(A) A steady-state availability protocol was used to assess the voltage-dependence of inactivation of TTX-S and TTX-R currents. Left panel: The protocol consisted of a 500 ms pre-pulse to potentials between −100 and +10 mV, followed by test pulse to 0 mV. Right panel: The fast component of the current evoked at 0 mV (open circles), was inactivated over a range of test potentials more hyperpolarized than the range of test potential over which the slow component of the current evoked at 0 mV (closed circles) was inactivated. (B) Left Panel, top traces: Current evoked during the test pulse in A (left panel), plotted on a shorter time scale to more clearly illustrate the fast and slow components of the current evoked at 0 mV. Because the fast component of the current was completely inactivated with a test pulse more hyperpolarized than that at which the slow component began to inactivate, it was possible to digitally isolate the fast component by subtracting the slow component (purple trace), from the total current. Conversely, because the fast component was completely inactivated within ~10 ms of the start of the test pulse, it was possible to generate an availability curve for the slow component across the entire range of pre-pulse potentials. The bottom traces are those of the fast component digitally isolated from the slow component. Right panel, top traces: In another neuron, the steady-state availability protocol used in A, was used to inactivate the fast component of the current evoked at 0 mV. Application of 300 nM TTX removed the same component of the total current as the test pulse to −40 mV. Bottom traces: The difference between the total current and the current evoked at −40 mV, or that evoked in the presence of 300 mM TTX is virtually identical. (C). Current-voltage (I-V) protocols were used to assess current activation, with pre-pulse potentials that were based on steady-state availability data. Thus, total current (top traces) was evoked following a 500 ms pre-pulse to a potential at which currents were fully available for activation (i.e., −100 mV). TTX-R currents (middle traces) were evoked following a 500 ms pre-pulse to a potential at which TTX-S currents were completely inactivated, but TTX-R currents were fully available for activation (i.e, −35 mV). It was then possible to digitally isolate TTX-S currents (Bottom traces) from the total current by subtracting TTX-R current from the total current.