Diethylcarbamazine elicits Ca2+ signals through TRP-2 channels that are potentiated by emodepside in Brugia malayi muscles

ABSTRACT Filarial nematode infections are a major health concern in several countries. Lymphatic filariasis is caused by Wuchereria bancrofti and Brugia spp. affecting over 120 million people. Heavy infections can lead to elephantiasis, which has serious effects on individuals’ lives. Although current anthelmintics are effective at killing microfilariae in the bloodstream, they have little to no effect against adult parasites found in the lymphatic system. The anthelmintic diethylcarbamazine is one of the central pillars of lymphatic filariasis control. Recent studies have reported that diethylcarbamazine can open transient receptor potential (TRP) channels in the muscles of adult female Brugia malayi, leading to contraction and paralysis. Diethylcarbamazine has synergistic effects in combination with emodepside on Brugia, inhibiting motility: emodepside is an anthelmintic that has effects on filarial nematodes and is under trial for the treatment of river blindness. Here, we have studied the effects of diethylcarbamazine on single Brugia muscle cells by measuring the change in Ca2+ fluorescence in the muscle using Ca2+-imaging techniques. Diethylcarbamazine interacts with the transient receptor potential channel, C classification (TRPC) ortholog receptor TRP-2 to promote Ca2+ entry into the Brugia muscle cells, which can activate Slopoke (SLO-1) Ca2+-activated K+ channels, the putative target of emodepside. A combination of diethylcarbamazine and emodepside leads to a bigger Ca2+ signal than when either compound is applied alone. Our study shows that diethylcarbamazine targets TRP channels to promote Ca2+ entry that is increased by emodepside activation of SLO-1 K+ channels.

L ymphatic filariasis (elephantiasis) is a neglected tropical disease caused by filarial nematode parasites that affects more than 120 million people worldwide, with 40 million people suffering from disfiguration or disability (1).Lymphatic filariasis is caused by the adults of the filarial parasites Wuchereria bancrofti, Brugia malayi, and Brugia timori.These parasites are transmitted between hosts through biting insects, and the adults live within the lymphatic vessels.In severe cases, the parasites block lymphatic ducts resulting in swelling of limbs and coarsening of the skin resulting in the condition known as elephantiasis.The swelling of limbs can result in disabilities, societal rejection, and prevent individuals from working.There are no effective vaccines against filarial parasites, nor have the measures to control the spread by their vectors been adequate.
Control of lymphatic filariasis relies on the use of chemotherapeutics to target and kill the microfilaria to prevent transmission between individuals by biting insects.The anthelmintics used to treat lymphatic filariasis include the following: benzimida zoles (albendazole) that bind to β-tubulin, inhibiting microtubule formation as well as metabolism (2); macrocyclic lactones (ivermectin), which target the glutamate-gated chloride channels (3); and diethylcarbamazine (DEC), a compound whose mode of action is not well characterized but have been reported to interact with transient receptor potential (TRP) channels (4,5).Each of these anthelmintics may be administered alone or in combination in mass drug administration (MDA) programs (6,7).However, the use of diethylcarbamazine in individuals who suffer from onchocerciasis is not recommended by the Centers for Disease Control and Prevention (CDC) or the World Health Organi zation (WHO) because of complications that could lead to aggravation of Onchocercainduced eye disease.Although effective at killing and clearing microfilaria, none of these anthelmintics kill all of the adult worms; the surviving parasites live for 6-8 years producing microfilaria.
Historically, diethylcarbamazine had been understood to act on the host immune system rather than the parasite itself (8)(9)(10).However, recent studies have shown that diethylcarbamazine can stimulate nematode TRP channels, including the TRPC ortholog TRP-2 and the transient receptor potential channel, M subtype (TRPM) orthologs GON-2 and CED-11 (4,5).In Brugia muscle cells, the effect of diethylcarbamazine opening TRP channels is an inward depolarizing current and contraction with spastic paraly sis.The inward current produced by diethylcarbamazine is followed by an outward current produced by the activation of Ca 2+ -activated SLO-1 K + channels.The anthelmintic emodepside (Emo), which activates nematode SLO-1 channels (11)(12)(13)(14) is a broad-spec trum antiparasitic drug that has been reported to target and kill adult filaria.However, the in vivo potency of emodepside against some filarial parasites, like Brugia, is limited.Combinations of diethylcarbamazine and emodepside may promote increased efficacy: previous studies have highlighted the synergism between diethylcarbamazine and emodepside on muscle membrane potentials in the soil-transmitted helminth Ascaris suum (12); and the long-term muscle paralysis in Brugia malayi which is dependent on TRP-2 channels (13,15).
In this study, we have utilized Ca 2+ imaging to identify the role of Ca 2+ in mediating diethylcarbamazine signaling and the role of the TRP-2 in promoting Ca 2+ entry.We show that TRP-2 is a major source of diethylcarbamazine-stimulated Ca 2+ entry as inhibition of the channels with SKF96365 and dsRNA knockdown inhibits the Ca 2+ signal.We have tested the effects of arachidonic acid (AA) and miconazole (MIC), and have observed that they increase Ca 2+ entry into the cytoplasm and TRP channel activation.Finally, we identify a synergistic relationship between diethylcarbamazine and emodepside in potentiating the Ca 2+ signal that explains the enhanced paralysis observed in motility assays by disrupting the homeostasis of Ca 2+ in the muscle cell.These results support previous observations that diethylcarbamazine interacts with TRP channels and that the combination of diethylcarbamazine and emodepside has potential for the treatment of individuals infected with Brugia malayi.

