DOI: 10.5061/dryad.hmgqnk9tq
title: A conifer metabolite corrects episodic ataxia type 1 by voltage sensor-mediated ligand activation of Kv1.1

description: Loss-of-function sequence variants in KCNA1, which encodes the
 voltage-gated potassium channel Kv1.1, cause Episodic Ataxia Type 1 (EA1)
 and epilepsy. Due to a paucity of drugs that directly rescue mutant Kv1.1
 channel function, current therapeutic strategies for KCNA1-linked
 disorders involve indirect modulation of neuronal excitability. Native
 Americans have traditionally used conifer extracts to treat paralysis,
 weakness, and pain, all of which may involve altered electrical activity
 and/or Kv1.1 dysfunction specifically. Here, screening conifer extracts,
 we found that Chamaecyparis pisifera increases wild-type Kv1.1
 activity, as does its prominent metabolite, the abietane diterpenoid
 pisiferic acid. Uniquely, pisiferic acid also restored function in 12/12
 EA1-linked mutant Kv1.1 channels tested in vitro. Crucially, pisiferic
 acid (1 mg/kg) restored wild-type function in Kv1.1E283K/+ mice, a new
 model of human EA1. Experimentally validated all-atom molecular dynamics
 simulations in a neuron-like membrane revealed that the Kv1.1
 voltage-sensing domain (VSD) also acts as a ligand-binding domain akin to
 those of classic ligand-gated channels; binding of pisiferic acid induces
 a conformational shift in the VSD that ligand-dependently opens the pore.
 Conifer metabolite pisiferic acid is a promising and versatile therapeutic
 lead for EA1 and other Kv1.1-linked disorders.
description: <strong>Plant extracts, electrophysiology and MD
 simulations</strong> Briefly, methanolic extract
 of conifers, including those collected under permit from Yosemite National
 Park (study # YOSE-00839); and C. pisifera metabolite pisiferic acid
 (presence in extract confirmed by HPLC and LC/MS), were tested for effects
 on Kv channel heterologous expressed in <em>Xenopus
 laevis</em> oocytes using two-electrode voltage-clamp
 electrophysiology. An atomic model for Kv1.1 was constructed from the
 predicted structure of the Kv1.1 monomer available in the AlphaFold
 database37 545 (UNIPROT: AF-Q09470-F1). The transmembrane segments of
 Kv1.1 including the S1–S6 helices were retained, and the disordered
 intracellular regions (residues 1-142 and 416-495) were removed from the
 model. An x-ray crystallographic structure for the Kv1.2 tetramer (PDB ID:
 2A79) was used as a template to obtain the quaternary protein-protein
 contacts that stabilize the Kv1.1 tetramer. MD simulations of pisiferic
 acid binding to Kv1.1 were conducted, and then validated using TEVC and
 site-directed mutagenesis. <strong>Mouse
 studies</strong> For mouse studies, pisiferic
 acid (Combi-Blocks, San Diego, CA, USA) was solubilized at 0.1 mg/mL in a
 vehicle containing 1% DMSO in sterile PBS. Isoproterenol hydrochloride
 (Sigma-Aldrich, St. Louis, MO, USA) was made up at 0.5 mg/mL free base in
 sterile PBS. All compounds were injected intraperitoneally at 10 mL/kg.
 The Kv1.1-E283K transgenic mouse line was created from C57BL/6 mice by
 CRISPR knock-in at the Chao Family Comprehensive Cancer Center Transgenic
 Mouse Facility at UCI. Experimental mice were bred by
 crossing Kv1.1E283K/+ mice with wild-type Kv1.1 mice (Kv1.1+/+). The
 offspring were genotyped by qPCR and approximated typical Mendelian
 inheritance. Kv1.1E283K/+ and littermate Kv1.1+/+ 561 transgenic mice were
 group-housed under a 12-hour light/dark cycle with access to food and
 water <em>ad libitum</em>. Unless otherwise noted, all of the
 behavioral experiments were conducted on adult, male mice between 2 and 4
 months of age with the genotypes being tested in a counterbalanced
 fashion. These behavioral paradigms were approved by the
 Institutional Animal Care and Use Committee at the University of
 California, Irvine.
