Kenji Okuse

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.

The TTX-Resistant Sodium Channel Nav1.8 (SNS/PN3): Expression and Correlation with Membrane Properties in Rat Nociceptive Primary Afferent Neurons

We have examined the distribution of the sensory neuron‐specific Na+ channel Nav1.8 (SNS/PN3) in nociceptive and non‐nociceptive dorsal root ganglion (DRG) neurons and whether its distribution is related to neuronal membrane properties. Nav1.8‐like immunoreactivity (Nav1.8‐LI) was examined with an affinity purified polyclonal antiserum (SNS11) in rat DRG neurons that were classified according to sensory receptive properties and by conduction velocity (CV) as C‐, Aδ‐ or Aα/β. A significantly higher proportion of nociceptive than low threshold mechanoreceptive (LTM) neurons showed Nav1.8‐LI, and nociceptive neurons had significantly more intense immunoreactivity in their somata than LTM neurons. Results showed that 89, 93 and 60 % of C‐, Aδ‐ and Aα/β‐fibre nociceptive units respectively and 88 % of C‐unresponsive units were positive. C‐unresponsive units had electrical membrane properties similar to C‐nociceptors and were considered to be nociceptive‐type neurons. Weak positive Nav1.8‐LI was also present in some LTM units including a C LTM, all Aδ LTM units (D hair), about 10 % of cutaneous LTM Aα/β‐units, but no muscle spindle afferent units. Nav1.8‐LI intensity was negatively correlated with soma size (all neurons) and with dorsal root CVs in A‐ but not C‐fibre neurons. Nav1.8‐LI intensity was positively correlated with action potential (AP) duration (both rise and fall time) in A‐fibre neurons and with AP rise time only in positive C‐fibre neurons. It was also positively correlated with AP overshoot in positive neurons. Thus high levels of Nav1.8 protein may contribute to the longer AP durations (especially in A‐fibre neurons) and larger AP overshoots that are typical of nociceptors.

Annexin II light chain regulates sensory neuron-specific sodium channel expression

The tetrodotoxin-resistant sodium channel NaV1.8/SNS is expressed exclusively in sensory neurons and appears to have an important role in pain pathways. Unlike other sodium channels, NaV1.8 is poorly expressed in cell lines even in the presence of accessory β-subunits. Here we identify annexin II light chain (p11) as a regulatory factor that facilitates the expression of NaV1.8. p11 binds directly to the amino terminus of NaV1.8 and promotes the translocation of NaV1.8 to the plasma membrane, producing functional channels. The endogenous NaV1.8 current in sensory neurons is inhibited by antisense downregulation of p11 expression. Because direct association with p11 is required for functional expression of NaV1.8, disrupting this interaction may be a useful new approach to downregulating NaV1.8 and effecting analgesia.

Regulation of expression of the sensory neuron-specific sodium channel SNS in inflammatory and neuropathic pain

Increased voltage-gated sodium channel activity may contribute to the hyperexcitability of sensory neurons in inflammatory and neuropathic pain states. We examined the levels of the transcript encoding the tetrodotoxin-resistant sodium channel SNS in dorsal root ganglion neurons in a range of inflammatory and neuropathic pain models in the rat. Local Freunds adjuvant or systemic nerve growth factor-induced inflammation did not substantially alter the total levels of SNS mRNA. When NGF-treated adult rat DRG neurons in vitro were compared with NGF-depleted control neurons, SNS total mRNA levels and the levels of membrane-associated immunoreactive SNS showed a small increase (17 and 25%, respectively), while CGRP levels increased fourfold. SNS expression is thus little dependent on NGF even though SNS transcript levels dropped by more than 60% 7-14 days after axotomy. In the streptozotocin diabetic rat SNS levels fell 25%, while in several manipulations of the L5/6 tight nerve ligation rat neuropathic pain model, SNS levels fell 40-80% in rat strains that are either susceptible or relatively resistant to the development of allodynia. Increased expression of SNS mRNA is thus unlikely to underlie sensory neuron hyperexcitability associated with inflammation, while lowered SNS transcript levels are associated with peripheral nerve damage.

