DOI: 10.5061/dryad.1rn8pk16j
title: Conifer metabolite pisiferic acid restores activity in human Kv1.2 potassium channels carrying pathogenic sequence variants

description: 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.
description: <strong>Channel subunit cRNA preparation and Xenopus laevis
 oocyte injection</strong> 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
 <em>in vitro</em> transcription using the mMessage mMachine
 kit (Thermo Fisher Scientific), after vector linearization, from cDNA
 subcloned into plasmids (pMAX) incorporating <em>Xenopus
 laevis</em> β-globin 5’ and 3’ UTRs flanking the coding region to
 enhance translation and cRNA stability.  We injected defolliculated stage
 V and VI <em>Xenopus laevis</em> oocytes (Xenoocyte, Dexter,
 MI, US) with Kv1 cRNAs (0.1-10 ng). We incubated the oocytes at 16
 <sup>o</sup>C in ND96 oocyte storage solution containing
 penicillin and streptomycin, with daily washing, for 1-2 days prior to
 two-electrode voltage-clamp (TEVC) recording.
 <strong>Two-electrode voltage clamp (TEVC)
 </strong> 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 MgCl<sub>2</sub>, 1
 CaCl<sub>2,</sub> 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 (V<sub>0.5</sub>), 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.
 <strong>In
 </strong><strong>silico</strong><strong>
 docking</strong> For <em>in
 silico</em> 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.
 <strong>S</strong><strong>tatistics and
 Reproducibility</strong> 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 <em>p</em> values were
 two-sided.
 <strong>Schematics</strong> The
 schematic in Figure 1a was generated using Biorender.com.
description: ## 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 &gt; Channel Investigated (including mutant
 and wild type (WT)) &gt; Condition &gt; 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.