Brugia supply and maintenance
Only female Brugia malayi worms were used for the study.Live adult female B. malayi were shipped overnight from the NIH/NIAID Filariasis Research Reagent Resource Center (FR3; College of Veterinary Medicine, University of Georgia, Athens, USA).B. malayi were maintained in non-phenol red Hyclone Roswell Park Memorial Institute (RPMI) 1640 media (Cytiva, USA) containing 10% heat-inactivated fetal bovine serum (FBS; Fisher-Sci entific) and 1% penicillin-streptomycin (Life Technologies, USA).Parasites were separated individually into a 24-well microtiter plate containing 2 mL of the RPMI media.Parasites were held in an incubator set at 37°C and 5% CO 2 .All parasites were used up to 5 days post-delivery.

Dissection of B. malayi
Dissection of B. malayi was performed as previously described (16)(17)(18).All dissections were performed at room temperature.Briefly, worms were cut into 1 cm pieces from the anterior region and single sections placed into the recording chamber filled with B. malayi bath solution (23 mM NaCl, 110 mM NaAc, 5 mM KCl, 1 mM CaCl 2 , 4 mM MgCl 2 , 5 mM N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES), 11 mM D-glucose, 10 mM sucrose, pH 7.2 using NaOH, ~320 mOsmol).The base of the chamber was a coverslip (24 × 50 mm) coated with a thin layer of Sylgard.The body piece was immobilized by gluing each end to the Sylgard pad using Glushield cyanoacrylate glue (Glustitch, BC, Canada) and immobilized by creating a wall down one side under the dissecting microscope.The body piece was cut longitudinally using a tungsten needle, and the "muscle flap" was glued to the coverslip along the cut edge exposing the muscle cells.The intestines and the uterus were removed using fine forceps and the prep was washed with bath solution to remove any eggs or debris.

