Persistent tetrodotoxin-sensitive sodium current resulting from U-to-C RNA editing of an insect sodium channel

Abstract

The persistent tetrodotoxin (TTX)-sensitive sodium current, detected in neurons of many regions of mammalian brains, is associated with many essential neuronal activities, including boosting of excitatory synaptic inputs, acceleration of firing rates, and promotion of oscillatory neuronal activities. However, the origin and molecular basis of the persistent current have remained controversial for decades. Here, we provide direct evidence that U-to-C RNA editing of an insect sodium channel transcript generates a sodium channel with a persistent current. We detected a persistent TTX-sensitive current in a splice variant of the cockroach sodium channel gene BgNa(v) (formerly para(CSMA)). Site-directed mutagenesis experiments revealed that an F-to-S change at the C-terminal domain of this variant was responsible for the persistent current. We demonstrated that this F-to-S change was the result of a U-to-C RNA editing event, which also occurred in the Drosophila para sodium channel transcript. Our work provides direct support for the hypothesis that posttranscriptional modification of a conventional transient sodium channel produces a persistent TTX-sensitive sodium channel.

Fig. 1.

Unique gating properties of BgNa_v4. (A) Sodium current traces from a representative oocyte expressing BgNa_v4. Currents were recorded from a 20-ms depolarization of a series of depolarizing voltages (-65–10 mV) from the holding potential of -120 mV. (B) Sodium current recorded from a 200-ms depolarization to -10 mV from the holding potential of -120 mV. The current was completely blocked by 20 nM TTX. (C) Voltage dependence of the persistent current. The persistent current was measured 15 ms into a 20-ms depolarization, normalized by the amplitude of peak current, and plotted against the depolarizing voltages. (D) Voltage dependence of activation of BgNa_v4 compared with BgNa_v1 (18). (E) Voltage dependence of steady-state inactivation of BgNa_v4 compared with BgNa_v1. (F) Recovery from fast inactivation of BgNa_v4 compared with BgNa_v1. The protocols for recording these gating properties are described in Materials and Methods.

Fig. 2.

S1919 at the C terminus is responsible for the persistent current and other unique properties identified in BgNa_v4. (A) Sodium current traces recorded from oocytes expressing BgNa_v4 and six single-amino-acid-substitution mutants, BgNa_v1 (containing F1919), Drosophila Para (containing F at 1950 equivalent to F1919 in BgNa_v1), and a Para mutant carrying S1950 for comparison. The current was measured by a 20-ms depolarization to -10 mV from the -120-mV holding potential. (B) Percentage of persistent currents. The persistent current was measured 15 ms into a 20-ms depolarization and was normalized by the amplitude of peak current. (C) Voltage dependence of activation of BgNa_v4 and six mutants. (D) Voltage dependence of steady-state inactivation of BgNa_v4 and six mutants. (E) Recovery from fast inactivation of BgNa_v4 and six mutants. The protocols for recording these gating properties are described in Materials and Methods.

Fig. 3.

Effects of amino acid substitutions at S1919 on persistent current in BgNa_v4. (A) Sodium currents from five amino acid substitutions, A, T, Y, D, and R, at S1919 of BgNa_v4. The F substitution at 1919 is shown in Fig. 2. (B) Percentage of persistent current. The persistent current was measured 15 ms into a 20-ms depolarization and was normalized by the amplitude of peak current. (C) Voltage dependence of activation of single-amino-acid-substituted BgNa_v4 mutants. (D) Voltage dependence of steady-state inactivation of single-amino-acid-substituted BgNa_v4 mutants.