cAMP-dependent phosphorylation of the tetrodotoxin-resistant voltage-dependent sodium channel, SNS

1 Protein kinase A (PKA) modulation of tetrodotoxin‐resistant (TTX‐r) voltage‐gated sodium channels may underly the hyperalgesic responses of mammalian sensory neurones. We have therefore examined PKA phosphorylation of the cloned α‐subunit of the rat sensory neurone‐specific TTX‐r channel SNS. Phosphorylation of SNS was compared with that of a mutant channel, SNS(SA), in which all five PKA consensus sites (RXXS) within the intracellular I‐II loop had been eliminated by site‐directed mutagenesis (serine to alanine). 2 In vitro PKA phosphorylation and tryptic peptide mapping of SNS and mutant SNS(SA) I‐II loops expressed as glutathione‐S‐transferase (GST) fusion proteins confirmed that the five mutated serines were the major PKA substrates within the SNS I‐II loop. 3 SNS and SNS(SA) channels were transiently expressed in COS‐7 cells and their electrophysiological properties compared. In wild‐type SNS channels, forskolin and 8‐bromo cAMP produced effects consistent with PKA phosphorylation. Mutant SNS(SA) currents, however, were not significantly affected by either agent. Thus, elimination of the I‐II loop PKA consensus sites caused a marked reduction in PKA modulation of wild‐type channels. 4 Under control conditions, the voltage dependence of activation of SNS(SA) current was shifted to depolarized potentials compared with SNS. This was associated with a slowing of SNS(SA) current inactivation at hyperpolarized potentials and suggested a tonic PKA phosphorylation of wild‐type channels under basal conditions. 5 We conclude that the major substrates involved in functional PKA modulation of the SNS channel are located within the intracellular I‐II loop.

trkA Is Expressed in Nociceptive Neurons and Influences Electrophysiological Properties via Nav1.8 Expression in Rapidly Conducting Nociceptors

To test the hypothesis that trkA (the high-affinity NGF receptor) is selectively expressed in nociceptive dorsal root ganglion (DRG) neurons, we examined the intensity of trkA immunoreactivity in single dye-injected rat DRG neurons, the sensory receptor properties of which were identified in vivo with mechanical and thermal stimuli. We provide the first evidence in single identified neurons that strong trkA expression in DRGs is restricted to nociceptive neurons, probably accounting for the profound influence of NGF on these neurons. Furthermore, we demonstrate that trkA expression is as high in rapidly conducting (Aα/β) as in more slowly conducting (Aδ and C) nociceptors. All Aα/β low-threshold mechanoreceptors (LTMs) are trkA negative, although weak but detectable trkA is present in some C and Aδ LTMs. NGF can influence electrophysiological properties of DRG neurons, probably by binding to trkA. We found positive correlations for single identified Aα/β (but not C or Aδ) nociceptors between trkA immunocytochemical intensity and electrophysiological properties typical of nociceptors, namely long action potential and afterhyperpolarization durations and large action potential amplitudes. Furthermore, for Aα/β (notCorAδ) nociceptors, trkA intensity is inversely correlated with conduction velocity. Similar relationships, again only in Aα/β nociceptors, between electrophysiological properties and trkA expression exist for sodium channel Nav1.8 but not Nav1.9 immunoreactivities. These findings suggest that in Aα/β nociceptors, influences of NGF on expression levels of Nav1.8 are related to, and perhaps limited by, expression levels of trkA. This view is supported by a positive correlation between immuno-intensities of trkA and Nav1.8 in A-fiber, but not C-fiber, nociceptors.

