Authors: Rían W. Manville, Lorenzo Foglia, Ryan Yoshimura, Daniel E. Santos, Alvin Yu, and Geoffrey W. Abbott

Associated manuscript: PNAS

Data collection period: 2023 - 2026

Repository: Dryad Digital Repository

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The dataset contains Excel files exported from the original analysis workbooks. All formulas were replaced by the resulting values during CSV export. These files were used to generate the corresponding panels for figures 1, 2, 3, 6, 7, and 9 in this manuscript. Each filename is structured to correspond to the figure in the manuscript:

Figure Number & panel > Channel Investigated > Condition > Parameter Measured

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The repository contains data obtained from cellular electrophysiology recordings, radioligand-binding experiments, and mouse behavioral studies. We used two-electrode voltage clamp (TEVC) electrophysiology and the Xenopus laevis oocyte expression system to record the electrical activity of voltage-gated potassium (Kv) channels Kv7.2, Kv7.3, Kv7.3* (expression-enhancing Kv7.3-A315T construct), and Kv7.2/3 channels in response to carnosic acid. Additionally, we generated Kv7.3 and Kv7.2 mutations to ascertain the binding site and selectivity profile of carnosic acid. Xenopus laevis oocytes were injected with cRNA encoding for the following human potassium channels (genes encoding these channels denoted in brackets):

• Kv7.2/3 (KCNQ2/3)
• Kv7.2 (KCNQ2)
• Kv7.3* (KCNQ3)

All injected oocytes were incubated at 16°C for 24–96 h before TEVC recordings. The subsequent measurements were used to characterize the biophysical responses of these channels to carnosic acid.

The parameters measured as part of this study are as follows:

Gmax

These data were used to generate conductance-voltage curves (Gmax). Conductance-voltage curves were generated by normalizing the raw value of the tail current (recorded at -30 mV) to the peak tail-current amplitude. These conductance-voltage curves were then plotted as a function of the prepulse voltage and fitted with a single Boltzmann function:

g=A1+((A2-A1))/{1 + exp [(V(1/2)-V)/Vs]}

g: normalized tail-current amplitude, used as a measure of normalized conductance

(A1): lower asymptote

(A2): upper asymptote

(V): prepulse voltage

(V{1/2}): voltage of half-maximal activation

(Vs): slope factor

Defined column headings included for these CSV files

• Voltage (mV) - Prepulse voltage applied during the activation protocol
• Control - Normalized conductance from biological replicate/oocyte 1 under control conditions
• 10 or 100 uM Carnosic acid - Normalized conductance from the same oocyte after 10 or 100 uM carnosic acid
• Mean - Arithmetic mean across biological replicates
• SEM - Standard error of the mean
• n - Number of biologically independent oocytes

The datasets pertaining to these measurements can be found in the following CSV files as part of this submission:

• Figure_2F_KCNQ3-S228A_10_uM_Carnosic_acid_-_Gmax.csv.xlsx
• Figure_2H_KCNQ3-R242A_10_uM_Carnosic_acid_-_Gmax.csv.xlsx
• Figure_2H_KCNQ3-R243A_10_uM_Carnosic_acid_-_Gmax.csv.xlsx
• Figure_2J_KCNQ3-R236A_10_uM_Carnosic_acid_-_Gmax.csv.xlsx
• Figure_2K_KCNQ3-L226A_10_uM_Carnosic_acid_-_Gmax.csv.xlsx

• Figure_3B_KCNQ3-L226F_10_uM_Carnosic_acid_-_Gmax.csv.xlsx
• Figure_3B_KCNQ3-L226V_10_uM_Carnosic_acid_-_Gmax.csv.xlsx
• Figure_3B_KCNQ3-L226W_10_uM_Carnosic_acid_-_Gmax.csv.xlsx

• Figure_3F_KCNQ3-L226Q_10_uM_Carnosic_acid_-_Gmax.csv.xlsx
• Figure_3F_KCNQ3-L226S_10_uM_Carnosic_acid_-_Gmax.csv.xlsx

• Figure_3H_KCNQ3-L226E_10_uM_Carnosic_acid_-_Gmax.csv.xlsx
• Figure_3H_KCNQ3-L226R_10_uM_Carnosic_acid_-_Gmax.csv.xlsx

• Figure_6E_KCNQ3L222F,-S225-A-L226_100_uM_Carnosic_acid_-_Gmax.csv.xlsx

• Figure_6E_KCNQ3-S225-A-L226_100_uM_Carnosic_acid_-_Gmax.csv.xlsx
• Figure_7D_KCNQ2-Q3_100_uM_Carnosic_acid_-_Gmax.csv.xlsx

