Bma-SLO-1F and Ovo-SLO-1A are highly conserved

Adult Brugia malayi expresses two slo-1 isoforms (slo-1a and slo-1f), whereas adult Onchocerca volvulus has five isoforms (slo-1a, b, c, d, and f). Furthermore, functional expression of these splice variants in the Xenopus oocyte expression system have demonstrated pharmacological differences in the sensitivity of B. malayi slo-1 splice variants to emodepside [7], but the sensitivities to emodepside of the different splice variants of O. volvulus slo-1 were not reported to be different [20]. We compared the amino acid sequences of B. malayi and O. volvulus slo-1 splice variants, to identify the highest sequence conservation between isoforms of the two filarial parasites. From our amino acid sequence analysis, we selected the B. malayi SLO-1F and O. Volvulus SLO-1A splice variants.

Fig 1 shows the amino acid sequence alignment of Bma-SLO-1F and Ovo-SLO-1A using the EMBOSS Needle pairwise sequence online alignment tool. Annotated are characteristic BK channel domains, namely, the N-terminal transmembrane domain, consisting of a voltage sensor domain (enclosed in orange box) and the pore domain (enclosed in blue box). Additionally, within the pore domain is the selectivity filter (light blue box). Fig 1 also shows the cytosolic domain (CTD) that includes two regulating domains for potassium (K⁺) conductance, RCK1 (pink box) and RCK2 (black box). Overall, the isoforms showed 96.2% identity between the two sequences.

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Fig 1. Amino acid sequence alignment of Bma-SLO-1F and Ovo-SLO-1A.

The voltage sensor domain (VSD; orange boxes), pore domain (PD; blue boxes) comprising the selectivity filter (light blue box) and two C-terminal domains for regulator of K⁺ conductance (RCK1; pink boxes and RCK2; black boxes) are indicated. Amino acids which are not identical between filarial species are highlighted by a black background. Gaps are indicated by “_” symbols for amino acid residues that are missing.

https://doi.org/10.1371/journal.ppat.1012946.g001

Further alignment of each individual domain revealed that the voltage sensor domain was 94.5% identical between both isoforms, with differences in eleven (11) amino acid residues (highlighted in black). In contrast, the pore domain and RCK1 domain had identical amino acid residues (100% identity), whereas the RCK2 domain was 98.6% identical with 5 differences in amino acid residues (highlighted in black). The region between the RCK1 and RCK2 domain was 73.5% identical, with Ovo-SLO-1A having 15 additional amino acid residues when compared to Bma-SLO-1F. There were also 12 residues that were not identical (highlighted in black).

Taken together, both Bma-SLO-1F and Ovo-SLO-1A are highly conserved in all domains. Nevertheless, of the four domains, the voltage sensor domain is least conserved with 11 amino acid differences which may be critical in influencing the opening and pharmacology of the individual receptors [21]. Furthermore, the presence or absence of residues between the RCK1 and RCK2 domain of Ovo-SLO-1A and Bma-SLO-1F respectively, may also result in small differences in the receptor conformation during the binding of a ligand or divalent cations and intracellular signaling molecules.

Ovo-SLO-1A is more sensitive to emodepside than Bma-SLO-1F

To test the functional expression of our cloned Bma-slo-1f and Ovo-slo-1a genes in the Xenopus laevis oocyte expression system, we conducted cumulative concentration-response experiments as described in the methods. Fig 2A shows representative traces produced from our recordings for Ovo-SLO-1A (top; pink trace) and Bma-SLO-1F (lower; blue trace) splice variants. We observed that perfusion of increasing concentrations of emodepside (0.1, 0.3, 1, 3, 10 µM), elicited channel activation, with a concentration-dependent increase in outward currents in oocytes expressing their respective ion-channel receptors.

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Fig 2. Emodepside (emo) concentration response relationships for Ovo-SLO-1A and Bma-SLO-1F.

A. Representative traces for two-electrode voltage-clamp recording showing outward currents for Ovo-SLO-1A (top; pink trace) and Bma-SLO-1F (lower; blue trace) channels, elicited in the presence of increasing concentrations of emodepside (0.1 to 10 µM) at a holding potential of +20 mV. B. Emodepside concentration-response relationships for Ovo-SLO-1A (pink) and Bma-SLO-1F. C. Emodepside EC₅₀ analysis (mean ± S.E.M) for Ovo-SLO-1A and Bma-SLO-1F channels. D: Maximum current responses (Rmax) (mean ± S.E.M) of emodepside for Ovo-SLO-1A and Bma-SLO-1F channels. Bottom was constrained to zero for curve fitting. Emodepside concentration-response curves were generated using n = 10 oocytes for Ovo-SLO-1 A and n = 10 oocytes for Bma-SLO-1F, pooled from three independent batches of oocytes to generate 10 biological replicates. EC₅₀ and R_max values were also prepared using the concentration response curve analysis. ***P < 0.001, ****P < 0.0001 significantly different as indicated; unpaired two-tailed student t-test.

https://doi.org/10.1371/journal.ppat.1012946.g002

Fig 2B shows emodepside concentration-response relationships for both channels. The EC₅₀ and maximum response (R_max) values for emodepside for Ovo-SLO-1A expressing oocytes were 0.40 ± 0.05 µM and 10762 ± 478 nA, (n = 10), while the EC₅₀ and maximum response (R_max) values for Bma-SLO-1F were 1.4 ± 0.2 µM and 6446 ± 287 nA, (n = 10). The EC₅₀ for Ovo-SLO-1A was significantly smaller than that for Bma-SLO-1F, thus suggesting that Ovo-SLO-1A is 3.5 times more sensitive to emodepside than Bma-SLO-1F (Fig 2C). Our analysis of the R_max values also show that Ovo-SLO-1A had a statistically significantly higher value in comparison to Bma-SLO-1F (Fig 2D). Finally, we observed that the Hillslope values for the Ovo-SLO-1A expressing oocytes was 1.2 ± 0.1 and oocytes expressing Bma-SLO-1F was 1.1 ± 0.1. These values showed little cooperativity suggesting only 1 molecule of emodepside was binding with the SLO-1 K channels.

Effects of emodepside on Ovo-SLO-1A and Bma-SLO-1F current-voltage relationships

To investigate the effects of emodepside on voltage-dependent currents, we conducted voltage step experiments on oocytes expressing Ovo-SLO-1A, Bma-SLO-1F and water injected oocytes (control). Mean current response for water injected oocytes in the absence or presence of emodepside were similar over the range of step potentials (Fig 3A and 3C). This showed that emodepside had little or no agonist effects on any endogenous channels of Xenopus laevis.

