Augmented sodium currents contribute to the enhanced excitability of small diameter capsaicin-sensitive sensory neurons isolated from Nf1+/⁻ mice

Abstract

Neurofibromin, the product of the Nf1 gene, is a guanosine triphosphatase activating protein (GAP) for p21ras (Ras) that accelerates conversion of active Ras-GTP to inactive Ras-GDP. Sensory neurons with reduced levels of neurofibromin likely have augmented Ras-GTP activity. We reported previously that sensory neurons isolated from a mouse model with a heterozygous mutation of the Nf1 gene (Nf1+/⁻) exhibited greater excitability compared with wild-type mice. To determine the mechanism giving rise to the augmented excitability, differences in specific membrane currents were examined. Consistent with the enhanced excitability of Nf1+/⁻ neurons, peak current densities of both tetrodotoxin-resistant sodium current (TTX-R I(Na)) and TTX-sensitive (TTX-S) I(Na) were significantly larger in Nf1+/⁻ than in wild-type neurons. Although the voltages for half-maximal activation (V(0.5)) were not different, there was a significant depolarizing shift in the V(0.5) for steady-state inactivation of both TTX-R and TTX-S I(Na) in Nf1+/⁻ neurons. In addition, levels of persistent I(Na) were significantly larger in Nf1+/⁻ neurons. Neither delayed rectifier nor A-type potassium currents were altered in Nf1+/⁻ neurons. These results demonstrate that enhanced production of action potentials in Nf1+/⁻ neurons results, in part, from larger current densities and a depolarized voltage dependence of steady-state inactivation for I(Na) that potentially leads to a greater availability of sodium channels at voltages near the firing threshold for the action potential.

Fig. 1.

Mouse sensory neurons exhibit 2 distinct phenotypes of I_K. A, top: I_Ks recorded from a Nf1+/− neuron that exhibited rapid activation with little time-dependent inactivation. Bottom: the currents obtained with the steady-state inactivation protocol for this same neuron. The prepulses for the steady-state inactivation are continuous steps but have been broken at the indicated times for presentation purposes. B, top: the I_Ks recorded from an Nf1+/− neuron that exhibited rapid activation with faster inactivation kinetics. Bottom: the currents obtained with the steady-state inactivation protocol for this neuron. The voltage protocols used are illustrated at the bottom of each panel. The lines labeled 0 represents the zero-current level. C, left: inactivation kinetics for I_K obtained for the step to +60 mV after prepulses to −100 mV from the neurons shown in A and B, bottom. Right: the subtraction of the slowly inactivating trace (A) from the more rapidly inactivating trace (B) and illustrates the rapidly inactivating current. The line through the data points represents the double exponential fit to the decay of the rapidly inactivating current wherein the fitting parameters were A1, 1,696; τ1, 97 ms; A2, 1,659; τ2, 33 ms; C, 0.59 nA; correlation coefficient, 0.989.

Fig. 2.

The peak and steady-state I_Ks in wild-type and Nf1+/− sensory neurons are not different. A, left: the current density-voltage relations obtained for the peak I_Ks from wild-type and Nf1+/− sensory neurons. Right: the current density-voltage relations obtained for the steady-state I_Ks from wild-type and Nf1+/− sensory neurons. The steady-state values were measured at the end of the voltage step. B: the conductance-voltage relations for the peak (left) and the steady-state (right) measurements. The points in B have been fitted by the Boltzmann relation and are shown as the continuous lines. The values in each panel of A and B represent the means ± SE obtained from 11 wild-type and 13 Nf1+/− neurons. C: the steady-state inactivation of I_K in 7 wild-type and 6 Nf1+/− sensory neurons. The steady-state inactivation voltage protocol is shown in Fig. 1, A and B. Currents were normalized to the maximal value of G obtained for the −100 mV prepulse. The data points have been fitted by the Boltzmann relation and are shown as ---.

Fig. 3.

The rapidly inactivating I_A current in wild-type and Nf1+/− neurons is not different. Representative current traces for a wild-type neuron exhibiting I_A are shown in A, top. The voltage protocol used to elicit the currents are shown below the traces; neurons were held at −100 mV, 500 ms steps were from −80 to +40 mV in 20 mV increments. B: representative current traces for the same neuron as in A except that a 4 s prepulse to −40 mV preceded the voltage steps. C: the A-type of I_K obtained by the subtraction of the traces in B from those in A. The G/G_max-voltage relations for activation and inactivation of I_A are summarized in D. The results for activation were obtained from 14 wild-type and 11 Nf1+/− neurons; inactivation from 8 wild-type and 6 Nf1+/− neurons. The data points have been fitted by the Boltzmann relation and are shown as continuous lines.

Fig. 4.

Representative traces of I_Na obtained from a wild-type (left) and Nf1+/− neuron (right). Top: total I_Na, middle: TTX sensitive (TTX-S) I_Na, bottom: TTX resistant (TTX-R) I_Na. TTX-S was obtained by digital subtraction of the traces for TTX-R I_Na from those of the total I_Na.

Fig. 5.

The current density of both TTX-R and TTX-S I_Na in Nf1+/− neurons is significantly larger than those in wild-type neurons. A: the current density for TTX-S I_Na was significantly larger in the 6 Nf1+/− neurons compared with 5 wild-type neurons. The values were significantly different between −20 and +20 mV (P < 0.05, Student's t-test). Right: the G/G_max-voltage relation for TTX-S I_Na in these neurons and shows that there is no difference in the voltage-dependence of activation; however, the steady-state inactivation of the 12 Nf1+/− neurons was shifted to more depolarizing potentials compared with the 10 wild-type neurons. The values for inactivation were significantly different for the prepulse voltages of −50 and −40 mV (P < 0.05, Student's t-test). The continuous lines through the points are the Boltzmann fits for the wild-type (black line) and Nf1+/− (gray line) neurons. B, left: the current density for TTX-R I_Na obtained from 9 Nf1+/− neurons was significantly larger compared with 8 wild-type neurons. The current values were significantly different between 0 and +25 mV (P < 0.05, Student's t-test). Right: the G/G_max-voltage relation for TTX-R I_Na in these neurons and shows that there is no difference in the voltage dependence of activation; however, the steady-state inactivation of the Nf1+/− neurons was shifted to more depolarizing potentials. The continuous lines through the points are the Boltzmann fits for the wild-type (black line) and Nf1+/− (gray line) neurons. The values of G/G_max for the Nf1+/− neurons were significantly different from the wild-type neurons for prepulse voltages from −60 to −30 mV (P < 0.05, Student's t-test). The fitting parameters are described in Table 2.

Fig. 6.

Nf1+/− sensory neurons exhibit larger persistent I_Na compared with wild-type neurons. A: representative current traces for the total I_Na obtained from a wild-type neuron (left) and from an Nf1+/− neuron (right). The currents were evoked by a series of 200 ms voltage pulses that ranged from −120 to +10 mV. The values for the persistent I_Na were obtained 100 ms after the onset of the prepulse (noted by the vertical bar labeled 100 ms). The peak currents have been truncated for clarity of the persistent I_Na. B: summary for the voltage dependence of the persistent I_Na measured as the percent of the maximum transient current for wild-type (n = 10) and Nf1+/− (n = 12) neurons. Left: the persistent current for the total I_Na; right: current remaining after exposure to TTX. *, significant difference between wild-type and Nf1+/− neurons (P < 0.05).