DOI: 10.5061/dryad.5dv41nsfc
title: Polymodal potassium channel modulation contributes to dual analgesic and anti-inflammatory actions of ancient botanical medicines

description: Pain and inflammation contribute immeasurably to reduced quality of life,
 yet modern analgesic and anti-inflammatory therapeutics can cause
 dependence and side effects. Here, we screened 1444 plant
 extracts, prepared primarily from native species in California
 and the United States Virgin Islands, against two voltage-gated K+
 channels - T38 cell expressed Kv1.3 and nociceptive-neuron
 expressed Kv7.2/7.3. A subset of extracts both inhibited Kv1.3,
 and activated Kv7.2/7.3 at hyperpolarized potentials,
 effects predicted to be anti-inflammatory and analgesic,
 respectively. Among the top dual hits were witch hazel and
 fireweed; polymodal modulation of multiple K+ channel types
 by hydrolysable tannins contributed to their dual
 anti-inflammatory, analgesic actions. In silico docking and
 mutagenesis data suggested pore-proximal extracellular linker sequence
 divergence underlies opposite effects of hydrolysable tannins on different
 Kv1 isoforms. The findings provide molecular insights into the enduring,
 widespread medicinal use of witch hazel and fireweed and demonstrate a
 screening strategy for discovering dual anti-inflammatory, analgesic small
 molecules.
description: <strong>Collection of plant
 samples</strong><br>We collected, between 2019 and 2022,
 aerial parts of plants under permit from Mojave National Preserve (study #
 MOJA-00321), Yosemite National Park (study # YOSE-00839), Santa
 Monica Mountains National Recreation Area (study # SAMO-00192), Muir Woods
 National Monument (study # MUWO-00035), Santa Cruz Island and Santa Rosa
 Island (study # CHIS-0023), and Boyd Deep Canyon (Indian Wells, CA)
 (permit through Philip L. Boyd Deep Canyon Desert Research Center,
 University of California, Riverside, CA) in California, US. In USVI,
 we collected from Virgin Islands National Park, St. John (study #
 VIIS-20001), Salt River Bay National Historical Park and Ecological
 Preserve, St. Croix (study # SARI-00056) and Buck Island Reef National
 Monument, St. Croix (study # BUIS-00103). Plant samples were collected
 in a manner designed to not kill the remaining plant, sealed in Ziploc
 bags (SC Johnson, Racine, WI, US), kept cold, and frozen as soon as
 possible. Other plant extracts were made from plants purchased from
 Crimson Sage Nursery (Orleans, CA) and grown in the senior author’s garden
 in Irvine CA, Mountain Rose Herbs (Eugene, OR) or Mother’s Market (Irvine,
 CA). Plant samples were stored in -20<sup> o</sup>C freezers
 until extraction. <strong>Preparation of plant
 extracts</strong><br>Leaves and flowers were pulverized using
 a bead mill with porcelain beads in batches in 50 ml tubes (Omni
 International, Kennesaw, GA, United States), then the homogenates
 resuspended in 80% methanol/20% water (100 ml per 5 g solid) and incubated
 for 48 hours at room temperature, with occasional inversion to resuspend
 the particulate matter. We then filtered the extracts through Whatman
 filter paper #1 (Whatman, Maidstone, UK), removed the methanol using
 evaporation in a fume hood for 24-48 hours at room temperature,
 centrifuged extracts for 10 minutes at 15 <sup>o</sup>C, 4000
 RCF to remove the remaining particulate matter, followed by storage (-20
 <sup>o</sup>C). On the day of electrophysiological recording,
 we thawed the extracts and diluted them 1:100 in bath solution (see below)
 immediately before use. <strong>High-throughput
 screening for Kv7.2/7.3 activation</strong><br>Plant extracts
 were applied to human Kv7.2/7.3 channels expressed in HEK293 cells using
 a FLIPR potassium assay kit and a Fluorescence Imaging Plate Reader
 (FLIPRTETRA™) instrument. All chemicals used in solution preparation were
 purchased from Sigma-Aldrich (St. Louis, MO) unless otherwise noted and
 were of ACS reagent grade purity or higher. Stock solutions of
 the positive controls were prepared in dimethyl sulfoxide (DMSO) or
 deionized water, aliquoted, and stored frozen. Stock solutions of the
 plant extracts were prepared in buffer and stored frozen after use. Test
 solutions of the test articles and the controls were prepared on the day
 of testing by diluting stock solutions into the assay buffers. The ability
 of each plant extract to act as an agonist of Kv7.2/7.3 channels was
 evaluated in a thallium (potassium ion surrogate, Molecular Devices) flux
 assay by Charles River Laboratories (Cleveland, OH, US). The assay
 was performed with the FLIPR potassium assay kit (Molecular Devices)
 according to the manufacturer’s instructions. For dye loading, growth
 media was removed and replaced with 20 μl of dye loading buffer for 60
 minutes at room temperature. For stimulation (agonist mode): 5x (5 μL)
 test, vehicle, or control article solutions prepared in the stimulation
 buffer (K<sup>+</sup>-free buffer with 5 mM
 Tl<sup>+</sup>) was added to each well for ~5 minutes. The
 agonist effects of test or control articles on Kv7.2/7.3 channels were
 evaluated. The positive control was Flupirtine 516 (9 concentrations).