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Fig 3. Effects of emodepside on current-voltage curves (IVCs) of the Ovo-SLO-1A and Bma-SLO-1F channels expressed in Xenopus laevis oocytes.

A. Basal currents (mean ± S.E.M) from oocytes expressing Ovo-SLO-1A (n = 6 biological replicates, black) or injected with water (n = 6 biological replicates, grey) in the absence of emodepside. Currents (mean ± S.E.M) obtained from oocytes expressing Ovo-SLO-1A (n = 6 biological replicates, pink) or injected with water (n = 6 biological replicates, red) in the presence of 1 µM emodepside. B. Slope conductance analysis (mean ± S.E.M) of Ovo-SLO-1A expressing oocytes perfused with recording solution and no drug (n = 6 biological replicates; black), Ovo-SLO-1A expressing oocytes exposed to 1 µM emo (n = 6 biological replicates; pink), water injected oocytes perfused with recording solution and no drug (grey; n = 6 biological replicates) and water injected oocytes exposed to 1 µM emodepside (red; n = 6 biological replicates). C. Basal currents (mean ± S.E.M) from oocytes expressing Bma-SLO-1F (n = 6 biological replicates, black) or injected with water (n = 6 biological replicates, grey) in the absence of emodepside. Currents (mean ± S.E.M) obtained from oocytes expressing Bma-SLO-1F (n = 6 biological, blue) or injected with water (n = 6 biological replicates, tan) in the presence of 1 µM emodepside. D. Slope conductance analysis (mean ± S.E.M) of Bma-SLO-1F expressing oocytes perfused with recording solution and no drug (n = 6 biological replicates; black), Bma-SLO-1F expressing oocytes exposed to 1 µM emo (n = 6 biological replicates; pink), water injected oocytes perfused with recording solution and no drug (grey; n = 6 biological replicates) and water injected oocytes exposed to 1 µM emodepside (red; n = 6 biological replicates). Biological replicates were pooled from two independent studies for water injected, Bma-SLO-1F and Ovo-SLO-1A injected oocytes.

https://doi.org/10.1371/journal.ppat.1012946.g003

However, in the absence of 1 µM emodepside, the slope of current-voltage relationships for oocytes expressing Ovo-SLO-1A channels showed an increase in conductance to 26 ± 2.9 µS compared to the water injected controls, 4.81 ± 1.2 µS, with a reversal potential of -71 mV, suggesting that some of the expressed Ovo-SLO-1A channels are open in the absence of emodepside (Fig 3A and 3B). The application of 1 µM emodepside produced an increase in inward current responses at potentials more negative than the reversal potential, -71 mV, and an increase in outward current at potentials positive than the reversal potential, (Fig 3A and 3B). There was an increase in the conductance to 61 ± 4.4 µS, but no change in the reversal potential (Fig 3A and 3B). Collectively, these observations demonstrate that emodepside increases the opening of the filarial Ovo-SLO-1A channels expressed in Xenopus oocytes.

Unlike the Ovo-SLO-1A channels that produced high current responses in the absence and presence of emodepside, Bma-SLO-1F channels showed smaller current responses. Firstly, we observed that the control (basal) currents for oocytes expressing Bma-SLO-1F were not significantly different from water injected oocytes in the absence of 1 µM emodepside (Fig 3C). The difference in conductance, 2.4 ± 0.3 µS, of the Bma-SLO-1F injected oocytes compared to the conductance, 1.6 ± 0.1 µS, of the water injected oocytes was not significant (Fig 3D). This indicates that there were fewer open Bma-SLO-1F channels present in the oocytes.

Secondly, the conductance change induced by the application of 1 µM emodepside was smaller. 1 µM emodepside, increased the conductance from 2.4 ± 0.3 µS to 12 ± 1.0 µS in the Bma-SLO-1F expressing oocytes. Again, 1 µM emodepside produced no detectable change in the conductance for water injected oocytes (Fig 3C and 3D). The reversal potential of all the current voltage plots was close to -72 mV and close to the reversal potential of the Ovo-SLO-1A expressed channels.

Thirdly, a reduction in the outward current response was notably different at +40 mV to +60 mV step potentials for Bma-SLO-1F (Fig 3C). This phenomenon was absent from the current-voltage curves of Ovo-SLO-1A. Despite having similar sequences, Ovo-SLO-1A and Bma-SLO-1F, showed clear detectable differences in their current-voltage relationship, suggesting that the differences in the voltage-sensitive regions of the channels affects the response to emodepside.

GoSlo-SR-5–69 is not an activator of Bma-SLO-1F and Ovo-SLO-1A channel

To explore the effects of additional synthetic BK channel activators on filarial SLO-1 K channels, we selected and tested the tetrahydro-2-napthalene derivative, GoSlo-SR-5–69 which is a mammalian BK channel opener. We observed in oocytes held at a steady-state potential of +20 mV, that application of 3 µM GoSlo-SR-5–69 to water injected oocytes and oocytes expressing Bma-SLO-1F and Ovo-SLO-1A receptors, produced slowly inactivating inward current, (Fig 4A, 4B and 4C). In contrast, subsequent application of 0.3 µM emodepside resulted in the activation of Bma-SLO-1F and Ovo-SLO-1A channels that produced outward K⁺ currents, (Fig 4B and 4C). Water injected oocytes yielded no response to emodepside. These results show that GoSlo-SR-5–69 does not by itself activate Bma-SLO-1F or Ovo-SLO-1A receptors.

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Fig 4. Effects of GoSlo-SR-5-69 on Bma-SLO-1F and Ovo-SLO-1A channels.

A. Sample trace of water injected oocytes (n = 6). B. Sample trace of oocytes expressing the Bma-SLO-1F channel (n = 6). C. Sample trace of Ovo-SLO-1A expressing channel (n = 6). Application of 3 µM GoSlo-SR-5-69 produced transient inactivating inward currents in oocytes injected with water, Bma-SLO-1F and Ovo-SLO-1A channels. Emodepside failed to activate water injected oocytes but produced outward currents in oocytes expressing Bma-SLO-1F and Ovo-SLO-1A channels. For each splice variant and water injected oocytes, 6 oocytes were tested from a single batch to generate 6 biological replicates.

https://doi.org/10.1371/journal.ppat.1012946.g004

GoSlo-SR-5–69 is a positive allosteric modulator of filarial nematode SLO-1 channels

Our experiments above demonstrated that GoSlo-SR-5–69 lacks agonist activity on Bma-SLO-1F and Ovo-SLO-1A receptors at a concentration of 3 µM. To test for allosteric emodepside modulating effects of GoSlo-SR-5–69, oocytes were perfused with 0.3 µM emodepside for channel activation, followed by co-application of 3 µM GoSlo-SR-5–69 in the continued presence of emodepside (Fig 5A). This was then followed by washing of the GoSlo-SR-5–69 and then washing of the 0.3 µM emodepside with recording solution.