Fluo-4 injection
To record Ca 2+ signals, muscles were injected with 5 µM Fluo-4 penta-potassium salt (Thermo Fisher Scientific, USA) as previously described and viewed with DIC optics on a Nikon Eclipse TE300 inverted light microscope (400×) (19).Briefly, the dissected worms were treated with 2 mg/mL collagenase (Type 1A, Gibco) for 20-30 s and washed several times with buffer to remove excess collagenase.Patch pipettes were pulled from capillary glass and fire-polished.The pipettes were filled with pipette solution (120 mM KCl, 20 mM KOH, 4 mM MgCl 2 , 5 mM Tris, 0.25 mM CaCl 2 , 4 mM NaATP, 5 mM ethylene glycol-bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid (EGTA), and 36 mM sucrose, pH 7.2 with KOH, ~315-330 mOsmol).Fluo-4 was added to the pipette solution at the start of each recording day at a concentration of 5 µM and was kept in a dark environment to prevent degradation of the dye.Pipettes with a resistance of 1.5-3 MΩ were used.Giga ohm seals were formed before breaking the membrane by suction.After breaking in, cells were left to allow the Fluo-4 solution to diffuse the entire muscle cell (~5 to 10 minutes) (Fig. 1A).

Ca 2+ imaging
All recordings were performed with a Nikon Eclipse TE300 microscope (20×/0.45Nikon PlanFluor objective), fitted with a Photometrics Retiga R1 Camera (Photometrics, Surrey, BC, Canada).Light control was achieved using a Lambda 10-2 two-filter wheel system with a shutter controller (Lambda Instruments, Switzerland).Filter wheel one was set on a green filter (510-560 nM bandpass, Nikon, USA) between the microscope and camera.Filter wheel two was set on the blue filter (460-500 nM, bandpass, Nikon, USA) between a Lambda LS Xenon bulb light box which delivered light via a fiber optic cable to the microscope (Lambda Instruments, Switzerland).Blue light emission was controlled using a shutter.All Ca 2+ signal recordings were acquired and analyzed using MetaFluor 7.10.2(MDS Analytical Technologies, Sunnyvale, CA, USA).Exposure times were 150 ms with 2× binning.Maximal Ca 2+ signal amplitudes (ΔF/F 0 , %) for all stimuli applied were calculated using the equation F − F 0 /F 0 where F is the fluorescent value and F 0 is the baseline fluorescent value, which was determined as the value immediately before the stimulus was applied to the sample for all recordings analyzed.Representative traces were generated using the same formula, with F 0 being the value before the application of the first stimulus.For 1 mM CaCl 2 bath solution control experiments (Fig. 1B and C), F 0 was determined to be the first value of the recording.Rise times were calculated by normaliz ing the trace during stimulus exposure until the application of the 10 mM CaCl 2 positive control, with the lowest fluorescence value being represented by 0% and the highest being 100%.The peak time was calculated by subtracting the time when the stimulus was applied from the time the signal reached 100%.

Application of compounds
The preparation was constantly perfused with all solutions being delivered to the chamber under gravity feed through solenoid valves controlled with a VC-6 six channel Valve Controller (Warner Instruments, Hamden, CT, USA) through an inline heater set at 37°C (Warner Instruments, Hamden, CT, USA) at a rate of 1.5 mL/min.New preparations were perfused with bath solution containing 1 mM CaCl 2 before being exposed to either 30 µM DEC for 5 minutes, 10 µM AA for 5 minutes, 10 µM MIC for 5 minutes or 1 µM Emo for 5 minutes.Exposure to diethylcarbamazine and SKF96365 was achieved by exposing muscle cells to 10 µM SKF96365 alone (SKF) for 5 minutes, followed by 10 µM SKF96365 and 30 µM diethylcarbamazine for 5 minutes (SKF + DEC), and finally to 30 µM DEC alone for 5 minutes giving a total exposure time of 15 minutes.For diethylcarbamazine and emodepside combination experiments, muscle cells were exposed to 30 µM DEC for 5 minutes and then 30 µM diethylcarbamazine + 1 µM emodepside (DEC + Emo) for 5 minutes, for a total of 10 minutes.To ensure that each of the preparations was viable after being exposed to any of the stimuli tested, all samples were subjected to 10 mM CaCl 2 for 1 minute to act as a positive control.Muscles that elicited Ca 2+ signals to 10 mM CaCl 2 (>10%) were considered viable, while those that failed to elicit response (<10%) were classified as non-viable.

