Enhancement of slow inactivation in the F1490L-M1493I channels. (a) Steady-state slow inactivation in hSkM1 (filled circles; n = 23), F1490L (filled diamonds; n = 9), M1493I (filled squares; n = 9), and F1490L-M1493I (open circles; n = 18) channels. The first step consists of holding the cells at potentials ranging from –130 to 10 mV in 10-mV steps for 50 seconds. A 30-millisecond recovery pulse to –100 mV and a 20-millisecond test pulse to –10 mV were given before the holding potential was incremented again. The holding potential was incremented by 10 mV immediately after each recovery pulse/test pulse sequence. We term this protocol sequential because the channels are not allowed to recover from slow inactivation for each test pulse; the expectation is that the sequential changes in holding potential will mimic changes in holding potential of longer durations. The short hyperpolarizing recovery pulses can be used to remove fast inactivation just before a test pulse to measure slow inactivation (31). The peak current elicited by the test pulse to –10 mV was plotted as a fraction of the maximum current. (b) A greater fraction of F1490L-M1493I current than WT current is slow inactivated at –60 mV. Cells were held for 50 seconds at –60 mV, allowed to recover for 30 millisecond at –100 mV, and then depolarized to –10 mV to determine the fraction of channels that are slow inactivated. For comparison, the total current available from a holding potential of –100 mV is also shown for WT and F1490L-M1493I channels. (c) Development of slow inactivation is shown to be faster for the F1490L-M1493I (open circles; n = 5) than for the WT channels (filled circles; n = 5). Cells were held at –100 mV for an increasing conditioning time. (d) Normalized Na⁺ current in representative WT (filled circles; n = 9), F1490L (filled diamonds; n = 8), M1493I (filled squares; n = 7), and F1490L-M1493I (open circles; n = 8) cells recovering from slow inactivation (conditioning pulse is –50 mV). The time axis is logarithmic. The recovery protocol required about 45 minutes to complete and had two phases: short recovery times were obtained with individual recovery pulses, and long recovery times were obtained in a continuous recording.