Regulation of the spontaneous augmentation of Na(V)1.9 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, Na(V)1.8 and Na(V)1.9. These isoforms play key roles in the pathophysiology of chronic pain. Of special interest is Na(V)1.9: our previous studies revealed a unique property of the Na(V)1.9 current, i.e., the Na(V)1.9 current shows a gradual and notable up-regulation of the peak amplitude during recording ("spontaneous augmentation of Na(V)1.9"). However, the mechanism underlying the spontaneous augmentation of Na(V)1.9 is still unclear. In this study, we examined the effects of protein kinases A and C (PKA and PKC), on the spontaneous augmentation of Na(V)1.9. The spontaneous augmentation of the Na(V)1.9 current was significantly suppressed by activation of PKA, whereas activation of PKA did not affect the voltage dependence of inactivation for the Na(V)1.9 current. On the contrary, the finding that activation of PKC can affect the voltage dependence of inactivation for Na(V)1.9 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 Na(V)1.9 was regulated directly by PKA, and indirectly by PKC.

Involvement of voltage-gated sodium channel Na(v)1.8 in the regulation of the release and synthesis of substance P in adult mouse dorsal root ganglion neurons.

This study was conducted to determine whether Na(v)1.8 contributes to the release and/or synthesis of substance P (SP) in adult mice dorsal root ganglion (DRG) neurons. The SP released from cultured DRG neurons of Na(v)1.8 knock-out mice exposed to either capsaicin or KCl was significantly lower than that from wild-type (C57BL/6) mice based on a radioimmunoassay. The SP level of L6 DRG in Na(v)1.8 knock-out mice was also lower than that in wild-type mice. After chronic constriction injury (CCI) of the sciatic nerve, the level of SP decreased in the L6 ipsilateral DRG of wild-type but not Na(v)1.8 knock-out mice. The preprotachykinin-A (PPT-A) mRNAs in L4 - 6 DRGs of Na(v)1.8 knock-out mice also fell to half their normally abundant levels of expression. There were significant increases in Na(v)1.8 expression of the L6 contralateral DRG from wild-type mice and in the percentage of neurons expressing neurokinin-1 receptor in the cytosol of L6 DRGs from wild-type or Na(v)1.8 knock-out mice. These findings suggest that Na(v)1.8 is involved in the regulation of the release and synthesis of SP in the DRG neurons of wild-type mice.

Prostaglandin E2 has no effect on two components of tetrodotoxin-resistant Na+ current in mouse dorsal root ganglion.

One possible mechanism underlying inflammation-induced sensitization of the primary afferent neuron is the upregulation of tetrodotoxin-resistant (TTX-R) Na(+) current by inflammatory mediators such as prostaglandins. This notion is based on reports that showed an augmentation of TTX-R Na(+) current following an application of prostaglandin E(2) (PGE(2)) in dorsal root ganglion (DRG) neurons. However, no information was available on the properties of the novel type of TTX-R Na(+) channel, Na(V)1.9, at times when these reports were published. Hence, the contribution of Na(V)1.9 to the PGE(2)-induced upregulation of TTX-R Na(+) current remains to be elucidated. To further examine the modulation of TTX-R Na(+) current by PGE(2), we recorded two components of TTX-R Na(+) current in isolation from small (<25 microm in diameter) DRG neurons using wild-type and Na(V)1.8 knock-out mice. Unexpectedly, neither the component mediated by Na(V)1.8 nor the persistent component mediated by Na(V)1.9 was affected by PGE(2) (1 and 10 microM). Our results raise a question regarding the well-known modulatory role of PGE(2) on TTX-R Na(+) current in inflammatory hyperalgesia.

Multiple types of Na(+) currents mediate action potential electrogenesis in small neurons of mouse dorsal root ganglia.