Chemicals
Emodepside was procured from Advanced ChemBlocks and SKF96365 was procured from Tocris; Sigma Aldrich supplied all other chemicals.All compounds were dissolved in either water or dimethyl sulfoxide (DMSO) and diluted in bath solution to obtain final concentrations.DMSO final concentration: 0.01%

Statistical analysis
Statistical analysis of all data was done using GraphPad Prism 9.0 (Graphpad Software, Inc., La Jolla, CA, USA).We repeated our experiments to ensure reproducibility.The number of experiments are provided in the results and figure legends.The concen trations and the duration of applications of diethylcarbamazine, arachidonic acid, miconazole, SKF96365, and emodepside are provided in the methods and legends of the figures.Analysis of Ca 2+ amplitudes was done using either unpaired or paired Student's t-tests with P values <0.05 considered as significant using Prism Graphpad version 9.0 software.

Diethylcarbamazine stimulates a detectable Ca 2+ response in Brugia muscles
Ca 2+ signals can be recorded in the muscles of Brugia malayi by directly injecting a fluorescent dye into the cell via a patch pipette (19).Here, we use Fluo-4, an analog of Fluo-3, which has increased fluorescence excitation and higher fluorescence signal levels according to the manufacturer.We loaded individual muscle cells with Fluo-4 until we were able to see key structures of the muscle cell including the muscle arms, nucleus, and the cell body (Fig. 1A).We exposed muscle cells to bath solutions containing 1 mM CaCl 2 and observed small (1.0% ± 0.3%, n = 6) fluctuations in the fluorescent signal (Fig. 1B and C, black bar) due to miniature end-plate potentials that are seen in the Brugia muscle cells.To verify that our muscles are physiologically active, we increased the CaCl 2 concentration by applying 10 mM CaCl 2 to the preparation.We observed rapid large reversible increases in fluorescence (27.4% ± 3.0%, n = 6) when 10 mM CaCl 2 was applied, and that fell when it was removed (Fig. 1B and C, gray bar).
We have reported previously that diethylcarbamazine inhibits motility and produces an initial spastic paralysis in B. malayi that is associated with inward currents on the muscle (4).This inward current was shown to be dependent on cation permeable TRP channels (4).We hypothesized that the activation of these channels would allow the entry of Ca 2+ into the cell, which we could detect using our Ca 2+ -imaging protocol.We exposed muscles cells loaded with Fluo-4 to 30 µM diethylcarbamazine for 5 minutes and observed a characteristic increase in the Ca 2+ signal which decreased when the diethylcarbamazine was removed (Fig. 2A).The average increase in fluorescence to diethylcarbamazine was 15% (15.4% ± 4.2%, n = 7; Fig. 2B, brown bar).The cells were then challenged with 10 mM CaCl 2 to ensure they were functioning physiologically, and we observed an increase in fluorescence (Fig. 2A and B, gray bar, 41.4% ± 11.9%, n = 7).We see that diethylcarbamazine simulates a reversible rise in cytoplasmic Ca 2+ in Brugia muscle cells that is consistent with the opening of TRP channels that are permeable to Ca 2+ .

