Figure 8. The time course and amplitude of I_NaR are recapitulated during repetitive brief depolarizing steps.

To determine if two competing inactivation states underlie the observed differences in I_NaT and I_NaR recovery from inactivation (illustrated in Figure 5), a protocol was developed to allow direct comparison of I_NaR recorded during a single (80 ms) hyperpolarizing voltage step to –45 mV (red), presented following a brief (5 ms) depolarization to 0 mV, with I_NaR recorded (in the same cell) at –45 mV during successive brief (2 ms) hyperpolarizing voltage steps interspersed with brief (5 ms) depolarizations to 0 mV (black). Representative records are shown in (A); the voltage-clamp paradigms are illustrated below the current records. Similar results were obtained in four additional Purkinje neurons. As is evident (A), the envelope of the currents generated using these two protocols superimpose, suggesting that the inactivation pathway responsible for I_NaR decay does not compete with fast inactivation. (B) Simulated current waveforms, generated using the same two voltage-clamp protocols illustrated in (A) with the novel kinetic state model presented in Figure 3A, are shown. (C) Gating state occupancies for simulated current traces are shown with black representing the closed state, blue representing the open state, green representing the IC1+ IC2 states, aqua representing the IF2 state, orange representing the IF1 state, and purple representing the IS state. For direct comparison of the results of the simulations using the voltage-clamp protocols illustrated in (A) with the Raman-Bean gating model (2001), see Figure 8—figure supplement 1.

Figure 8—figure supplement 1. Gating state occupancies/transitions produced in the Raman-Bean model (with the voltage-clamp protocols used in Figure 8).

The Raman-Bean model (presented in Figure 7A) does not reproduce the Nav currents recorded from a Purkinje neuron that is depolarized (to 0 mV) and repolarized (to –45 mV) repeatedly (see Figure 8A); the voltage-clamp protocol and the simulated currents produced are shown in (A) as solid black lines. Nav current waveforms, simulated using the Raman-Bean model, in response to a sustained hyperpolarization to –45 mV following a brief (5 ms) depolarizing voltage step to 0 mV are also shown; the voltage-clamp protocol and the simulated currents are shown in (A) as dashed red lines. The gating state occupancy plot (B), presented on the same time scale as the voltage-clamp records, is shown; colors denote the various kinetic states with black representing the closed state, blue representing the open state, red representing the open-blocked (OB) state, and green representing the inactivated (I1–I6) states. The dashed lines are the gating state transitions associated with the dashed (red) current records in (A), and the solid lines are the gating state transitions associated with the solid (black) current records in (A).