Simon Tate

Transient receptor potential channels: targeting pain at the source

Pain results from the complex processing of neural signals at different levels of the central nervous system, with each signal potentially offering multiple opportunities for pharmacological intervention. A logical strategy for developing novel analgesics is to target the beginning of the pain pathway, and aim potential treatments directly at the nociceptors — the high-threshold primary sensory neurons that detect noxious stimuli. The largest group of receptors that function as noxious stimuli detectors in nociceptors is the transient receptor potential (TRP) channel family. This Review highlights evidence supporting particular TRP channels as targets for analgesics, indicates the likely efficacy profiles of TRP-channel-acting drugs, and discusses the development pathways needed to test candidates as analgesics in humans.

Two sodium channels contribute to the TTX-R sodium current in primary sensory neurons

We have cloned a second tetrodotoxin-resistant (TTX-R) sodium channel α subunit, SNS2, with the same amino-acid sequence as a putative sodium channel α subunit NaN (ref. 1). SNS2 expression in HEK293T cells produced a TTX-R voltage-gated sodium current with faster kinetics and a lower TTX IC50 than the previously cloned sensory-neuron-specific sodium channel, SNS/PN3 (Refs 2, 3). SNS2 was co-expressed with SNS/PN3 in small DRG neurons.

The Voltage-Gated Sodium Channel Nav1.9 Is an Effector of Peripheral Inflammatory Pain Hypersensitivity

We used a mouse with deletion of exons 4, 5, and 6 of the SCN11A (sodium channel, voltage-gated, type XI, α) gene that encodes the voltage-gated sodium channel Nav1.9 to assess its contribution to pain. Nav1.9 is present in nociceptor sensory neurons that express TRPV1, bradykinin B2, and purinergic P2X3 receptors. In Nav1.9−/− mice, the non-inactivating persistent tetrodotoxin-resistant sodium TTXr-Per current is absent, whereas TTXr-Slow is unchanged. TTXs currents are unaffected by the mutation of Nav1.9. Pain hypersensitivity elicited by intraplantar administration of prostaglandin E2, bradykinin, interleukin-1β, capsaicin, and P2X3 and P2Y receptor agonists, but not NGF, is either reduced or absent in Nav1.9−/− mice, whereas basal thermal and mechanical pain sensitivity is unchanged. Thermal, but not mechanical, hypersensitivity produced by peripheral inflammation (intraplanatar complete Freunds adjuvant) is substantially diminished in the null allele mutant mice, whereas hypersensitivity in two neuropathic pain models is unchanged in the Nav1.9−/− mice. Nav1.9 is, we conclude, an effector of the hypersensitivity produced by multiple inflammatory mediators on nociceptor peripheral terminals and therefore plays a key role in mediating peripheral sensitization.

Diversity of Expression of the Sensory Neuron-Specific TTX-Resistant Voltage-Gated Sodium Ion Channels SNS and SNS2

The differential distribution of two tetrodotoxin resistant (TTXr) voltage-gated sodium channels SNS (PN3) and SNS2 (NaN) in rat primary sensory neurons has been investigated. Both channels are sensory neuron specific with SNS2 restricted entirely to those small dorsal root ganglion (DRG) cells with unmyelinated axons (C-fibers). SNS, in contrast, is expressed both in small C-fiber DRG cells and in 10% of cells with myelinated axons (A-fibers). All SNS expressing A-fiber cells are Trk-A positive and many express the vanilloid-like receptor VRL1. About half of C-fiber DRG neurons express either SNS or SNS2, and in most, the channels are colocalized. SNS and SNS2 are found both in NGF-responsive and GDNF-responsive C-fibers and many of these cells also express the capsaicin receptor VR1. A very small proportion of small DRG cells express either only SNS or only SNS2. At least four different classes of A- and C-fiber DRG neurons exist, therefore, with respect to expression of these sodium channels.

Voltage-gated sodium channels as therapeutic targets

Voltage-gated sodium channels (VGSCs) play a central role in the generation and propagation of action potentials in neurons and other cells. VGSC modulators have their origins in empirical pharmacology and are being used as local anaesthetics, antiarrhythmics, analgesics and antiepileptics, and for other disorders. However, the identification of a multigene family of VGSCs, along with tools to study the different subtypes in pathophysiology, is now providing a rational basis for selective intervention. Recent advances have addressed the technical challenges of expressing and assaying these complex proteins, enabling the correlation of empirical pharmacology to subtypes and the screening of individual subtypes for novel inhibitors with increased potency and selectivity.