 Data acquisition was performed via the ScreenWorks FLIPR control software
 that is supplied with the FLIPR System (MDS-AT). Data were analyzed using
 Microsoft Excel 2013 (Microsoft Corp., Redmond, WA). For each well, the
 raw kinetic data were reduced to the maximum or Area Under Curve
 fluorescence after subtracting bias and possibly applying the negative
 control correction. Reduced data were analyzed as follows:
 For each assay plate, a Z’ factor and Signal Window (SW) were
 calculated: Z’ factor = (([Agonist Control mean] – 3 x
 [Agonist Control STDEV]) – ([Vehicle Control mean] + 3 x [Vehicle Control
 STDEV])) / ([Agonist Control mean] – [Vehicle Control mean]) SW =
 (([Agonist Control mean] – 3 x [Agonist Control STDEV]) – ([Vehicle
 Control mean] + 3 x [Vehicle Control STDEV])) / [Agonist Control STDEV]
 Where the stimulation buffer was dispensed to Vehicle Control wells and a
 high concentration of agonist positive control was dispensed to Agonist
 Control wells. Concentration-response curves were fitted to the agonist
 positive control. Reduced data from test article wells were normalized to
 the vehicle and agonist control means on each plate and expressed as
 normalized percent activation: Normalized % Activation = ([individual well
 RLU] – [Vehicle Control mean]) / ([Agonist Control mean] – [Vehicle
 Control mean]) Where individual well RLU are the relative light units for
 each well to which test article is dispensed. A
 significance threshold of 3 standard deviations from the vehicle control
 mean was calculated: Significance Threshold = 3 x
 [Vehicle Control STDEV] / ([Agonist Control mean] – [Vehicle Control
 mean]) Concentration-response curves for positive agonist controls for
 each plate were also conducted. The positive control results confirmed the
 sensitivity of the test systems to agonists. The test and control samples
 were prepared in the stimulation buffer (a combination of low Cl- buffer,
 5 mM Tl2SO4 and water). The signal elicited in the presence of the
 positive agonist control (30 or 100 μM Flupirtine) was set to 100%
 activation and the signal from the vehicle (stimulation buffer) was set to
 0% activation. <strong>High-throughput screening
 for Kv1.3 inhibition</strong><br>Chemicals used in solution
 preparation were purchased from Sigma-Aldrich unless otherwise noted and
 were of ACS reagent grade purity or higher. Stock solutions of plant
 extracts and the positive controls were prepared in water and stored
 frozen, unless otherwise specified. Reference compound concentrations were
 prepared fresh daily by diluting stock solutions into a HEPES-buffered
 physiological saline (HB-PS) (composition in mM): NaCl, 137; KCl, 4.0;
 CaCl2, 4.8; MgCl2, 1; HEPES, 10; Glucose, 10; pH adjusted to 7.4 with
 NaOH. To minimize run-down of the Kv1.3 channel currents 0.3% DMSO was
 added in all reference, plant extract and control solutions. The plant
 extracts (diluted to 2% and 0.2%) were loaded into 384-well polypropylene
 compound plates and placed in the plate well of an automated patch-clamp
 (APC) system, SyncroPatchTM 384PE (SP384PE; Nanion Technologies,
 Livingston, NJ) immediately before application to Chinese Hamster Ovary
 (CHO) cells (stain source, ATCC Manassas, VA; sub strain source, ChanTest
 Corporation, Cleveland, OH, US) expressing human Kv1.3. Screening was
 conducted by Charles River Laboratories. Extracellular
 buffer was loaded into the wells of the Nanion 384-well Patch Clamp
 (NPC-384) chips (60 μl per well). Then, cell suspension was pipetted into
 the wells (20 μL per well) of the NPC-384 chip. After establishment of a
 whole-cell patch-clamp configuration, membrane currents were recorded
 using the patch clamp amplifier in the SP384PE system. Plant extracts were
 applied to naïve cells (n = 3, where n = the number cells/concentration).