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Fig 5. Effects of GoSlo-SR-5-69 on Bma-SLO-1F and Ovo-SLO-1A-mediated emodepside responses.

A. Representative traces for Bma-SLO-1F, Ovo-SLO-1A and water injected oocytes perfused with 0.3 µM emodepside, followed by 3 µM GoSlo-SR-5-69 in the continued presence of emodepside and an initial wash with 0.3 µM emodepside with a final wash with oocyte recording solution. B. Mean current responses (in nA) generated in response to 0.3 µM emodepside alone and in combination with 3 µM GoSlo-SR-5-69 for oocytes expressing the Bma-SLO-1F channel. Blue bar: 0.3 µM emodepside alone (n = 6 biological replicates, pooled from two independent experiments). Black bar: 3 µM GoSlo-SR-5-69 co-applied with 0.3 µM emodepside (n = 6 biological replicates, pooled from two independent experiments). C. Mean current responses (in nA) generated in response to 0.3 µM emodepside alone and in combination with 3 µM GoSlo-SR-5-69 for oocytes expressing the Ovo-SLO-1A channel. Pink bar: 0.3 µM emodepside alone (n = 6 biological replicates, pooled from two independent experiments). Black bar: 3 µM GoSlo-SR-5-69 in combination with 0.3 µM emodepside (n = 6 biological replicates, pooled from two independent experiments). D. Percentage (%) increase analyses of currents produced by emodepside and GoSlo-SR-5-69 co-application for Bma-SLO-1F (white, n = 6) and Ovo-SLO-1A (grey, n = 6). Data are plotted as mean ± S.E.M; ****P < 0.0001 significantly different as indicated; paired two-tailed student t-test; **P < 0.001, significantly different as indicated, unpaired two-tailed student t-test.

https://doi.org/10.1371/journal.ppat.1012946.g005

We observed that activating both Bma-SLO-1F and Ovo-SLO-1A with emodepside (0.3 µM), then co-applying 3 µM GoSlo-SR-5–69 with emodepside, produced an additional increase in the outward current amplitude response for both channels (Fig 5A). Washing off the GoSlo-SR-5–69 reduced the amplitude of the current response of both receptors. Water injected oocytes did not show activation with emodepside or potentiation when GoSlo-SR-5–69 and emodepside were co-applied (Fig 5A). These results provide validation that the observed potentiation of channel openings of Bma-SLO-1F and Ovo-SLO-1A is elicited by GoSlo-SR-5–69.

Using traces of experiments like those shown in Fig 5A, we quantified current responses for both receptors in the presence of 0.3 µM emodepside alone, and in combination with 3 µM GoSlo-SR-5–69. The current (I_max) responses for oocytes expressing Bma-SLO-1F challenged with 0.3 µM emodepside alone were 954 ± 355 nA, (n = 6); in the combined presence of 3 µM GoSlo-SR-5–69 and 0.3 µM emodepside, they were 2799 ± 100 nA, (n = 6), (Fig 5B). For Ovo-SLO-1A injected oocytes, the current responses were 4170 ± 355 nA, (n = 6) in the sole presence of emodepside and 6735 ± 272 nA, (n = 6) in the presence of 3 µM GoSlo-SR-6–69 (Fig 5C). Our analysis confirmed that GoSlo-SR-5–69 significantly increased the emodepside responses of both Bma-SLO-1F and Ovo-SLO-1A receptors.

We also calculated the percentage increases in peak currents produced by 3 µM GoSlo-SR-5–69 on the 0.3 µM emodepside response for Bma-SLO-1F and Ovo-SLO-1A. Our analyses revealed that Bma-SLO-1F percentage increase in peak currents was 2.6 times larger than that of Ovo-SLO-1A, namely 200% and 65.2% respectively (Fig 5D). This implies a greater potency of GoSlo-SR-5–69 on Bma-SLO-1F. These findings are notable and highlight the positive allosteric effect of GoSlo-SR-5–69 on SLO-1 K channels isoforms of two filarial nematode species.

Extracellular Ca²⁺ is not required for GoSlo-SR-5–69 potentiation of emodepside

We have observed that GoSlo-SR-5–69 produces a slowly inactivating inward current in water injected oocytes and oocytes expressing Bma-SLO-1F or Ovo-SLO-1A channels. In addition, we have also observed that activation of the filarial nematodes SLO-1 K splice variant channels with emodepside, followed by co-application with GoSlo-SR-5–69 led to a significant potentiation of emodepside responses. If the transient inward currents produced by GoSlo-SR-5–69 were due to activation of Ca²⁺ permeable channels, then the entry of Ca²⁺ may lead to the activation of Bma-SLO-1F and Ovo-SLO-1A channels. To test this possibility, we replaced Ca²⁺ with equimolar Co²⁺ in our oocyte recording solution to inhibit the entry of Ca²⁺ during our recordings.

The inward current response to GoSlo-SR-5–69 persisted unchanged when extracellular Ca²⁺ was replaced by Co²⁺ in the water injected oocytes and oocytes expressing the Bma-SLO-1F or Ovo-SLO-1A channels, as shown in S1A, S1B and S1C Fig. These currents were not Ca²⁺ currents and emodepside still activated outward currents when Ca²⁺ was replaced by Co²⁺. We did not investigate these GoSlo-SR-5–69 currents further that were produced by the Xenopus oocytes and were not associated with the expression of Bma-SLO-1F or Ovo-SLO-1A channels.

To determine the influence of extracellular Ca²⁺ on the GoSlo-SR-5–69 potentiation of emodepside, we compared results from oocytes expressing Bma-SLO-1F and Ovo-SLO-1A that were exposed to extracellular Ca²⁺ with oocytes that had Ca²⁺ replaced with equimolar Co²⁺. GoSlo-SR-5–69 still potentiated the emodepside responses in the absence of Ca²⁺ (Fig 6A, 6B, 6D and 6E). Although the mean currents appeared smaller in the absence of extracellular Ca²⁺, the differences in percentage increase in the emodepside currents by GoSlo-SR-5–69 were not significant (Fig 6C and 6F). These observations suggest that GoSlo-SR-5–69 potentiation is not mediated by entry of extracellular Ca²⁺ that could increase SLO-1 K channel opening.

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Fig 6. Effects of extracellular Ca²⁺ on GoSlo-SR-5-69 potentiation of Bma-SLO-1F and Ovo-SLO-1A-mediated emodepside responses.

