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Investigating dynamic protocol-dependence of hERG potassium channel inhibition at 37 degrees C: Cisapride versus
James T Milnes1, Harry J Witchel, Joanne L Leaney
1Department of Physiology and Pharmacology, School of Medical Sciences, University Walk, Bristol, BS8 1TD, UK.
Insights
Cisapride shows dynamic binding to the human ether-à-go-go-related gene (hERG) channel, unlike dofetilide, impacting drug safety assessments. These findings are crucial for understanding hERG channel inhibition in drug development.
Area of Science:
- Cardiovascular Pharmacology
- Ion Channel Electrophysiology
- Drug Safety Assessment
Background:
- Inhibition of cardiac potassium channels, specifically the human ether-à-go-go-related gene (hERG) channel, is linked to dangerous heart rhythm abnormalities like QT interval prolongation and torsades de pointes.
- Electrophysiological assays assessing hERG channel inhibition are critical for the safety evaluation of new drug candidates before clinical use.
Purpose of the Study:
- To compare hERG channel blockade by high-affinity inhibitors dofetilide and cisapride using action potential (AP) waveforms versus conventional screening waveforms (step and step-ramp).
- To investigate the dynamic, pulse-by-pulse protocol-dependence of hERG channel inhibition by these drugs.
Main Methods:
- Whole-cell patch-clamp recordings were performed on hERG-expressing HEK 293 cells at 37°C.
- Half-maximal inhibitory concentrations (IC50 values) for I(hERG) blockade were determined using conventional voltage clamp and action potential clamp with digitized ventricular and Purkinje fiber AP waveforms.
Main Results:
- Cisapride exhibited greater variability in IC50 values (7–72 nM) compared to dofetilide (4–15 nM) across different waveforms, with higher IC50s observed using AP waveforms.
- While both drugs showed voltage-dependent blockade initially, only cisapride maintained voltage-dependence at steady-state.
- Cisapride demonstrated significant pulse-by-pulse dissociation and reassociation from the hERG channel, a characteristic not observed with dofetilide.
Conclusions:
- The dynamic binding of cisapride to hERG channels, revealed by AP clamp protocols, offers a valuable approach for future drug interaction studies.
- Digitized ventricular and Purkinje fiber AP waveforms are provided for use in AP clamp experiments, aiding further research in hERG channel dynamics.
Introduction:
Pharmacological inhibition of cardiac potassium channels encoded by hERG (human ether-à-go-go-related gene) is associated with QT interval prolongation and torsades de pointes arrhythmia. Electrophysiological assays of hERG channel inhibition are integral to the safety testing of novel drug candidates. This study was conducted to compare, for the high affinity hERG inhibitors dofetilide and cisapride, hERG blockade between action potential (AP) and conventional (step and step-ramp) screening waveforms. Furthermore, it evaluated dynamic (pulse-by-pulse) protocol-dependence of hERG channel inhibition by these drugs.
Methods:
Whole-cell patch-clamp recordings were made at 37 degrees C from hERG-expressing HEK 293 cells. Half-maximal inhibitory concentrations (IC(50) values) for I(hERG) blockade were obtained using conventional voltage clamp and action potential clamp, using previously digitised ventricular and Purkinje fibre (PF) AP waveforms.
Results:
A more marked variation in IC(50) values with different command waveforms was observed for cisapride (ranging from 7 to 72 nM) than for dofetilide (ranging from 4 to 15 nM), with higher IC(50)s obtained with AP than step or step-ramp commands. The two drugs differed little from one another in effects on voltage-dependent activation; however, I(hERG) blockade by each drug was initially voltage-dependent, but at steady-state was only voltage-dependent for cisapride. There was comparatively little difference between the two drugs in effects on I(hERG) availability or time constants of development of inactivation. Features of time-dependence of blockade and the use of protocols employing varying rest periods in drug or commands of alternating duration highlighted a pronounced ability of cisapride, but not dofetilide, to dissociate and reassociate from hERG on a pulse-by-pulse basis.
Discussion:
Protocols described here that demonstrated dynamic variation (drug dissociation/reassociation) in hERG channel current blockade at 37 degrees C for cisapride may have future value for investigating drug interactions with the hERG channel. Downloadable digitised ventricular and PF AP waveforms that can be used in AP clamp experiments also accompany this article.
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