 Each application consisted of addition of 40 μl of 2x concentrated test
 article solution to the total 80 μl of final volume of the extracellular
 well of the NPC-384 chip. Duration of exposure to each test
 article concentration was five (5) minutes. The intracellular solution was
 (in mM): KCl, 70; KF, 70; MgCl2, 2; EGTA, 2.5; HEPES, 10; pH adjusted to
 7.2 with KOH. In preparation for a recording session, the intracellular
 solution was loaded into the intracellular 569 compartment of the
 NPC-384 chip. The extracellular solution was the HB-PS solution described
 above. Kv1.3 channel currents were elicited using test pulses with fixed
 amplitudes: depolarization pulse to +20 mV amplitude, 200 ms duration from
 the holding potential of –90 mV. The test pulses were repeated with
 frequency 0.1 Hz: 3 min before (baseline) and 5 min after test
 articles addition. Kv1.3 channel current amplitudes were measured at the
 peak and at the end of the step to +20 mV. The positive control antagonist
 used was 4-aminopyridine, prepared as a 1 M stock in water; test
 concentrations were 1, 3, 10, 30, 100, 300, 1000 and 3000 μM.
 <strong>Chemical analysis of fireweed and witch hazel bark
 extract</strong><br>Chemicals: (+)-Catechin hydrate, ellagic
 acid, gallic acid, propionyl chloride, tannic acid and vanillin were from
 Sigma-Aldrich; 1-O-Galloyl-beta-d-glucose was from Combi-Blocks, Inc
 (San Diego, CA). Quercetin was obtained from Synaptent LLC (Chicago, IL).
 Conc. hydrochloric acid (35-38% in water) was from Fisher. Methanol
 (MeOH), ethyl acetate (EtOAc) and acetonitrile (ACN) were HPLC or LC/MS
 grade from Fisher or VWR International. Water was 18.2 mΩ-cm from a
 Barnstead NANOpure DiamondTM system. Trifluoroacetic acid was obtained
 from EMD Millipore. Methyl gallate was synthesized from gallic acid as
 described in the literature 77. A solution of gallic acid in MeOH
 containing sulfuric acid was refluxed overnight. Once at rt, the reaction
 was added to ice-water and extracted with EtOAc (3 x 25 mL). The pooled
 organic layers were washed twice with water, once with brine and conc in
 vacuo affording the methyl ester as a light-yellow solid. MS/MS with
 negative ionization mode gave m/z 183 (M-H+ with daughter ions 168 and
 124). <em>Chromatography</em>: Thin layer
 chromatography (TLC) employed Analtech GHLF UV254 UniplateTM silica gel
 plates from Miles Scientific (Newark, DE). Preparative HPLC separations
 were carried out using a Shimadzu system consisting of two LC-8A pumps, a
 fraction collector (FRC-10A), a SIL-10AP auto sampler, a diode array
 detector (CPD-M20A) and a CBM-20A communication module. The separations
 employed a Waters PREP Nova-Pak® HR C18 6 μM 60Å 40 x 100 mm reversed
 phase column with a 40 x 10 mm Guard-Pak insert and a Waters PrepLC
 Universal Base. The solvent systems employed were MeOH/water gradients
 both containing 0.1 % TFA or ACN/water gradients also with added 0.1 %
 TFA. Fractions were collected based on their response at 254 nm.
