Using Patchxpress to Screen Blockers of the Cardiac Sodium Channel (Nav1.5) for Effects on Late INA, Peak INA, and Channel Kinetics

Abstract

The Nav1.5 channel is responsible for the upstroke of the action potential in cardiac myocytes. Congenital mutations in hNav1.5 have been shown to impair fast inactivation, leading to an increase in persistent sodium current (late INa) and LQT3. Growing evidence suggests that an increase in late INa can contribute to the pathogenesis of several cardiac diseases. With the goal of finding small molecules that selectively target cardiac late INa over peak INa and have rapid unbinding kinetics, we developed three novel assays on PatchXpress using HEK-293 cells stably expressing hNav1.5. The first assay measures block of late INa generated by tefluthrin, a pyrethroid that impairs fast inactivation. We chose tefluthrin over another late INa activator, the sea anemone toxin ATX-II, because with tefluthrin late INa reaches steady state faster and more reliably. Importantly, block obtained with tefluthrin and ATX-II is usually similar. The second assay measures tonic and use-dependent block of peak INa stimulated at 0.1 Hz and 3 Hz, respectively. To reduce voltage clamp errors on PatchXpress due to large inward peak currents and access resistance, we used 50% series resistance compensation and reduced Na+ in the extracellular solution to 20 mM. The third assay is designed to determine the rate at which compounds dissociate from the channel and is calculated from the time-course of the decrease in peak INa when the stimulation frequency is reduced from 10 to 1 Hz. All three assays on PatchXpress were validated by comparing IC50s or off-rates for several standard Nav1.5 blockers (eg. flecainide, mexiletine, quinidine and lidocaine) to those obtained from manual patch clamp. IC50s agree within two-fold and off-rate rankings are identical.