Nitric oxide increases persistent sodium current in rat hippocampal neurons

Abstract

1. The effects of nitric oxide (NO) donors on whole-cell, TTX-sensitive sodium currents and single sodium channels in excised patches were examined in rat hippocampal neurons. The whole-cell sodium current consisted of a large transient component (INa,t) and a smaller, inactivation-resistant, persistent component (INa,p). 2. In acutely dissociated neurons, the amplitude of the whole-cell INa, p increased by 60-80 % within a few minutes of exposure to either of two NO donors, sodium nitroprusside (SNP, 100 microM) or S-nitroso-N-acetyl-DL-penicillamine (SNAP, 100 microM). 3. The amplitude of INa,t was not changed significantly by the same concentrations of SNP and SNAP, indicating that NO had a selective effect on INa,p. 4. Both NO donors significantly increased the mean persistent current in excised inside-out patches from cultured hippocampal neurons. SNP at 10-100 microM increased average mean persistent current at a pipette potential (Vp) of +30 mV from -0.010 +/- 0.014 pA (control) to -2.91 +/- 1.41 pA (n = 10). SNAP at 3-100 microM increased the average mean inward current in six inside-out patches from -0.07 +/- 0.02 to -0.30 +/- 0.08 pA (Vp = +30 mV). 5. The increase in persistent Na+ channel activity recorded in inside-out patches in the presence of SNP or SNAP could be reversed by the reducing agent dithiothreitol (DTT, 2-5 mM) or by lidocaine (1-10 microM). 6. The average mean current recorded in the presence of SNP was 10-fold higher than that elicited by SNAP. The time delay before an increase was observed was shorter with SNP (4.0 +/- 0.8 min, n = 8) than with SNAP (8.4 +/- 1.6 min, n = 7). 7. A component of the SNP molecule added on its own, 5 mM sodium cyanide (NaCN), increased mean current in excised inside-out patches (Vp = +30 mV) from -0.06 +/- 0.04 to -0.58 +/- 0.21 pA (n = 19). This increase in channel activity could be blocked by 10 microM lidocaine and 2-5 mM DTT. 8. These results suggest that NO may directly increase the activity of neuronal persistent Na+ channels, but not transient Na+ channels, through an oxidizing action directly on the channel protein or on a closely associated regulatory protein in the plasma membrane.

Figure 1. Stability of measurements of I_Na,p over time

The amplitude of averaged TTX-subtracted I_Na,p amplitudes (from 8 hippocampal neurons) measured at the end of 400 ms depolarizing pulses relative to the amplitude of I_Na,p recorded when first obtaining whole-cell seal (Normalised current) plotted against time from the beginning of the whole-cell recording. There was no significant change in the amplitude for more than 15 min of recording.

Figure 2. Effect of NO donors on whole-cell Na⁺ currents in CA1 neurons

Currents were generated by a depolarising step to -30 mV (A) or -20 mV (B and C) from a holding potential of -100 mV. The traces shown are TTX subtracted (see Methods). A, currents recorded from a neuron before (upper trace) and after exposure to 0.1 mm SNP for 7.5 min and after 12 min of wash-out (lower traces). In this neuron, 12 min of wash-out did not reverse the effect of SNP. B, TTX-sensitive currents recorded from a neuron before (upper trace) and after 3 and 10 min of exposure to 0.1 mm SNAP (lower traces). An increase in the amplitude of I_Na,p was seen after 3 min and the amplitude of I_Na,p was doubled after 10 min of 0.1 mm SNAP perfusion. C, same neuron as in B but at a lower vertical amplification to show the lack of comparable effect of 0.1 mm SNAP on I_Na,t.