 <em>Mass Spectroscopy</em>: Mass spectroscopy
 employed a Thermo Scientific TSQ Quantum Ultra triple stage quadrupole
 mass spectrometer. Heated-electrospray ionization (H-ESI) was used
 in negative or positive ionization mode depending on the structure of the
 analyte. Automatic methods for the optimization of instrument parameters
 were used to maximize sensitivity. Samples were analyzed by direct
 injection in MeOH or MeOH/water (TFA conc kept at 0.01% or less) using a
 syringe pump. Aqueous plant extracts were diluted 100-fold with MeOH
 before analysis. Gallic acid was identified from parent m/z 169 (M-H+) and
 daughter ion m/z 125 in negative ionization mode. Ellagic acid showed m/z
 (M-H+) in negative ionization mode and its presence was confirmed by
 daughter ion analysis to distinguish it from quercetin, also m/z 301
 (M-H+). <em>Transesterification Analysis for
 Hydrolysable Tannins</em>: Methanolysis was initially
 accomplished by treating samples with a 10% v/v sulfuric acid solution in
 MeOH as described by Harztfeld et al. Aqueous plant extracts (1 mL) were
 concentrated in vacuo with a Büchi Rotavapor R-205 (water bath temp 40oC)
 connected to a DryFast Ultra® pump, model 2031B-01, from Welch Rietschle
 Thomas. The residues obtained were dissolved in the H2SO4/MeOH solution
 (1-2 mL) and heated at 85 <sup>o</sup>C in a sealed tube under
 N2 overnight. Once at rt, the reactions were added to ice-water/EtOAc. The
 organic layer was separated and washed twice with water and conc
 to dryness. This method was replaced with the operationally simpler method
 of Newsome et al. except that acetyl chloride was replaced with an
 equivalent molar amount of propionyl chloride. Plant
 extracts were concentrated to dryness as above and the residues w 623 ere
 treated with 1-2 ml of the 2.75 M methanol-HCl solution under N2. The
 resulting solutions were heated at 85 <sup>o</sup>C for 6 h.
 Once at rt, the solvent was removed in vacuo and the residues were
 reconstituted in MeOH and injected onto the preparative HPLC system. The
 methanolysis method was tested with tannic acid. Heating as above gave
 methyl gallate that was identified by HPLC retention time (RT), TLC Rf and
 mass spectrum. <em>Vanillin Assay for Condensed
 Tannins:</em> The concentration of condensed tannins in the
 bark samples was determined using the vanillin hydrochloric acid assay as
 described by Makkar et al. Aqueous plant extracts were diluted with an
 equal volume of MeOH and then 100 μL of the resulting solutions (in
 triplicate) were diluted with 0.6 mL of a 4% w/v solution of vanillin in
 MeOH and 0.3 mL of conc hydrochloric acid. The samples were vortexed
 briefly and then allowed to stand in the dark for 20 min. The absorbance
 at 490 nm was measured in a 96-well plate using a KC Junior plate reader
 (Bio-Tek Instruments, Vermont, USA). (+)-Catechin hydrate was used to
 generate a standard curve. The condensed tannin content of the extracts
 is expressed as (+)-catechin equivalents (μg/mL and μg/mg). The absorbance
 of a blank with no (+)-catechin was subtracted from the standard curve and
 from the results of the plant extracts.
 <strong>Channel subunit cRNA preparation and
 <em>Xenopus laevis </em>oocyte injection for manual two
 </strong><strong>electrode voltage-clamp (TEVC)
 electrophysiology</strong><br>We generated cRNA transcripts
 encoding human Kv1.1, Kv1.3, Kv7.2, Kv7.3, Kv7.5 and TREK-1 by in vitro
 transcription using the mMessage mMachine kit (Thermo Fisher Scientific,
 Waltham, MA, USA) according to manufacturer’s instructions, after vector
 linearization, from cDNA subcloned into expression vectors (pTLNx, pXOOM
 and 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 Xenopus laevis
 oocytes (Xenoocyte, Dexter, MI, USA) with the channel cRNAs (0.5-10 ng)
 and incubated the oocytes at 16 oC in ND96 oocyte storage solution
 containing penicillin and streptomycin, with daily washing, for 1-4 days
 prior to two electrode voltage-clamp (TEVC) recording. Mutant channel
 cDNAs were generated by GenScript Biotech (Piscataway, NJ).
