current value (pA) of five subsequent traces for the respective cell and individual currents for each trace below.
• cell 4: Mean peak current value (pA) of five subsequent traces for the respective cell and individual currents for each trace below.
• cell 5: Mean peak current value (pA) of five subsequent traces for the respective cell and individual currents for each trace below.
• cell 6: Mean peak current value (pA) of five subsequent traces for the respective cell and individual currents for each trace below.
• Exponential fit to current decay: Description of parameter described here
• Trace Vh=-44mV: Voltage condition and cell identifier
• peak (pA): Peak current (pA)
• peak time (ms): Time at which the peak current was reached (ms)
• max rise (pA/ms): Maximal slope of the rise of light-induced currents(pA/ms) and mean values and sem below
• tdecay (ms): Decay time of light-induced currents(ms) and mean values and sem below
• S. E.: Standard error of the decay time parameter
• Correlation: Correlation values of the decay time fit

File: Figure_S11c-d.csv

Description: Representative photocurrents of RoCK2.1 and WiChR-mScarlet in cardiomyocytes.

Variables

• Figure S11c: Figure identifier
• Time (ms)(1): Time (ms) of the experiment
• Current (pA)(1): Photocurrents in a RoCK-expressing cell
• Figure S11d: Figure identifier
• Time (ms)(2): Time (ms) of the experiment
• Current (pA)(2): Photocurrents in a RoCK-expressing cell

File: Figure_S11e-f.csv

Description: Quantification of normalized peak currents and number of transported charges (N = 2, n = 32 cells for RoCK and n = 12 cells for WiChR).

Variables

• Figure S11e: Figure identifier, description of parameter described here, and experimental condition
• Cell #: Cell identifier
• RoCK-expressing cardiomyocytes(1): Peak currents normalized to the capacitance of the cell (pA/pF)
• WiChR-expressing cardiomyocytes(1): Peak currents normalized to the capacitance of the cell (pA/pF)
• Figure S11f: Figure identifier, description of parameter described here, and experimental condition
• cell #: Cell identifier
• RoCK-expressing cardiomyocytes(2): Number of transported charges normalized to the capacitance of the cell charges (pC/pF)
• WiChR-expressing cardiomyocytes(2): Number of transported charges normalized to the capacitance of the cell charges (pC/pF)

File: Figure_S1b.csv

Description: All points histogram of a five-channel patch activated by a saturating concentration of cAMP or cGMP. Cyclic cAMP-dependent open probability of the SthK C-helix channel (n = 8 experiments).

Variables

• Figure S1b: Figure identifier
• cGMP: Ligand used
• current (pA)(1): Current bin (pA), 0.1 pA bin width
• counts(1): respective counts of currents within the respective bin
• cAMP: Ligand used
• current (pA)(2) : Current bin (pA), 0.1 pA bin width
• counts(2): respective counts of currents within the respective bin
• dose response: Description of parameter described here
• Conc cAMP (M): concentration of cAMP (M)
• Mean: Mean value of normalized currents activated at the respective cAMP concentration
• SD: Standard deviation

File: Figure_S1a.csv

Description: SthK C-helix(cGMP) currents of an excised inside-out patch in the absence or the presence of cGMP or cAMP.

Variables

• Figure S1a: Figure identifier
• cGMP: ligand used
• time (msec)(1): experiment time (ms)
• current trace( pA)(1): Current response (pA) during activation with cGMP
• cAMP: ligand used
• time (msec)(2): experiment time (ms)
• current trace( pA)(2): Current response (pA) during activation with cAMP
• Control: no ligand present, control
• time (msec)(3): experiment time (ms)
• current trace( pA)(3): Current response (pA) in the absence of ligand

File: Figure_S1d.csv

Description: Mean ratio of fractional current IcGMP/IcAMP (after background subtraction, n=6

experiments, saturating concentrations of cAMP and cGMP).

Variables

• Figure S1d: Figure identifier
• exp: Experiment identifier and mean and SD indicatior
• C helix IcGMP/IcAMP: Ratio of currents IcGMP/IcAMP for individual cells and mean values and Standard deviation.

File: Figure_S1c.csv

Description: Current responses of an excised inside-out patch containing many SthK C-helix channels. The patch was superfused with either control solution, 1 mM cGMP or with 1 mM cAMP.

Variables

• Figure S1c: Figure identifier
• control: no ligand present, control
• Time(s)(1): experiment time (ms)
• current(pA)(1): Current response (pA) in the absence of ligand
• cGMP: 1 mM cGMP present
• Time(s)(2): experiment time (ms)
• current(pA)(2): Current response (pA) in 1 mM cGMP
• cAMP: 1 mM cAMP present
• Time(s)(3): experiment time (ms)
• current(pA)(3): Current response (pA) in 1 mM cAMP

File: Figure_S3b.csv

Description: Maximal open probability Pomax for SthK1.0, SthK1.1, SthK2.0 and SthK2.1 for cAMP and cGMP.

