DOI: 10.5061/dryad.jdfn2z3pr
title: Data from: Effects of emodepside on single-channel properties of <em>Onchocerca volvulus</em> SLO-1A (BK) potassium channels

description: Control of onchocerciasis (river blindness of humans due to infection with
 the filarial nematode, Onchocerca volvulus) remains a challenge because of
 the lack of effective adulticides and vaccines. Emodepside is a
 broad-spectrum veterinary anthelmintic that has been found to inhibit
 nematode muscle activity by activating their tetrameric SLO-1K
 channels. Emodepside has adulticidal activity and is being
 trialed for onchocerciasis treatment, but the molecular mode of action of
 emodepside is still being elucidated. Here we examine the single-channel
 currents of Ovo-SLO-1A, a SLO-1K splice variant from O. volvulus, and
 explore how emodepside modulates the dynamics of the opening of the
 channel. Ovo-SLO-1A was expressed in HEK 293 cells, and patch clamp
 electrophysiology techniques were used to record currents. Single-channel
 currents were recorded in a symmetrical 132 mM K⁺ solution to determine
 the main open-state channel conductance. Emodepside’s effects were tested
 at 0.3 µM and 1.0 µM. Ovo-SLO-1A had a main open-state conductance of 110
 ± 3 pS and frequent flickering sub-conductance states. The presence of the
 flickering sub-conductance states suggests that there is limited
 cooperativity between the tetrameric channel subunits required for opening
 to the main open-state. Emodepside increased mean current amplitudes.
 Emodepside also increased open-burst times, and open probability.
 Verruculogen (1 µM) inhibited channel opening in the presence or absence
 of emodepside. This study successfully expressed Ovo-SLO-1A in HEK 293
 cells, measured the conductance of the main open-state and detected the
 presence of sub-conductance states, and flickering openings. The increased
 amplitudes of the single-channel currents, open-burst times and open
 probabilities provide insights into how emodepside increases Slo-1K
 currents and illustrate dynamic actions of emodepside on Ovo-SLO-1A.
description: <strong>Methods</strong>
 <strong>Cloning</strong>
 <em>O. volvulus</em> <em>slo-1a</em> was
 synthesized by Life Technologies GeneArt (BioPark Regensburg, Germany).
 Primers for the gene were designed with sequences flanking the pcDNA
 3.1-T2A-GFP expression vector that included the restriction site (Nhe1).
 PCR was conducted on the <em>O. volvulus slo-1a</em>.
 Subsequently, the amplicon was separated on a 1% Agarose SYBR Safe gel,
 purified using NucleoSpin Gel and PCR Clean-up Kit (Macherey-Nagel,
 Allentown, PA, USA), and then cloned into the pcDNA 3.1-T2A-GFP vector by
 using the Infusion HD Cloning Kit (Takara Bio USA, Inc, San Jose, CA, USA)
 according to the manufacturer’s protocols. Once cloned, the plasmids were
 verified by sequencing. <strong>HEK 293 Cell
 culture and transfection</strong> HEK 293 cells
 were maintained in DMEM (GIBCO, ThermoFisher Scientific Waltham MA, USA)
 with 10% FBS (GIBCO ThermoFisher Scientific Waltham MA, USA) and 1%
 penicillin/streptomycin antibiotics in a cell culture flask (CORNING, AZ,
 USA) at 37°C in a humidified environment with 5%
 CO<sub>2</sub>. Media was changed every two days. Once cells
 reached 80–90% confluency, they were sub-cultured to smaller 50ml culture
 flasks. Transfections were carried out in these smaller flasks when the
 cells reached 80–90% confluency, following standard protocol. Briefly, the
 medium was removed and replaced with Opti-MEM ® reduced serum medium
 (GIBCO ThermoFisher Scientific Waltham MA, USA) 1 hour prior to
 transfection. The pcDNA 3.1-<em>Ovo-slo1a</em>-T2A-GFP
 construct was transiently transfected into HEK 293 cells using the
 Lipofectamine™ 2000 (Invitrogen, Carlsbad, CA, USA) protocol. On separate
 flasks, transfections with empty plasmid (pcDNA 3.1-T2A-GFP) for control
 recordings were done in the same ratios as the transfections with the gene
 of interest. The GFP in the plasmid construct served as a transfection
 marker. Cells were incubated with transfection mixtures for 6-12 hours,
 after which the medium was replaced with DMEM/10% FBS and incubated at
 29°C, 5% CO<sub>2</sub>. Cells were detached from the petri
 dish using trypsin after 24 - 48 hours, seeded on coverslips coated with
 poly-D-lysine and incubated for 2 hours at 37°C, 5%
 CO<sub>2</sub> prior to imagery and patch recordings. The
 fluorescent image was captured at 20X using the BZ 800 viewer from a
 Keyence digital microscope, equipped with a blue-to-green GFP filter box
 (EX 470/40 DM 495 BA 525/50, Osaka, Japan).
