Conifer metabolite pisiferic acid restores activity in human Kv1.2 potassium channels carrying pathogenic sequence variants
Data files
Jul 16, 2025 version files 1.46 MB
-
Figure_2A__B_-_Kv1.2_WT_-_IV.xlsx
16.82 KB
-
Figure_2A__B_-_Kv1.2-F333V__T374A__T401I___P405S_-_IV.xlsx
39.82 KB
-
Figure_2A__B_-_Kv1.2-H18R__R147K___I177V_-_IV.xlsx
33.06 KB
-
Figure_2A__B_-_Kv1.2-H196R__Q214K__I263T___S289P_-_IV.xlsx
40.26 KB
-
Figure_2A__B_-_Kv1.2-P405L__V408A__R419Q__N488S_-_IV.xlsx
40.34 KB
-
Figure_2A__B_-_Kv1.2-R294H__R297Q__L298F___H310R_-_IV.xlsx
40.77 KB
-
Figure_2C__D_-_Kv1.2_WT_-_Gmax.xlsx
19.19 KB
-
Figure_2C__D_-_Kv1.2-F333V__T374A__T401I___P405S_-_Gmax.xlsx
45.20 KB
-
Figure_2C__D_-_Kv1.2-H18R__R147K___I177V_-_Gmax.xlsx
44.18 KB
-
Figure_2C__D_-_Kv1.2-H196R__Q214K__I263T___S289P_-_Gmax.xlsx
49.60 KB
-
Figure_2C__D_-_Kv1.2-P405L__V408A__R419Q__N488S_-_Gmax.xlsx
46.35 KB
-
Figure_2C__D_-_Kv1.2-R294H__R297Q__L298F___H310R_-_Gmax.xlsx
50.72 KB
-
Figure_4A__B_-_Kv1.2_WT_12_uM_Pisiferic_acid_-_IV.xlsx
26.34 KB
-
Figure_4A__C_-_Kv1.2_WT_12_uM_Pisiferic_acid_-_Gmax.xlsx
33.49 KB
-
Figure_4E_-_Kv1.2_WT_12_uM_Pisiferic_acid_-_Activation.xlsx
23.64 KB
-
Figure_4F__G_-_Kv1.2_WT_12_uM_Pisiferic_acid_-_Deactivation.xlsx
23.81 KB
-
Figure_5A__B__E_-_Mutant_Baseline_vs_WT_-_Current_Magnitude_-20_mV.xlsx
9.84 KB
-
Figure_5A__B__F_-_Kv1.2___Mutants_12_uM_Pisiferic_acid_-_Fold_increase_-20_mV.xlsx
10.18 KB
-
Figure_5A__C__G_-_Mutants_Baseline_vs_WT_-_V0.5_Shift.xlsx
9.68 KB
-
Figure_5A__C__H_-_Kv1.2___Mutants_12_uM_Pisiferic_acid_-_V0.5_Shift.xlsx
9.81 KB
-
Figure_6A__B_-_Kv1.1-Kv1.2_WT_-_IV.xlsx
16.40 KB
-
Figure_6A__B_-_Kv1.2-F333V__P405L__P405S__R419Q___N488S_-_IV.xlsx
49.24 KB
-
Figure_6A__B_-_Kv1.2-H18R__R147K__I177V___H196R_-_IV.xlsx
40.20 KB
-
Figure_6A__B_-_Kv1.2-Q214K__I263T__S289P__R294H___H310R_-_IV.xlsx
49.69 KB
-
Figure_6C__D_-_Kv1.1-Kv1.2_WT_-_Gmax.xlsx
18.40 KB
-
Figure_6C__D_-_Kv1.2-F333V__P405L__P405S__R419Q___N488S_-_Gmax.xlsx
61.05 KB
-
Figure_6C__D_-_Kv1.2-H18R__R147K__I177V___H196R_-_Gmax.xlsx
53.88 KB
-
Figure_6C__D_-_Kv1.2-Q214K__I263T__S289P__R294H___H310R_-_Gmax.xlsx
65.83 KB
-
Figure_7A__B__E_-_Mutant_Baseline_vs_WT_-_Current_Magnitude_-20_mV.xlsx
9.89 KB
-
Figure_7A__B__F_-_Kv1.2___Mutants_12_uM_Pisiferic_acid_-_Fold_increase_-20_mV.xlsx
10.08 KB
-
Figure_7A__C__G_-_Mutants_Baseline_vs_WT_-_V0.5_Shift.xlsx
10.08 KB
-
Figure_7A__C__H_-_Kv1.2___Mutants_12_uM_Pisiferic_acid_-_V0.5_Shift.xlsx
10.13 KB
-
Figure_8A__B_-_Kv1.2_WT___Mutants_12_uM_Pisiferic_acid_-_Inactivation.xlsx