Variables

• Figure S3b: Figure identifier
• max open probability of mutants: parameter displayed here
• SthK 1.0(cGMP): max open probability of the particular mutant in saturating concentrations of cGMP and mean values with standard deviation thereof
• SthK 1.0(cAMP): max open probability of the particular mutant in saturating concentrations of cAMP and mean values standard deviation thereof
• SthK 1.1(cGMP): max open probability of the particular mutant in saturating concentrations of cGMP and mean values standard deviation thereof
• SthK 1.1(cAMP): max open probability of the particular mutant in saturating concentrations of cAMP and mean values standard deviation thereof
• SthK 2.0(cGMP): max open probability of the particular mutant in saturating concentrations of cGMP and mean values standard deviation thereof
• SthK 2.0(cAMP): max open probability of the particular mutant in saturating concentrations of cAMP and mean values standard deviation thereof
• SthK 2.1(cGMP): max open probability of the particular mutant in saturating concentrations of cGMP and mean values standard deviation thereof
• SthK 2.1(cAMP): max open probability of the particular mutant in saturating concentrations of cAMP and mean values standard deviation thereof

File: Figure_S3c.csv

Description: All points histograms (top) and corresponding currents (bottom) of patches activated by saturating concentration of cNMPs for SthK1.0.

Variables

• Figure S3c: Figure identifier
• cGMP: ligand used
• Time ms: Experiment time (ms)
• current pA: Current (pA)
• current bin pA: Current bin (pA), 0.1 pA bin width
• Counts x 100(cGMP): Counts of currents within the respective current bin
• cAMP: ligand used
• Time ms: Experiment time (ms)
• current pA: Current (pA)
• current bin pA: Current bin (pA), 0.1 pA bin width
• counts x 100(cAMP): Counts of currents within the respective current bin

File: Figure_S3d.csv

Description: All points histograms (top) and corresponding currents (bottom) of patches activated by saturating concentration of cNMPs for SthK1.1.

Variables

• Figure S3d: Figure identifier
• cGMP: ligand used
• Time ms: Experiment time (ms)
• current pA: Current (pA)
• current bin pA: Current bin (pA), 0.1 pA bin width
• Counts x 100(cGMP): Counts of currents within the respective current bin
• cAMP: ligand used
• Time ms: Experiment time (ms)
• current pA: Current (pA)
• current bin pA: Current bin (pA), 0.1 pA bin width
• counts x 100(cAMP): Counts of currents within the respective current bin

File: Figure_S3e.csv

Description: All points histograms (top) and corresponding currents (bottom) of patches activated by saturating concentration of cNMPs for SthK2.0.

Variables

• Figure S3e: Figure identifier
• cGMP: ligand used
• Time ms: Experiment time (ms)
• current pA: Current (pA)
• current bin pA: Current bin (pA), 0.1 pA bin width
• Counts x 100(cGMP): Counts of currents within the respective current bin
• cAMP: ligand used
• Time ms: Experiment time (ms)
• current pA: Current (pA)
• current bin pA: Current bin (pA), 0.1 pA bin width
• counts x 100(cAMP): Counts of currents within the respective current bin

File: Figure_S3f.csv

Description: All points histograms (top) and corresponding currents (bottom) of patches activated by saturating concentration of cNMPs for SthK2.1.

Variables

• Figure S3f: Figure identifier
• cGMP: ligand used
• Time ms: Experiment time (ms)
• current pA: Current (pA)
• current bin pA: Current bin (pA), 0.1 pA bin width
• Counts x 100(cGMP): Counts of currents within the respective current bin
• cAMP: ligand used
• Time ms: Experiment time (ms)
• current pA: Current (pA)
• current bin pA: Current bin (pA), 0.1 pA bin width
• counts x 100(cAMP): Counts of currents within the respective current bin

File: Figure_S5a_d.csv

Description: Characterization of RoCK1.0, RoCK1.1, RoCK2.0, and RoCK2.1 in ND7/23 cells. Normalized currents and maximal rise slopes determined at different light intensities to activate the respective RoCK tool. For some cells at the lowest intensities, the current rise was too small for an analysis. Those events are marked nd.

Variables

• Fig. S5 a, d: Figure identifier and illumination conditions
• RoCK1.0: RoCK variant used in the experiment
• Cell RoCK1.0: Cell identifier
• Capacity (pF)(RoCK1.0): capacity (in pF) of the particular cell
• Intensity (mW/mm2)(RoCK1.0): light intensity used
• Peak Current (pA)(RoCK1.0): Peak current (pA) of the experiment
• Rise Slope (5%-50%)(RoCK1.0): maximal slope (pA/ms) of the current rise of the experiment
• RoCK1.1: RoCK variant used in the experiment
• Cell RoCK1.1: Cell identifier
• Capacity (pF)(RoCK1.1): capacity (in pF) of the particular cell
• Intensity (mW/mm2)(RoCK1.1): light intensity used
• Peak Current (pA)(RoCK1.1): Peak current (pA) of the experiment
• Rise Slope (5%-50%)(RoCK1.1): maximal slope (pA/ms) of the current rise of the experiment
• RoCK2.0: RoCK variant used in the experiment
• Cell RoCK2.0: Cell identifier
• Capacity (pF)(RoCK2.0): capacity (in pF) of the particular cell
• Intensity (mW/mm2)(RoCK2.0): light intensity used
• Peak Current (pA)(RoCK2.0): Peak current (pA) of the experiment
• Rise Slope (5%-50%)(RoCK2.0): maximal slope (pA/ms) of the current rise of the experiment
• RoCK2.1: RoCK variant used in the experiment
• Cell RoCK2.1: Cell identifier
• Capacity (pF)(RoCK2.1): capacity (in pF) of the particular cell
• Intensity (mW/mm2)(RoCK2.1): light intensity used
• Peak Current (pA)(RoCK2.1): Peak current (pA) of the experiment
• Rise Slope (5%-50%)(RoCK2.1): maximal slope (pA/ms) of the current rise of the experiment