Small (<25 microm in diameter) neurons of the dorsal root ganglion (DRG) express multiple voltage-gated Na(+) channel subtypes, two of which being resistant to tetrodotoxin (TTX). Each subtype mediates Na(+) current with distinct kinetic property. However, it is not known how each type of Na(+) channel contributes to the generation of action potentials in small DRG neurons. Therefore, we investigated the correlation between Na(+) currents in voltage-clamp recordings and corresponding action potentials in current-clamp recordings, using wild-type (WT) and Na(V)1.8 knock-out (KO) mice, to clarify the action potential electrogenesis in small DRG neurons. We classified Na(+) currents in small DRG neurons into three categories on the basis of TTX sensitivity and kinetic properties, i.e., TTX-sensitive (TTX-S)/fast Na(+) current, TTX-resistant (TTX-R)/slow Na(+) current, and TTX-R/persistent Na(+) current. Our concurrent voltage- and current-clamp recordings from the same neuron revealed that the action potentials in WT small DRG neurons were mainly dependent on TTX-R/slow Na(+) current mediated by Na(V)1.8. It was surprising that a large portion of TTX-S/fast Na(+) current was switched off in WT small DRG neurons due to a hyperpolarizing shift of the steady-state inactivation (h (infinity)), whereas in KO small DRG neurons which are devoid of TTX-R/slow Na(+) current, the action potentials were generated by TTX-S/fast Na(+) current possibly through a compensatory shift of h (infinity) in the positive direction. We also confirmed that TTX-R/persistent Na(+) current mediated by Na(V)1.9 actually regulates subthreshold excitability in small DRG neurons. In addition, we demon strated that TTX-R/persistent Na(+) current can carry an action potential when the amplitude of this current was abnormally increased. Thus, our results indicate that the action potentials in small DRG neurons are generated and regulated with a combination of multiple mechanisms that may give rise to unique functional properties of small DRG neurons.

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 Na(v)1.9 that mediates a tetrodotoxin-resistant (TTX-R) Na+ current. We investigated properties of the Na+ current mediated by Na(v)1.9 (I(NaN)) using the whole-cell, patch-clamp recording technique. To isolate I(NaN) from heterogeneous TTX-R Na+ currents that also contain another type of TTX-R Na+ current mediated by Na(v)1.8, we used Na(v)1.8-null mutant mice. When F- was used as an internal anion in the patch pipette solution, both the activation and inactivation kinetics for I(NaN) 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 I(NaN) declined with time after cell dissociation and recovered during culture, implying that Na(v)1.9 may be regulated dynamically by trophic factors or depend on subtle environmental factors for its survival. During whole-cell recordings, the peak amplitude of I(NaN) 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 Na(v)1.9. These peculiar properties of I(NaN) may provide an insight into the plasticity of Na+ channels that are related to pathological functions of Na+ channels accompanying abnormal pain states.

Use-dependent removal of the 4-aminopyridine-induced block of the transient K+ current in rat dorsal root ganglia.

Effects of 4-aminopyridine (4-AP) on the transient K(+) current (I(A)) was studied in rat sensory neurons using the whole cell patch-clamp technique. The amplitude of I(A) was reduced by 4-AP. The steady-state inactivation curve for I(A) was shifted in the positive direction by 4-AP, suggesting that the blocking action of 4-AP may be attenuated by membrane depolarization. When two I(A)s were evoked with variable intervals, the peak amplitude of the I(A) induced by the second pulse was augmented in the presence of 4-AP. These results indicate that the action of 4-AP can be modulated by concurrent neuronal activities.

Molecular diversity of structure and function of the voltage-gated Na+ channels.

A variety of different isoforms of voltage-sensitive Na+ channels have now been identified. The recent three-dimensional analysis of Na+ channels has unveiled a unique and unexpected structure of the Na+ channel protein. Na+ channels can be classified into two categories on the basis of their amino acid sequence, Nav1 isoforms currently comprising nine highly homologous clones and Nax that possesses structure diverging from Nav1, especially in several critical functional motifs. Although the functional role of Nav1 isoforms is primarily to form an action potential upstroke in excitable cells, recent biophysical studies indicate that some of the Nav1 isoforms can also influence subthreshold electrical activity through persistent or resurgent Na+ currents. Nav1.8 and Nav1.9 contain an amino acid sequence common to tetrodotoxin resistant Na+ channels and are localized in peripheral nociceptors. Recent patch-clamp experiments on dorsal root ganglion neurons from Nav1.8-knock-out mice unveiled an additional tetrodotoxin-resistant Na+ current. The demonstration of its dependence on Nav1.9 provides evidence for a specialized role of Nav1.9, together with Nav1.8, in pain sensation. Although Nax has not been successfully expressed in an exogenous system, recent investigations using relevant native tissues combined with gene-targeting have disclosed their unique "concentration"-sensitive but not voltage-sensitive roles. In this context, these emerging views of novel functions mediated by different types of Na+ channels are reviewed, to give a perspective for future research on the expanding family of Na+ channel clones.