155.07 KB
-
Figure_9D__E_-_Kv1.2-E183A_12_uM_Pisiferic_acid_-_Gmax.xlsx
36.80 KB
-
Figure_9D__E_-_Kv1.2-L293A_12_uM_Pisiferic_acid_-_Gmax.xlsx
35.84 KB
-
Figure_9D__E_-_Kv1.2-R189A_12_uM_Pisiferic_acid_-_Gmax.xlsx
39.74 KB
-
Figure_9D__E_-_Kv1.2-R294A_12_uM_Pisiferic_acid_-_Gmax.xlsx
40.24 KB
-
Figure_9D__E_-_Kv1.2-R297A_12_uM_Pisiferic_acid_-_Gmax.xlsx
35.58 KB
-
Figure_9F_-_Kv1.2_12_uM_Pisiferic_acid_-_RMP.xlsx
14.40 KB
-
Figure_9F_-_Kv1.2-E183A_12_uM_Pisiferic_acid_-_RMP.xlsx
15.22 KB
-
Figure_9F_-_Kv1.2-L293A_12_uM_Pisiferic_acid_-_RMP.xlsx
15.19 KB
-
Figure_9F_-_Kv1.2-R189A_12_uM_Pisiferic_acid_-_RMP.xlsx
15.14 KB
-
Figure_9F_-_Kv1.2-R294A_12_uM_Pisiferic_acid_-_RMP.xlsx
15.32 KB
-
Figure_9F_-_Kv1.2-R297A_12_uM_Pisiferic_acid_-_RMP.xlsx
15.21 KB
-
Figure_9G__H_-_Kv1.2_WT___Binding_Site_Mutants_12_uM_Pisiferic_acid_-_Delta_V0.5___RMP.xlsx
11.05 KB
-
README.md
7.87 KB
Abstract
Sequence variants in KCNA2, the gene encoding voltage-gated potassium channel Kv1.2, cause epilepsy, intellectual disability, and movement disorders. Drugs that directly correct mutant Kv1.2 function are lacking. Kv1.2 downregulation is also implicated in pain and amyotrophic lateral sclerosis (ALS). We recently found that the abietane diterpenoid pisiferic acid (PA) from conifer Chamaecyparis pisifera beneficially restores activity in pathogenic loss-of-function (LOF)-variant Kv1.1 channels. Here, using cellular electrophysiology, we classified 19 human Kv1.2 gene variants (pathogenic or of unknown significance) into LOF, gain of function (GOF), or mixed LOF/GOF. By hyperpolarizing their voltage dependence of activation, PA improved function in 13/13 LOF and 1/1 LOF/GOF pathogenic Kv1.2 variants tested, using cRNA ratios representative of autosomal dominant KCNA2 disorders. In silico docking, mutagenesis, and electrophysiology identified a PA binding site in the Kv1.2 voltage sensor. Given its in vitro efficacy and low preclinical toxicity, PA is a promising lead compound for Kv1.2 LOF disorders.
Conifer metabolite pisiferic acid restores activity in human Kv1.2 potassium channels carrying pathogenic sequence variants
The datasets included are the original Excel files used to generate each panel for figures 2-9 in this manuscript. The title of each Excel file is labeled to directly correspond to the panel of each figure in the manuscript:
Figure Number & panel > Channel Investigated (including mutant and wild type (WT)) > Condition > Parameter Measured.
