Simultaneous Measurement of Cardiac Nav1.5 Peak and Late Currents in an Automated QPatch Platform
High throughput in vitro profile of the cardiac NaV1.5 peak sodium current (INa) is widely used in cardiac safety screening. However, there is no standardized high throughput method to measure late INa. 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-NaV1.5), (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 INa from the NaV1.5 channel (human SCN5A gene) or ΔKPQ-NaV1.5 mutant channel expressed in mammalian cells.
When measured at the maximal response during the ramp of the voltage protocol, the ΔKPQ-NaV1.5 mutant produced a small late INa (41.9 ± 5.4 pA). Veratridine and ATX-II-induced concentration-dependent increases in the late INa. The amplitude of late INa 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 INa
(IC50 = 84.1 ± 10.5 μM) and altered the biophysical properties of the INa. ATX-II showed minimal effects on the peak INa and preserved the biophysical properties of the INa. In the presence of 100 nM ATX-II, potencies of 25 clinical INa blockers on peak and late INa were characterized. In addition, the IC50 values of these clinical INa blockers on peak INa correlated well with and without ATX-II. The results also demonstrated that the potency of a compound blocking late INa could be either overestimated or underestimated if the late INa 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 INa could be assessed simultaneously under a single protocol. Our results suggest that late INa may be best assessed using the maximum response obtained during the ramp after 200 ms depolarizing pulse at 40 mV.