Simultaneous assessment of compound activity on cardiac Nav1.5 peak and late currents in an automated patch clamp...

High throughput in vitro profiling of the cardiac Nav1.5 peak sodium current (I_Na) is widely used in cardiac safety screening. However, there is no standardized high throughput method to measure late I_Na. This study assessed the pharmacological and biophysical properties of veratridine and ATX-II, as well as the channel mutation (Nav1.5-∆KPQ) on the late I_Na. We describe a method for simultaneous measurement of both peak and late I_Na.

The planar patch clamp technique (QPatch) was applied to record the peak and late I_Na.

The Nav1.5-∆KPQ mutant produced a small late I_Na (41.9 ± 5.4 pA) not large enough to enable compound profiling. In contrast in wild type Nav1.5 expressing cells veratridine (100 μM) and ATX-II (100 nM) enhanced concentration-dependent increases in the late I_Na (maximum responses of 1162.2 ± 258.5 pA and 392.4 ± 71.3 pA, respectively). Veratridine inhibited, whereas, ATX-II had a minimal effect, on the peak I_Na and preserved the current-voltage curve. Peak and late I_Na inhibition was characterized for 25 clinical I_Na blockers in the presence of ATX-II. Compound IC₅₀ values for peak I_Na generated in the absence or presence of ATX-II correlated. The potency of the late I_Na block was found to be dependent on whether it was measured at the end of the depolarizing pulse or during the ramp.

In the presence of ATX-II, both peak and late I_Na could be assessed simultaneously. Late I_Na may be best assessed using the maximum response obtained during the ramp of the voltage protocol.