A. Peak current (in nA) amplitudes produced by Bma-SLO-1F expressing oocytes in response to 0.3 µM emodepside alone and in combination with 3 µM GoSlo-SR-5-69 in the presence of normal recording solution consisting of 1.8 mM added Ca²⁺. Blue bar: 0.3 µM emodepside alone; Black bar: 3 µM GoSlo-SR-5-69 co-applied with 0.3 µM emodepside (n = 6 biological replicates, pooled from three independent experiments). ****P < 0.0001 significantly different as indicated; paired two-tailed student t-test B. Peak current (in nA) amplitudes produced by oocytes expressing the Bma-SLO-1F channel in response to 0.3 µM emodepside alone and in combination with 3 µM GoSlo-SR-5-69 in the absence of 1.8 mM added Ca²⁺. Blue bar: 0.3 µM emodepside alone; Black bar: 3 µM GoSlo-SR-5-69 co-applied with 0.3 µM emodepside (n = 6 biological replicates, pooled from three independent experiments). ***P < 0.001, significantly different as indicated, paired two-tailed student t-test. C. Percentage (%) increase in currents produced by emodepside and GoSlo-SR-5-69 co-application for Bma-SLO-1F expressing oocytes perfused with normal recording solution, containing 1.8 mM added Ca²⁺ (White bar with brown border, n = 6), or modified recording solution lacking added Ca²⁺(grey bar with black border, n = 6). P > 0.05, no statistical significance (ns) as indicated, unpaired two-tailed student t-test. D. Peak current (in nA) amplitudes produced by oocytes expressing the Ovo-SLO-1A channel in response to 0.3 µM emodepside alone and in combination with 3 µM GoSlo-SR-5-69 in the presence of 1.8 mM added Ca²⁺. Pink bar: 0.3 µM emodepside alone; Black bar: 3 µM GoSlo-SR-5-69 co-applied with 0.3 µM emodepside (n = 6 biological replicates, pooled from three independent experiments). E. Peak current (in nA) amplitudes produced by Ovo-SLO-1A expressing oocytes in response to 0.3 µM emodepside alone and in combination with 3 µM GoSlo-SR-5-69 in the absence of 1.8 mM added Ca²⁺. Pink bar: 0.3 µM emodepside alone; Black bar: 3 µM GoSlo-SR-5-69 co-applied with 0.3 µM emodepside (n = 6 biological replicates, pooled from three independent experiments). **P < 0.0001 significantly different as indicated; paired two-tailed student t-test; ***P < 0.01, significantly different as indicated, paired two-tailed student t-test. F. Percentage (%) increase in currents produced by emodepside and GoSlo-SR-5-69 co-application for Ovo-SLO-1A expressing oocytes in the presence of 1.8 mM added Ca²⁺ (White bar with orange border, n = 6), or modified recording solution lacking added Ca²⁺(Grey bar with black border, n = 6). P > 0.05, no statistical significance (ns) as indicated, unpaired two-tailed student t-test.

https://doi.org/10.1371/journal.ppat.1012946.g006

GoSlo-SR-5–69 increases emodepside potency and efficacy for Bma-SLO-1F

Our observation of GoSlo-SR-5–69 potentiating emodepside responses in our previous experiments prompted us to investigate further the positive allosteric modulation. We compared the effect of GoSlo-SR-5–69 on the concentration-response relationships of emodepside on oocytes expressing the Bma-SLO-1F channel with the concentration effect of emodepside alone. For the experiments we alternated between oocytes tested with emodepside alone and those pre-treated with GoSlo-SR-5–69. A representative trace of emodepside current responses alone and in the presence of 3 µM GoSlo-SR-5–69 is shown in S2A and S2B Fig.

Our analysis revealed a left shift in the sigmoidal concentration-response curve for emodepside in the presence of GoSlo-SR-5–69 (Fig 7A). The EC₅₀ was 1.40 ± 0.15 µM and R_max was 2679 ± 318 nA, n = 6, for emodepside in the absence of GoSlo-SR-5–69. The EC₅₀ was 0.20 ± 0.02 µM and the R_max value was 4493 ± 433 nA, n = 6 for emodepside in the presence of 3 µM GoSlo-SR-5–69, n = 6.

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Fig 7. Effects of GoSlo-SR-5-69 as a positive allosteric modulator on Bma-SLO-1F and Ovo-SLO-1A channels on emodepside mediated response.

A. Concentration-response plots for emodepside alone, (blue) and emodepside in the presence of 3 µM GoSlo-SR-5-69 (black) for the Bma-SLO-1F channel, (n = 6 biological replicates, pooled from three independent experiments). B. Emodepside concentration-response plots for Ovo-SLO-1A in the presence of emodepside alone, pink) and emodepside in the presence of 3 µM GoSlo-SR-5-69 (black), (n = 6 biological replicates, pooled from three independent experiments). Bottom was constrained to zero for curve fitting.

https://doi.org/10.1371/journal.ppat.1012946.g007

The sensitivity of the Bma-SLO-1F channel was increased 7-fold to emodepside in the presence of 3 µM GoSlo-SR-5–69. Moreover, the 1.7-fold increase in R_max also implies an increased efficacy of emodepside when GoSlo-SR-5–69 is present.

GoSlo-SR-5–69 increases emodepside efficacy for Ovo-SLO-1A

The potentiation of emodepside responses were also seen for oocytes expressing the Ovo-SLO-1A channel. Using a similar approach as mentioned previously for Bma-SLO-1F, we investigated the concentration-response relationships on of emodepside Ovo-SLO-1A in the presence of 3 µM GoSlo-SR-5–69. A representative trace of emodepside current responses alone and in the presence of 3 µM GoSlo-SR-5–69 is shown in S3A, S3B Fig. respectively.

The concentration response plots showed GoSlo-SR-5–69 is also a positive allosteric modulator of Ovo-SLO-1A (Fig 7B). The EC₅₀ was 0.50 ± 0.09 µM and the R_max was 2454 ± 87 nA, (n = 6), for emodepside in the absence of GoSlo-SR-5–69: the EC₅₀ was 0.40 ± 0.03 µM and R_max was 4928 ± 830 nA, (n = 6) in the presence of 3 µM GoSlo-SR-5–69. GoSlo-SR-5–69 did not cause a significant shift in EC₅₀, but significantly increased the R_max, confirming that GoSlo-SR-5–69 is also a positive allosteric modulator of the Ovo-SLO-1A receptor.