Arachidonic acid and miconazole that stimulate TRP channels also promote Ca 2+ entry
Nematodes produce poly-unsaturated fatty acids (PUFAs) by converting arachidonic acid into biologically active and inactive PUFAs via ω-hydroxylases and epoxygenases enzymes.Verma et al. (4) showed that arachidonic acid has a similar effect on the amplitude of the inward current on B. malayi muscles as diethylcarbamazine, but the signal was slower than diethylcarbamazine in onset and reaching a peak suggesting that arachidonic acid metabolites were responsible for the activation of the TRP channels.Here, we investigated the role of arachidonic acid on the Ca 2+ signal by exposing the muscles to arachidonic acid.10 µM arachidonic acid stimulated a Ca 2+ signal that had a similar profile to that of diethylcarbamazine (Fig. 3A, compare blue trace to black trace) and a similar overall amplitude (13.3% ± 1.9%, n = 6 vs Fig. 3B, blue bar).Although similar in amplitude, the arachidonic acid signals had a significantly slower rise to the peak of the Ca 2+ signal compared to diethylcarbamazine (5.6 minutes ± 1.0, n = 6 vs 2.6 minutes, ± 0.8, n = 7; Fig. 3C, blue bar), a phenotype that mimics the slower electrophysi ology responses to arachidonic acid (4).
Miconazole is an inhibitor of epoxygenase CYP450 enzymes that mimics the effects of arachidonic acid, which is explained by diverting the metabolism of arachidonic acid to active PUFAs that open the TRP channels in Brugia muscle; like arachidonic acid, miconazole produces a slow opening of TRP channels current during its application (4).
We applied 10 µM miconazole to Brugia muscles and recorded the Ca 2+ signals, and observed Ca 2+ signals with similar peak amplitudes to our responses to diethylcarbama zine and arachidonic acid (11.1% ± 2.1%, n = 7; Fig. 3A, green trace and Fig. 3B, green bar).The times to peak for the miconazole response were slower than our diethylcarba mazine responses (4.4 minutes ± 0.7, n = 7; Fig. 3C, green bar) although the difference did not reach statistical significance.Nonetheless, these results, taken together, further strengthen the idea that TRP channels play a key role in allowing the entry of Ca 2+ into the muscle cells and that endogenous PUFAs can open TRP channels that yield similar Ca 2+ amplitudes as the diethylcarbamazine-mediated responses.

TRP-2 mediates the diethylcarbamazine-induced Ca 2+ signal
The TRPC nematode ortholog channel, TRP-2, has been identified as a key channel in mediating diethylcarbamazine effects, particularly effects on muscle membrane currents and motility (4,15).To test if the observed diethylcarbamazine Ca 2+ signal is mediated by Ca 2+ entering through TRP-2 channels, we treated dissected muscles with the TRPC specific antagonist SKF96365, a compound which we have previously used to inhibit TRP channel activity in both Brugia and the gastrointestinal parasite Ascaris suum (4,5).We exposed muscles to 10 µM SKF96365 for 5 minutes and observed no changes in the Ca 2+ profile (Fig. 4A).We then exposed the muscle to 30 µM diethylcarbamazine in the presence of SKF96365 for an additional 5 minutes and observed that the Ca 2+ signal was inhibited (1.9% ± 0.8%, n = 9; Fig. 4A and B, light brown bar).We also noticed that the inhibition was maintained after washing the preparation in the continued presence of 30 µM diethylcarbamazine for a final 5 minutes and that the effects of SKF96365 were not readily reversed (Fig. 4A).We observed no inhibition to the control CaCl 2 signal (33.2% ± 7.0%, n = 9; Fig. 4B, gray bar).This suggests that the TRPC channel TRP-2 is a major source of the diethylcarbamazine-stimulated Ca 2+ signal in Brugia muscles.

