Characterization of the Reverse Use Dependent Block of Voltage Gated Sodium Channels

Abstract

Some clinically used drugs interact via state-dependent inhibition of voltage-gated sodium channels. For example, cocaine, procaine or lidocaine preferentially interact with, and stabilize the inactivated conformation of the channel. Upon repetitive high frequency activation they cause a progressive inhibition during the pulse train which is termed use-dependent inhibition.Here we describe compounds that show the opposite behaviour, ie the inhibition is diminished during the pulse train.To adequately determine the state-dependent interactions of drugs with sodium channels, we developed a high-throughput electrophysiological assay using the IonWorks(r) Quattro(tm) PPC platform. Compounds were tested against the brain Nav1.3 sodium channel expressed in CHO cells. A train of 10 depolarizing voltage steps from −90mV to 0mV for 20ms (10Hz frequency) was applied before and after compound addition. To evaluate the tonic block, inhibition of the peak current at the first pulse was measured while the use-dependent block was determined as the inhibition at the 10th pulse. Lidocaine shows the expected use-dependent inhibition. Surprisingly, we found compounds with the opposite profile: the compound with the most pronounced effect blocked the 1st and 10th pulses by 72.3±6.1 % and 42.9±6.9 % (mean±SD, n=5) at 10uM. In a second instance these compounds were tested against the cardiac Nav1.5 and the peripheral nervous and neuroendocrine systems Nav1.7 observing similar effects. An in-depth comparison between use-dependent and reverse use-dependent blockers was performed for parameters such as voltage-dependent activation and inactivation, recovery from inactivation, and frequency dependency. These data provide biophysical insights in the mechanism of reverse use-dependent inhibition for Nav channels.