Molecular docking proposes favorable binding mode of emodepside at S6 pocket in Ovo-SLO-1A and Bma-SLO-1F channels

Our molecular docking of emodepside in the SLO-1 K channel for both B. malayi and O. volvulus suggests that it adopts a similar pose, consistent with the findings for the cryo-EM structure of D. melanogaster SLO-1 K channel [16] (Fig 8A and 8B). The favorable hydrophobic pocket at the S6 site suitably accommodates emodepside, suggested by GLIDE to have very favorable docking scores, and an improvement on those of the D. melanogaster SLO-1 conformation, at -9.3 kcal/mol for Ovo-SLO-1A, and -9.7 kcal/mol for Bma-SLO-1F compared to -8.6 kcal/mol for D. melanogaster.

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Fig 8. Identified poses of emodepside and GoSlo-SR-5-69 in Ovo-SLO-1A and Bma-SLO-1F channels.

A. Emodepside bound at the S6 pocket for Ovo-SLO-1A channel. B. GoSlo-SR-5-69 bound at the RCK1 pocket for Ovo-SLO-1A channel. C. Emodepside bound at the S6 pocket for Bma-SLO-1F channel D. GoSlo-SR-5-69 bound at the RCK1 pocket for Bma-SLO-1F channel. For both channels, emodepside is bound below the selectivity filter, indicating the π-π stacking between F342 and emodepside. Displayed are also the stabilizing ligand-receptor interactions between GoSlo-SR-5-69 and the respective filarial nematode splice variant channels.

https://doi.org/10.1371/journal.ppat.1012946.g008

The top poses identified by GLIDE for both channels indicated a stabilizing π-π interaction between F342 and the phenyl rings of emodepside (Fig 8A and 8C), but these interactions were not found with the corresponding ligand F329 on the D. melanogaster binding pocket. After energy minimization, the RMSD difference in the emodepside structures between Bma-SLO-1F and Ovo-SLO-1A was low at 1.1 Å. The difference in the F342 residue position between the Bma-SLO-1F and Ovo-SLO-1A was 0.2 Å, with marginally better orientation between the phenyl rings for Ovo-SLO-1A. The RMSD between D. melanogaster and each of the nematode BK channels for the emodepside binding position was 6.5 Å for Ovo-SLO-1A and 6.4 Å for Bma-SLO-1F.

Molecular docking suggests the RCK1 binding site for GoSlo-SR-5–69

To elucidate the potential positive allosteric modulation mechanism of GoSlo-SR-5–69 in the SLO-1 K channels of Ovo-SLO-1A and Bma-SLO-1F, we probed the additional drug-binding pockets, RCK1 and RCK2 previously identified [16] using molecular docking. We identified binding poses for the GoSlo-SR-5–69 in both the RCK1 and RCK2 pockets; but the docking scores for the RCK1 pocket were much more favorable for both channels compared to those of the RCK2 pocket, with an average GlideScore difference of 2.4kcal/mol between two pockets. The GlideScores for the RCK1 pocket were -3.0 kcal/mol for Bma-SLO-1F and -2.5 kcal/mol for Ovo-SLO-1A. More non-covalent and π interactions are present within the RCK1 pocket, with the following key interactions between the ligand and channels for both Bma-SLO-1F and Ovo-SLO-1A: H-Bond (Bma-SLO-1F: H406, E463, N454, Ovo-SLO-1A: H406, R470, E463, N454), Salt bridge (Bma-SLO-1F: R407, R470, Ovo-SLO-1A: R470), π-π stacking (Ovo-SLO-1A: Y377) (Fig 8B and 8D). Overall, this suggests that the RCK1 pocket is the preferred binding site for GoSlo-SR-5–69 over RCK2.

Emodepside is more potent on Ovo-SLO-1A than previously reported

Here we demonstrate that emodepside is more potent on Ovo-SLO-1A than Bma-SLO-1F. This differs from previous reports in two independent studies that show a higher emodepside EC₅₀ for Ovo-SLO-1A [20] and a lower EC₅₀ for Bma-SLO-1F [7]. The discrepancy in potency between these studies and our findings may be attributed to differences in the duration of emodepside application. Emodepside is highly lipophilic, thus resulting in membrane partitioning prior to reaching its target site. Consequently, the activation of SLO-1 K channels by emodepside has a long-time course to achieve maximal effect.

Muscle tension recordings have shown that emodepside produces a much slower inhibitory action on Ascaris suum muscle contraction in contrast to gamma-amino butyric acid (GABA) that caused a rapid inhibitory response [22]. Furthermore, the same study also showed that emodepside produces a slow hyperpolarization of A. suum muscle cells in current clamp recordings [22]. In another study, emodepside activation of A. suum SLO-1 K channels elicited currents that were slow in onset, gradually increasing over a longer period exceeding 10 minutes [23].

In our experiments, we also observed that the effects of emodepside on heterologously expressed Bma-SLO-1F and Ovo-SLO-1A splice variant channels were slow, with current responses increasing gradually to a peak amplitude over a period of 5 minutes. Hence, perfusion of emodepside for a longer (5 minutes) rather than shorter (30 seconds or 1 minute) period, allows the elicited current responses to achieve plateau at each concentration. Consequently, concentration-response curves were produced that displayed the maximal effects of emodepside. Taken together, extending the duration of emodepside application is critical for improving the estimation of emodepside potency and efficacy on nematode SLO-1 K channel isoforms.

Explanation of differences in Bma-SLO-1F and Ovo-SLO-1A sensitivity to emodepside

Our homology modeling revealed that emodepside can bind in the S6 pocket of the pore domain (PD) below the selectivity filter of Ovo-SLO-1A and Bma-SLO-1F, Fig 9A and 9C. Remarkably, this is similar to previous docking studies of emodepside with cryo-EM structures of the D. melanogaster Slo (dSlo1) channel [16].The PD is 100% identical between both channel isoforms and emodepside interacts with the same amino acid residues (S4 Fig). This suggests that the difference in emodepside potency should be attributed to allosteric effects of other structural domains such as the cytosolic domain (CTD) and voltage sensor domain (VSD) that modulate SLO-1 K channel function.

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Fig 9. Summary diagram of the putative mechanism of GoSlo-SR-5-69 alone and in combination with emodepside on Bma-SLO-1F and Ovo-SLO-1A channels.

