A conifer metabolite corrects episodic ataxia type 1 by voltage sensor-mediated ligand activation of Kv1.1
Data files
Dec 16, 2024 version files 1.23 MB
-
Figure_10C__D_-_E187A_12_uM_Pisiferic_acid_-_Gmax.xlsx
33.03 KB
-
Figure_10C__D_-_K193A___R295A_12_uM_Pisiferic_acid_-_Gmax.xlsx
34 KB
-
Figure_10C__D_-_K193A_12_uM_Pisiferic_acid_-_Gmax.xlsx
35.67 KB
-
Figure_10C__D_-_L291A_12_uM_Pisiferic_acid_-_Gmax.xlsx
30.92 KB
-
Figure_10C__D_-_R292A_12_uM_Pisiferic_acid_-_Gmax.xlsx
31.09 KB
-
Figure_10C__D_-_R295A_12_uM_Pisiferic_acid_-_Gmax.xlsx
34.65 KB
-
Figure_10E_-_All_Binding_site_mutants_-_V0.5_shift.xlsx
10.63 KB
-
Figure_1C__D_-_Kv1.1_C._pisifera_extract_-_Gmax.xlsx
29.50 KB
-
Figure_1C__D_-_Kv1.1_P._albicaulis_extract_-_Gmax.xlsx
28.98 KB
-
Figure_1C__D_-_Kv1.1_P._contorta_1_-_Gmax.xlsx
27.43 KB
-
Figure_1C__D_-_Kv1.1_P._ponderosa_-_Gmax.xlsx
27.72 KB
-
Figure_1C__D_-_Kv1.1_P.concorta_2_-_Gmax.xlsx
28.34 KB
-
Figure_1C__D_-_Kv1.1_T._mertensiana_-_Gmax.xlsx
26.39 KB
-
Figure_1E_-_Kv1.1_All_pine_extracts_-_RMP.xlsx
10.05 KB
-
Figure_2A_-_Kv1.1_All_pine_extracts_-_V0.5_Shift.xlsx
9.96 KB
-
Figure_2B_-_Kv1.1_All_pine_extracts_-_Activation.xlsx
58.84 KB
-
Figure_2C__D_-_Kv1.2_C._pisifera_extract_-_Gmax.xlsx
28.47 KB
-
Figure_2C__D_-_Kv1.2_P._ponderosa_-_Gmax.xlsx
26.10 KB
-
Figure_2E_-_Kv1.1_Kv1.2_C.pisifera_-_RMP.xlsx
9.50 KB
-
Figure_2E_-_Kv1.2_C.pisifera___P._ponderosa_-_RMP.xlsx
9.60 KB
-
Figure_2F__G_-_Kv1.1_Kv1.2_C._pisifera_-_Gmax.xlsx
29.78 KB
-
Figure_2I__J_-_Kv1.1_Kv1.1-V408A_C._pisifera_-_Gmax.xlsx
27.15 KB
-
Figure_2K_-_Kv1.1_Kv1.1-V408A_C.pisifera_-_RMP.xlsx
9.47 KB
-
Figure_3A__B_-_Kv1.1_10_uM_Pisiferic_acid_-_IV.xlsx
31.32 KB
-
Figure_3C__D_-_Kv1.1_Pisiferic_acid_dose_response_-_Gmax.xlsx
91.02 KB
-
Figure_3E_-_Kv1.1_10_uM_Pisiferic_acid_-_RMP.xlsx
9.76 KB
-
Figure_3F_-_Kv1.1_Pisiferic_acid_dose_response_-_V0.5_Shift.xlsx
11.48 KB
-
Figure_3G_-_Kv1.1_Pisiferic_acid_dose_response_-_RMP_Shift.xlsx
12.56 KB
-
Figure_3H_-_Kv1.1_12_uM_Pisiferic_acid_-_Activation.xlsx
27.37 KB
-
Figure_3I_-_Kv1.1_12_uM_Pisiferic_acid_-_Deactivation.xlsx
22.23 KB
-
Figure_3J__K_-_Kv1.2_10_uM_Pisiferic_acid_-_IV.xlsx
29.81 KB
-
Figure_3L__M_-_Kv1.2_Pisiferic_acid_dose_response_-_Gmax.xlsx
82.05 KB
-
Figure_3N_-_Kv1.2_10_uM_Pisiferic_acid_-_RMP.xlsx
9.64 KB
-
Figure_3O_-_Kv1.2_Pisiferic_acid_dose_response_-_V0.5_Shift.xlsx
11.26 KB
-
Figure_3P_-_Kv1.2_Pisiferic_acid_dose_response_-_RMP_Shift.xlsx
12.43 KB
-
Figure_3R__S_-_Kv1.1_Kv1.2_12_uM_Pisiferic_acid_-_Gmax.xlsx
28.96 KB
-
Figure_3T_-_Kv1.1_Kv1.2_12_uM_Pisiferic_acid_-_RMP.xlsx
9.65 KB
-
Figure_4A__B_-_Kv1.1-E283K-Kv1.2_12_uM_Pisiferic_acid_-_Gmax.xlsx
30.61 KB
-
Figure_4A__B_-_Kv1.1-R307C-Kv1.2_12_uM_Pisiferic_acid_-_Gmax.xlsx
28.18 KB
-
Figure_4A__B_-_Kv1.1-V408A-Kv1.2_12_uM_Pisiferic_acid_-_Gmax.xlsx
30.46 KB
-
Figure_4C_-_Kv1.1-E283K-Kv1.2_12_uM_Pisiferic_acid_-_RMP.xlsx
11 KB
-
Figure_4C_-_Kv1.1-R307C-Kv1.2_12_uM_Pisiferic_acid_-_RMP.xlsx
11.01 KB
-
Figure_4C_-_Kv1.1-V408A-Kv1.2_12_uM_Pisiferic_acid_-_RMP.xlsx
11.01 KB
-
Figure_4D_-_All_mutants_-_Fraction_of_WT_current.xlsx
10.34 KB
-
Figure_4E_-_All_mutants_-_12_uM_Pisiferic_acid_current_fold_increase.xlsx
10.36 KB
-
Figure_4F_-_All_mutants_-_V0.5_shift_vs_WT.xlsx
10.37 KB
-
Figure_4G_-_All_mutants_-_V0.5_shift_12_uM_Pisiferic_acid.xlsx
10.26 KB
-
Figure_6C__D_-_Kv1.13M_12_uM_Pisiferic_acid_-_IV.xlsx
25.65 KB
-
Figure_6E_-_Kv1.13M_12_uM_Pisiferic_acid_-_Gmax.xlsx
32.82 KB
-
Figure_6F_-_Kv1.33M_12_uM_Pisiferic_acid_-_RMP.xlsx
9.72 KB
-
README.md
8.36 KB
Abstract
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.
README: 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 > Channel Investigated > Condition > 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.
Methods
Plant extracts, electrophysiology and MD simulations
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 Xenopus laevis 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.
Mouse studies
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 ad libitum. 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.