Figure 1.
INaP activation and time dependent inactivation.
A, INaP evoked in a representative neuron by means of a slow voltage ramp compared with the current trace evoked in the presence of TTX, revealing a small outward current activating at more positive potentials. B, kinetics of development of INaP time-dependent inactivation assessed with and without TTX subtraction, which completely overlap. The data points are fit with a bi-exponential function. The inset illustrates the stimulus protocol. C, examples of INaP traces with or without TTX subtraction evoked after inactivating prepulses to 0 mV lasting from 100 ms to 10 seconds. The traces are shown partially overlapped to better compare peak amplitude.
Table 1.
Parameters of activation, voltage-dependence and time-dependent inactivation under control conditions and in the presence of PHT.
Figure 2.
INaP recorded in a representative pyramidal neuron with 50 mVs−1 voltage ramps.
Current traces are shown under control conditions, in the presence of 100 µM PHT and during PHT washout.
Figure 3.
Effect of PHT on the voltage-dependence of INaP inactivation.
A, effect of 100 µM PHT on the activation curve; the inset shows the stimulus protocol (prepulses were 10 s in duration; ramps had a slope of 50 mV s−1). Panels B, C and D show the current peaks evoked after different prepulses in a representative neuron. In the presence of PHT, the currents evoked after inactivating prepulses at −40 and −20 mV are clearly reduced with respect to those evoked under control conditions, and partially recover during PHT washout.
Figure 4.
Effect of PHT on the development of INaP inactivation.
Effect of 100 µM PHT on INaP evoked without (leftmost traces) and with inactivating prepulses to −20 mV lasting from 100 ms to 10 seconds in a representative layer V neuron. The arrows indicate the peak current evoked using depolarizing ramp stimuli under control conditions (left) and in the presence of 100 µM PHT (right). Development of INaP inactivation in layers II/III (B) and V (C) at −20 mV and in layer V at +40 mV (D) on a semi-logarithmic scale, under control conditions (open triangles) and in the presence of 100 PHT µM (black triangles); the data points were fit to bi-exponential functions with a baseline (see Table 1).
Figure 5.
Effect of different concentrations of PHT on INaP evoked using inactivating prepulses of different durations.
Panels A and B show the dose response curves with prepulses of 200 ms (A) or 500 ms (B); higher concentrations of PHT have not been used because of its solubility limits, and the maximal block was not reached. The solid lines are fit to rectangular hyperbolas that gave apparent IC50 values of 28 and 18 µM and apparent maximal block of 24 and 26% respectively. All of the PHT concentrations used induced a statistically significant reduction of INaP peak amplitude with both 200 and 500 ms inactivating prepulses, as shown in C (**p<0.01; *** = p<0.001; ANOVA test).
Figure 6.
INaP evoked by ramps with different slopes.
Current traces are shown under control conditions (A) and in the presence of PHT (100 µM) (B). The amplitude of the current decreased with the duration of the ramps and the inhibitory effect of phenytoin became clear and significant for INaP evoked by slower ramps (arrows). The inset shows a graphical comparison of INaP amplitude in the different conditions. The graph in C displays the mean values of the INaP peaks measured on five neurons in response to 100, 50 and 10 mVs−1 ramps under control conditions, in the presence of PHT and after PHT wash out.
Figure 7.
INaP evoked by a 10 s step stimulus to −50 mV.
Current traces are shown under control conditions (gray line) and in the presence of 100 µM PHT (black line) in a representative layer V neuron (A); note that the amplitude of the current in the first few hundred milliseconds of the trace is unaffected by PHT (inset in A). The average of the currents obtained in five layer V neurons and normalized to the maximum (B) shows that the decay time constant describing the current inactivation is accelerated and enhanced in the presence of PHT (middle panel). The decay was fit with a mono-exponential relationship (tick line); Note that in these experiments the effect of PHT is particularly small because at −50 mV INaP inactivation is minimal. See text for details.
Figure 8.
Effect of PHT on recovery from INaP inactivation.
