Simultaneous Measurement of Cardiac Nav1.5 Peak and Late Currents in an Automated QPatch Platform

High throughput in vitro profile of the cardiac Na_V1.5 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. A variety of differing protocols used across industry and academia may contribute to variation in the data.
The objectives of this study were to (1) assess pharmacology and biophysical properties of veratridine- and ATX-II-induced late INa together with that induced by a mutation in the channel (ΔKPQ-Na_V1.5), (2) develop a protocol to allow simultaneous measurement of both peak and late INa under a single protocol using automated QPatch system. The planar patch clamp technique (QPatch) was applied to record the peak and late I_Na from the Na_V1.5 channel (human SCN5A gene) or ΔKPQ-Na_V1.5 mutant channel expressed in mammalian cells.
When measured at the maximal response during the ramp of the voltage protocol, the ΔKPQ-Na_V1.5 mutant produced a small late I_Na (41.9 ± 5.4 pA). Veratridine and ATX-II-induced concentration-dependent increases in the late INa. The amplitude of late I_Na were 1162.2 ± 258.5 pA and 392.4 ± 71.3 pA in the presences of 100 μM veratridine and 100 nM ATX-II, respectively. Veratridine inhibited the peak I_Na
(IC50 = 84.1 ± 10.5 μM) and altered the biophysical properties of the INa. ATX-II showed minimal effects on the peak I_Na and preserved the biophysical properties of the I_Na. In the presence of 100 nM ATX-II, potencies of 25 clinical I_Na blockers on peak and late INa were characterized. In addition, the IC₅₀ values of these clinical INa blockers on peak I_Na correlated well with and without ATX-II. The results also demonstrated that the potency of a compound blocking late I_Na could be either overestimated or underestimated if the late I_Na was measured at the end of the depolarizing pulse versus during the ramp.
In conclusion, in the presence of ATX-II, both peak and late I_Na could be assessed simultaneously under a single protocol. Our results suggest that late I_Na may be best assessed using the maximum response obtained during the ramp after 200 ms depolarizing pulse at 40 mV.