A. Emodepside (gold ring) binds within the pore domain (PD) beneath the selectivity filter (light blue inverted block arc) and directly activates either Bma-SLO-1F or Ovo-SLO-1A channels resulting in the translocation of K⁺ ions (red circles) out of the cell (outward current). B. Activation of Bma-SLO-1F or Ovo-SLO-1A by emodepside (gold ring) in the PD. GoSlo-SR-5-69 (green octagon) then binds to a putative allosteric site (RCK1 domain) of the channel giving rise to stabilization of the channel gating ring in the open state. Subsequently, K⁺ permeation and translocation are further increased leading to greater current amplitude and the potentiation of emodepside response. The removal of GoSlo-SR-5-69 results in ion translocation almost back to normal levels previously seen in the presence of emodepside binding alone.

https://doi.org/10.1371/journal.ppat.1012946.g009

The CTD is connected to the C-terminus of the PD and serves as an intracellular sensor of Ca²⁺, Mg²⁺ and other intracellular ligands. In addition to its chemo-mechanical role, the CTD also possesses regulatory regions that impact SLO-1K channel function. We observed that oocytes expressing Ovo-SLO-1A channels produced higher currents and greater slope conductance’s than Bma-SLO-1F. Previous studies have reported a direct correlation between the membrane conductance and the singel channel conductances, the number of ion channels expressed, and the probability of the channels being open [24,25]. 1) the probability of the Ovo-SLO-1A channels being open may be higher than the Bma-SLO-1F channels or 2) the single channel conductance of the Ovo-SLO-1A channels may be bigger than the Bma-SLO-1F channels.

Variation in the tension of the RCK1-RCK2 loop in the CTD influences SLO-1 K activation. We found that the RCK1-RCK2 loop for Bma-SLO-1F was shorter than that of Ovo-SLO-1A with a total length of 87 and 102 amino acids, respectively. Mammalian SLO-1 K channels generally have at least 101 amino acids separating the two RCK domains. Reducing the length of the RCK1-RCK2 loop affects SLO-1 K channel function and consequently macroscopic currents [26]. A minimum length of approximately 70 amino acids is required in the connecting loop for the channel to be functional [26]. This shorter loop for Bma-SLO-1F is predicted to reduce the channel openings due to a stronger tension on the gate that is needed for openings. The shorter amino acid length separating the RCK1-RCK-2 loop is predicted to reduce the overall lower currents for Bma-SLO-1F produced in the absence and presence of emodepside.

Differences in emodepside sensitivity between Bma-SLO-1F and Ovo-SLO-1A may also be attributed to non-conserved amino acid residues in the VSD. The PD is surrounded by four VSDs that forge strong electromechanical coupling [27,28]. Changes in membrane potential induces conformational changes in the central cavity of the channel, resulting in its activation or deactivation [29,30]. We identified 11 amino acid residue differences in the VSD of Ovo-SLO-1A and Bma-SLO-1F. Despite these differences in the VSD, our voltage step analysis showed that emodepside produced increased currents for both channel isoforms in the absence of increased intracellular Ca²⁺ and hyperpolarized potentials. This provides further evidence that emodepside deregulates voltage and Ca²⁺ sensitivity of SLO-1 K channels [16]. Nevertheless, the elicited emodepside currents were inhibited at potentials exceeding +20 mV for Bma-SLO-1F, in a manner like that seen for the Caenorhabditis elegans SLO-1A channel [31]. The non-conserved residues in the VSD may alter the structural architecture of the VSD, thus influencing interactions with the PD during channel pore activation. However, further studies are needed to test these assumptions.

The significance and mode of action of the potentiator, GoSlo-SR-5–69

Drug combination therapies have been widely used for the treatment of malaria, tuberculosis, and HIV [32–37]. Despite the therapeutic potential of emodepside for the treatment of human onchocerciasis, its use as an adulticidal treatment of lymphatic filariasis has not been pursued due to reduced emodepside potency on Brugia spp. [6,8]. Increasing the potency of emodepside on the less sensitive Brugia spp. could offer a significant therapeutic advance.

A putative candidate for drug combination and toxicity testing are the negatively charged activators (NCAs) like GoSlo-SR-5–69. The GoSlo-SR family of compounds have been reported to activate mammalian SLO-1 K channels through interactions with amino acid residues in the transmembrane domain [17,19]. Additionally, their negatively charged sulphonate group has been predicted to attract numerous K⁺ ions to the channel pore, increasing ion occupancy and consequently channel conductance, based on the mechanism of other NCAs [38].

GoSlo-SR-5–69 is one of the most potent and efficacious mammalian BK channel activators, having an EC₅₀ of 251 nM [17]. However, its effects on the human SLO-1 K channels (KCNMA) are unknown. In addition to GoSlo-SR-5–69, several BK channel activators have been synthesized for the past decades and studied in animal models. However, their therapeutic potential for treating diseases that affect humans has been diminished due to failure to clear phase III clinical trials [39]. This could possibly be attributed to their lack of effect at physiological membrane potentials. Hence, lack of efficacy in humans, while enhancing the potency and efficacy of anthelmintics in combination is desired for treating filarial diseases.

We found in our experiments that GoSlo-SR-5–69 does not activate the channels of Bma-SLO-1F or Ovo-SLO-1A. Nevertheless, we observed clear effects of GoSlo-SR-5–69 in combination with emodepside, that produced emodepside potentiation. Remarkably, the sensitivity of Bma-SLO-1F to emodepside, measured by its EC₅₀ was increased 7-fold, while the EC₅₀ of Ovo-SLO-1A remained unchanged, although there was an increase in the R_max.

Our homology modelling revealed that GoSlo-SR-5–69 can bind to the RCK1 pocket of both channels, at the Rossmann-fold subdomain (βA-βF) in the Ca²⁺ bound open state. The Rossmann-fold subdomain is known to form the central core of the gating ring [28]. Therefore, we propose a mechanism for emodepside potentiation that involves the binding of emodepside at the pore domain, leading to channel activation. Access of GoSlo-SR-5–69 to the channel in its open conformation allows favorable binding of the molecule at the RCK1 domain where it stabilizes the open-state of the channel, thereby potentiating emodepside response. GoSlo-SR-5–69 would not have access to this binding site in the closed conformation and by itself does not open the channel.

We also note that the greater level of emodepside potentiation by GoSlo-SR-5–69 on Bma-SLO-1F could be attributed to minor differences in GoSlo-SR-5–69 interactions with the channels. GoSlo-SR-5–69 was found to dock to the same residues except for R407 and P409 for Ovo-SLO-1A (S4 Fig). Hence, slight differences in binding site may influence the degree of potentiation.

In conclusion, our data has highlighted the pharmacological diversity of two highly conserved filarial nematode SLO-1 K channels. We demonstrate that both Bma-SLO-1F and Ovo-SLO-1A are activated by emodepside. Bma-SLO-1F is less sensitive to emodepside than Ovo-SLO-1A, providing an explanation for the lack of emodepside efficacy on Brugia spp. in contrast to Onchocerca spp. We also show that GoSlo-SR-5–69 is not an activator of the splice variant channels but acts as a positive allosteric modulator: 1) increasing the potency and efficacy of emodepside on Bma-SLO-1F channels; and 2) increasing emodepside efficacy on Ovo-SLO-1A. The identification of emodepside and GoSlo-SR-5–69 binding site and provides a proposed mechanism of action for GoSlo-SR-5–69 potentiation of emodepside. We provide support for the concept of increasing emodepside potency on B. malayi by using drug combinations.