 <strong>Two-electrode voltage clamp
 (TEVC)</strong><br>We conducted TEVC at room temperature with
 an OC-725C amplifier (Warner Instruments, Hamden, CT, USA) and pClamp10
 software (Molecular Devices, Sunnyvale, CA, USA) 2-4 days after cRNA
 injection. Oocytes, in a small-volume oocyte bath (Warner), were viewed
 with a dissection microscope for cellular electrophysiology. We purchased
 chemicals from Sigma-Aldrich (St. Louis, MO). We studied the effects of
 plant extracts and constituents solubilized directly 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 extracts or
 compounds into the oocyte recording bath by gravity perfusion at
 a constant flow of 1 ml per minute for 3 minutes prior to recording.
 Pipettes (1-2 MΩ resistance) were filled with 3 M KCl. We recorded
 currents in response to voltage pulses between -120 mV or -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. We analyzed
 data using Clampfit (Molecular Devices) and Graphpad Prism software
 (GraphPad, San Diego, CA, USA), stating values as mean ± SEM. We
 calculated the voltage dependence of activation (V0.5) by
 measuring currents at a voltage pulse of -30 mV (Kv7) or -50 mV (Kv1)
 immediately following prepulse voltages between -80 mV and + 40 mV. We
 plotted raw or normalized tail currents versus prepulse voltage and fitted
 with a single Boltzmann function. For wild type and mutant Kv1.3 channels
 we calculated conductance-voltage curves from current-voltage
 relationships recorded in response to voltage pulses between -80 mV to +40
 mV in 10 mV intervals from a holding potential of -80 mV. The conductance
 was calculated by subtracting the driving force (EK) and the subsequent
 value was then divided by the current recorded at the relevant voltage to
 give the conductance corrected for EK. This value was then divided against
 the peak conductance. <strong>Formalin paw lick
 assay</strong><br>Adult, male C57BL/6 mice (Charles River,
 Wilmington, MA) were group housed under a 12-hour light:dark cycle and
 allowed access to food and water ad libitum. Mice were tested in
 the formalin paw lick assay between 9 and 12 weeks of age. The mouse study
 was performed under an approved Institutional Animal Care and Use
 Committee protocol at the University of California, Irvine. The fireweed
 plant extract was prepared by diluting to 0.2%, 0.6%, or 2% in sterile
 saline and titrating each to pH 7.4. The dilutions used for in vivo
 testing were estimated from the concentration required to modulate
 electrophysiological responses in Kv7.2/7.3 channels expressed in
 <em>Xenopus laevis</em> oocytes. Neutral buffered formalin
 (Sigma-Aldrich, St. Louis, MO) was diluted in sterile saline to a
 concentration of 5%. Experimental solutions were prepared by combining an
 equal volume of the 5% formalin solution with the diluted plant extract,
 resulting in a final solution containing 2.5% formalin and 0.1%, 0.3%, or
 1% plant extract. The vehicle control solution was prepared by combining
 an equal volume of formalin with sterile saline, resulting in a final
 solution with only 2.5% formalin. These solutions were prepared fresh
 daily. Mice were habituated to the procedure room in their home cages for
 700 at least one hour prior to testing. Either 2.5% formalin alone or 2.5%
 formalin with 0.1%, 0.3%, or 1% plant extract was injected into the dorsal
 surface of the left hindpaw and then the animal was immediately placed in
 a large, clear polymethylpentene beaker for observation. The amount of
 time spent licking the injected paw was recorded in 5-minute bins over 60
 minutes by individuals blinded to the treatment received. Paw licking was
 separated into an early, acute phase (0–5 min post-formalin) and a late,
 inflammatory phase (10–60 min post-formalin).