Example:
Figure 2A, B - Kv1.2 12 uM Pisiferic acid - IV.xlsx
The data contained within this repository are those obtained from cellular electrophysiology recordings. We used two-electrode voltage clamp (TEVC) electrophysiology and the Xenopus laevis oocyte expression system to record the electrical activity of wild-type Kv1.2 and Kv1.1/Kv1.2 channels in response to pisiferic acid. We also studied nineteen Kv1.2 disease-linked mutations. Using TEVC, we biophysically characterized homomeric homozygous patient Kv1.2 mutants versus wild-type Kv1.2 to establish whether the mutants were gain-of-function (GOF), loss-of-function (LOF), or a combination of both (Figures 2 and 3). Next, we investigated whether 12 uM pisiferic acid could rescue some or all of the homomeric homozygous patient Kv1.2 mutants' function using TEVC. We then biophysically characterized heterozygous Kv1.2 mutant heteromeric Kv1.1/Kv1.2 channels versus Kv1.1/Kv1.1 heteromers and tested whether 12 uM pisiferic acid could rescue some or all function. Finally, guided by in-silico docking of pisiferic acid to the AlphaFold predicted structure of Kv1.2 and site-directed mutagenesis, we uncovered the binding site of pisiferic acid on Kv1.2. These investigations were conducted using Xenopus laevis oocytes injected with cRNA encoding for Kv1.2, Kv1.1/Kv1.2, and Kv1.2 mutant channels. Xenopus oocytes were incubated at 16 degrees for 24-48 hrs prior to recording using TEVC. To study the action of pisiferic acid, we made a 250 mM stock solution in dimethyl sulfoxide (DMSO), which was diluted to a 12 uM working concentration in 4 mM extracellular potassium recording solution on the day of each experiment.
The parameters measured to characterize the biophysical properties of Kv1.2 mutants and the action of pisiferic acid were as follows:
Current-voltage (IV) curve
This is a graph representing the relationship between the electrical current (flow of ions) and voltage applied across a device (the cell membrane). In electrophysiology, I-V curves are used to study the activity of biological cells, in this case Xenopus oocytes expressing wild-type and mutant channels. The data contained in the I-V curve Excel files were measured from the peak of the prepulse current generated by a voltage protocol that starts at a holding potential of -80 mV and starts from -120/-80 and increases in +10 mV increments until +40 mV. All raw values are in microamps (uA).
Gmax
These data were used to generate conductance-voltage curves. Graphs were generated by taking measurements from the tail current (recorded at -40 mV) immediately following the prepulse current as described above. For channels without a discernible tail current, Gmax graphs were plotted from the IV, correcting for driving force and normalizing to the peak conductance. These data enable us to determine the shift in voltage-dependence of activation of the channel in response to the plant extracts and pisiferic acid. Non-normalized values are in microamps (uA).
Activation
These data were used to determine the time constant of activation in response to plant extracts or pisiferic acid. The activation kinetics were fitted by selecting the currents generated from voltages between -30 to +40 mV. Two cursors were used. The first was set at the beginning of the current trace at each of these voltages, the start of the channel opening. The second was placed where the current plateaued, where steady-state current was achieved. The current traces were then fitted with a single exponential term to measure the change in channel opening. All raw values are in milliseconds (ms).
Deactivation
These data were used to determine the time constant of deactivation in response to pisiferic acid. The deactivation kinetics were fitted by selecting the currents generated from voltages between -120 to -60 mV. Two cursors were used. The first was set at the beginning of the current trace at each of these voltages, the start of the channel closing. The second was placed where the current plateaued, the end of channel closure. The current traces were then fitted with a single exponential term to measure the change in channel closing. All raw values are in milliseconds (ms).
Resting membrane potential
The resting membrane potential (RMP) 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 (EM) of unclamped Xenopus laevis oocytes expressing the above channels and reported the values in millivolts (mV).
Current magnitude comparisons: Mutants vs WT
These graphs were generated by taking the currents recorded at either -30 or -20 mV from the I-V curves generated for Kv1.2 mutant channels. The change in current magnitude was then represented as a fraction of the WT current at the same voltages. These values were then displayed as a bar graph. These measurements enable us to observe what effect the different Kv1.2 mutations have on the ability of the channel to generate currents compared to wild-type. All raw values are in microamps (uA).
Current magnitude comparisons: Control vs 12 uM Pisiferic acid
These graphs were generated by taking the currents recorded at either -30 or -20 mV from the I-V curves generated for Kv1.2 mutant channels in control solution and after application of 12 uM pisiferic acid. The change in current magnitude was then calculated by Idrug/Icontrol, giving us the fold change. These values were then displayed as a bar graph. These measurements enable us to observe whether pisiferic acid can increase the currents of different Kv1.2 mutations, rescuing the effect of the EA1 mutations on channel current density at these voltages. All raw values are in microamps (uA).
V0.5 shift: Mutants vs WT
These graphs were generated by taking the V0.5 shifts obtained from the Gmax of Kv1.2 mutant channels. The mutant V0.5 values were then subtracted from the V0.5 value for WT channels. These values were then displayed as a bar graph. These data enable us to observe the effect the Kv1.2 mutations have on the voltage-dependence of activation compared to wild-type. This informs us how these Kv1.2 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).
