Drug potency on inhibiting late Na+ current is sensitive to gating modifier and current region where drug effects were measured

Min Wu, P. N. Tran, Jiansong Sheng, Aaron L. Randolph, Wendy Wu

Research output: Contribution to journalArticle

Abstract

Introduction: Cardiac late Na+ current (INaL) contributes to ventricular action potential duration. Pathological increase in INaL is arrhythmogenic, and inhibition of INaL offers protection against ventricular repolarization disturbance. Recently, two INaL datasets generated by different laboratories that assessed current inhibition by a panel of clinical drugs as a part of the Comprehensive in vitro Proarrhythmia Assay (CiPA) initiative were published. The results revealed a surprising degree of data variability despite of the use of a standardized voltage protocol. This study investigated whether remaining procedural differences related to experimental methods and data analysis associated with these datasets can produce differences in INaL pharmacology. Methods: Whole cell voltage clamp recordings were performed on cells expressing NaV1.5 α- and β1-subunits to study: 1) the impact of gating modifiers used to augment INaL (ATX-II vs. veratridine), internal solution composition (with vs. without ATP and GTP), and recording temperature (23 °C vs 37 °C) on stability of INaL measured across the duration of a patch clamp experiment; 2) mechanisms of each gating modifier on Na+ channels; and 3) effects of six drugs (lidocaine, mexiletine, chloroquine, ranolazine, ritonavir, and verapamil) on INaL induced by either gating modifier. Results: Stability of INaL is affected by the choice of gating modifier, presence of nucleotides in the internal solution, and recording temperature. ATX-II and veratridine produced different changes in Na+ channel gating, inducing mechanistically distinct INaL. Drug potencies on inhibiting INaL were dependent on the choice of gating modifier and current region where drug effects were measured. Discussion: INaL pharmacology can be impacted by all experimental factors examined in this study. The effect of gating modifier and current region used to quantify drug inhibition alone led to 30× difference in half inhibitory concentration (IC50) for ritonavir, demonstrating that substantial difference in drug inhibition can be produced. Drug potencies on inhibiting INaL derived from different patch clamp studies may thus not be generalizable. For INaL pharmacology to be useful for in silico modeling or interpreting drug-induced changes in cardiac action potentials or ECG, standardizing INaL experimental procedures including data analysis methods is necessary to minimize data variability.

Original languageEnglish (US)
Article number106605
JournalJournal of Pharmacological and Toxicological Methods
Volume100
DOIs
StatePublished - Nov 1 2019

Fingerprint

Pharmaceutical Preparations
Clamping devices
Veratridine
Ritonavir
Pharmacology
Action Potentials
Mexiletine
Temperature
Bioelectric potentials
Chloroquine
Electric potential
Verapamil
Lidocaine
Guanosine Triphosphate
Electrocardiography
Computer Simulation
Inhibitory Concentration 50
Assays
Nucleotides
Adenosine Triphosphate

Keywords

  • Cardiac safety pharmacology
  • CiPA
  • In vitro electrophysiology
  • Ion channel voltage protocol
  • Na1.5
  • SCN5A
  • Voltage clamp

ASJC Scopus subject areas

  • Toxicology
  • Pharmacology

Cite this

Drug potency on inhibiting late Na+ current is sensitive to gating modifier and current region where drug effects were measured. / Wu, Min; Tran, P. N.; Sheng, Jiansong; Randolph, Aaron L.; Wu, Wendy.

In: Journal of Pharmacological and Toxicological Methods, Vol. 100, 106605, 01.11.2019.

Research output: Contribution to journalArticle

@article{fd006b2a10ec456d9975b472375e5e69,
title = "Drug potency on inhibiting late Na+ current is sensitive to gating modifier and current region where drug effects were measured",
abstract = "Introduction: Cardiac late Na+ current (INaL) contributes to ventricular action potential duration. Pathological increase in INaL is arrhythmogenic, and inhibition of INaL offers protection against ventricular repolarization disturbance. Recently, two INaL datasets generated by different laboratories that assessed current inhibition by a panel of clinical drugs as a part of the Comprehensive in vitro Proarrhythmia Assay (CiPA) initiative were published. The results revealed a surprising degree of data variability despite of the use of a standardized voltage protocol. This study investigated whether remaining procedural differences related to experimental methods and data analysis associated with these datasets can produce differences in INaL pharmacology. Methods: Whole cell voltage clamp recordings were performed on cells expressing NaV1.5 α- and β1-subunits to study: 1) the impact of gating modifiers used to augment INaL (ATX-II vs. veratridine), internal solution composition (with vs. without ATP and GTP), and recording temperature (23 °C vs 37 °C) on stability of INaL measured across the duration of a patch clamp experiment; 2) mechanisms of each gating modifier on Na+ channels; and 3) effects of six drugs (lidocaine, mexiletine, chloroquine, ranolazine, ritonavir, and verapamil) on INaL induced by either gating modifier. Results: Stability of INaL is affected by the choice of gating modifier, presence of nucleotides in the internal solution, and recording temperature. ATX-II and veratridine produced different changes in Na+ channel gating, inducing mechanistically distinct INaL. Drug potencies on inhibiting INaL were dependent on the choice of gating modifier and current region where drug effects were measured. Discussion: INaL pharmacology can be impacted by all experimental factors examined in this study. The effect of gating modifier and current region used to quantify drug inhibition alone led to 30× difference in half inhibitory concentration (IC50) for ritonavir, demonstrating that substantial difference in drug inhibition can be produced. Drug potencies on inhibiting INaL derived from different patch clamp studies may thus not be generalizable. For INaL pharmacology to be useful for in silico modeling or interpreting drug-induced changes in cardiac action potentials or ECG, standardizing INaL experimental procedures including data analysis methods is necessary to minimize data variability.",
keywords = "Cardiac safety pharmacology, CiPA, In vitro electrophysiology, Ion channel voltage protocol, Na1.5, SCN5A, Voltage clamp",
author = "Min Wu and Tran, {P. N.} and Jiansong Sheng and Randolph, {Aaron L.} and Wendy Wu",
year = "2019",
month = "11",
day = "1",
doi = "10.1016/j.vascn.2019.106605",
language = "English (US)",
volume = "100",
journal = "Journal of Pharmacological and Toxicological Methods",
issn = "1056-8719",
publisher = "Elsevier Inc.",

}

TY - JOUR

T1 - Drug potency on inhibiting late Na+ current is sensitive to gating modifier and current region where drug effects were measured

AU - Wu, Min

AU - Tran, P. N.