Sequence analysis

Bma-SLO-1F and Ovo-SLO-1A amino acid sequences were acquired from the B. malayi and O. volvulus genome using WormBase ParaSite (parasite.wormbase.org). Sequence alignment was conducted using EMBOSS Needle pairwise sequence alignment tools, with EBLOSUM62 matrix, a default gap penalty of 10 and extension penalty of 0.5 [40], to determine sequence identity and similarity between both species and isoforms.

Sequence annotation was achieved using previously published alignment information by highlighting the voltage sensor domain (VSD), pore domain (PD) and the regulator of potassium conductance domains (RCK1 and RCK2) [16]. To further estimate conservation of each individual domain between isoforms, alignments and sequence identity analysis were also conducted for each domain individually.

Cloning of Brugia malayi slo-1f and Onchocerca volvulus slo-1a

Primers for Brugia malayi slo-1f and Onchocerca volvulus slo-1a isoforms were designed with sequences flanking the pT7TS-rich expression vector that included the restriction site (NheI). PCR amplification was conducted on B. malayi slo-1f that was previously cloned in the pCDNA3.1 vector. In contrast, Onchocerca volvulus slo-1a was synthesized by Life Technologies GeneArt. Subsequently, both amplicons were separated on a 1% Agarose SYBR Safe gel, purified using NucleoSpin Gel and PCR Clean-up Kit (Macherey-Nagel) and cloned into the pT7TS-rich vector by using Infusion HD Cloning Kit (Takara Bio USA, Inc) according to the manufacturer’s protocols. Once cloned, the plasmids were verified by sequencing.

In vitro transcription of Bma-slo-1f and Ovo-slo-1a

The pT7TS-rich plasmids containing cloned products of either Bma-slo-1f or Ovo-Slo-1a were linearized by SmaI and BamHI respectively and purified. Capped cRNAs were then synthesized from the linearized vectors containing the B. malayi and O. volvulus Slo-1 isoforms previously mentioned using the T7 mMessage mMachine Kit (Ambion, USA). The cRNAs were stored at -80°C until further use.

Heterologous expression of Bma-SLO-1F and Ovo-SLO-1F receptors in Xenopus laevis oocytes

Defolliculated Xenopus laevis oocytes were purchased from Ecocyte Bioscience (Austin, TX, USA) and Xenopus 1 Corp (Dexter, MI, USA). Heterologous expression of the Bma-SLO-1F receptor was achieved by injecting 15 ng of cRNA in a total volume of 50 nL in nuclease-free water. Each oocyte was microinjected into the cytoplasm of the animal pole region using a Drummond Nanoject II microinjector (Drummond Scientific, Broomall, PA, USA). After injection, oocytes were incubated at 17°C in a sterile 96-well culture plate containing 300 μl of incubation solution (100 mM NaCl, 2 mM KCl, 1.8 mM CaCl₂.2H₂O, 1 mM MgCl₂.6H₂O, 5 mM HEPES, 2.5 mM Na pyruvate, 100 U/mL penicillin and 100 μg/mL streptomycin, pH 7.5) in each well. Incubation solution was changed daily during the period of incubation. The same procedure was also conducted for the Ovo-SLO-1A receptor. Experiments were performed on oocytes within 5 – 6 days post injection.

Two-microelectrode voltage clamp (TEVC) electrophysiology

TEVC was conducted at room temperature by impaling oocytes with two microelectrodes; a current injecting electrode, Im, used to inject the required current for holding the membrane at a set voltage, and a voltage sensing electrode, Vm. The microelectrodes were pulled using a Flaming/Brown horizontal electrode puller (Model P-97; Sutter Instruments, Novato, CA, USA) and filled with 3 M KCl. Each electrode tip was broken with a piece of Kimwipe paper (Kimtech Science, Fisher) to achieve a resistance of 2 – 5 MΏ in recording solution (88 mM NaCl, 2.5 mM KCl, 1 mM MgCl₂.6H₂O, 1.8 mM CaCl₂.2H₂O and 5 mM HEPES, at pH 7.4). To investigate the concentration-response relationship of emodepside on the expressed Bma-SLO-1F or Ovo-SLO-1A receptors, oocytes were voltage clamped at a steady-state potential of +20 mV with an Axoclamp 2B amplifier (Molecular Devices, Sunnyvale, CA, USA). Amplified signals were converted from analog to digital format by a Digidata 1322A digitizer (Molecular Devices, CA, USA) and all data were acquired on a desktop computer with the Clampex 10.3 data acquisition software (Molecular Devices, Sunnyvale, CA, USA). In addition, the same protocol was also used to test the effects of GoSlo-SR-5–69 alone or in combination with emodepside.

Voltage step electrophysiology

Voltage step experiments were conducted using the two-electrode voltage-clamp technique to determine current-voltage relationships of each receptor in the absence and presence of 1 µM emodepside. Briefly, oocytes expressing either Bma-SLO-1F or Ovo-SLO-1A channels were impaled and subjected to a current-voltage protocol that consisted of 500 ms voltage steps from -120 to + 60 mV in 20 mV increments, starting from a holding position of -70 mV for 1 s between each step. Plateau currents were recorded at a frequency of 5000 Hz during clamping and perfused with recording solution: (88 mM NaCl, 2.5 mM, KCl, 1 mM MgCl₂.6H₂O, 1.8 mM CaCl₂.2H₂O and 5 mM HEPES, at pH 7.4). The results of the voltage steps were evaluated and analyzed using the ClampFit 10.3 software (Molecular Devices), whereas current-voltage curves (IVCs) were prepared using Graphpad Prism 10.1.1(GraphPad Software, Inc., USA).

Chemicals

Emodepside was purchased from Advanced ChemBlock Inc (Hayward, CA, USA). GoSlo-SR-5–69 was purchased from Tocris Bioscience (Bristol, UK). Stock solutions of emodepside were prepared at 0.1, 0.3, 1, 3, 10 and 30 mM in dimethyl sulfoxide (DMSO) solutions prior to experimentation, then diluted in recording solution. Stock solutions of GoSlo-SR-5–69 were made in DMSO, at 50 mM, then diluted in recording solution to the required concentrations (3 µM). The final DMSO concentration did not exceed 0.1% in the experimental solutions.

