Sulayman D. Dib-Hajj

Sodium Channels in Normal and Pathological Pain

Nociception is essential for survival whereas pathological pain is maladaptive and often unresponsive to pharmacotherapy. Voltage-gated sodium channels, Na(v)1.1-Na(v)1.9, are essential for generation and conduction of electrical impulses in excitable cells. Human and animal studies have identified several channels as pivotal for signal transmission along the pain axis, including Na(v)1.3, Na(v)1.7, Na(v)1.8, and Na(v)1.9, with the latter three preferentially expressed in peripheral sensory neurons and Na(v)1.3 being upregulated along pain-signaling pathways after nervous system injuries. Na(v)1.7 is of special interest because it has been linked to a spectrum of inherited human pain disorders. Here we review the contribution of these sodium channel isoforms to pain.

Spinal sensory neurons express multiple sodium channel α-subunit mRNAs

Abstract The expression of sodium channel α-, β1- and β2-subunit mRNAs was examined in adult rat DRG neurons in dissociated culture at 1 day in vitro and within sections of intact ganglia by in situ hybridization and reverse transcription polymerase chain reaction (RT-PCR). The results demonstrate that sodium channel α-subunit mRNAs are differentially expressed in small ( 45 μm diam.) cultured DRG neurons at 1 day in vitro (div). Sodium channel mRNA I is expressed at higher levels in large neurons than small DRG neurons, while sodium channel mRNA II is variably expressed, with most cells lacking or exhibiting low levels of detectable signal of these mRNAs and limited numbers of neurons with moderate expression levels. DRG neurons generally exhibit negligible or low levels of hybridization signal for sodium channel mRNA III. Sodium channel mRNAs Na6 and NaG show similar patterns of expression, with most large and many medium DRG neurons exhibiting high levels of expression. The mRNA for the rat cognate of human sodium channel hNE-Na is detected in virtually every DRG neuron; most cells in all size classes exhibit moderate or high levels of hNE-Na expression. Sodium channel SNS mRNA is expressed in all size classes of DRG neurons, but shows greater expression in small and medium DRG neurons than in large neurons. The mRNA for the rat cognate of mouse sodium channel mNav2.3 is not detected, or is detected at low levels, in most DRG neurons, regardless of size, although moderate expression is detected in some neurons. Sodium channel β1- and β2-subunit mRNAs exhibit similar expression patterns; they are detected in most DRG neurons, although the level of expression tends to be greater in large neurons than in small neurons. RT-PCR and in situ hybridization of intact adult DRG showed a similar pattern of expression of sodium channel mRNAs to that observed in DRG neurons in vitro. These results demonstrate that adult DRG neurons express multiple sodium channel mRNAs in vitro and in situ and suggest a molecular basis for the biophysical heterogeneity of sodium currents observed in these cells.

Sodium channel α-subunit mRNAs I, II, III, NaG, Na6 and hNE (PN1): different expression patterns in developing rat nervous system

The expression of sodium channel alpha-subunit mRNAs I, II, III, NaG, Na6 and hNE (PN1) was examined in developing (E17-P30) hippocampus, cerebellum, spinal cord and dorsal root ganglia using non-isotopic in situ hybridization cytochemistry. The results showed distinct patterns of expression for each of the sodium channel mRNAs with maturation of the nervous system. In the hippocampus, sodium channel mRNA I was not detected at any developmental time, while mRNA II showed increasing hybridization signal between E17 and P30. Sodium channel mRNA III was more prevalent at late embryonic and early postnatal times, and was barely detectable at P30. The transcript for NaG showed transient expression between P2 and P15, being expressed at low levels at E17 and not being detectable at P30. Sodium channel mRNA Na6 exhibited a high level of expression between E17 and P15 in the hippocampal formation, with an attenuation of the signal by P30. hNE (PN1) mRNA was not detected in the hippocampus at any time examined. In the cerebellum, sodium channel mRNA I was not detected at E17 or P2, but became detectable in Purkinje cells at P15 and continued to show a low level of expression in these cells at P30. mRNA I was not detected at any time examined in granule cells of the cerebellum. Sodium channel mRNA II exhibited increasing expression in the developing cerebellum, and showed increasing signal in Purkinge cells beginning on P2 and granule cells on P15. Sodium channel mRNA III was down-regulated with development in the cerebellum, although mRNA III was readily detected at E17, it was not detected in any layers of the cerebellum by P15. NaG mRNA showed a peak of expression at P2, and was present at low levels at E17 and P15 and not detectable at P30. Na6 mRNA was highly expressed in the E17 cerebellum; this mRNA was present at high levels in Purkinje cells throughout development, although in granule cells the signal was attenuated at P15-P30. Sodium channel hNE (PN1) mRNA was not detected in the cerebellum at any time in development. In the spinal cord, sodium channel mRNA I showed increasing expression beginning at P2 and was highly expressed, particularly in ventral motor neurons, by P30. Sodium channel II mRNA was detected at all stages of development in the spinal cord; in contrast, mRNA III was detected at E17 and P2, but showed very low levels of expression by P30. NaG mRNA exhibited a transient expression in spinal cord at P2, but was not detectable at E17 and P30. Na6 mRNA was detectable at very low levels at E17 and became highly expressed at P2, prior to a reduction of the signal at P15 and P30. hNE (PN1) mRNA was not detected in the spinal cord at any time in development. In the dorsal root ganglia, sodium channel I mRNA hybridization signal was detected in DRG neurons at P2, with slightly increased levels at P15 and P30. Sodium channel II mRNA exhibited a relatively constant, moderate level of expression at all developmental ages. Sodium channel III mRNA was highly expressed in DRG neurons at E17 but was down-regulated with further development so that it was not detectable by P30. NaG mRNA was strongly expressed by some DRG neurons at all stages of development from E17 to P30; in general the level of NaG labelling was greater in larger neurons than in smaller neurons. Na6 mRNA showed increasing expression with development in DRG neurons; at E17, low levels of Na6 mRNA were detected and by P15 to P30 high levels of expression were present in some neurons. hNE (PN1) mRNA was present in DRG neurons at P2, and was up-regulated with further development so that by P30 hNE (PN1) was expressed in all DRG neurons sizes. These results demonstrate that sodium channel alpha-subunit mRNAs I, II, III, NaG, Na6 and hNE (PN1) exhibit distinct spatial and temporal patterns of expression in nervous tissue, and suggest that the expression of the sodium channel alpha-subunits is differentially regulated. (ABSTRACT TRUNCATED)

