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.

Collection of plant samples
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 ^oC freezers until extraction.

Preparation of plant extracts
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 ^oC, 4000 RCF to remove the remaining particulate matter, followed by storage (-20 ^oC). On the day of electrophysiological recording, we thawed the extracts and diluted them 1:100 in bath solution (see below) immediately before use.

High-throughput screening for Kv7.2/7.3 activation
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⁺-free buffer with 5 mM Tl⁺) 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.

High-throughput screening for Kv1.3 inhibition
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.

Chemical analysis of fireweed and witch hazel bark extract
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).

Chromatography: 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.

Mass Spectroscopy: 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+).

Transesterification Analysis for Hydrolysable Tannins: 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 ^oC 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 ^oC 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.

Vanillin Assay for Condensed Tannins: 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.

Channel subunit cRNA preparation and Xenopus laevis oocyte injection for manual two electrode voltage-clamp (TEVC) electrophysiology
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 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, 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).

Two-electrode voltage clamp (TEVC)
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 MgCl₂, 1 CaCl₂, 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.

Formalin paw lick assay
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 Xenopus laevis 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).

Mesenteric artery myography
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 CaCl₂, 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.

Activated human T cells
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.

Human CD4 T cell proliferation assay
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 >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 ^oC 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.

Patch-clamp recordings
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, CaCl₂ 2, MgCl₂ 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).

In silico docking
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.