Emodepside potentiates diethylcarbamazine Ca 2+ signals
Emodepside is a current anthelmintic used in veterinary medicine that targets different parasitic nematodes and has significant potential for human use (26,27).Emodepside functions by targeting the Ca 2+ -activated K + channel SLO-1 which causes paralysis of the nematode (11,28).We have demonstrated that emodepside produces flaccid paralysis in Brugia, by opening SLO-1 potassium channels and hyperpolarizes the muscle membrane of Ascaris (12,13,15).Interestingly there is a synergistic relationship between emodep side and diethylcarbamazine resulting in potentiation of flaccid paralysis that is depend ent on TRP-2 in Brugia (15) and which increases in the membrane potential hyperpolarization in the muscles of Ascaris suum (12).
We sought to determine if emodepside alone had any effect on the Ca 2+ signal in our Brugia muscle cells.We predicted that there would be no effect of emodepside alone which by activating SLO-1 potassium channels would cause hyperpolarization (12) and thereby to close any voltage-sensitive Ca 2+ channels.We applied 1 µM emodepside and observed no changes in the Ca 2+ signal amplitude (0.9% ± 0.4%, n = 5), while there was no effect on the control 10 mM CaCl 2 Ca 2+ signal (31.8% ± 8.1%, n = 5; Fig. 6A and B).
Subsequently, we applied 30 µM diethylcarbamazine for 5 minutes and observed the characteristic rise in Ca 2+ that plateaued with an average amplitude of 8% (8.0% ± 1.4%, n = 5; Fig. 6C and D, light brown bar) and then added 1 µM emodepside for 5 minutes on top of diethylcarbamazine; this generated a significant increase in the Ca 2+ signal, 24% (24.2% ± 3.4%, n = 5) which failed to decline after emodepside and diethylcarbamazine were removed (Fig. 6C and D, red bar).This observation may explain the persistence of reduced motility in parasites treated with both diethylcarbamazine and emodepside (15).Our results illustrate a synergistic relationship between diethylcarbamazine and emodepside that together increases the entry of Ca 2+ to disrupt the Ca 2+ homeostasis within the muscles of Brugia malayi.

Use of diethylcarbamazine and emodepside
Diethylcarbamazine, ivermectin, and albendazole are recommended by WHO (1) for lymphatic filariasis MDA in areas without onchocerciasis.Diethylcarbamazine is a drug of choice for the treatment of lymphatic filariasis (7).It kills microfilaria and is active against adult worms, although its effects are less pronounced.Diethylcarbamazine is generally well tolerated but side effects can occur when there are higher numbers of microfilaria in the blood.Diethylcarbamazine should not be used when humans are infected with onchocerciasis because it can make eye disease worse due to the reactions of the larvae on the eye.There is also a concern of a possible severe reaction with individuals infected with Loa loa microfilaria and the development of encephalopathy.
Given that the existing registered antifilarial drugs (diethylcarbamazine, albendazole, and ivermectin) do not kill all adult worms, there are concerns that those that survive administration of therapeutic doses will enhance the rate of development of resistance.Also, if a therapeutic MDA program were able to kill all adults so that microfilariae are no longer produced, elimination would be speeded up.This is because adults can survive for 6-8 years producing millions of microfilariae maintaining the life cycle (29,30) if they are not eliminated.

Interaction of diethylcarbamazine and emodepside involves TRP-2 and SLO-1 K + channels
We have observed in Ascaris suum (12) and in adult Brugia malayi (4, 15) that diethylcar bamazine potentiates the effects of emodepside: 1 µM diethylcarbamazine shifts the effective concentration for 50% of maximum effect (EC50) of emodepside in female Brugia malayi from 395 nM to 114 nM an increase in potency of 3.5-fold (15).This potentiation was hypothesized to involve the activation of TRP-2 channels that allow entry of Ca 2+ to increase cytosolic Ca 2+ and thereby increase emodepside activation of the Ca 2+ -sensitive SLO-1 K + channels.Here, we have seen that application of diethylcar bamazine increases cytosolic Ca 2+ concentrations by activation of the TRP-2 channel.The involvement of TRP-2 channels mediating the effects of diethylcarbamazine on cytosolic Ca 2+ is revealed by inhibition of TRP-2 by the TRP-C antagonist, SKF96365, and by RNAi knockdown of trp-2.Furthermore, we were able to observe that not only does diethylcarbamazine increase cytosolic Ca 2+ directly but that activation of SLO-1 K + channels by emodepside enhances the Ca 2+ response to diethylcarbamazine (Fig. 6 and 7).The positive interaction between TRP channels and SLO-1 K + channels has been described previously in mammalian systems (40).The opening by emodepside of potassium channels in the muscle membrane hyperpolarizes the muscle membrane and increases the driving potential for the entry of Ca 2+ into the muscle cell through the TRP channels opened by diethylcarbamazine.Thus, there is a positive feed-back loop with diethylcarbamazine enhancing the effects of emodepside on SLO-1 K + channels and emodepside enhancing the entry of Ca 2+ produced by diethylcarbamazine (Fig. 7).This positive feed-back loop can explain the synergistic effect of diethylcarbamazine and emodepside on inhibition of motility which can be seen at 1 µM concentrations of diethylcarbamazine (15).