Gain of function NaV1.7 mutations in idiopathic small fiber neuropathy

Small nerve fiber neuropathy (SFN) often occurs without apparent cause, but no systematic genetic studies have been performed in patients with idiopathic SFN (I‐SFN). We sought to identify a genetic basis for I‐SFN by screening patients with biopsy‐confirmed idiopathic SFN for mutations in the SCN9A gene, encoding voltage‐gated sodium channel NaV1.7, which is preferentially expressed in small diameter peripheral axons.

Electrophysiological Properties of Mutant Nav1.7 Sodium Channels in a Painful Inherited Neuropathy

Although the physiological basis of erythermalgia, an autosomal dominant painful neuropathy characterized by redness of the skin and intermittent burning sensation of extremities, is not known, two mutations of Nav1.7, a sodium channel that produces a tetrodotoxin-sensitive, fast-inactivating current that is preferentially expressed in dorsal root ganglia (DRG) and sympathetic ganglia neurons, have recently been identified in patients with primary erythermalgia. Nav1.7 is preferentially expressed in small-diameter DRG neurons, most of which are nociceptors, and is characterized by slow recovery from inactivation and by slow closed-state inactivation that results in relatively large responses to small, subthreshold depolarizations. Here we show that these mutations in Nav1.7 produce a hyperpolarizing shift in activation and slow deactivation. We also show that these mutations cause an increase in amplitude of the current produced by Nav1.7 in response to slow, small depolarizations. These observations provide the first demonstration of altered sodium channel function associated with an inherited painful neuropathy and suggest that these physiological changes, which confer hyperexcitability on peripheral sensory and sympathetic neurons, contribute to symptom production in hereditary erythermalgia.

The Na V 1.7 sodium channel: from molecule to man

The voltage-gated sodium channel NaV1.7 is preferentially expressed in peripheral somatic and visceral sensory neurons, olfactory sensory neurons and sympathetic ganglion neurons. NaV1.7 accumulates at nerve fibre endings and amplifies small subthreshold depolarizations, poising it to act as a threshold channel that regulates excitability. Genetic and functional studies have added to the evidence that NaV1.7 is a major contributor to pain signalling in humans, and homology modelling based on crystal structures of ion channels suggests an atomic-level structural basis for the altered gating of mutant NaV1.7 that causes pain.

Plasticity of sodium channel expression in DRG neurons in the chronic constriction injury model of neuropathic pain

Previous studies have shown that transection of the sciatic nerve induces dramatic changes in sodium currents of axotomized dorsal root ganglion (DRG) neurons, which are paralleled by significant changes in the levels of transcripts of several sodium channels expressed in these neurons. Sodium currents that are resistant to tetrodotoxin (TTX-R) and the transcripts of two TTX-R sodium channels are significantly attenuated, while a rapidly repriming tetrodotoxin-sensitive (TTX-S) current emerges and the transcripts of alpha-III sodium channel, which produce a TTX-S current when expressed in oocytes, are up-regulated. We report here on changes in sodium currents and sodium channel transcripts in DRG neurons in the chronic constriction injury (CCI) model of neuropathic pain. CCI-induced changes in DRG neurons, 14 days post-surgery, mirror those of axotomy. Transcripts of NaN and SNS, two sensory neuron-specific TTX-R sodium channels, are significantly down-regulated as is the TTX-R sodium current, while transcripts of the TTX-S alpha-III sodium channel and a rapidly repriming TTX-S Na current are up-regulated in small diameter DRG neurons. These changes may provide at least a partial basis for the hyperexcitablity of DRG neurons that contributes to hyperalgesia in this model.