Figure 3. Current-voltage and conductance-voltage relationships for I_Na,p before and after 0.1 mm SNAP

A, current-voltage relationship of the TTX-sensitive persistent Na⁺ current before (^) and after a 10 min 0.1 mm SNAP exposure (•) (same cell as in Fig. 2B and C). B, conductance-voltage relationship of the TTX-sensitive persistent Na⁺ current before (^) and after 10 min of 0.1 mm SNAP (•) (same cell as in A). The amplitude of I_Na,p was divided by V - V₀ to obtain the conductance (V₀ is the potential at which I= 0). The lines through the data points were the best fits of the equation G = G_max/(1 + exp((V’ - V)/k)). The line of best fit gave a G_max of 1.5 nS before SNAP exposure and 2.4 nS in the presence of SNAP. V’ and slope factors were -41 and 12 mV in control and -42 and 13 mV in the presence of SNAP. Further details are given in the text.

Figure 5. Increases in I_Na,p produced by SNP and SNAP in inside-out patches

The histograms show mean current amplitudes. The vertical lines denote ± 1 s.e.m.A, mean data from 6 patches before (control, -0.07 ± 0.02 pA) and after 3-100 μm SNAP (-0.30 ± 0.08 pA, P= 0.02). B, mean data from 10 patches before (control, -0.010 ± 0.014 pA, n= 10) and after 10-100 μm SNP (-2.91 ± 1.41 pA, n= 10, P= 0.05).

Figure 4. Effect of SNAP on I_Na,p activity in an excised inside-out patch

The representative 400 ms traces shown were obtained from 3-4 min recordings from a patch held at V_p=+30 mV. The accompanying all-points histograms were calculated from the full 3-4 min current record sampled at 10 kHz. A, control activity. B, increased activity after 5 min exposure to 0.1 mm SNAP. C, SNAP-induced activity was completely blocked after 4-5 min exposure to 10 μm lidocaine and 0.1 mm SNAP.

Figure 6. Effects of SNP, DTT and lidocaine on an inside-out patch

Current traces were obtained at a holding potential of -30 mV (V_p=+30 mV) and are all from the same patch. A, control current records before exposure to SNP. B, after approximately 4 min in the presence of 10 μm SNP. C, inhibition of channel activity after 2-3 min of perfusion with 2 mm DTT (with 10 μm SNP). D, the return of activity after 1.5 min of washing out DTT with a solution containing 10 μm SNP. E, abolition of SNP-elicited channel activity by 10 μm lidocaine (with 10 μm SNP, 10 min).

Figure 7. Average effects of DTT and lidocaine on current activity produced by SNP or SNAP

Histograms show averaged data and the vertical bars denote 1 s.e.m.A, mean current was measured from 5 patches before (-0.04 ± 0.02 pA), and after exposure to 3-100 μm SNP or SNAP (≈5.5 min exposure, -2.46 ± 1.70 pA) and then in the presence of 10 μm lidocaine plus NO donor (-0.07 ± 0.04 pA). B, mean current was measured in 4 excised patches before (-0.03 ± 0.02 pA), and after approximately 7.5 min exposure to SNP or SNAP (-0.45 ± 0.07 pA) and then after 2-3 min perfusion with 2-5 mm DTT plus NO donor (-0.03 ± 0.04 pA).

Figure 8. Effect of NaCN on Na⁺ channel activity

Representative traces are from an inside-out patch held at V_p=+30 mV. A, traces before drug application. B, after a 5 min application of 5 mm NaCN. C, inhibition of activity by 10 μm lidocaine.

Figure 9. The effect of 5 mm NaCN on mean persistent Na⁺ current

Mean current recorded from 19 inside-out patches in control (-0.06 ± 0.04 pA), after approximately 5 min of exposure to 5 mm NaCN (-0.58 ± 0.21 pA) and after lidocaine exposure (-0.01 ± 0.01 pA, n= 4). NaCN significantly increased inward persistent channel activity which could be abolished by 10 μm lidocaine. The difference between control and 5 mm NaCN is statistically significant (n= 19, P= 0.02) as is the difference between 5 mm NaCN and 10 μm lidocaine (n= 4, P= 0.01).

Figure 10. Effect of 1 mm DTNB on inside-out hippocampal patch

A sulfhydryl specific reagent, DTNB, caused an increase in persistent Na⁺ channel activity in a patch showing no channel activity over a previous 4 min recording period. A, before 1 mm DTNB was perfused. B, after about 6 min of exposure to 1 mm DTNB.