The macrolide drug erythromycin does not protect the hERG channel from inhibition by thioridazine and terfenadine

Figures

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  Figure 1. Sensitivity of IhERG to erythromycin (a), terfenadine (b) and thioridazine (c). Top panels show representative traces for IhERG recorded at 37ºC before and during exposure to 3 or 30 µM erythromycin (Ery) (ai), 100 nM terfenadine (Terf) (bi) or 100 nM thioridazine (Thior) (ci). Currents were elicited using the protocol shown in the lower panels. Bottom panels show the isochronal concentration–response relationships obtained for erythromycin (aii) (IC50 94 µM (CI 38.4–230.1 µM); n = 5 to 8 to cells per concentration), terfenadine (bii) (IC50 128.5 nM (CI 68.0–243.3 nM); n = 4 to 6 to cells per concentration) and thioridazine (cii) (IC50 62 nM (CI 52.7–73.1 nM); n = 5 to 7 cells per concentration). Respective nH values yielded from fit were of 0.40 (CI 0.25–0.54), 0.57 (CI 0.36–0.77), 0.55 (CI 0.50–0.61) for erythromycin, for terfenadine and for thioridazine

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  Figure 2. Effect of a pretreatment with 3 µM erythromycin on IhERG block by terfenadine (a) and thioridazine (b). Left panels show representative current traces for effects of 100 nM terfenadine (ai) or 100 nM thioridazine (bi) on IhERG after a 15‐min preincubation with 3 µM erythromycin. Currents were elicited using the protocol shown in the lower panels. Right panels show isochronal concentration–response relationships for terfenadine inhibition of IhERG (aii) or thioridazine (bii) in the presence of 3 µM erythromycin. Fit to the concentration–response curves yielded an IC50 of 4 nM ((CI 0.63–25.6 nM); n = 5 cells per concentration) for terfenadine and 46.4 nM ((CI 36.3–59.3 nM); n = 4 to 6 cells per concentration) for thioridazine. Respective nH were of 0.30 (CI 0.14–0.47) for terfenadine and of 0.73 (CI 0.58–0.90) for thioridazine. Asterisks in Aii denote statistical significance: ** at p < .01 and *p at < .05 (two‐way ANOVA with Bonferroni post hoc test)

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  Figure 3. Effect of mutation at F656 on IhERG block by erythromycin (a) Representative traces for WT IhERG (ai) and F656V IhERG (aii) before (control) and after exposure to 600 µM erythromycin (Ery) at 37ºC, using the voltage protocols in the panels below. (b) Bar charts comparing the level of block produced by 600 µM erythromycin of WT (white bar; n = 5) and F656V (gray bar; n = 5) IhERG tail on repolarization to −40 mV following a 2 s depolarization from −80 mV to +20 mV. Asterisks in B denote statistical significance: ** p < .01 (unpaired t test)

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  Figure 4. Effects of 3 µM erythromycin on IhERG tail sensitivity to 30nM BeKm‐1. (a) Representative current traces for effects of 30 nM BeKm‐1 on IhERG in the absence (ai) or after superfusion of 3 µM erythromycin (aii). Currents were elicited using the protocol shown in the lower panels. (b) Bar charts showing the level of block by 30 nM BeKm‐1 of WT IhERG tail in the absence (white bar; n = 10) and presence of 3 µM erythromycin (gray bar; n = 6). (c) Bar charts comparing thalfvalues of inhibition of WT IhERG tail in the absence (white bar; n = 10) and presence of 3µM erythromycin (gray bar; n = 6). BeKm‐1 effect on IhERG tail recorded on repolarization to −40mV was calculated as the mean fractional block and plotted against time to derive the value for time to half inhibition (thalf) of IhERG tail. ** denotes statistically significant difference from WT at p < .01 (unpaired t‐test)

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  Figure 5. Effects of mutation F656V on erythromycin modulation of IhERG sensitivity to 30 nM BeKm‐1. (a) Bar charts showing the level of block by 30 nM BeKm‐1 of F656V IhERG tail in the absence (white bar; n = 5) and presence of 3 µM erythromycin (gray bar; n = 5; p > .05 vs. in the absence of erythromycin 3 µM [unpaired t test]). (b) Bar charts comparing thalf values of inhibition of F656V IhERG tails in the absence (white bar; n = 5) and presence of 3 µM erythromycin (gray bar; n = 5; p > .05 vs. in the absence of erythromycin 3 µM [unpaired t test]). BeKm‐1 effect on IhERG tail recorded on repolarization to −40mV was calculated as the mean fractional block and plotted against time to derive the value for time to half inhibition thalf of IhERG tail. The F656V mutation increased hERG sensitivity to BeKm‐1 in the two experimental conditions tested but did not affect the rate of block (cf. Figure 4b and 4c)

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  Figure 6. Sensitivity of IhERG tail to 30 nM BeKm‐1 in the presence of 3 µM disopyramide. (a) Representative traces for IhERG before and during exposure to 30 nM BeKm‐1 in the continuous presence of 3 µM disopyramide. Currents were elicited using the protocol shown in the lower panel. (b) Bar charts comparing thalf values of inhibition by 30 nM BeKm‐1 of WT IhERG tail in the absence (white bar; n = 10) and presence of 3 µM disopyramide (gray bar; n = 7). Asterisks in B denote statistical significance: **p < .01 (unpaired t test)