• Figure_7F_KCNQ2-A196del_100_uM_Carnosic_acid_-_Gmax.csv.xlsx
• Figure_7F_KCNQ2-F192L__A196del_100_uM_Carnosic_acid_-_Gmax.csv.xlsx
• Figure_7F_KCNQ2-F192L_100_uM_Carnosic_acid_-_Gmax.csv.xlsx

• Figure_9C_KCNQ3_5_uM_Carnosic_acid_-_Gmax.csv.xlsx
• Figure_9F_KCNQ2-Q3_100_uM_Carnosic_acid_-_Gmax.csv.xlsx

• Figure_9I_KCNQ3_5_uM_Retigabine__5_uM_Retigabine___5_uM_Carnosic_acid_-_Gmax.csv.xlsx
• Figure_9L_KCNQ2-Q3_5_uM_Retigabine__5_uM_Retigabine___5_uM_Carnosic_acid_-_Gmax.csv.xlsx

Resting membrane potential
The resting membrane potential (E_M) is the electrical potential difference across a cell's membrane at rest. The RMP is determined by the concentration of ions across the membrane and the membrane permeability to each type of ion. Here, we measured the RMP (E_M) of unclamped Xenopus laevis oocytes expressing the above channels and reported the values in millivolts (mV).

Defined column headings included for these CSV files

• Voltage (mV) - Prepulse voltage applied during the activation protocol
• Control - Resting membrane potential from biological replicate/oocyte 1 under control conditions
• 10 or 100 uM Carnosic acid - Resting membrane potential from the same oocyte after 10 or 100 uM carnosic acid
• Mean - Arithmetic mean across biological replicates
• SEM - Standard error of the mean
• n - Number of biologically independent oocytes

The datasets pertaining to these measurements can be found in the following CSV files as part of this submission:

• Figure_6F_KCNQ3-L222F__S225-A-L226_100_uM_Carnosic_acid_-_EM.csv.xlsx

• Figure_6F_KCNQ3-S225-A-L226_100_uM_Carnosic_acid_-_EM.csv.xlsx

• Figure_7H_KCNQ2_Mutants_100_uM_Carnosic_acid_-_Delta_EM_Shift.csv.xlsx

• Figure_9D_KCNQ3_5_uM_Carnosic_acid_-_EM.csv.xlsx

• Figure_9G_KCNQ2-Q3_100_uM_Carnosic_acid_-_EM.csv.xlsx

• Figure_9J_KCNQ3_5_uM_Retigabine__5_uM_Retigabine___5_uM_Carnosic_acid_-_EM.csv.xlsx

• Figure_9M_KCNQ2-Q3_5_uM_Retigabine__5_uM_Retigabine___5_uM_Carnosic_acid_-_EM.csv.xlsx

Dose responses (ΔV_0.5 shift) (mV)
These data were used to determine the potency and efficacy of carnosic acid at Kv7.3*, Kv7.3-L226A and Kv7.3-R236A channels. Carnosic acid was applied to Xenopus laevis oocytes expressing these channels at concentrations ranging from 0.01 - 100 uM. The tail current was then measured (as described above). All raw values are in millivolts (mV). The control V_0.5 was subtracted from the V_0.5 measured at each carnosic acid concentration. Negative delta V_0.5 values represent hyperpolarizing shifts:

ΔV_0.5​ = V_0.5,drug​ − V_0.5, control​

Defined column headings included for these CSV files

• Voltage (mV) - Prepulse voltage applied during the activation protocol
• Control - Normalized conductance from biological replicate/oocyte 1 under control conditions
• 0.01-100 uM Carnosic acid - Normalized conductance from the same oocyte after carnosic acid
• Mean - Arithmetic mean across biological replicates
• SEM - Standard error of the mean
• n - Number of biologically independent oocytes

The datasets pertaining to these measurements can be found in the following CSV files as part of this submission:

• Figure_1E-G_KCNQ3_Carnosic_acid_Dose_Response_-_Gmax.csv.xlsx

• Figure_2M_KCNQ3-R236A_Carnosic_acid_Dose_Response_-_Gmax.csv.xlsx

• Figure_2N_KCNQ3-L226A_Carnosic_acid_Dose_Response_-_Gmax.csv.xlsx

Delta V_0.5 shift: Mutants vs WT
These graphs were generated by taking the V_0.5 shifts obtained from the Gmax of Kv7.3* alanine mutant channels. The mutant V_0.5 values were then subtracted from the V_0.5 value for WT Kv7.3* channels:

Δ_V0.5 = V_0.5,mutant ​− V_0.5, wild type​

These values were then displayed as a scatter graph. These data enable us to observe the effect Kv7.3* alanine mutations have on the voltage-dependence of activation compared to wild-type Kv7.3*. This informs us how these alanine mutations change the ability of the channel to open in response to changes in voltage across the cell membrane. All values are in millivolts (mV). The datasets pertaining to these measurements can be found in the following CSV files as part of this submission:

• Figure_2A_KCNQ3_Alanine_Mutants_-_Delta_Voltage-dependence_Shift_vs_WT.csv.xlsx

• Figure_3C_Baseline_Voltage-dependence_Shift_vs_KCNQ3.csv.xlsx

• Figure_3I_Baseline_Voltage-dependence_Shift_vs_KCNQ3.csv.xlsx

Delta V_0.5 shift: Control vs 10 uM Carnosic acid

These graphs were generated by taking the V_0.5 shifts obtained from the Gmax of Kv7.3* alanine mutant channels in control solution and after application of 10 uM carnosic acid. These data were then displayed as a scatter plot. These data enable us to observe the effect of carnosic acid on the voltage-dependence of activation on the Kv7.3* alanine mutant channels. All values are in millivolts (mV). The datasets pertaining to these measurements can be found in the following CSV files as part of this submission:

• Figure_2B_KCNQ3_Alanine_Mutants_-_10_uM_Carnosic_acid_Delta_Voltage-dependence_Shift.csv.xlsx
• Figure_3D_10_uM_Carnosic_acid_-_Delta_Voltage-dependence_Shift.csv.xlsx

• Figure_7G_KCNQ2_Mutants_100_uM_Carnosic_acid_-_Delta_Voltage-dependence_Shift.csv.xlsx

Tritiated GABA [3H GABA] Binding

These graphs were generated by assessing the radioactivity (Counts Per Minute; CPM) of cells injected with either water or Kv7.3* after incubation with tritiated GABA. Cells injected with Kv7.3* were either incubated in tritiated GABA alone or with 100 uM non-radioactive carnosic acid. These data help to inform whether GABA and carnosic acid are competing for the same binding site on the Kv7.3* channel. All values are in counts per minute (CPM).

Defined column headings included for these CSV files

• Water-injected - Radioactivity measured after 30 minutes incubation with [3H GABA]. Values are defined as counts per minute (CPM)
• Kv7.3* [3H GABA] - Radioactivity measured after 30 minutes incubation with [3H GABA]. Values are defined as counts per minute (CPM)
• Kv7.3* [3H GABA] + 100 uM Carnosic acid - Radioactivity measured after 30 minutes incubation with [3H GABA] + 100 uM carnosic acid. Values are defined as counts per minute (CPM)

The datasets pertaining to these measurements can be found in the following CSV files as part of this submission:

• Figure_9A_Tritiated_GABA_vs_Carnosic_acid.csv.xlsx

Mouse Behavioral Studies
Chemoconvulsant seizure studies were conducted on mice using pentylenetetrazol (PTZ). Mice were injected with either vehicle (DMSO), carnosic acid, retigabine, or carnosic acid + retigabine 20 minutes prior to being injected with PTZ. Mice were then assessed for time to first clonic (seconds) or tonic seizure (seconds). Additionally, survival was monitored for 60 minutes after PTZ injection. Scores of 1 "survival" or 0 "deceased" were used. The data collected from these experiments help us assess whether carnosic acid and retigabine can synergize and protect against PTZ-induced seizures.

Defined column headings included for these CSV files

• Vehicle - Mice injected intraperitoneally with DMSO
• CA 1 mg/kg - Mice injected intraperitoneally with 1 mg/kg carnosic acid
• RET 10 mg/kg - Mice injected intraperitoneally with 10 mg/kg retigabine
• CA + RET - Mice injected intraperitoneally with a combination of 1 mg/kg carnosic acid and 10 mg/kg retigabine

The datasets pertaining to these measurements can be found in the following CSV files as part of this submission:

• Figure_9N_Time_to_first_clonic_seizure.csv.xlsx
• Figure_9O_Time_to_first_tonic_seizure.csv.xlsx
• Figure_9P_Survival_Curves.csv.xlsx

Statistics

Paired two-tailed Student’s t-tests were used for within-oocyte comparisons between control and drug conditions. One-way ANOVA followed by Dunnett’s multiple-comparisons test was used when multiple treatments or mutants were compared with a single control. Statistical results are reported in the associated manuscript; the deposited files contain the underlying observations only.

Additional Information

'n.a.' indicates that a measurement was not applicable and was therefore not collected.

Software required
The deposited files can be opened using any plain-text editor or spreadsheet/data-analysis software. GraphPad Prism was used for statistical analysis and graphing. Clampfit version 11.4 was used to analyze electrophysiological recordings. No proprietary software is required to access the deposited files.