 <strong>Electrophysiology</strong>
 <em><strong>Recording
 Conditions</strong></em> Borosilicate glass
 electrodes (1.50-mm OD Clark Electromedical Instruments, UK) were pulled
 with a Narishige PC-100 Vertical puller (Narishige, Tokyo, Japan). The
 pipette tips were coated with Sylgard and fire polished using a Narishige
 Micro Forge MF-900 (Narishige CO., Ltd, Tokyo, Japan). Pipette resistances
 between 4 and 12 MΩ were used for recordings. Coverslips were transferred
 to a cell chamber 2 hours after plating and mounted on the stage of a
 stabilized inverted epifluorescence microscope (Nikon ECLIPSE TE 2000-U,
 Tokyo, Japan). Fluorescent cells were identified for patching by observing
 at 20X and 40X using the SOLA light engine (Lumencor light engine
 Beaverton, OR, USA) equipped with a band pass blue to green GFP filter.
 Patch-clamp recordings were done in the whole-cell, and inside-out
 configurations. All experiments were conducted at room
 temperature. For whole cell experiments, the drug
 solution was delivered to the chamber under gravity feed through solenoid
 valves controlled using a VC-8 Eight-Channel Valve Controller (Warner
 Instruments, Hamden, CT, USA). For inside-out recordings, drugs were
 manually added to the bath and the cells exposed for 5 to 30 mins
 (depending on the drug) to allow enough time for diffusion from the point
 of application to the channels in the patches. All experiments where we
 observed channel rundown or membrane breakdown were excluded. After each
 experiment where drugs were applied to the bath, the coverslip in the
 experimental chamber was replaced with another containing a new sample of
 cells. To investigate the drug effects on the
 single-channel properties of <em>Ovo</em>-SLO-1A, we recorded
 events in the absence of drug for approximately 2 mins and then added the
 first drug (either emodepside or verruculogen). For experiments where both
 drugs were added, once the effect of the first drug added was observed,
 the second drug was added and recordings continued for 20 mins. While we
 added emodepside to patches with initial low activity, we added
 verruculogen to patches with high initial channel activity so the effect
 of each drug could be easily identified.
 <em><strong>Recording
 Solutions</strong></em> For experiments to
 determine the conductance of the channel, we used inside-out patches with
 solutions that were symmetrical on both sides of the membrane (6 mM NaCl,
 132 mM KCl, 1.2 mM MgCl<sub>2</sub>, 1 mM
 CaCl<sub>2</sub>, 11 mM Glucose and 10 mM HEPES, pH 7.4). For
 whole-cell experiments, the pipette solution contained 140 mM KCl, 1.2 mM
 MgCl<sub>2</sub>, 5.4 mM CaCl<sub>2</sub>, 5 mM
 EGTA, 2 mM dipotassium ATP and 10 mM HEPES, pH 7.2, giving a free
 (Ca<sup>2</sup>⁺) of 100 µM; and 137 mM NaCl, 5.9 mM KCl, 1.2
 mM MgCl2, 2.2 mM CaCl<sub>2</sub>, 14 mM Glucose and 10 mM
 HEPES, pH 7.4 in the bath. For investigation drug effects on inside-out
 patches we used solutions containing: 150 mM NaCl, 4 mM KCl, 2 mM
 MgCl<sub>2</sub>, 2 mM CaCl<sub>2</sub> and 10 mM
 HEPES, pH 7.4 in the pipette; and 150 mM KCl, 9.77 mM
 CaCl<sub>2</sub>,10 mM EGTA, and 10 mM HEPES, pH 7.2. The bath
 (Ca<sup>2</sup>⁺) was adjusted to be 100 µM using the MAX
