Inhibition of oxidative metabolism increases persistent sodium current in rat CA1 hippocampal neurons

Abstract

1. Whole-cell patch-clamp recordings from freshly dissociated rat CA1 neurons revealed a large transient Na+ current (INa,T) and a smaller, inactivation-resistant persistent Na+ current (INa,P). Both currents could be blocked with TTX. 2. The average current densities of INa,T and INa,P in thirty cells were 111.0 +/- 9.62 and 0.87 +/- 0.13 pA pF-1, respectively. 3. Inhibiting oxidative phosphorylation by adding 5 mM sodium cyanide to the pipette solution significantly increased the amplitude of INa,P but had no significant effect on the amplitude of INa,T. 4. Exposing CA1 neurons to hypoxia for more than 7 min caused an increase in the amplitude of INa,P. There was also a delayed decrease in the amplitude of INa,T. 5. INa,P was more sensitive to the Na+ channel blockers TTX and lidocaine than INa,T. The IC50 for the effect of TTX on INa,P was 9.1 +/- 1.2 nM whereas the IC50 for INa,T was 37.1 +/- 1.2 nM, approximately 4-fold higher. Lidocaine (lignocaine; 1 microM) reduced INa,P to 0.24 +/- 0.15 of control (n = 4) whereas INa,T was essentially unaffected (0.99 +/- 0. 11, n = 4). 6. These results show that INa,P is increased when oxidative metabolism is blocked in CA1 neurons. The persistent influx of Na+ through non-inactivating Na+ channels can be blocked by concentrations of Na+ channel blockers that do not affect INa,T.

Figure 1. Recording TTX-sensitive current

Currents shown were recorded with an electrode containing 5 mm sodium cyanide 15 min after the whole-cell seal was obtained. A, the two traces show currents recorded before (lower trace) and after exposure of the neuron to 0.5 μm TTX. The currents were generated by a voltage step to -30 mV from a holding potential of -100 mV. B, the TTX-sensitive current obtained by subtracting the two traces in A. The transient current is truncated to show the persistent current in more detail. The scale bars apply for both A and B. The dashed line denotes the subtracted current level before the depolarizing pulse (zero current).

Figure 2. Inhibition of oxidative metabolism by hypoxia or cyanide increases persistent Na⁺ current

A, TTX-sensitive currents recorded from a neuron after 10 and 60 min of internal sodium cyanide exposure. Normally (in 12 out of 15 cells) the increase was large and stabilized within 10-15 min after the whole-cell seal was obtained. Currents shown were generated by a depolarizing step to -30 mV from a holding potential of -100 mV. B, TTX-sensitive currents recorded from a neuron before (top trace) and after exposure to hypoxic solution for 12 min (middle trace) and 23 min (lower trace). Currents were generated by a voltage step to -20 mV from a holding potential of -100 mV. The dashed line denotes the subtracted current level before the depolarizing pulse (zero current).

Figure 3. Current-voltage and conductance-voltage relationships before and after hypoxia

A, current-voltage relationship of the TTX-sensitive persistent Na⁺ current during normoxia (•) and after 12-15 min of hypoxia (^). The lines through the data points were obtained as described in the text. B, conductance-voltage relationship of the TTX-sensitive persistent Na⁺ current during normoxia (•) and after 12-15 min of hypoxia (^). The amplitude of I_Na,P was measured at the end of a 400 ms depolarizing pulse and divided by V - E₀ to obtain the conductance (E₀ is the potential at which I = 0). The lines through the data points were the best fits of the equation: G(V) = G_max/(1 + exp((V′ - V)/k)). Further details are given in the text.

Figure 4. Low concentrations of TTX and lidocaine block the persistent Na⁺ current more than the transient Na⁺ current

A, effects of 5 and 50 nM TTX on the persistent Na⁺ current generated by a voltage step from -100 to -30 mV 15-25 min after whole-cell seal was obtained. The pipette contained 5 mm cyanide. B, the persistent Na⁺ current after approximately 25 min of sodium cyanide exposure is depressed by 30 nM lidocaine. Currents were generated by a voltage pulse to -30 mV from a holding potential of -100 mV. C and D, the relationship between the concentration of TTX (C) or lidocaine (D) and depression of the normalized amplitudes (I′ = I/I_control) of the transient () and persistent (▪) Na⁺ current. The bars show the average normalized amplitude of the current and the vertical lines denote 1 s.e.m.