A, representative traces in control (above) and with 100 μM PHT (below) recorded after a 20 s-long inactivating prepulse to −10 mV and a recovery period at −80 mV of 1 ms, 1000 ms, 4000 ms, 10000 ms and 40000 ms (see stimulus in B). B, plot showing average recovery in control (black triangles) and with 100 μM PHT (hollow triangles). The lines are single exponentials obtained averaging the parameters of the fits of the single cells (see text for details).
Figure 9.
Effect of PHT on depolarized plateau recorded in current clamp.
Depolarized plateau induced by the intracellular injection of a brief (40 ms) depolarizing pulse in a representative neuron recorded in current clamp configuration. Long-lasting depolarizations following the action potential were observed in the presence of K+ and Ca2+ channel blockers and are most likely sustained by the INaP flowing after the fast inactivation of the transient Na+ current. The long depolarized plateau observed under control conditions (A) were shortened in the presence of PHT (100 µM) (B). In five neurons, the decay of the depolarizing plateau was fit by bi-exponential functions; the values of the time-constants were comparable to those describing the time-dependent inactivation of INaP and were significantly shortened by PHT. Panel C shows the mean plateau potentials normalized to their maximal value and the bi-exponential fitting obtained averaging the parameters of the fits of the single cells.
Figure 10.
Inferences on the mechanism of action.
A; simplified gating schemes illustrating the action of PHT, excluding an interaction with the open state of the channel. The scheme on the left depicts PHT as a pure inactivated state stabilizer that binds to the inactivated state of the channel; the scheme on the right is an interpretation of our results and depicts PHT as an inactivated state stabilizer that binds to the inactivated state and to a hypothetical intermediate in the inactivation pathway. C, O, OI and CI are the closed, open, open-inactivated and closed-inactivated states of the Na+ channel; P is PHT, which in both cases has much higher affinity to the I state than to the C (resting) state. The black parts of the schemes are the transitions induced by depolarized potentials: the dimension of the arrowhead indicates the value of the rate constants and the dashed oval indicates the absorbing state at depolarized potentials. The gray parts are the transitions induced by repolarizations and the dotted oval is the absorbing state in these conditions. The main difference between the two schemes is the fact that on the left PHT binds to channels already in the inactivated state stabilizing it, whereas on the right it can also accelerate the kinetics of inactivation by binding to a hypothetical intermediate, similarly to a catalyst in a chemical reaction. B; the simulated curve of development of INaP inactivation shows that the effect of PHT cannot be obtained with a simple intrinsic slow binding of PHT to inactivated channels as in scheme A, left. The dotted line is a simple simulation of the maximal effect of PHT on development of INaP inactivation at +40 mV in Layer V neurons according to the scheme in A, left. It is assumed that that the action of PHT is intrinsically slow, developing with a time constant of 1 s (Kuo and Bean, 1994; Kuo et al, 1997), and that it binds irreversibly to inactivated channels. Solid and dashed lines are the fits of the experimental data in control and with PHT respectively, which are shown in Figure 4 and in Table 1. The simulated curve (hDt, development of inactivation in the presence of PHT) has been obtained with the following equation: hDt = ht{1-[(1-ht) dt]}, in which ht is the curve of development in control (fraction of channels available as a function of time at +40 mV) and dt the fractional binding of PHT (kinetics of PHT binding). It is evident that the simulated curve does not approximate the experimental curve in the presence of PHT (compare the dashed line with the dotted one). In order to quantitatively compare the parameters with those of the experimental curves (Table 1), the simulated curve was fit with a double exponential relationship that gave the following parameters: τ1 = 0.12 s, A1 = 0.25, τ2 = 2.4 s, A2 = 0.6, baseline = 0.15, which are different in comparison with those obtained from the experimental curve in the presence of PHT (Table 1). Thus, PHT effect cannot be simulated with a simple intrinsic slow binding of PHT to the channels in the inactivated conformation; an acceleration of the rate constants of development of INaP inactivation as in scheme A, right, is more consistent with the experimental data (see text).