The pattern of expression of the voltage-gated sodium channels Nav1.8 and Nav1.9 does not change in uninjured primary sensory neurons in experimental neuropathic pain models

&NA; A spared nerve injury of the sciatic nerve (SNI) or a segmental lesion of the L5 and L6 spinal nerves (SNL) lead to behavioral signs of neuropathic pain in the territory innervated by adjacent uninjured nerve fibers, while a chronic constriction injury (CCI) results in pain sensitivity in the affected area. While alterations in voltage‐gated sodium channels (VGSCs) have been shown to contribute to the generation of ectopic activity in the injured neurons, little is known about changes in VGSCs in the neighboring intact dorsal root ganglion (DRG) neurons, even though these cells begin to fire spontaneously. We have now investigated changes in the expression of the TTX‐resistant VGSCs, Nav1.8 (SNS/PN3) and Nav1.9 (SNS2/NaN) by immunohistochemistry in rat models of neuropathic pain both with an intermingling of intact and degenerated axons in the nerve stump (SNL and CCI) and with a co‐mingling in the same DRG of neurons with injured and uninjured axons (sciatic axotomy and SNI). The expression of Nav1.8 and Nav1.9 protein was abolished in all injured DRG neurons, in all models. In intact DRGs and in neighboring non‐injured neurons, the expression and the distribution among the A‐ and C‐fiber neuronal populations of Nav1.8 and Nav1.9 was, however, unchanged. While it is unlikely, therefore, that a change in the expression of TTX‐resistant VGSCs in non‐injured neurons contributes to neuropathic pain, it is essential that molecular alterations in both injured and non‐injured neurons in neuropathic pain models are investigated.

Small and intermediate conductance Ca2+-activated K+ channels confer distinctive patterns of distribution in human tissues and differential cellular localisation in the colon and corpus cavernosum

The SK/IK family of small and intermediate conductance calcium-activated potassium channels contains four members, SK1, SK2, SK3 and IK1, and is important for the regulation of a variety of neuronal and non-neuronal functions. In this study we have analysed the distribution of these channels in human tissues and their cellular localisation in samples of colon and corpus cavernosum. SK1 mRNA was detected almost exclusively in neuronal tissues. SK2 mRNA distribution was restricted but more widespread than SK1, and was detected in adrenal gland, brain, prostate, bladder, liver and heart. SK3 mRNA was detected in almost every tissue examined. It was highly expressed in brain and in smooth muscle-rich tissues including the clitoris and the corpus cavernosum, and expression in the corpus cavernosum was upregulated up to 5-fold in patients undergoing sex-change operations. IK1 mRNA was present in surface-rich, secretory and inflammatory cell-rich tissues, highest in the trachea, prostate, placenta and salivary glands. In detailed immunohistochemical studies of the colon and the corpus cavernosum, SK1-like immunoreactivity was observed in the enteric neurons. SK3-like immunoreactivity was observed strongly in smooth muscle and vascular endothelium. IK1-like immunoreactivity was mainly observed in inflammatory cells and enteric neurons of the colon, but absent in corpus cavernosum. These distinctive patterns of distribution suggest that these channels are likely to have different biological functions and could be specifically targeted for a number of human diseases, such as irritable bowel syndrome, hypertension and erectile dysfunction.