V0.5 shift: Control vs 12 uM Pisiferic acid
These graphs were generated by taking the V0.5 shifts obtained from the Gmax of Kv1.2 mutant channels in 4 mM extracellular potassium solution and after application of 12 uM pisiferic acid. These data were then displayed as a bar graph. These data enable us to observe the effect of pisiferic acid on the voltage-dependence of activation on the Kv1.2 mutant channels. All values are in millivolts (mV).
Statistics
All statistical analyses were conducted as either a paired t-test, one-way ANOVA, or two-way ANOVA with either Dunnett's or Bonferroni corrections for multiple comparisons.
Additional Information
Excel files with cells with 'n.a' means not applicable. No data was obtained for this cell or voltage.
Software version numbers
Clampex 8.2
Clampfit 11.4
Microsoft Excel 365
GraphPad Prism 10.5.0
Data License: This dataset is shared under a CC0 1.0 Public Domain Dedication.
Channel subunit cRNA preparation and Xenopus laevis oocyte injection
Wild-type Kv1.1 and wild-type and mutant Kv1.2 cDNAs were generated (Genscript, Piscataway, NJ) in the pMAX and pTLNx expression vectors. As previously described, we generated cRNA transcripts encoding human Kv1.2 (wild-type and mutant) by in vitro transcription using the mMessage mMachine kit (Thermo Fisher Scientific), after vector linearization, from cDNA subcloned into plasmids (pMAX) incorporating Xenopus laevis β-globin 5’ and 3’ UTRs flanking the coding region to enhance translation and cRNA stability. We injected defolliculated stage V and VI Xenopus laevis oocytes (Xenoocyte, Dexter, MI, US) with Kv1 cRNAs (0.1-10 ng). We incubated the oocytes at 16 oC in ND96 oocyte storage solution containing penicillin and streptomycin, with daily washing, for 1-2 days prior to two-electrode voltage-clamp (TEVC) recording.
Two-electrode voltage clamp (TEVC)
We performed TEVC at room temperature using an OC-725C amplifier (Warner Instruments, Hamden, CT) and pClamp10 software (Molecular Devices, Sunnyvale, CA) 2 days after cRNA injection as described in the section above and as before. For recording, oocytes we placed in a small-volume oocyte bath (Warner) and viewed with a dissection microscope. We sourced chemicals from Combi Blocks. We studied the effects of pisiferic acid, which was solubilized in DMSO at a stock concentration of 250 mM before being diluted to 12 µM in bath solution (in mM): 96 NaCl, 4 KCl, 1 MgCl2, 1 CaCl2, 10 HEPES (pH 7.6). We introduced pisiferic acid into the oocyte recording bath by gravity perfusion at a constant flow of 1 ml per minute for 3 minutes prior to recording. Pipettes were of 1-2 MΩ resistance when filled with 3 M KCl. We recorded currents in response to voltage pulses between -80 mV and +40 mV at 10 mV intervals from a holding potential of -80 mV, to yield current-voltage relationships and examine activation kinetics. To calculate the voltage dependence of activation (V0.5), tail currents were recorded at a voltage pulse of -40 mV immediately following prepulse voltages between -80 mV and +40 mV. We analyzed data using Clampfit (Molecular Devices) and Graphpad Prism software (GraphPad, San Diego, CA, USA), stating values as mean ± SEM. We plotted raw or normalized tail currents versus prepulse voltage and fitted them with a single Boltzmann function. To calculate the voltage dependence of inactivation, we recorded currents in response to 15-second pulses from -100 mV to + 40 mV in 10 mV increments, followed by a 100-ms pulse to +40 mV, from a holding potential of -80 mV. The current magnitude was measured from the 100-ms pulse to +40 mV, where all values were then normalized to the peak current.
In silico docking
For in silico ligand docking predictions of pisiferic acid binding to Kv1.2, we performed unguided docking to predict potential binding sites, using SwissDock with CHARMM force fields. We used AlphaFold to generate a predicted structure for Kv1.2. We prepared the channel structure for docking using DockPrep in UCSF Chimera https://www.rbvi.ucsf.edu/chimera), with which we also generated docking figures.
Statistics and Reproducibility
All values are expressed as mean ± SEM. Multiple comparison statistics were conducted using a One-way ANOVA with a post-hoc Tukey HSD. Comparison of the two groups was conducted using a t-test; all p values were two-sided.
Schematics
The schematic in Figure 1a was generated using Biorender.com.