Mark D. Baker

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.

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.

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.

Neurological perspectives on voltage-gated sodium channels.

The activity of voltage-gated sodium channels has long been linked to disorders of neuronal excitability such as epilepsy and chronic pain. Recent genetic studies have now expanded the role of sodium channels in health and disease, to include autism, migraine, multiple sclerosis, cancer as well as muscle and immune system disorders. Transgenic mouse models have proved useful in understanding the physiological role of individual sodium channels, and there has been significant progress in the development of subtype selective inhibitors of sodium channels. This review will outline the functions and roles of specific sodium channels in electrical signalling and disease, focusing on neurological aspects. We also discuss recent advances in the development of selective sodium channel inhibitors.

GTP‐induced tetrodotoxin‐resistant Na+ current regulates excitability in mouse and rat small diameter sensory neurones

Peripheral pain thresholds are regulated by the actions of inflammatory mediators. Some act through G‐protein‐coupled receptors on voltage‐gated sodium channels. We have found that a low‐threshold, persistent tetrodotoxin‐resistant Na+ current, attributed to NaV1.9, is upregulated by GTP and its non‐hydrolysable analogue GTP‐γ‐S, but not by GDP. Inclusion of GTP‐γ‐S (500 μm) in the internal solution led to an increase in maximal current amplitude of > 300 % within 5 min. In current clamp, upregulation of persistent current was associated with a more negative threshold for action potential induction (by 15–16 mV) assessed from a holding potential of −90 mV. This was not seen in neurones without the low‐threshold current or with internal GDP (P < 0.001). In addition, persistent current upregulation depolarized neurones. At −60 mV, internal GTP‐γ‐S led to the generation of spontaneous activity in initially silent neurones only when persistent current was upregulated. These findings suggest that regulation of the persistent current has important consequences for nociceptor excitability.

Nociceptor-specific gene deletion using heterozygous NaV1.8-Cre recombinase mice.

&NA; NaV1.8 is a voltage‐gated sodium channel expressed only in a subset of sensory neurons of which more than 85% are nociceptors. In order to delete genes in nociceptive neurons, we generated heterozygous transgenic mice expressing Cre recombinase under the control of the NaV1.8 promoter. Functional Cre recombinase expression replicated precisely the expression pattern of NaV1.8. Cre expression began at embryonic day 14 in small diameter neurons in dorsal root, trigeminal and nodose ganglia, but was absent in non‐neuronal or CNS tissues into adulthood. Sodium channel subtypes were normal in isolated DRG neurons. Pain behaviour in response to mechanical or thermal stimuli, and in acute, inflammatory and neuropathic pain was also normal. These data demonstrate that the heterozygous NaV1.8‐Cre mouse line is a useful tool to analyse the effects of deleting floxed genes on pain behaviour.

Nerve injury induces robust allodynia and ectopic discharges in Nav1.3 null mutant mice.

Changes in sodium channel activity and neuronal hyperexcitability contribute to neuropathic pain, a major clinical problem. There is strong evidence that the re-expression of the embryonic voltage-gated sodium channel subunit Nav1.3 underlies neuronal hyperexcitability and neuropathic pain.Here we show that acute and inflammatory pain behaviour is unchanged in global Nav1.3 mutant mice. Surprisingly, neuropathic pain also developed normally in the Nav1.3 mutant mouse. To rule out any genetic compensation mechanisms that may have masked the phenotype, we investigated neuropathic pain in two conditional Nav1.3 mutant mouse lines. We used Nav1.8-Cre mice to delete Nav1.3 in nociceptors at E14 and NFH-Cre mice to delete Nav1.3 throughout the nervous system postnatally. Again normal levels of neuropathic pain developed after nerve injury in both lines. Furthermore, ectopic discharges from damaged nerves were unaffected by the absence of Nav1.3 in global knock-out mice. Our data demonstrate that Nav1.3 is neither necessary nor sufficient for the development of nerve-injury related pain.

Small RNAs Control Sodium Channel Expression, Nociceptor Excitability, and Pain Thresholds

To examine the role of small RNAs in peripheral pain pathways, we deleted the enzyme Dicer in mouse postmitotic damage-sensing neurons. We used a Nav1.8-Cre mouse to target those nociceptors important for inflammatory pain. The conditional null mice were healthy with a normal number of sensory neurons and normal acute pain thresholds. Behavioral studies showed that inflammatory pain was attenuated or abolished. Inflammatory mediators failed to enhance excitability of Nav1.8+ sensory neurons from null mutant mice. Acute noxious input into the dorsal horn of the spinal cord was apparently normal, but the increased input associated with inflammatory pain measured using c-Fos staining was diminished. Microarray and quantitative real-time reverse-transcription PCR (qRT-PCR) analysis showed that Dicer deletion lead to the upregulation of many broadly expressed mRNA transcripts in dorsal root ganglia. By contrast, nociceptor-associated mRNA transcripts (e.g., Nav1.8, P2xr3, and Runx-1) were downregulated, resulting in lower levels of protein and functional expression. qRT-PCR analysis also showed lowered levels of expression of nociceptor-specific pre-mRNA transcripts. MicroRNA microarray and deep sequencing identified known and novel nociceptor microRNAs in mouse Nav1.8+ sensory neurons that may regulate nociceptor gene expression.

GTP up-regulated persistent Na+ current and enhanced nociceptor excitability require NaV1.9.

Persistent tetrodotoxin‐resistant (TTX‐r) sodium currents up‐regulated by intracellular GTP have been invoked as the site of action of peripheral inflammatory mediators that lower pain thresholds, and ascribed to the NaV1.9 sodium channel. Here we describe the properties of a global knock‐out of NaV1.9 produced by replacing exons 4 and 5 in SCN11A with a neomycin resistance cassette, deleting the domain 1 voltage sensor and introducing a frameshift mutation. Recordings from small (< 25 μm apparent diameter) sensory neurones indicated that channel loss eliminates a TTX‐r persistent current. Intracellular dialysis of GTP‐γ‐S did not cause an up‐regulation of persistent Na+ current in NaV1.9‐null neurones and the concomitant negative shift in voltage‐threshold seen in wild‐type and heterozygous neurones. Heterologous hNaV1.9 expression in NaV1.9 knock‐out sensory neurones confirms that the human clone can restore the persistent Na+ current. Taken together, these findings demonstrate that NaV1.9 underlies the G‐protein pathway‐regulated TTX‐r persistent Na+ current in small diameter sensory neurones that may drive spontaneous discharge in nociceptive nerve fibres during inflammation.

Voltage-gated sodium channels

Increased knowledge of the molecular diversity of sodium channel alpha- and beta-subunits, and their distribution of expression have been highlights of the past year. The development of subtype-specific channel blockers remains elusive, but the discovery of selective inhibitors such as mu-conotoxins promises useful antagonists in the near future.