File: Figure_S5e-f.csv

Description: Action spectra of RoCK2.1 for 10 ms flashes with equal photon density from 410 nm to 670

nm, in 20 nm steps and current density of RoCK2.1 elicited by a 10 ms light pulse at 525

nm (10.1 mW mm^-2 ) and a 50 ms light pulse at 595 nm (3.8 mW mm^-2 )

Variables

• Fig. S5 e, f: Figure identifier
• peak current (pA): parameter studied here
• Wavelength (nm): In the first row, the wavelength of the experiment is given, and cell identifier
• 410: Peak current (pA) at the particular wavelength
• 430: Peak current (pA) at the particular wavelength
• 450: Peak current (pA) at the particular wavelength
• 470: Peak current (pA) at the particular wavelength
• 490: Peak current (pA) at the particular wavelength
• 510: Peak current (pA) at the particular wavelength
• 530: Peak current (pA) at the particular wavelength
• 550: Peak current (pA) at the particular wavelength
• 570: Peak current (pA) at the particular wavelength
• 590: Peak current (pA) at the particular wavelength
• 610: Peak current (pA) at the particular wavelength
• 630: Peak current (pA) at the particular wavelength
• 650: Peak current (pA) at the particular wavelength
• 670: Peak current (pA) at the particular wavelength
• RoCK2.1 (10ms 525nm): Illumination conditions for comparison of green vs red illumination
• exp: cell identifier
• Peak Current (pA): Peak current (pA) of the particular cell at the given illumination conditions
• Capacity (pF): capacity of the particular cell

File: Figure_S7.csv

Description: Biophysical properties of cultured hippocampal neurons expressing RoCK2.1. Comparison of resting membrane potential and (b) membrane time constant (c) between RoCK2.1 expressing and control neurons.

Variables

• Figure S7: Figure identifier and experiment info
• Control: untransduced neurons were used for this dataset
• File Name: identifier
• A: parameter describing the initial amplitude of the fit
• S.E.(A): Standard error of the parameter
• Tau: membrane time constant tau (ms) of the particular cell
• S.E.(Tau): Standard error of the parameter
• Tau average (ms): average time constant tau (ms) from different experiments on the same cell
• C1: parameter describing the final amplitude of the fit
• S.E.(C1): Standard error of the parameter
• Resting Mem (mV): recorded resting membrane potential
• Resting Mem corr (mV): Resting membrane potential (mV) after liquid junction potential correction
• RoCK2.1: RoCK variant used in these experiments
• file name: identifier
• a: parameter describing the initial amplitude of the fit
• s.e.(a): Standard error of the parameter
• tau: membrane time constant tau (ms) of the particular cell
• s.e.(tau): Standard error of the parameter
• tau average: average time constant tau (ms) from different experiments on the same cell
• C: parameter describing the final amplitude of the fit
• S.E.(C): Standard error of the parameter
• Vm (mV): recorded resting membrane potential
• Vm corr: Resting membrane potential (mV) after liquid junction potential correction

File: Figure_S9.csv

Description: Relative cell dispersion, calculated as ratio of the thickness of the pyramidal cell layer of RoCK2.1-injected versus either non-injected (n=4 animals) or eGFP-injected (n=4 animals) controls of the contralateral side. Right panel: Relative GFAP intensity, calculated as ratio of mean greyscale values of regions of interest of the injected CA1 region versus either non-injected (n=4 animals) or eGFP-injected (n=4 animals) control values of the contralateral side.

Variables

• Figure S9: Figure identifier
• GFAP staining: description of parameter displayed here
• Animals 1-4: animal identifier
• GFAPRoCK/GFAPeGFP: Ratio of GFAP staining intensity of RoCK vs eGFP injected hippocampus
• Animals 5-8: animal identifier
• GFAPRoCK/GFAPuninjected: Ratio of GFAP staining intensity of RoCK vs uninjected hippocampus
• thickness d of the pyramidal cell layer: description of parameter displayed here
• animals 1-4: animal identifier
• mean(d)Rock/mean(d)eGFP: Ratio of thickness of the pyramidal cell layer of RoCK vs eGFP injected hippocampus
• animals 5-8: animal identifier
• mean(d)Rock/mean(d)uninjected: Ratio of thickness of the pyramidal cell layer of RoCK vs uninjected hippocampus