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 INaL 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(V)1.5 alpha- and beta 1-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 degrees C vs 37 degrees 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 30x 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.