DOI: 10.5061/dryad.k98sf7mm2
title: Optogenetic silencing by combining a rhodopsin cyclase with an engineered cGMP-gated potassium channel

description: Since the advent of optogenetics, significant progress has been made in
 developing tools to modulate and detect cellular activity using light. We
 present a novel two-component optogenetic silencing tool, RoCK (Rhodopsin
 Cyclase/K+ channel), which pairs the rhodopsin guanylyl cyclase
 CaRhGC with customized SthK K⁺ channels that are engineered to open
 selectively upon cGMP binding. By enhancing the cGMP sensitivity and open
 probability of SthK mutants, we obtained four channel variants suited for
 different levels of cGMP concentration. CaRhGC’s membrane-bound nature
 enables localized cGMP production, and the lack of dark activity reduces
 the risk for off-target effects. Optimized RoCK effectively modulated
 cellular activity in mouse hippocampal neurons, in acute hippocampal
 slices, and in rabbit cardiomyocytes. In zebrafish, RoCK silenced motor
 neurons in vivo, suppressing the characteristic coiling behavior of
 embryos, thus highlighting its potential for behavioral studies. In
 summary, RoCK expands our optogenetic toolkit threefold for fast
 cGMP-production, fast cGMP-sensing, and K⁺-based cell silencing.
description: # Optogenetic silencing by combining a rhodopsin cyclase with an
 engineered cGMP-gated potassium channel Dataset DOI:
 [10.5061/dryad.k98sf7mm2](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*L^n^/(K1/2^n^+L^n^) 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 10^5^): 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/cm^2^). 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/mm^2^)(1):
 illumination intensity (mW/mm^2^) * 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/mm^2^) *
 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/mm^2^) * 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/mm^2^) * 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/mm^2^) * 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](cGMP): Experiment time (ms) * current [pA](cGMP): Current
 (pA) * current bin [pA](cGMP): 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](cAMP): Experiment time (ms) * current [pA](cAMP):
 Current (pA) * current bin [pA](cAMP): 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](cGMP): Experiment time (ms) *
 current [pA](cGMP): Current (pA) * current bin [pA](cGMP): 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](cAMP): Experiment
 time (ms) * current [pA](cAMP): Current (pA) * current bin [pA](cAMP):
 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](cGMP): Experiment time (ms) * current [pA](cGMP): Current
 (pA) * current bin [pA](cGMP): 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](cAMP): Experiment time (ms) * current [pA](cAMP):
 Current (pA) * current bin [pA](cAMP): 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](cGMP): Experiment time (ms) *
 current [pA](cGMP): Current (pA) * current bin [pA](cGMP): 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](cAMP): Experiment
 time (ms) * current [pA](cAMP): Current (pA) * current bin [pA](cAMP):
 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