 <strong>Mesenteric artery
 myography</strong><br>In accordance with the methods of
 killing animals described in annex IV of the EU Directive 2010/63EU on the
 protection of animals used for scientific purposes, male Wistar rats, 12
 weeks old (Janvier Labs, France), were made unconscious by a single,
 percussive blow to the head. Immediately after the onset of
 unconsciousness, cervical dislocation was performed. Rats
 were group-housed with regular 12-hour light/dark cycles, in clear plastic
 containers with ad libitum access to food and water and underwent at least
 one week of habituation. After euthanasia, the intestines were removed,
 and third-order mesenteric arteries were dissected in
 ice-cold physiological saline solution containing (in mM): 121 NaCl, 2.8
 KCl, 1.6 CaCl<sub>2</sub>, 25 NaHCO3, 1.2 KH2PO4, 1.2 MgSO4,
 0.03 EDTA, and 5.5 glucose. Segments, 2 mm in length, of mesenteric artery
 were mounted on 40 μm stainless steel wires in a myograph (Danish Myo
 Technology, Aarhus, Denmark) for isometric tension recordings. The
 chambers of the myograph contained PSS maintained at 37°C and aerated with
 95% O2/5% CO2. Changes in tension were recorded by PowerLab and Chart
 software (ADInstruments, Oxford, United Kingdom). The arteries
 were equilibrated for 30 minutes and normalized to passive force. Artery
 segments were precontracted with 10 μM methoxamine (Sigma; Copenhagen,
 Denmark) in the absence or presence of linopirdine (10 μM) (Sigma;
 Copenhagen, Denmark), before application of increasing concentrations of
 witch hazel bark extract. <strong>Activated human
 T cells</strong><br>Human CD4+ T cells from frozen PBMCs
 (STEMCELL Technologies) were isolated with EasySep negative isolation kit
 (STEMCELL Technologies, cat. #17952) according to manufacturers’
 protocols. After isolation cells were plated on anti-CD3 (BioLegend, clone
 OKT3, 2.5 μg/ml), anti-CD28 (BioLegend, clone CD28.2, 2.5 μg/ml) -coated
 6-well plate dish at 1.5×106 cells per well in 3 ml RPMI medium (Gibco)
 supplemented with 10% FBS (Omega Scientific), 1% L-glutamine (Gibco), 1%
 nonessential amino acids (Gibco), 1% sodium pyruvate (Gibco),
 1% penicillin–streptomycin–amphotericin B (Gibco) and 50 μM,
 β-mercaptoethanol (Sigma), 30 U/ml recombinant human IL-2 (BioLegend, cat.
 #589102). Cells were incubated during 48-144 hours in 5% CO2 + 95% O2
 atmosphere at 37 °C, harvested and plated on poly-lysine
 coated coverslips; 30-60 minutes later cells were used in the
 experiment. <strong>Human CD4 T cell
 proliferation assay</strong><br>Whole blood from de-identified
 healthy donors was obtained from the Institute for Clinical
 and Translational Science (ICTS) research Blood Donor Program at the
 University of California, Irvine approved and reviewed by the
 Institutional Review Boards of UCI (IRB protocol HS#2001-2058). Peripheral
 blood mononuclear cells (PBMC) were isolated by density
 gradient separation using lymphocyte separation media (Corning, Cat#
 25-072-CV) and SepMate tubes (STEMCELL Technologies, Cat# 85450). Blood
 was first diluted (1:3) in phosphate-buffered saline (PBS) containing 2%
 fetal bovine serum (FPS), then overlaid on top of lymphocyte separation
 media (2:1). CD4+ T cells were isolated from PBMCs using magnetic
 separation (EasySep Human CD4+ T cell isolation kit, STEMCELL
 Technologies, Cat# 17952), post isolation purity is typically &gt;95%
