Tz1 effects on inactivation-deficient NaV channels. (A) Current traces of NaV1.4–IFC channels at −20 mV in the presence of 200 µM of intracellular LY before (ctrl) and 200 s after illumination with blue light. The arrow indicates 15 ms, the time at which the removal of fast inactivation was analyzed. (B) Time course of the loss of fast inactivation of NaV1.4–IFC mutant channels. The ratio of current after 15 ms and the peak current was monitored at an interval of 5 s and plotted as a function of time. The continuous line is a single-exponential description of the data. Closed circles mark the data points obtained from the current traces shown in A. During irradiation, NaV1.4–IFC progressively lost fast inactivation, characterized by a time constant of 79.6 ± 6.4 s; inactivation loss saturated at 89.1 ± 2.2% (n = 6). NaV1.4–IFC–G658N lost inactivation with a time constant of 92.2 ± 12.9 s and saturated at 93.5 ± 2.5% (n = 6). (C and D) Representative current–voltage plots of inactivation-deficient NaV1.4–IFC and the inactivation-deficient double mutant NaV1.4–IFC–G658N in the absence (open circles) and presence (closed symbols) of 25 µM Tz1 without (gray) or with (black) a prepulse. Continuous curves are data fits according to Eq. 1. (E and F) Current traces from independent experiments obtained for 100-ms depolarizations to the indicated voltages in the absence (black) and presence (gray) of 25 µM Tz1 without (−pp) or with (+pp) a prepulse.