Conclusion
We have seen that diethylcarbamazine has a rapid effect on increasing cytosolic Ca 2+ that is maintained during its application.The increased cytosolic Ca 2+ will activate the contractile machinery of the muscle cells and limit the normal vibrating muscle activity that is seen in healthy worms.Emodepside enhances the effects of diethylcarbamazine with a positive feed-back loop: diethylcarbamazine opens TRP-2 channels, increasing cytosolic Ca 2+ , and activating SLO-1 K + channels; and emodepside hyperpolarizes the membrane potential of the muscle, increasing the driving force for the entry of Ca 2+ .The combination of diethylcarbamazine and emodepside may be useful where the potency of either drug is limited by species of parasite or location of the parasite.

FIG 1
FIG 1 Fluo-4 induced Ca 2+ signals in Brugia muscles.(A) Micrograph of a fluorescing dissected Brugia muscle injected with 5 µM Fluo-4 under blue light.Key structures of the body, nucleus, and muscle arms are indicated with the arrows.The red box indicates the region of interest (ROI) where we take the calcium recording.ROI is determined as the clearest part of the muscle cell with good Fluo-4 fluorescence.(B) Representative control trace of Fluo-4 fluorescence in a muscle being washed with bath solution containing 1 mM CaCl 2 followed by an increase to 10 mM CaCl 2 for 1 minute (gray box).(C) Total amplitudes of Fluo-4 fluorescence in response to constant 1 mM CaCl 2 bath solution perfusion (black bar) and to 10 mM CaCl 2 (gray bar).*** indicates significantly different to 1 mM CaCl 2 bath solution (bath solution vs CaCl 2 ).P < 0.001, t = 8.817, df = 5, paired t-test.n = 6, individual muscles from six individual Brugia females.All values are represented as means ± standard error of the mean (SEM).

FIG 4 FIG 5
FIG 4 Inhibition of TRP-2 ablates diethylcarbamazine sensitivity: (A) Representative trace showing the application of 10 µM SKF96365 (light brown bar) for 5 minutes alone followed by the combination of SKF96365 and of 30 µM diethylcarbamazine (light brown box) for 5 minutes.SKF96365 is removed leaving diethylcarbamazine alone for a final 5-min exposure.Ten millimolar CaCl 2 control was applied for 1 minute (gray box) as a positive control.(B) Total Ca 2+ amplitudes in response to SKF96365 + diethylcarbamazine (SKF + DEC) (light brown bar) and CaCl 2 control (gray bar).* indicates significantly different to SKF96365 + diethylcarbamazine.SKF96365 + diethylcarbamazine (SKF + DEC) vs CaCl 2 : P < 0.002, t = 4.480, df = 8, paired t-test.n = 9 for diethylcarbamazine + SKF96365 responses and n = 9 for CaCl 2 responses from nine individual muscles from nine individual Brugia.All values are represented as means ± SEM.

FIG 7
FIG 7 Summary diagram of the positive feed-back loop produced by diethylcarbamazine and emodepside.Diethylcarbamazine stimulates Ca 2+ entry into Brugia muscle cells via TRP-2 and enhances the activation of SLO-1 K + channels by emodepside that by hyperpolarizing the muscle cell further increases Ca 2+ entry.There is a synergistic relationship between the actions of diethylcarbamazine and emodepside produced by a positive feed-back loop.Diethylcarbamazine activates the TRP-2 channel and emodepside activates the SLO-1 K + channels.