Drug application

Emodepside is known to be lipophilic, thus making it difficult to wash off completely from the Xenopus laevis oocyte preparation after application. Additionally, emodepside concentrations exceeding 10 µM showed evidence of precipitation and a limit of solubility. To estimate EC₅₀ values, we utilized a cumulative concentration-response protocol (no wash steps between drug application) and a maximum concentration of 10 µM emodepside. For our recordings, the times for drug applications were selected to allow the currents recorded to reach a stable plateau. At the beginning of experiments the Xenopus oocytes were perfused with drug free recording solution for 1 min, to obtain stable initial resting currents. For our drug applications we used successive applications of increasing concentrations of emodepside (0.1 – 10 µM) for 5 mins per concentration. Washing of the Xenopus oocytes then followed for 3 mins.

To investigate the effects of GoSlo-SR-5–69 (a mammalian BK channel activator), on Bma-SLO-1F and Ovo-SLO-1A channels, oocytes were perfused with recording solution for 1 min followed by application of 3 µM GoSlo-SR-5–69 for 5 mins and subsequently 0.3 µM emodepside (positive control) for 5 mins with a final wash step of recording solution for 3 mins.

To determine the effects of GoSlo-SR-5–69 in combination with emodepside, we employed a protocol for oocytes that involved 1 min perfusion of recording solution to obtain the control current levels. This was followed by the application of 0.3 µM emodepside for 5 mins, then co-application of 3 µM GoSlo-SR-5–69 in the continued presence of 0.3 µM emodepside for 5 mins and immediate wash off with 0.3 µM emodepside for 5 mins and a final wash with recording solution for 3 mins.

Finally, to evaluate the effects of 3µM GoSlo-SR-5–69 on emodepside concentration-response relationships, recording solution was applied for 1 min to each oocyte, followed by 3 µM GoSlo-SR-5–69 until evoked currents were stabilized. Next, 5 mins applications of increasing concentrations of emodepside (0.1 – 10 µM) were perfused in the continued presence of 3 µM GoSlo-SR-5–69. A 3 min wash off time was allowed at the end of the final concentration of emodepside.

Homology modelling and molecular docking

SLO-1 channels for O. volvulus and B. malayi were constructed from FASTA sequences obtained from the WormBase genome projects (Bma-SLO-1F and Ovo-SLO-1A). Homology models were constructed using SWISS-MODEL, with the Drosophila melanogaster SLO channels in Ca²⁺ bound state (RCSB: 7PXE) as template [16]. The structure of emodepside was obtained from PubChem (CID6918632), and pre-docking structures of the channels were energy-minimized using GROMACS 2023.2, solvated in water and neutralized with potassium (K⁺) ions [41]. AMBER99SB-ILDN force field was used, and water molecules were parametrized with SPC/E [42]. Emodepside was parameterized with GAFF using Antechamber version 17.3 [43,44]. The structure of GoSlo-SR-5–69 was obtained from PubChem (CID56944133), and prepared for docking using LigPrep in the Schrödinger package version 2023–4 at pH 7.0, with OPLS4 FF.

Docking was performed using GLIDE module in Schrödinger, docked to the homology models of SLO-1A for O. volvulus, SLO-1F for B. malayi and the cryo-EM structure for D. melanogaster SLO [45]. Docking for all structures was performed at Extended Precision (XP) level. The S6 emodepside pocket was defined as the position emodepside adopted in the energy-minimized structure. The RCK1 binding site was defined as the centroid of G430, M433, Y377, D411 for O. volvulus and B. malayi and for the RCK2, L777, H491, Y490, R781 for B. malayi and L792, H491, Y490, and R796 for O. volvulus.

Analysis of docking results was performed in the Schrödinger Maestro suite to identify non-covalent interactions between ligand and receptor, RMSD was calculated by aligning the structures in PyMOL (v.2.5.4).

Data analysis

Our emodepside concentration-response experiments involved the use of Clampfit 10.3 (Molecular Devices, Sunnyvale, CA, USA) to measure peak current responses for each drug concentration (0.1 - 10 µM) per oocyte. GraphPad Prism 10.1.1 software (GraphPad Software Inc., USA) was used to generate Concentration-response curves using the log agonist vs. response equation (variable slope) to estimate EC₅₀, R_max and Hillslope (nH) values for both Bma-SLO-1F and Ovo-SLO-1A channels. We also used the unpaired two-tailed Student’s t-test to test for statistical significance. A p value < 0.05 was deemed significant. The analyzed results were expressed as mean ± S.E.M.

To obtain current-voltage curves (IVCs) from our voltage steps experiments, plateau currents elicited at each step potential (-120 to + 60 mV) were acquired for individual oocytes in Clampfit 10.3 (Molecular Devices, Sunnyvale, CA, USA). Mean currents for all replicate oocytes were plotted against their corresponding voltage step potentials to obtain IVCs using Graphpad Prism 10.1.1 software (GraphPad Software, Inc., USA). Mean currents between each treatment group of oocytes were compared for each step potential using two-way ANOVA and Tukey’s multiple comparison test to test for significance. To obtain and compare conductance changes in the absence and presence of 1 µM emodepside for Bma-SLO-1F and Ovo-SLO-1A channels, IVCs were analyzed for slopes between step potentials of -120 and +60 mV using linear regression analysis in Graphpad Prism 10.1.1(GraphPad Software, Inc., USA). Subsequently, statistical differences for slope values among treatment groups were analyzed using ANOVA, followed by the Tukey multiple comparison post-hoc test. Results were expressed as mean ± S.E.M.

To determine statistical significance of emodepside potentiation by GoSlo-SR-5–69, we measured the mean currents evoked by 0.3 µM emodepside alone and compared it to the subsequent application of 3 µM GoSlo-SR-5–69 in combination with 0.3 µM emodepside on each oocyte using the unpaired two-tailed Student’s t-test in GraphPad Prism 10.1.1. Similar analyses were also conducted for the effect of extracellular Ca²⁺ on GoSlo-SR-5–69 potentiation of emodepside response involving recordings conducted in normal recording solution (1.8 mM added Ca²⁺) and modified recording solution (Ca²⁺-free). The results were expressed as the mean ± S.E.M.

Analysis involving the determination of GoSlo-SR-5–69 effects on Bma-SLO-1F and Ovo-SLO-1A mediated emodepside concentration-response relationship were conducted in a similar manner as previously described for the emodepside concentration-response experiments. Student’s t-tests were also used for comparing EC_50s and R_max for emodepside alone and emodepside in combination with GoSlo-SR-5–69 using GraphPad Prism 10.1.1 software (GraphPad Software, Inc., USA). Results were expressed as the mean ± S.E.M.