Physiological and genetic analysis of multiple sodium channel variants in a model of genetic absence epilepsy.

In excitatory neurons, SCN2A (NaV1.2) and SCN8A (NaV1.6) sodium channels are enriched at the axon initial segment. NaV1.6 is implicated in several mouse models of absence epilepsy, including a missense mutation identified in a chemical mutagenesis screen (Scn8a(V929F)). Here, we confirmed the prior suggestion that Scn8a(V929F) exhibits a striking genetic background-dependent difference in phenotypic severity, observing that spike-wave discharge (SWD) incidence and severity are significantly diminished when Scn8a(V929F) is fully placed onto the C57BL/6J strain compared with C3H. Examination of sequence differences in NaV subunits between these two inbred strains suggested NaV1.2(V752F) as a potential source of this modifier effect. Recognising that the spatial co-localisation of the NaV channels at the axon initial segment (AIS) provides a plausible mechanism for functional interaction, we tested this idea by undertaking biophysical characterisation of the variant NaV channels and by computer modelling. NaV1.2(V752F) functional analysis revealed an overall gain-of-function and for NaV1.6(V929F) revealed an overall loss-of-function. A biophysically realistic computer model was used to test the idea that interaction between these variant channels at the AIS contributes to the strain background effect. Surprisingly this modelling showed that neuronal excitability is dominated by the properties of NaV1.2(V752F) due to "functional silencing" of NaV1.6(V929F) suggesting that these variants do not directly interact. Consequent genetic mapping of the major strain modifier to Chr 7, and not Chr 2 where Scn2a maps, supported this biophysical prediction. While a NaV1.6(V929F) loss of function clearly underlies absence seizures in this mouse model, the strain background effect is apparently not due to an otherwise tempting Scn2a variant, highlighting the value of combining physiology and genetics to inform and direct each other when interrogating genetic complex traits such as absence epilepsy.

Nav1.9 expression in magnocellular neurosecretory cells of supraoptic nucleus.

Osmoregulation in mammals is tightly controlled by the release of vasopressin and oxytocin from magnocellular neurosecretory cells (MSC) of the supraoptic nucleus (SON). The release of vasopressin and oxytocin in the neurohypophysis by axons of MSC is regulated by bursting activity of these neurons, which is influenced by multiple sources, including intrinsic membrane properties, paracrine contributions of glial cells, and extrinsic synaptic inputs. Previous work has shown that bursting activity of MSC is tetrodotoxin (TTX)-sensitive, and that TTX-S sodium channels Nav1.2, Nav1.6 and Nav1.7 are expressed by MSC and upregulated in response to osmotic challenge in rats. The TTX-resistant sodium channels, NaV1.8 and Nav1.9, are preferentially expressed, at relatively high levels, in peripheral neurons, where their properties are linked to repetitive firing and subthreshold electrogenesis, respectively, and are often referred to as "peripheral" sodium channels. Both sodium channels have been implicated in pain pathways, and are under study as potential therapeutic targets for pain medications which might be expected to have minimal CNS side effects. We show here, however, that Nav1.9 is expressed by vasopressin- and oxytocin-producing MSC of the rat supraoptic nucleus (SON). We also show that cultured MSC exhibit sodium currents that have characteristics of Nav1.9 channels. In contrast, Nav1.8 is not detectable in the SON. These results suggest that Nav1.9 may contribute to the firing pattern of MSC of the SON, and that careful assessment of hypothalamic function be performed as NaV1.9 blocking agents are studied as potential pain therapies.

Nav1.7-related small fiber neuropathy: impaired slow-inactivation and DRG neuron hyperexcitability.

OBJECTIVES: Although small fiber neuropathy (SFN) often occurs without apparent cause, the molecular etiology of idiopathic SFN (I-SFN) has remained enigmatic. Sodium channel Na(v)1.7 is preferentially expressed within dorsal root ganglion (DRG) and sympathetic ganglion neurons and their small-diameter peripheral axons. We recently reported the presence of Na(v)1.7 variants that produce gain-of-function changes in channel properties in 28% of patients with painful I-SFN and demonstrated impaired slow-inactivation in one of these mutations after expression within HEK293 cells. Here we show that the I739V Na(v)1.7 variant in a patient with biopsy-confirmed I-SFN impairs slow-inactivation within DRG neurons and increases their excitability. METHODS: A patient with SFN symptoms including pain, and no identifiable underlying cause, was evaluated by skin biopsy, quantitative sensory testing, nerve conduction studies, screening of genomic DNA for variants in SCN9A, and functional analysis. RESULTS: Voltage-clamp analysis following expression within DRG neurons revealed that the Na(v)1.7/I739V substitution impairs slow-inactivation, depolarizing the midpoint (V(1/2)) by 5.6 mV, and increasing the noninactivating component at 10 mV from 16.5% to 22.2%. Expression of I739V channels within DRG neurons rendered these cells hyperexcitable, reducing current threshold and increasing the frequency of firing evoked by graded suprathreshold stimuli. CONCLUSIONS: These observations provide support, from a patient with biopsy-confirmed SFN, for the suggestion that functional variants of Na(v)1.7 that impair slow-inactivation can produce DRG neuron hyperexcitability that contributes to pain in SFN. Na(v)1.7 channelopathy-associated SFN should be considered in the differential diagnosis of cases of SFN in which no other cause is found.

