Characterization of Compound-Specific, Concentration-Independent Biophysical Properties of Sodium Channel Inhibitor Mechanism of Action Using Automated Patch-Clamp Electrophysiology

Pesti, Krisztina; Foldi, M; Zboray, Katalin; Toth, Adam; Lukács, Péter; Mike, Arpad

doi:10.3389/fphar.2021.738460

Cited by 2 publications

(1 citation statement)

References 40 publications

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“…The holding potential was set to −150 mV to minimize the voltage-dependent rundown of the current and to ensure that all channels are in the resting state. We used a voltage-clamp protocol that was optimized for high information content and high temporal resolution ( Lukacs et al, 2021 ; Pesti et al, 2021 ). It consisted of 17 depolarizing pulses ( Figure 3A ).…”

Section: Methodsmentioning

confidence: 99%

The Bradycardic Agent Ivabradine Acts as an Atypical Inhibitor of Voltage-Gated Sodium Channels

et al. 2022

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Background and purpose: Ivabradine is clinically administered to lower the heart rate, proposedly by inhibiting hyperpolarization-activated cyclic nucleotide-gated cation channels in the sinoatrial node. Recent evidence suggests that voltage-gated sodium channels (VGSC) are inhibited within the same concentration range. VGSCs are expressed within the sinoatrial node and throughout the conduction system of the heart. A block of these channels thus likely contributes to the established and newly raised clinical indications of ivabradine. We, therefore, investigated the pharmacological action of ivabradine on VGSCs in sufficient detail in order to gain a better understanding of the pro- and anti-arrhythmic effects associated with the administration of this drug.Experimental Approach: Ivabradine was tested on VGSCs in native cardiomyocytes isolated from mouse ventricles and the His-Purkinje system and on human Nav1.5 in a heterologous expression system. We investigated the mechanism of channel inhibition by determining its voltage-, frequency-, state-, and temperature-dependence, complemented by a molecular drug docking to the recent Nav1.5 cryoEM structure. Automated patch-clamp experiments were used to investigate ivabradine-mediated changes in Nav1.5 inactivation parameters and inhibition of different VGSC isoforms.Key results: Ivabradine inhibited VGSCs in a voltage- and frequency-dependent manner, but did not alter voltage-dependence of activation and fast inactivation, nor recovery from fast inactivation. Cardiac (Nav1.5), neuronal (Nav1.2), and skeletal muscle (Nav1.4) VGSC isoforms were inhibited by ivabradine within the same concentration range, as were sodium currents in native cardiomyocytes isolated from the ventricles and the His-Purkinje system. Molecular drug docking suggested an interaction of ivabradine with the classical local anesthetic binding site.Conclusion and Implications: Ivabradine acts as an atypical inhibitor of VGSCs. Inhibition of VGSCs likely contributes to the heart rate lowering effect of ivabradine, in particular at higher stimulation frequencies and depolarized membrane potentials, and to the observed slowing of intra-cardiac conduction. Inhibition of VGSCs in native cardiomyocytes and across channel isoforms may provide a potential basis for the anti-arrhythmic potential as observed upon administration of ivabradine.

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Section: Methodsmentioning

confidence: 99%

The Bradycardic Agent Ivabradine Acts as an Atypical Inhibitor of Voltage-Gated Sodium Channels

et al. 2022

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An Advanced Automated Patch Clamp Protocol Design to Investigate Drug—Ion Channel Binding Dynamics

et al. 2021

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Standard high throughput screening projects using automated patch-clamp instruments often fail to grasp essential details of the mechanism of action, such as binding/unbinding dynamics and modulation of gating. In this study, we aim to demonstrate that depth of analysis can be combined with acceptable throughput on such instruments. Using the microfluidics-based automated patch clamp, IonFlux Mercury, we developed a method for a rapid assessment of the mechanism of action of sodium channel inhibitors, including their state-dependent association and dissociation kinetics. The method is based on a complex voltage protocol, which is repeated at 1 Hz. Using this time resolution we could monitor the onset and offset of both channel block and modulation of gating upon drug perfusion and washout. Our results show that the onset and the offset of drug effects are complex processes, involving several steps, which may occur on different time scales. We could identify distinct sub-processes on the millisecond time scale, as well as on the second time scale. Automated analysis of the results allows collection of detailed information regarding the mechanism of action of individual compounds, which may help the assessment of therapeutic potential for hyperexcitability-related disorders, such as epilepsies, pain syndromes, neuromuscular disorders, or neurodegenerative diseases.

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Characterization of Compound-Specific, Concentration-Independent Biophysical Properties of Sodium Channel Inhibitor Mechanism of Action Using Automated Patch-Clamp Electrophysiology

Cited by 2 publications

References 40 publications

The Bradycardic Agent Ivabradine Acts as an Atypical Inhibitor of Voltage-Gated Sodium Channels

The Bradycardic Agent Ivabradine Acts as an Atypical Inhibitor of Voltage-Gated Sodium Channels

An Advanced Automated Patch Clamp Protocol Design to Investigate Drug—Ion Channel Binding Dynamics

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