description: # A conifer metabolite corrects episodic ataxia type 1 by voltage
 sensor-mediated ligand activation of Kv1.1 The datasets included are the
 original Excel files used to generate each panel for figures 1-10 in this
 manuscript. The title of each Excel file is labeled to directly correspond
 to the figure in the manuscript: **Figure Number & panel &gt; Channel
 Investigated &gt; Condition &gt; Parameter Measured** --- 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 Kv1.1, Kv1.2, and Kv1.1/Kv1.2
 channels in response to 1:50 dilutions of conifer extracts and the active
 metabolite from C. pisifera, pisiferic acid. Additionally, twelve Kv1.1
 episodic ataxia type 1 (EA1) linked mutations were recorded in response to
 12 uM pisiferic acid. *Xenopus laevis* oocytes were injected with cRNA
 encoding for each of these channels and were incubated at 16 degrees for
 24 hrs prior to recording using TEVC. The subsequent measurements allow us
 to characterize the biophysical responses of these channels to these
 extracts and pisiferic acid. These data not only represent the first
 characterization of the effects of these plant extracts and pisiferic acid
 on wild Kv1.1, Kv1.2, and Kv1.1/Kv1.2 channels, but also the ability of
 pisiferic acid to rescue EA1 Kv1.1-linked patient mutations. The
 parameters that we measured to characterize the effect of conifer extracts
 and pisiferic acid on Kv1.1, Kv1.2, Kv1.1/Kv1.2, and Kv1.1 EA1 mutants are
 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 starting 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 -30 mV) immediately following the
 prepulse current as described above. For channels without a discernable
 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).
 #### **Dose responses (V0.5 shift)** These data were used to determine the
 potency and efficacy of pisiferic acid at Kv1.1 and Kv1.2 channels.
 Pisiferic acid was applied to *Xenopus laevis* oocytes expressing these
 channels at concentrations of 0.1, 1, 3, 10, 30, and 100 uM. The tail
 current was then measured (as described above) for the control and each
 subsequent concentration of pisiferic acid to generate V0.5 values. These
 values were then subtracted from the V0.5 value for the channel under
 control conditions and plotted as a function of voltage. All raw values
 are in millivolts (mV). #### **Dose responses (Resting membrane
 potential)** These data were used to determine the potency and efficacy of
 pisiferic acid at Kv1.1 and Kv1.2 channels. Pisiferic acid was applied to
 *Xenopus laevis* oocytes expressing these channels at concentrations of
 0.1, 1, 3, 10, 30, and 100 uM. The RMP (EM) of unclamped *Xenopus laevis*
 oocytes was then measured (as described above) for the control and each
 subsequent concentration of pisiferic acid. These values were then
 subtracted from the RMP value for the channel under control conditions and
 plotted as a function of voltage. All raw values are in millivolts (mV).
 #### **Current density 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 EA1 mutant channels. The change in current
 density 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 EA1 mutations
 have on the ability of the channel to generate currents compared to
 wild-type. All raw values are in microamps (uA). #### **Current density
 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 EA1 mutant channels in control solution and after
 application of 12 uM pisiferic acid. The change in current density 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 EA1
 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 EA1 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 EA1 mutations have on the voltage-dependence of activation
 compared to wild-type. This informs us how these EA1 mutations change the
 ability of the channel to open in response to changes in voltage across
 the cell membrane. All values are in microamps (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 EA1 mutant channels in control
 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 EA1
 mutant channels, rescuing the effect of the EA1 mutations on the
 voltage-dependence of activation. All values are in microamps (mV). ####
 **Statistics** All statistical analysis were conducted as either 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.