Effects of tramadol on amplitude of partially inactivated Nav1.5 current in HEK293 cells. (A and B) Block of fast-inactivated (A) and slow-inactivated (B) Nav1.5 current by tramadol in a concentration-dependent manner. Inset: voltage clamp protocol used to measure (left panel) and typical Nav1.5 currents under baseline conditions and in the presence of 1–100 μm tramadol (middle panel). We achieved partial fast inactivation (A) by applying a −90 mV prepulse and studied the effect of partial fast inactivation by applying the test pulse immediately following this prepulse. We achieved partial slow inactivation (B) by applying a −40 mV prepulse, during which Nav1.5 channels enter into both a fast-inactivated and a slow-inactivated state. This was followed by a brief (20 ms long) repolarizing pulse to −120 mV, which allows almost full recovery from fast inactivation, but not slow inactivation. Thus, the ensuing test pulse activates only Nav1.5 channels that have recovered from fast inactivation, but not Nav1.5 channels that are still in a slow inactivation state. Cycle length was 5 s. Solid lines are Hill fits to the average data. Values are normalized to the values measured under baseline conditions. Numbers near symbols indicated the number of cells (n) measured at the given concentrations.
