NaV1.5 big late : An inactivation deficient mutant of NaV1.5 as screening tool for late sodium currents of the cardiac action potential

Torsades de pointes (TdP) is a potentially fatal type of a ventricular tachycardia associated with delayed repolarization of the cardiac action potential. The major reason for pharmacologically induced TdP is the blockade of the voltage-gated potassium channel (KV11.1 or hERG current, I_Kr).
Therefore, the main focus of pre-clinical in vitro tests has been set on detection of I_Kr blockade to effectively discard drugs with a propensity to induce TdP. However, not all compounds that block I_Kr will eventually induce tachyarrhythmia and, therefore, a detected block of I_Kr alone is not specifically
predictive for delayed repolarization and TdP. Not all known I_Kr blockers cause significant arrhythmia because effects caused by induced reduction of potassium outward currents may be
counterbalanced by a reduced calcium inward current (ICaL) or late inward sodium current (I_NaL). hERGand L-type calcium currents (Ca_V1.2) can easily be assessed in in-vitro systems using electrophysiology methods (e.g. patch-clamping). Since the physiological late sodium current exhibits only tiny current amplitudes, I_NaL needs to be increased for drug screening by decreasing or slowing the inactivation of Na_V1.5 channels. This can be pharmacologically achieved by adding a sea anemone toxin II (ATX-II), which binds to the extracellular linker of segments S3-S4 of domain IV or by using inactivation modifying mutations.

For this study, a cell line stably expressing a mutated NaV1.5 (CW) channel was generated and validated using known I_NaL blockers. The substitution L409C/A410W was found to lead to an inactivation-deficient mutation. The mutation is localized in D1S6 and presumably prevents access of the intrinsic fast inactivation particle to the inner cavity. During pharmacological validation using manual and automated (QPatch™) patch-clamping, IC₅₀ values differed by less than a factor of two between ATXII stimulated and CW mutated Na_V1.5 channels. Besides shorter duration of I_NaL experiments and larger current amplitudes, also the observed conserved sensitivity to ATXII and overall reduced assay costs are strong arguments to
screen late sodium currents in mutated rather than in pharmacologically stimulated Na_V1.5 channels.