 CHELATOR program: https://somapp.ucdmc.ucdavis.edu/pharmacology/bers/maxchelator/CaMgATPEGTA-TS.htm. <strong>Drugs</strong> Emodepside and verruculogen were purchased from Advanced ChemBlock Inc (Hayward, CA, USA). Stock solutions of emodepside (3 mM) and verruculogen (10 mM) for inside-out recordings were prepared in dimethyl sulfoxide (DMSO) and then diluted in the recording solution (to 3 µM for emodepside and 10 µM for verruculogen) prior to experimentation. Thus, the final concentration of DMSO in the experimental solutions was kept below 0.1%. We added a small volume of the drugs to the bath and allowed approximately 15 minutes for equilibration so that the final concentration in the bath of emodepside was 0.3 µM and the final concentration of verruculogen was 1 µM. For the whole cell recording with emodepside, the stock solution was prepared at 1 mM and diluted in recording solution to 1 µM. <strong>Data Analysis</strong> Data were collected using an Axopatch 200B amplifier (Molecular Devices, LLC. San Jose, CA, USA), filtered at 2 kHz with an 8-pole Bessel filter, and sampled at 10 kHz with a Digidata 1550B (Molecular Devices, LLC. San Jose, CA, USA); and the pCLAMP Software versions 10.7.0 and 11.1.0 (Molecular Devices, LLC. San Jose, CA, USA). Openings or closings shorter than 0.5 ms were not well resolved. To determine the main open-state channel conductance, currents were measured at four different membrane potentials (±50 mV and ±75 mV), in the symmetrical solutions with 132 mM K⁺. A current-voltage plot was generated, and the channel conductance was determined from the slope. For whole-cell recordings, peak currents responses elicited by the drug were measured, from baseline current prior to drug addition, using Clampfit. GraphPad Prism 10.3.0 software (GraphPad Software Inc., San Diego, CA, USA) was used to generate histograms. Unpaired Student’s <em>t-test</em> to test for statistical significance was used. A <em>p</em> value &lt; 0.05 was considered significant, and results were expressed as mean ± S.E.M. Single-channel analysis (amplitudes, open burst-times and probability of being open) was performed using Clampfit 11.1.0 and Clampfit 11.7.0. For Fig. 2A, absolute maximum amplitudes were calculated while for Fig. 5, the mean current amplitude values and open burst-times were calculated from values generated over a defined recording segment in Clampfit. Data were sorted using Microsoft excel and histograms generated in Clampfit 11.1.0 and GraphPad Prism 10.3.3. Gaussian functions were fitted to describe and illustrate the presence of the sub-conductance in the presence of emodepside. The open burst-times were defined as a single opening, or groups of openings separated by a close period of ≥ 0.5 ms. Probability of being open was calculated using the formula: Where <em>N</em> is the number of channels in the patch, <em>L</em> is the number of channels open, <em>To<sub>L</sub></em> is the total time <em>L</em> channels are open, and <em>T</em> is the duration of the recording analyzed. Burst-times were not normally distributed (Shapiro-Wilk test), so we used the Wilcoxon signed-rank test which is a non-parametric test to compare burst-times before and after the addition of emodepside. All error bars represent Standard Error of the Mean (SEM), and paired t-tests were utilized where appropriate. The burst-time distribution histograms ≥ 0.5 ms were fitted to a single exponential with the Clampfit software.