Upregulation of the Voltage-Gated Sodium Channel β2 Subunit in Neuropathic Pain Models: Characterization of Expression in Injured and Non-Injured Primary Sensory Neurons

The development of abnormal primary sensory neuron excitability and neuropathic pain symptoms after peripheral nerve injury is associated with altered expression of voltage-gated sodium channels (VGSCs) and a modification of sodium currents. To investigate whether the β2 subunit of VGSCs participates in the generation of neuropathic pain, we used the spared nerve injury (SNI) model in rats to examine β2 subunit expression in selectively injured (tibial and common peroneal nerves) and uninjured (sural nerve) afferents. Three days after SNI, immunohistochemistry and Western blot analysis reveal an increase in the β2 subunit in both the cell body and peripheral axons of injured neurons. The increase persists for >4 weeks, although β2 subunit mRNA measured by real-time reverse transcription-PCR and in situ hybridization remains unchanged. Although injured neurons show the most marked upregulation,β2 subunit expression is also increased in neighboring non-injured neurons and a similar pattern of changes appears in the spinal nerve ligation model of neuropathic pain. That increased β2 subunit expression in sensory neurons after nerve injury is functionally significant, as demonstrated by our finding that the development of mechanical allodynia-like behavior in the SNI model is attenuated in β2 subunit null mutant mice. Through its role in regulating the density of mature VGSC complexes in the plasma membrane and modulating channel gating, the β2 subunit may play a key role in the development of ectopic activity in injured and non-injured sensory afferents and, thereby, neuropathic pain.

Differential expression of tetrodotoxin-resistant sodium channels Nav1.8 and Nav1.9 in normal and inflamed rats.

In an attempt to understand mechanisms underlying peripheral sensitization of primary afferent fibers, we investigated the presence of the tetrodotoxin-resistant Na+ channel subunits Nav1.8 (SNS) and Nav1.9 (SNS2) on axons in digital nerves of normal and inflamed rat hindpaws. In normal animals, 14.3% of the unmyelinated and 10.7% of the myelinated axons labeled for the Nav1.8 subunit. These percentages significantly increased in 48 h inflamed animals to 22.0% (1.5-fold increase) and 57.5% (6-fold increase) for unmyelinated and myelinated axons, respectively. In normal animals, Nav1.9 labeled 9.9% of the unmyelinated and 2.1% of the myelinated axons and following inflammation, the proportion of Nav1.9-labeled unmyelinated axons significantly decreased to 3.0% with no change in the proportion of labeled myelinated axons. These data indicate that Nav1.8 and Nav1.9 subunits are transported to the periphery in normal animals and are differentially regulated during inflammation. The massive increase in Nav1.8 expression in myelinated axons suggests that these may contribute to peripheral sensitization and inflammatory hyperalgesia.

Heterologous expression and functional analysis of rat NaV1.8 (SNS) voltage-gated sodium channels in the dorsal root ganglion neuroblastoma cell line ND7–23

The voltage-gated sodium channel NaV1.8 (SNS, PN3) is thought to be a molecular correlate of the dorsal root ganglion (DRG) tetrodotoxin resistant (TTX-R) Na+ current. TTX-R/NaV1.8 is an attractive therapeutic drug target for inflammatory and neuropathic pain on the basis of its specific distribution in sensory neurones and its modulation by inflammatory mediators. However, detailed analysis of recombinant NaV1.8 has been hampered by difficulties in stably expressing the functional protein in mammalian cells. Here, we show stable expression and functional analysis of rat NaV1.8 (rNaV1.8) in the rat DRG/mouse N18Tg2 neuroblastoma hybridoma cell line ND7-23. Rat NaV1.8 Na+ currents were recorded (789 +/- 89 pA, n=62, over 20-cell passages) that qualitatively resembled DRG TTX-R in terms of gating kinetics and voltage-dependence of activation and inactivation. The local anaesthetic drug tetracaine produced tonic inhibition of rNaV1.8 (mean IC50 value 12.5 microM) and in repeated gating paradigms (2-10 Hz) also showed frequency-dependent block. There was a correlation between the ability of several analogues of the anticonvulsant/analgesic compound lamotrigine to inhibit TTX-R and rNaV1.8 (r=0.72, P<0.001). RT-PCR analysis of wild type ND7-23 cells revealed endogenous expression of the beta1 and beta3 accessory Na+ channel subunits-the possibility that the presence of these subunits assists and stabilises expression of rNaV1.8 is discussed. We conclude that the neuroblastoma ND7-23 cell line is a suitable heterologous expression system for rNaV1.8 Na+ channels in that it allows stable expression of a channel with biophysical properties that closely resemble the native TTX-R currents in DRG neurones. This reagent will prove useful in the search for pharmacological inhibitors of rNaV1.8 as novel analgesics.