AU - Sheng, Jiansong

AU - Randolph, Aaron L.

AU - Wu, Wendy

PY - 2019/11/1

Y1 - 2019/11/1

N2 - Introduction: Cardiac late Na+ current (INaL) contributes to ventricular action potential duration. Pathological increase in INaL is arrhythmogenic, and inhibition of INaL offers protection against ventricular repolarization disturbance. Recently, two INaL datasets generated by different laboratories that assessed current inhibition by a panel of clinical drugs as a part of the Comprehensive in vitro Proarrhythmia Assay (CiPA) initiative were published. The results revealed a surprising degree of data variability despite of the use of a standardized voltage protocol. This study investigated whether remaining procedural differences related to experimental methods and data analysis associated with these datasets can produce differences in INaL pharmacology. Methods: Whole cell voltage clamp recordings were performed on cells expressing NaV1.5 α- and β1-subunits to study: 1) the impact of gating modifiers used to augment INaL (ATX-II vs. veratridine), internal solution composition (with vs. without ATP and GTP), and recording temperature (23 °C vs 37 °C) on stability of INaL measured across the duration of a patch clamp experiment; 2) mechanisms of each gating modifier on Na+ channels; and 3) effects of six drugs (lidocaine, mexiletine, chloroquine, ranolazine, ritonavir, and verapamil) on INaL induced by either gating modifier. Results: Stability of INaL is affected by the choice of gating modifier, presence of nucleotides in the internal solution, and recording temperature. ATX-II and veratridine produced different changes in Na+ channel gating, inducing mechanistically distinct INaL. Drug potencies on inhibiting INaL were dependent on the choice of gating modifier and current region where drug effects were measured. Discussion: INaL pharmacology can be impacted by all experimental factors examined in this study. The effect of gating modifier and current region used to quantify drug inhibition alone led to 30× difference in half inhibitory concentration (IC50) for ritonavir, demonstrating that substantial difference in drug inhibition can be produced. Drug potencies on inhibiting INaL derived from different patch clamp studies may thus not be generalizable. For INaL pharmacology to be useful for in silico modeling or interpreting drug-induced changes in cardiac action potentials or ECG, standardizing INaL experimental procedures including data analysis methods is necessary to minimize data variability.

AB - Introduction: Cardiac late Na+ current (INaL) contributes to ventricular action potential duration. Pathological increase in INaL is arrhythmogenic, and inhibition of INaL offers protection against ventricular repolarization disturbance. Recently, two INaL datasets generated by different laboratories that assessed current inhibition by a panel of clinical drugs as a part of the Comprehensive in vitro Proarrhythmia Assay (CiPA) initiative were published. The results revealed a surprising degree of data variability despite of the use of a standardized voltage protocol. This study investigated whether remaining procedural differences related to experimental methods and data analysis associated with these datasets can produce differences in INaL pharmacology. Methods: Whole cell voltage clamp recordings were performed on cells expressing NaV1.5 α- and β1-subunits to study: 1) the impact of gating modifiers used to augment INaL (ATX-II vs. veratridine), internal solution composition (with vs. without ATP and GTP), and recording temperature (23 °C vs 37 °C) on stability of INaL measured across the duration of a patch clamp experiment; 2) mechanisms of each gating modifier on Na+ channels; and 3) effects of six drugs (lidocaine, mexiletine, chloroquine, ranolazine, ritonavir, and verapamil) on INaL induced by either gating modifier. Results: Stability of INaL is affected by the choice of gating modifier, presence of nucleotides in the internal solution, and recording temperature. ATX-II and veratridine produced different changes in Na+ channel gating, inducing mechanistically distinct INaL. Drug potencies on inhibiting INaL were dependent on the choice of gating modifier and current region where drug effects were measured. Discussion: INaL pharmacology can be impacted by all experimental factors examined in this study. The effect of gating modifier and current region used to quantify drug inhibition alone led to 30× difference in half inhibitory concentration (IC50) for ritonavir, demonstrating that substantial difference in drug inhibition can be produced. Drug potencies on inhibiting INaL derived from different patch clamp studies may thus not be generalizable. For INaL pharmacology to be useful for in silico modeling or interpreting drug-induced changes in cardiac action potentials or ECG, standardizing INaL experimental procedures including data analysis methods is necessary to minimize data variability.

KW - Cardiac safety pharmacology

KW - CiPA

KW - In vitro electrophysiology

KW - Ion channel voltage protocol

KW - Na1.5

KW - SCN5A

KW - Voltage clamp

UR - http://www.scopus.com/inward/record.url?scp=85069967155&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=85069967155&partnerID=8YFLogxK

U2 - 10.1016/j.vascn.2019.106605

DO - 10.1016/j.vascn.2019.106605

M3 - Article

C2 - 31255744

AN - SCOPUS:85069967155

VL - 100

JO - Journal of Pharmacological and Toxicological Methods

JF - Journal of Pharmacological and Toxicological Methods

SN - 1056-8719

M1 - 106605

ER -