John N. Wood

An SCN9A channelopathy causes congenital inability to experience pain.

The complete inability to sense pain in an otherwise healthy individual is a very rare phenotype. In three consanguineous families from northern Pakistan, we mapped the condition as an autosomal-recessive trait to chromosome 2q24.3. This region contains the gene SCN9A, encoding the α-subunit of the voltage-gated sodium channel, Nav1.7, which is strongly expressed in nociceptive neurons. Sequence analysis of SCN9A in affected individuals revealed three distinct homozygous nonsense mutations (S459X, I767X and W897X). We show that these mutations cause loss of function of Nav1.7 by co-expression of wild-type or mutant human Nav1.7 with sodium channel β1 and β2 subunits in HEK293 cells. In cells expressing mutant Nav1.7, the currents were no greater than background. Our data suggest that SCN9A is an essential and non-redundant requirement for nociception in humans. These findings should stimulate the search for novel analgesics that selectively target this sodium channel subunit.

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.

SCN9A Mutations in Paroxysmal Extreme Pain Disorder: Allelic Variants Underlie Distinct Channel Defects and Phenotypes

Paroxysmal extreme pain disorder (PEPD), previously known as familial rectal pain (FRP, or OMIM 167400), is an inherited condition characterized by paroxysms of rectal, ocular, or submandibular pain with flushing. A genome-wide linkage search followed by mutational analysis of the candidate gene SCN9A, which encodes hNa(v)1.7, identified eight missense mutations in 11 families and 2 sporadic cases. Functional analysis in vitro of three of these mutant Na(v)1.7 channels revealed a reduction in fast inactivation, leading to persistent sodium current. Other mutations in SCN9A associated with more negative activation thresholds are known to cause primary erythermalgia (PE). Carbamazepine, a drug that is effective in PEPD, but not PE, showed selective block of persistent current associated with PEPD mutants, but did not affect the negative activation threshold of a PE mutant. PEPD and PE are allelic variants with distinct underlying biophysical mechanisms and represent a separate class of peripheral neuronal sodium channelopathy.

Warm-coding deficits and aberrant inflammatory pain in mice lacking P2X3 receptors

ATP activates damage-sensing neurons (nociceptors) and can evoke a sensation of pain. The ATP receptor P2X3 is selectively expressed by nociceptors and is one of seven ATP-gated, cation-selective ion channels. Here we demonstrate that ablation of the P2X3 gene results in the loss of rapidly desensitizing ATP-gated cation currents in dorsal root ganglion neurons, and that the responses of nodose ganglion neurons to ATP show altered kinetics and pharmacology resulting from the loss of expression of P2X2/3 heteromultimers. Null mutants have normal sensorimotor function. Behavioural responses to noxious mechanical and thermal stimuli are also normal, although formalin-induced pain behaviour is reduced. In contrast, deletion of the P2X3 receptor causes enhanced thermal hyperalgesia in chronic inflammation. Notably, although dorsal-horn neuronal responses to mechanical and noxious heat application are normal, P2X3-null mice are unable to code the intensity of non-noxious ‘warming’ stimuli.

Purinergic receptors: Their role in nociception and primary afferent neurotransmission

The recent discovery of a P2X purinoceptor (a ligand-gated ion channel triggered by ATP) that is selectively expressed by small-diameter sensory neurons has led to the exploration of the sources of ATP involved in the initiation of different types of nociception and pain, including sympathetic nerves, endothelial cells and tumour cells. In addition, the anti-nociceptive actions of adenosine via prejunctional P1(A1) purinoceptors in the spinal cord and the pain-enhancing actions of adenosine via P1(A2) purinoceptors in the periphery have generated great interest in the development of P1 agonists and antagonists, as well as P2X antagonists as potential analgesic drugs.

The cell and molecular basis of mechanical, cold, and inflammatory pain.

Peripheral pain pathways are activated by a range of stimuli. We used diphtheria toxin to kill all mouse postmitotic sensory neurons expressing the sodium channel Nav1.8. Mice showed normal motor activity and low-threshold mechanical and acute noxious heat responses but did not respond to noxious mechanical pressure or cold. They also showed a loss of enhanced pain responses and spontaneous pain behavior upon treatment with inflammatory insults. In contrast, nerve injury led to heightened pain sensitivity to thermal and mechanical stimuli indistinguishable from that seen with normal littermates. Pain behavior correlates well with central input from sensory neurons measured electrophysiologically in vivo. These data demonstrate that Nav1.8-expressing neurons are essential for mechanical, cold, and inflammatory pain but not for neuropathic pain or heat sensing.

Sensory neuron sodium channel Nav1.8 is essential for pain at low temperatures.

Sensory acuity and motor dexterity deteriorate when human limbs cool down, but pain perception persists and cold-induced pain can become excruciating. Evolutionary pressure to enforce protective behaviour requires that damage-sensing neurons (nociceptors) continue to function at low temperatures. Here we show that this goal is achieved by endowing superficial endings of slowly conducting nociceptive fibres with the tetrodotoxin-resistant voltage-gated sodium channel (VGSC) Nav1.8 (ref. 2). This channel is essential for sustained excitability of nociceptors when the skin is cooled. We show that cooling excitable membranes progressively enhances the voltage-dependent slow inactivation of tetrodotoxin-sensitive VGSCs. In contrast, the inactivation properties of Nav1.8 are entirely cold-resistant. Moreover, low temperatures decrease the activation threshold of the sodium currents and increase the membrane resistance, augmenting the voltage change caused by any membrane current. Thus, in the cold, Nav1.8 remains available as the sole electrical impulse generator in nociceptors that transmits nociceptive information to the central nervous system. Consistent with this concept is the observation that Nav1.8-null mutant mice show negligible responses to noxious cold and mechanical stimulation at low temperatures. Our data present strong evidence for a specialized role of Nav1.8 in nociceptors as the critical molecule for the perception of cold pain and pain in the cold.

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.

A Gain-of-Function Mutation in TRPA1 Causes Familial Episodic Pain Syndrome

Summary Human monogenic pain syndromes have provided important insights into the molecular mechanisms that underlie normal and pathological pain states. We describe an autosomal-dominant familial episodic pain syndrome characterized by episodes of debilitating upper body pain, triggered by fasting and physical stress. Linkage and haplotype analysis mapped this phenotype to a 25 cM region on chromosome 8q12–8q13. Candidate gene sequencing identified a point mutation (N855S) in the S4 transmembrane segment of TRPA1, a key sensor for environmental irritants. The mutant channel showed a normal pharmacological profile but altered biophysical properties, with a 5-fold increase in inward current on activation at normal resting potentials. Quantitative sensory testing demonstrated normal baseline sensory thresholds but an enhanced secondary hyperalgesia to punctate stimuli on treatment with mustard oil. TRPA1 antagonists inhibit the mutant channel, promising a useful therapy for this disorder. Our findings provide evidence that variation in the TRPA1 gene can alter pain perception in humans. Video Abstract

Involvement of Na+ channels in pain pathways.

Voltage-dependent Na+ channels in sensory nerves contribute to the control of membrane excitability and underlie action potential generation. Na+ channel subtypes exhibit a neurone-specific and developmentally regulated pattern of expression, and changes in both channel expression and function are caused by disease. Recent evidence implicates specific roles for Na+ channel subtypes Na(v)1.3 and Na(v)1.8 in pain states that are associated with nerve injury and inflammation, respectively. Insight into the role of Na(v)1.8 in pain pathways has been gained by the generation of a null mutant. Although drugs discriminate poorly between subtypes, the molecular diversity of channels and subtype-specific modulation might provide opportunities to target pain pathways selectively.