Nobukuni Ogata

The tetrodotoxin-resistant sodium channel SNS has a specialized function in pain pathways

Many damage-sensing neurons express tetrodotoxin (TTX)-resistant voltage-gated sodium channels. Here we examined the role of the sensory-neuron-specific (SNS) TTX-resistant sodium channel α subunit in nociception and pain by constructing sns-null mutant mice. These mice expressed only TTX-sensitive sodium currents on step depolarizations from normal resting potentials, showing that all slow TTX-resistant currents are encoded by the sns gene. Null mutants were viable, fertile and apparently normal, although lowered thresholds of electrical activation of C-fibers and increased current densities of TTX-sensitive channels demonstrated compensatory upregulation of TTX-sensitive currents in sensory neurons. Behavioral studies demonstrated a pronounced analgesia to noxious mechanical stimuli, small deficits in noxious thermoreception and delayed development of inflammatory hyperalgesia. These data show that SNS is involved in pain pathways and suggest that blockade of SNS expression or function may produce analgesia without side effects.

Electrophysiological characterization of the tetrodotoxin-resistant Na+ channel, Na(v)1.9, in mouse dorsal root ganglion neurons.

Small dorsal root ganglion neurons express preferentially the Na+ channel isoform NaV1.9 that mediates a tetrodotoxin-resistant (TTX-R) Na+ current. We investigated properties of the Na+ current mediated by NaV1.9 (INaN) using the whole-cell, patch-clamp recording technique. To isolate INaN from heterogeneous TTX-R Na+ currents that also contain another type of TTX-R Na+ current mediated by NaV1.8, we used NaV1.8-null mutant mice. When F− was used as an internal anion in the patch pipette solution, both the activation and inactivation kinetics for INaN shifted in the hyperpolarizing direction with time. Such a time-dependent shift of the kinetics was not observed when Cl− was used as an internal anion. Functional expression of INaN declined with time after cell dissociation and recovered during culture, implying that NaV1.9 may be regulated dynamically by trophic factors or depend on subtle environmental factors for its survival. During whole-cell recordings, the peak amplitude of INaN increased dramatically after a variable delay, as if inactive or silent channels had been “kindled”. Such an unusual increase of the amplitude could be prevented by adding ATP to the pipette solution or by recording with the nystatin-perforated patch-clamp technique, suggesting that the rupture of patch membrane affected the behaviour of NaV1.9. These peculiar properties of INaN may provide an insight into the plasticity of Na+ channels that are related to pathological functions of Na+ channels accompanying abnormal pain states.

GABAB-mediated upregulation of the high-voltage-activated Ca2+ channels in rat dorsal root ganglia.

Abstract The mechanism underlying the enhancement of the high-voltage-activated (HVA) Ca2+ current (ICa) after application of baclofen, a GABAB agonist, in neurones of the rat dorsal root ganglia was studied by a combined use of the nystatin perforated patch clamp recording and our rapid superfusion system. Baclofen (50 μM) decreased the peak amplitude of HVA ICa and slowed the onset of the current, i.e. produced a typical G-protein-mediated inhibition of ICa. However, when baclofen was rapidly removed from the medium, the amplitude of the current was rather augmented, exceeding the control value obtained before application of the drug. This enhancement was not due to a shift of the voltage dependence of Ca2+ channel activation or a change in ionic permeability to other ions. The enhancement of HVA ICa by baclofen was sensitive to pertussis toxin treatment. The enhancement was evident during superfusion of baclofen. Since the inhibitory effect of baclofen on HVA ICa was not attenuated, even after a continuous application of baclofen for 10 min, the enhancement was not due to relief from tonic G-protein-mediated inhibition of the current or a desensitization of the GABAB receptor–effector system. An extremely prolonged time course of the enhancement of HVA ICa by baclofen strongly suggests an involvement of some intracellular signal transduction system.

Single-channel analysis of two types of Na+currents in rat dorsal root ganglia

The properties of voltage-gated Na+ channels were studied in neurones isolated from rat dorsal root ganglia using the outside-out configuration of the patch-clamp technique. Two types of single-channel currents were identified from the difference in unit amplitudes. Neither type was evoked in the medium in which extracellular Na+ ions were replaced by an equimolar amount of tetramethylammonium ions. The two types of single-channel currents differed in their sensitivity to tetrodotoxin (TTX). The smaller channel current was insensitive to 1 μM TTX (referred to as TTX-I), while the larger channel current was blocked by 1 nM TTX (TTX-S). The unit amplitudes measured during a step depolarization to −30 mV (1.4 mM internal and 250 mM external Na+ concentrations) were 1.16 pA for TTX-S and 0.57 pA for TTX-I, respectively. The slope conductance measured at −30 mV was 16.3 pS for TTX-S and 8.5 pS for TTX-I. TTX-S could be activated by step depolarizations positive to −60 mV, while TTX-I could be activated at potentials positive to −40 mV. When the test pulse was preceded by a depolarizing prepulse, the prepulse positive to −50 mV preferentially inactivated TTX-S with a minimal effect on TTX-I. Activation and inactivation time courses of the averaged ensemble currents computed from TTX-S showed remarkable resemblances to the time courses of the macroscopic TTX-sensitive Na+ current. Similarly, the ensemble currents of TTX-I mimicked the macroscopic TTX-insensitive Na+ current. It was concluded that the two types of Na+ channels in rat dorsal root ganglia differ not only in their sensitivity to TTX, but also in their single-channel conductances.

Regulation of the Spontaneous Augmentation of NaV1.9 in Mouse Dorsal Root Ganglion Neurons: Effect of PKA and PKC Pathways

Sensory neurons in the dorsal root ganglion express two kinds of tetrodotoxin resistant (TTX-R) isoforms of voltage-gated sodium channels, NaV1.8 and NaV1.9. These isoforms play key roles in the pathophysiology of chronic pain. Of special interest is NaV1.9: our previous studies revealed a unique property of the NaV1.9 current, i.e., the NaV1.9 current shows a gradual and notable up-regulation of the peak amplitude during recording (“spontaneous augmentation of NaV1.9”). However, the mechanism underlying the spontaneous augmentation of NaV1.9 is still unclear. In this study, we examined the effects of protein kinases A and C (PKA and PKC), on the spontaneous augmentation of NaV1.9. The spontaneous augmentation of the NaV1.9 current was significantly suppressed by activation of PKA, whereas activation of PKA did not affect the voltage dependence of inactivation for the NaV1.9 current. On the contrary, the finding that activation of PKC can affect the voltage dependence of inactivation for NaV1.9 in the perforated patch recordings, where the augmentation does not occur, suggests that the effects of PMA are independent of the augmentation process. These results indicate that the spontaneous augmentation of NaV1.9 was regulated directly by PKA, and indirectly by PKC.

Paradoxical Facilitation of the Voltage-Dependent Calcium Current Following Activation of GABAB Receptors

The mechanisms underlying the enhancement of the calcium current (ICa) after application of baclofen, a GABAB agonist, were studied in neurons of the rat dorsal root ganglia using nystatin perforated patch clamp recording. Baclofen (50 μM) decreased ICa and slowed the onset of ICa. However, when baclofen was rapidly washed out from the medium, the amplitude of ICa was paradoxically augmented exceeding the control value measured before application of the drug. This enhancement of ICa by baclofen was not due to desensitization of GABAB receptors or a liberation from tonic G protein-mediated inhibition of ICa. From its extremely prolonged time course, an involvement of some intracellular signal transduction system was strongly suggested.