description: # Data from: Effects of emodepside on single-channel properties of
 *Onchocerca volvulus* SLO-1A (BK) potassium channels Dataset DOI:
 [10.5061/dryad.jdfn2z3pr](10.5061/dryad.jdfn2z3pr) ## Description of the
 data and file structure **Fig 1C** Data Set consists of a list from
 different cells of peak current responses to the application of 1
 micromolar emodepside to A: non-transfected HEK293 cells and B:
 transfected HEK293 cells. **Fig 2C** Data set is a list of main state
 channel open amplitudes at the -75mV, -50mV, +50mV, and +75mV to determine
 by linear regression the slope conductance of the emodepside activated
 channels. **Fig 4B** Data set for the representative channel open
 amplitude histogram in the absence of emodepside. **Fig 4C** Data set for
 the representative channel open amplitude histogram in the presence of 0.3
 micromolar emodepside that was fitted to three Gaussian functions to
 sub-conductance openings (S1: 1.26 ± 0.02 pA; S2: 1.76 ± 0.03 pA; S3: 2.45
 ± 0.06 pA). **Fig 4D** Data set for the representative burst-times
 histogram for without emodepside with the distribution ≥ 0.5 ms fitted to
 a single exponential with a time-constant, *ϯ,* of 0.12 ms. **Fig 4E**
 Data set for Representative burst-times histogram following the addition
 of 0.3 µM emodepside with the distribution fitted to a single exponential
 with time a constant of 1.96 ms.  **Fig 5A** Data sets for mean current
 before (Mean ± SEM; 2.17 ± 0.16 pA) and after adding 0.3 µM emodepside
 (Mean ± SEM; 2.48 ± 0.21 pA); *p* = 0.03 (paired *t*-test). **Fig 5B**
 Data sets for mean burst-time before (Mean ± SEM; 1.27 ± 0.27 ms) and
 after adding 0.3 µM emodepside (Mean ± SEM; 1.87 ± 0.23 ms); *p* = 0.02
 (Wilcoxon signed-rank test). **Fig 5C** Data sets for *NPopen* before
 (Mean ± SEM; 0.01 ± 0.01) and after adding 0.3 µM emodepside (Mean ± SEM;
 0.15 ± 0.04); *p* = 0.02 (paired *t*-test). ### Files and variables ####
 File: Fig_1C_Histogram_Data_Set.xlsx **Description:** Peak current
 responses, nA to 1 micromolar emodepside at +20 mV holding potential for
 non-transcribed HEK203 cells and transcribed HEK 293 cells ##### Variables
 * peak current responses, nA #### File:
 Fig_2B_I_V_plot_single_channel__data_set.xlsx
 **Description:** Current-Voltage plot for single channel open states
 recordings from HEK 293 cells transfected with pcDNA 3.1(+) *Ovo* *slo-1a*
 T2A GFP plasmid; slope conductance was 110 ± 3 pS, r2 = 0.96 #####
 Variables * Membrane potential: mV and current amplitude, pA. #### File:
 Fig_4B_and_C_channel_amplitudes_data_sets.xlsx **Description:** **B:**
 Representative amplitude histogram without emodepside. **C:**
 Representative amplitude histogram for with emodepside ##### Variables *
 open channel amplitudes, pA #### File:
 Fig_4D_and_E_burst_times_data_sets.xlsx **Description:** **D:**
 Representative burst-times data set for the histogram for without
 emodepside with the distribution ≥ 0.5 ms fitted to a single exponential
 with a time-constant, ϯ, of 0.12 ms. **E:** Representative burst-times
 data set histogram for without emodepside with the distribution ≥ 0.5 ms
 fitted to a single exponential with a time-constant, ϯ, of 0.12 ms #####
 Variables * Burst times ms #### File:
 Fig_5A__B___C_Data_Sets_Effects_of_0.3_emodepside.xlsx
 **Description:** Effect of emodepside on *Ovo*-SLO-1A channel kinetics.
 **A**. Data set for Mean current before (Mean ± SEM; 2.17 ± 0.16 pA) and
 after adding 0.3 µM emodepside (Mean ± SEM; 2.48 ± 0.21 pA); *p* = 0.03
 (paired *t*-test). **B**. Data set for Mean burst-time before (Mean ± SEM;
 1.27 ± 0.27 ms) and after adding 0.3 µM emodepside (Mean ± SEM; 1.87 ±
 0.23 ms); *p* = 0.02 (Wilcoxon signed-rank test). **C**. Data set for
 *NPopen* before (Mean ± SEM; 0.01 ± 0.01) and after adding 0.3 µM
 emodepside (Mean ± SEM; 0.15 ± 0.04); *p* = 0.02 (paired *t*-test), n = 8
 for all experiments. ##### Variables * Open channel amplitudes at +20mV
 pA; burst-times in ms; and probability of a channel being open. ##
 Code/software Excel