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.

Methods

Cloning

O. volvulus slo-1a 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 O. volvulus slo-1a. 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.

HEK 293 Cell culture and transfection

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% CO₂. 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-Ovo-slo1a-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% CO₂. 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% CO₂ 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).

Electrophysiology

Recording Conditions

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 Ovo-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.

Recording Solutions

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 MgCl₂, 1 mM CaCl₂, 11 mM Glucose and 10 mM HEPES, pH 7.4). For whole-cell experiments, the pipette solution contained 140 mM KCl, 1.2 mM MgCl₂, 5.4 mM CaCl₂, 5 mM EGTA, 2 mM dipotassium ATP and 10 mM HEPES, pH 7.2, giving a free (Ca²⁺) of 100 µM; and 137 mM NaCl, 5.9 mM KCl, 1.2 mM MgCl2, 2.2 mM CaCl₂, 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 MgCl₂, 2 mM CaCl₂ and 10 mM HEPES, pH 7.4 in the pipette; and 150 mM KCl, 9.77 mM CaCl₂,10 mM EGTA, and 10 mM HEPES, pH 7.2. The bath (Ca²⁺) was adjusted to be 100 µM using the MAX CHELATOR program: https://somapp.ucdmc.ucdavis.edu/pharmacology/bers/maxchelator/CaMgATPEGTA-TS.htm.

Drugs

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.

Data Analysis

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 t-test to test for statistical significance was used. A p value < 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 N is the number of channels in the patch, L is the number of channels open, To_L is the total time L channels are open, and T 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.