From allergy to arrhythmia – electrophysiological basis for the antiarrhythmic properties of antazoline

Figures

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  FIGURE 1. Effects of antazoline on key cardiac ion channels heterologously expressed in Xenopus laevis oocytes. Representative current traces are shown for the indicated channels under control conditions (grey) and following 30 min perfusion with 100 µM antazoline (colored), along with the corresponding pulse protocols. Test pulses associated with the presented trace are highlighted in pink. Dotted lines represent the zero current level and scalebars are provided as insets. Center: Relative current inhibition was quantified for each channel. Antazoline induced significant inhibition of hK2P9.1, hK2P18.1, hKir3.1/3.4, hKV1.5, hKVLQT1/MinK and hERG channels, whereas hK2P17.1 and rKV1.4 exhibited significant activation. The chemical structure of antazoline is shown beneath the bar graph. Data are presented as mean ± SEM of n = 5–13 measurements; *, p < 0.05; **, p < 0.01; ***; p < 0.001 derived from two-tailed Student’s t-tests.

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  FIGURE 2. Concentration–response relationships for antazoline-induced inhibition of cardiac ion channel currents. Concentration–response curves were constructed by measuring the relative inhibition of current amplitudes following 30 min incubation with increasing concentrations of antazoline. Data were fitted using the Hill equation to determine the half-maximal inhibitory concentration (IC50) and Hill coefficient (n H ). (A) hKir3.1/3.4 currents: IC50 = 52.8 µM; n H = −1.3 (n = 5 cells per concentration). (B) hERG end-pulse currents: IC50 = 43.3 µM; n H = −0.9 (n = four to six cells per concentration). (C) hERG peak-tail currents: IC50 = 162.6 µM; n H = −1.4 (n = four to six cells per concentration). Values are presented as mean ± SEM.

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  FIGURE 3. Modulation of hK ir 3.1/3.4 currents by antazoline. (A) Representative current traces recorded at −100 mV under control conditions (grey) and following 30 min incubation with 100 µM antazoline (purple). Dotted lines represent the zero current level and scalebars are provided as insets. (B) Time course of relative current inhibition at −100 mV during antazoline perfusion and washout (n = 10 cells). (C) Bar graph showing absolute current amplitudes at −100 mV before (grey) and after (purple) 30 min antazoline application (n = 10 cells). (D) Representative current traces recorded at test potentials from −140 to +60 mV in 20 mV increments under control conditions and following 30 min antazoline perfusion. (E) Current–voltage (I–V) relationship of hKir3.1/3.4-mediated currents under control conditions (grey) and after 30 min antazoline exposure (purple), corresponding to the voltage steps shown in (D) (n = 10 cells). Data are provided as mean ± SEM of n = 10 measurements; *, p < 0.05; **, p < 0.01; ***; p < 0.001 derived from single (C) or multiple (E) two-tailed Student’s t-tests, followed by Bonferroni-Dunn correction.

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  FIGURE 4. hERG current inhibition by antazoline. (A) Representative current traces recorded under control conditions (grey) and after 30-min incubation with 100 µM antazoline (orange), shown with the corresponding double-step pulse protocol. (B) Time course of antazoline-induced relative current inhibition (100 μM; 30 min as indicated) as well as washout (44 min), measured for both the end-pulse (i.e., the steady-state/depolarization current; light orange) and the peak-tail (repolarizing current; dark orange) current components (n = 8 cells). (C) Scatter plot of absolute end-pulse and peak-tail current amplitudes before (light and dark grey) and after (light and dark orange) 30 min antazoline treatment (n = 8 cells). (D) Representative macroscopic current traces illustrating hERG voltage-dependent activation under control conditions and following 30 min antazoline perfusion, together with the applied pulse protocol. (E) End-pulse current–voltage (I–V) relationships under control conditions (light grey) and after antazoline exposure (light orange); left panel: absolute current amplitudes; right panel: values normalized to the respective maximal current (n = 8 cells). (F) Peak-tail I–V…

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  FIGURE 5. Biophysical characterization of hERG channel blockade by antazoline. (A,B) Block of hERG channels in the activated state by antazoline. (A) Representative current traces were recorded under control conditions and after 30 min of superfusion with 100 µM antazoline, during which the oocyte was clamped at −80 mV. The control recording (grey) and the first pulse measured after the incubation period (orange) are displayed. (B) Time course of current inhibition under the same conditions. Current inhibition increased time-dependently, illustrating that channel block occurs in the activated (i.e., the open and/or the inactivated channel state) and not in the preceding closed state. Similar results were observed in n = 6 experiments. (C,D) Assessment of hERG inhibition by antazoline in the inactivated state (n = 6 cells). (C) hERG channels were first driven into the inactivated state by a depolarizing step to +80 mV, followed by a second step to 0 mV to reopen the channels. The corresponding relative current inhibition during the 0 mV step is shown in (D). Representative current traces before (grey) and after (orange) 30 min antazoline application, along with the corresponding voltage…

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  FIGURE 6. Effects of Y652A and F656A mutations on hERG channel inhibition by 100 µM antazoline. (A) Time course of end-pulse current inhibition by 100 µM antazoline in hERG wild-type (WT) (light orange), hERG-Y652A (blue), and hERG-F656A (yellow) channels. (B) Relative end-pulse current amplitudes after 30 min of antazoline perfusion are depicted for hERG-WT, hERG-Y652A, and hERG-F656A channels (n = 8–15 cells, respectively). (C) Time course of peak-tail current inhibition by 100 µM antazoline in hERG-WT (dark orange), hERG-Y652A (blue), and hERG-F656A (yellow) channels. (D) Respective relative peak-tail current amplitudes after 30 min antazoline (100 µM) application. Both hERG-Y652A and hERG-F656A mutations significantly attenuated the inhibitory effect of antazoline on hERG currents. Dotted lines indicate the zero current level. Values are presented as mean ± SEM. Statistical significance, derived from one-way ANOVA with Tukey’s multiple comparisons test: ns = not significant; *, p < 0.05; **, p < 0.01; ***, p < 0.001.