SNS Na+ channel expression increases in dorsal root ganglion neurons in the carrageenan inflammatory pain model

It has been suggested that hyperexcitability in dorsal root ganglion (DRG) neurons due to altered sodium channel expression contributes to some chronic pain syndromes. To understand the role of the voltage-gated sodium channel α-SNS in inflammatory pain, we investigated the expression of α-SNS mRNA and tetrodotoxin-resistant (TTX-R) sodium current in small DRG neurons, which include nociceptive cells, following injection of carrageenan into the hind paw of the rat using in situ hybridization and patch-clamp recording. α-SNS mRNA expression in DRG neurons projecting to the inflamed limb was significantly increased 4 days following carrageenan injection, compared with DRG neurons from the contralateral side or naive (uninjected) rats (mean ± s.d. optical density ratio: ipsilateral/contralateral, 1.77 ± 0.17; ipsilateral/naive, 1.88 ± 0.36). The amplitude of the TTX-R sodium current in small DRG neurons projecting to the inflamed limb was significantly larger than on the contralateral side 4 days post-injection (31.7 ± 3.3 vs 20.0 ± 2.1 nA). The TTX-R current density was also significantly increased. These results demonstrate the increased expression of α-SNS sodium channels in small DRG neurons following injection of carrageenan into their projection field, and suggest that α-SNS is involved in the development of hyperexcitability associated with inflammation.

De Novo Pathogenic SCN8A Mutation Identified by Whole-Genome Sequencing of a Family Quartet Affected by Infantile Epileptic Encephalopathy and SUDEP

Individuals with severe, sporadic disorders of infantile onset represent an important class of disease for which discovery of the underlying genetic architecture is not amenable to traditional genetic analysis. Full-genome sequencing of affected individuals and their parents provides a powerful alternative strategy for gene discovery. We performed whole-genome sequencing (WGS) on a family quartet containing an affected proband and her unaffected parents and sibling. The 15-year-old female proband had a severe epileptic encephalopathy consisting of early-onset seizures, features of autism, intellectual disability, ataxia, and sudden unexplained death in epilepsy. We discovered a de novo heterozygous missense mutation (c.5302A>G [p.Asn1768Asp]) in the voltage-gated sodium-channel gene SCN8A in the proband. This mutation alters an evolutionarily conserved residue in Nav1.6, one of the most abundant sodium channels in the brain. Analysis of the biophysical properties of the mutant channel demonstrated a dramatic increase in persistent sodium current, incomplete channel inactivation, and a depolarizing shift in the voltage dependence of steady-state fast inactivation. Current-clamp analysis in hippocampal neurons transfected with p.Asn1768Asp channels revealed increased spontaneous firing, paroxysmal-depolarizing-shift-like complexes, and an increased firing frequency, consistent with a dominant gain-of-function phenotype in the heterozygous proband. This work identifies SCN8A as the fifth sodium-channel gene to be mutated in epilepsy and demonstrates the value of WGS for the identification of pathogenic mutations causing severe, sporadic neurological disorders.

Distinct repriming and closed-state inactivation kinetics of Nav1.6 and Nav1.7 sodium channels in mouse spinal sensory neurons

While large, myelinated dorsal root ganglion (DRG) neurons are capable of firing at high frequencies, small unmyelinated DRG neurons typically display much lower maximum firing frequencies. However, the molecular basis for this difference has not been delineated. Because the sodium currents in large DRG neurons exhibit rapid repriming (recovery from inactivation) kinetics and the sodium currents in small DRG neurons exhibit predominantly slow repriming kinetics, it has been proposed that differences in sodium channels might contribute to the determination of repetitive firing properties in DRG neurons. A recent study demonstrated that Nav1.7 expression is negatively correlated with conduction velocity and DRG cell size, while the Nav1.6 voltage‐gated sodium channel has been implicated as the predominant isoform present at nodes of Ranvier of myelinated fibres. Therefore we characterized and compared the functional properties, including repriming, of recombinant Nav1.6 and Nav1.7 channels expressed in mouse DRG neurons. Both Nav1.6 and Nav1.7 channels generated fast‐activating and fast‐inactivating currents. However recovery from inactivation was significantly faster (˜5‐fold at ‐70 mV) for Nav1.6 currents than for Nav1.7 currents. The recovery from inactivation of Nav1.6 channels was also much faster than that of native tetrodotoxin‐sensitive sodium currents recorded from small spinal sensory neurons, but similar to that of tetrodotoxin‐sensitive sodium currents recorded from large spinal sensory neurons. Development of closed‐state inactivation was also much faster for Nav1.6 currents than for Nav1.7 currents. Our results indicate that the firing properties of DRG neurons can be tuned by regulating expression of different sodium channel isoforms that have distinct repriming and closed‐state inactivation kinetics.