 based on CD4 and CD3 staining. Purified CD4+ T cells were labeled with 1.6
 μM of CellTrace CFSE in AIM V Serum-Free Medium (Gibco Cat# 12055-083) for
 8 minutes as described previously 80. CFSE labeled CD4 T cells were
 stimulated with anti CD3 – and anti CD28 – coated Dynabeads (Thermo Fisher
 Scientific, Cat# 111310) at 1:1 ratio in a round-bottom 96-well plate at
 37°C and 5% CO2 in the dark for 753 96 hours in T cell culture medium
 (RPMI with 10% FCS, L-glutamine, non-essential amino acids, sodium
 pyruvate, β-mercaptoethanol, and penn-strep with amphotericin B). On day 4
 cells were stained with Fixable Viability Dye eFluor 780 (FVD-780, Thermo
 Fisher Scientific, Cat# 65-0865-14) in PBS for 20 min at 4
 <sup>o</sup>C to identify dead cells. Cells were then washed
 with 1X PBS + 1% FBS twice and transferred to a round-bottom 96-deep well
 plate (Greiner, Cat# 07-000-119) for flow cytometry. Fluorescence
 intensity data were acquired using NovoCyte Quanteon (Agilent
 Technologies) flow cytometer. FSC files were used for gating and analysis
 using FlowJo analysis software (FlowJo LLC, Ashland, Oregon). Gating
 strategy: Density-based clustering was used to identify lymphocytes using
 forward scatter (FSC) and side scatter (SSC) bi-variate plots. Doublets
 were excluded using area and height of intensity FSC channel (FSC-A vs
 FSC-H); and single cells negative for FVD-780 were identified as live
 cells. CFSE+ cells within live cell gating were used to assess the dye
 dilution and model cell proliferation to calculate the Division Index (#
 Divisions/ # cells at the start of culture), a measure of T cell
 activation. <strong>Patch-clamp
 recordings</strong><br>The patch clamp setup was described
 elsewhere85. The pipette solution composition was following (in mM): KF
 140, K4EGTA 10, MgCl2 1.5, HEPES 10, pH = 7.2 adjusted by KOH, osmolality
 300 mOsm. The external solution contained (in mM): NaCl 129, KCl 4.5,
 CaCl<sub>2</sub> 2, MgCl<sub>2</sub> 1, HEPES 10,
 glucose 10, pH = 7.4 adjusted by NaOH, osmolality 280 mOsm. The
 pipette resistance was 2 ÷ 4 MΩ. Both pipette and cell capacitance were
 fully compensated, and series resistance was compensated by 80%. The
 liquid junction potential between the pipette and bath solutions was
 corrected by -7 mV. The Kv1.3 currents were recorded in response to 600 ms
 step pulse from holding potential -80 mV to +40 mV acquired every 30
 seconds. Once Kv1.3 current was stabilized, the tested compounds (witch
 hazel extract or tannic acid) diluted in bath solution at sequentially
 increasing concentrations were applied via local perfusion system. The
 recorded currents were analyzed with Patch Master (HEKA Electronics) and
 Origin Pro (Origin Lab). <strong><em>In
 silico</em> docking</strong><br>We plotted and viewed
 chemical structures and electrostatic surface potential using Jmol,
 an open-source Java viewer for chemical structures in 3D:
 http://jmol.org/. For in silico ligand docking predictions of binding to
 Kv1 channels, we performed unguided docking to predict potential binding
 sites, using SwissDock with CHARMM forcefields, the Kv1.2 pore module from
 the X-ray crystallography-derived paddle-chimera structure and the
 cryo-EM-derived Kv1.3 structure. We prepared channel structures for
 docking using DockPrep in UCSF
 Chimera (https://www.rbvi.ucsf.edu/chimera), with which we also generated
 docking figures.
