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

Introduction: Cardiac late Na⁺ current (I_NaL) contributes to ventricular action potential duration. Pathological increase in I_NaL is arrhythmogenic, and inhibition of I_NaL offers protection against ventricular repolarization disturbance. Recently, two I_NaL 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 I_NaL pharmacology. Methods: Whole cell voltage clamp recordings were performed on cells expressing Na_V1.5 α- and β1-subunits to study: 1) the impact of gating modifiers used to augment I_NaL (ATX-II vs. veratridine), internal solution composition (with vs. without ATP and GTP), and recording temperature (23 °C vs 37 °C) on stability of I_NaL 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 I_NaL induced by either gating modifier. Results: Stability of I_NaL 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 I_NaL. Drug potencies on inhibiting I_NaL were dependent on the choice of gating modifier and current region where drug effects were measured. Discussion: I_NaL 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 I_NaL derived from different patch clamp studies may thus not be generalizable. For I_NaL pharmacology to be useful for in silico modeling or interpreting drug-induced changes in cardiac action potentials or ECG, standardizing I_NaL experimental procedures including data analysis methods is necessary to minimize data variability.