Annexin II Light Chain p11 Promotes Functional Expression of Acid-sensing Ion Channel ASIC1a

Acid-sensing ion channels (ASICs) have been implicated in a wide variety of physiological functions. We have used a rat dorsal root ganglion cDNA library in a yeast two-hybrid assay to identify sensory neuron proteins that interact with ASICs. We found that annexin II light chain p11 physically interacts with the N terminus of ASIC1a, but not other ASIC isoforms. Immunoprecipitation studies confirmed an interaction between p11 and ASIC1 in rat dorsal root ganglion neurons in vivo. Coexpression of p11 and ASIC1a in CHO-K1 cells led to a 2-fold increase in expression of the ion channel at the cell membrane as determined by membrane-associated immunoreactivity and cell-surface biotinylation. Consistent with these findings, peak ASIC1a currents in transfected CHO-K1 cells were up-regulated 2-fold in the presence of p11, whereas ASIC3-mediated currents were unaffected by p11 expression. Neither the pH dependence of activation nor the rates of desensitization were altered by p11, suggesting that its primary role in regulating ASIC1a activity is to enhance cell-surface expression of ASIC1a. These data demonstrate that p11, already known to traffic members of the voltage-gated sodium and potassium channel families as well as transient receptor potential and chloride channels, also plays a selective role in enhancing ASIC1a functional expression.

TRANS-SPLICING OF A VOLTAGE-GATED SODIUM CHANNEL IS REGULATED BY NERVE GROWTH FACTOR

Mammalian sensory neurons express a voltage‐gated sodium channel named SNS. Here we report the identification of an SNS transcript (SNS‐A) that contains an exact repeat of exons 12, 13 and 14 encoding a partial repeat of domain II. Because the exons 12–14 are present in single copies in genomic DNA, the SNS‐A transcript must arise by trans‐splicing. Nerve growth factor, which regulates pain thresholds, and the functional expression of voltage‐gated sodium channels increases the levels of the SNS‐A transcript several‐fold both in vivo and in vitro as measured by RNase protection methods, as well as RT‐PCR. These data demonstrate a novel regulatory role for the nerve growth factor and are the first example of trans‐splicing in the vertebrate nervous system.

Comparison of dorsal root ganglion gene expression in rat models of traumatic and HIV-associated neuropathic pain.

To elucidate the mechanisms underlying peripheral neuropathic pain in the context of HIV infection and antiretroviral therapy, we measured gene expression in dorsal root ganglia (DRG) of rats subjected to systemic treatment with the anti‐retroviral agent, ddC (Zalcitabine) and concomitant delivery of HIV‐gp120 to the rat sciatic nerve. L4 and L5 DRGs were collected at day 14 (time of peak behavioural change) and changes in gene expression were measured using Affymetrix whole genome rat arrays. Conventional analysis of this data set and Gene Set Enrichment Analysis (GSEA) was performed to discover biological processes altered in this model. Transcripts associated with G protein coupled receptor signalling and cell adhesion were enriched in the treated animals, while ribosomal proteins and proteasome pathways were associated with gene down‐regulation. To identify genes that are directly relevant to neuropathic mechanical hypersensitivity, as opposed to epiphenomena associated with other aspects of the response to a sciatic nerve lesion, we compared the gp120+ddC‐evoked gene expression with that observed in a model of traumatic neuropathic pain (L5 spinal nerve transection), where hypersensitivity to a static mechanical stimulus is also observed. We identified 39 genes/expressed sequence tags that are differentially expressed in the same direction in both models. Most of these have not previously been implicated in mechanical hypersensitivity and may represent novel targets for therapeutic intervention. As an external control, the RNA expression of three genes was examined by RT‐PCR, while the protein levels of two were studied using western blot analysis.

A single serine residue confers tetrodotoxin insensitivity on the rat sensory-neuron-specific sodium channel SNS

Sensory neurons express a sodium channel (SNS) that is highly resistant to block by tetrodotoxin (IC50=60 μM). SNS is 65% homologous to the cardiac sodium channel, in which a single hydrophilic residue in the SS2 segment is critical for tetrodotoxin resistance. By site‐directed mutagenesis, we have substituted phenylalanine for serine at the equivalent position in SNS: this mutated (S356F) SNS channel is functionally similar to wild‐type SNS when expressed in Xenopus oocytes, but is potently blocked by tetrodotoxin and saxitoxin with IC50s of 2.8 nM and 8.2 nM, respectively. These data provide clues to the rational design of selective blockers of SNS with potential as analgesic drugs.