description: ## Source Data for manuscript: Polymodal potassium channel modulation
 contributes to dual analgesic and anti-inflammatory actions of ancient
 botanical medicines. The datasets included are the original Excel files
 used to generate each panel for figures 1-7 & 9-10 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 types of files
 included within this repository are as follows: &gt; Plant Screen &gt;
 This is a database providing the full details of all 1444 plant extracts
 screened for activity against Kv7.2/7.3 and Kv1.3 as summarized by the
 dot-plot in Figure 1A and Figure 1B. The database contains columns
 providing identifying information for each plant where appropriate. The
 information provided in each column is as follows: Plant ID: Unique
 Identifier of the plant sample. The code corresponds to the region the
 sample was obtained and the order for that day. For example - BDC-01 means
 Boyd Deep Canyon, plant number 01. Collection Date: The date the samples
 were collected Macro Region: The precise territory/national reserve the
 samples were collected. Region Curated: The location of the
 territory/national reserve. Region Geotag: The county and state where the
 samples were collected. Genus-species: Latin name of the plant species
 Common Name: English name of the plant species \[2% extract] KCNQ2/3
 Normalized % activation: Degree of thalium-flux mediated fluorescence \[2%
 extract] Kv1.3 Peak % inhibition (at +20 mV): Percentage inhibition of
 current density at +20 mV Plants highlighted in yellow indicate those
 summarized in Figure 1B. Cells where 'not applicable' is present
 indicate that either the information is unknown or is not pertinent to
 this study. &gt; Exemplar traces &gt; These data are the values used to
 generate exemplar traces of ion channel recordings. Data represents the
 current in microamps (uA) sampled every 100 microseconds (us) as a
 function of voltage (mV). &gt; IV &gt; These data were used to generate
 current-voltage relationships. Data in these files were measured from the
 peak of the prepulse current. All values are in microamps (uA). &gt; Gmax
 &gt; These data were used to generate conductance-voltage curves. Graphs
 were generated by taking measurements from the tail current (-30 mV)
 immediately following the prepulse current. For channels without a
 discernable tail current, Gmax graphs were plotted from the IV correcting
 for driving force and normalizing to the peak conductance. Non-normalized
 values are in microamps (uA). &gt; Resting membrane potential &gt; These
 data points represent the resting membrane potential (EM) of unclamped
 Xenopus laevis oocytes expressing the channel of interest in normal 4 mM
 extracellular potassium or in the presence of plant extracts or compounds.
 Resting membrane potential values are in millivolts (mV). &gt; Dose
 responses (Idrug/Icontrol - current desnity) &gt; These data were taken
 from values measured at 0 mV from the prepulse for control and each
 subsequent concentration. Raw values are in microamps (uA). &gt; Dose
 responses (Idrug/Icontrol - Resting membrane potential) &gt; These data
 were taken from unclamped oocytes for control and each subsequent
 concentration. Raw values are in millivolts (mV). &gt; Activation &gt;
 These data were used to determine the time constants in milliseconds (ms)
 of activation were fitted using either a single or double term. &gt;
 Percentage Inactivation &gt; These data were generated by measuring the
 current density at the beginning of pulse and the final current density at
 the end of the pulse. Pulses were either 0 or +40 mV. Raw values are in
 microamps (uA). &gt; C-type inactivation &gt; These data were derived from
 a consecutive pulse protocol from -80 to + 40 mV at intervals of either
 200ms or 2s. Additionally, the efficacy of Kv1.3 inhibition in high
 extracellular potassium was also measured using a single pulse protocol
 from -80 to +40 mV. Raw values are in microamps (uA). &gt; Myography &gt;
 Raw data for extract-mediated relaxation of precontracted tone in a
 mesenteric artery from adult male rats. Raw values are in milli-newton
 (mN). &gt; T cell activation & proliferation &gt; Raw data for the
 above assays taken directly from FlowJo: Division Index: The average
 number of cell divisions that a cell in the original population has
 undergone. This is an average even for cells which never divided (i.e.,
 includes the undivided peak). Proliferation Index: The total number of
 divisions divided by the the number of cells that went into division. The
 proliferation index only takes into account the cells that underwent at
 least one division, that is, only responding cells are reflected in the
 proliferation index. This is probably a more useful value to compare from
 sample to sample, as it considers only the fraction of responding cells.
 &gt; Formalin paw lick assay &gt; These data describe scores obtained for
 a pain assay in the presence or absence of fireweed extract (0.1, 0.3, and
 1%).The amount of time spent licking the injected paw was recorded in
 5-minute bins over 60 minutes by individuals blinded to the treatment
 received. Paw licking was separated into an early, acute phase (0–5 min
 post-formalin) and a late, inflammatory phase (10–60 min post-formalin).
 &gt; Additional Information &gt; All statistical analysis were conducted
 as either paired t-test or one-way ANOVA with Dunnett's correction
 for multiple comparison. Excel files with 'null' or
 'n/a' indicates no data points were obtained for these
 measurements.