Can robots patch-clamp as well as humans? Characterization of a novel sodium channel mutation.

Ion channel missense mutations cause disorders of excitability by changing channel biophysical properties. As an increasing number of new naturally occurring mutations have been identified, and the number of other mutations produced by molecular approaches such as in situ mutagenesis has increased, the need for functional analysis by patch-clamp has become rate limiting. Here we compare a patch-clamp robot using planar-chip technology with human patch-clamp in a functional assessment of a previously undescribed Nav1.7 sodium channel mutation, S211P, which causes erythromelalgia. This robotic patch-clamp device can increase throughput (the number of cells analysed per day) by 3- to 10-fold. Both modes of analysis show that the mutation hyperpolarizes activation voltage dependence (8 mV by manual profiling, 11 mV by robotic profiling), alters steady-state fast inactivation so that it requires an additional Boltzmann function for a second fraction of total current (approximately 20% manual, approximately 40% robotic), and enhances slow inactivation (hyperpolarizing shift--15 mV by human,--13 mV robotic). Manual patch-clamping demonstrated slower deactivation and enhanced (approximately 2-fold) ramp response for the mutant channel while robotic recording did not, possibly due to increased temperature and reduced signal-to-noise ratio on the robotic platform. If robotic profiling is used to screen ion channel mutations, we recommend that each measurement or protocol be validated by initial comparison to manual recording. With this caveat, we suggest that, if results are interpreted cautiously, robotic patch-clamp can be used with supervision and subsequent confirmation from human physiologists to facilitate the initial profiling of a variety of electrophysiological parameters of ion channel mutations.

NaV1.7 gain-of-function mutations as a continuum: A1632E displays physiological changes associated with erythromelalgia and paroxysmal extreme pain disorder mutations and produces symptoms of both disorders.

Gain-of-function mutations of Na(V)1.7 have been shown to produce two distinct disorders: Na(V)1.7 mutations that enhance activation produce inherited erythromelalgia (IEM), characterized by burning pain in the extremities; Na(V)1.7 mutations that impair inactivation produce a different, nonoverlapping syndrome, paroxysmal extreme pain disorder (PEPD), characterized by rectal, periocular, and perimandibular pain. Here we report a novel Na(V)1.7 mutation associated with a mixed clinical phenotype with characteristics of IEM and PEPD, with an alanine 1632 substitution by glutamate (A1632E) in domain IV S4-S5 linker. Patch-clamp analysis shows that A1632E produces changes in channel function seen in both IEM and PEPD mutations: A1632E hyperpolarizes (-7 mV) the voltage dependence of activation, slows deactivation, and enhances ramp responses, as observed in Na(V)1.7 mutations that produce IEM. A1632E depolarizes (+17mV) the voltage dependence of fast inactivation, slows fast inactivation, and prevents full inactivation, resulting in persistent inward currents similar to PEPD mutations. Using current clamp, we show that A1632E renders dorsal root ganglion (DRG) and trigeminal ganglion neurons hyperexcitable. These results demonstrate a Na(V)1.7 mutant with biophysical characteristics common to PEPD (impaired fast inactivation) and IEM (hyperpolarized activation, slow deactivation, and enhanced ramp currents) associated with a clinical phenotype with characteristics of both IEM and PEPD and show that this mutation renders DRG and trigeminal ganglion neurons hyperexcitable. These observations indicate that IEM and PEPD mutants are part of a physiological continuum that can produce a continuum of clinical phenotypes.

Gain-of-function mutation in Nav1.7 in familial erythromelalgia induces bursting of sensory neurons.

Erythromelalgia is an autosomal dominant disorder characterized by burning pain in response to warm stimuli or moderate exercise. We describe a novel mutation in a family with erythromelalgia in SCN9A, the gene that encodes the Na(v)1.7 sodium channel. Na(v)1.7 produces threshold currents and is selectively expressed within sensory neurons including nociceptors. We demonstrate that this mutation, which produces a hyperpolarizing shift in activation and a depolarizing shift in steady-state inactivation, lowers thresholds for single action potentials and high frequency firing in dorsal root ganglion neurons. Erythromelalgia is the first inherited pain disorder in which it is possible to link a mutation with an abnormality in ion channel function and with altered firing of pain signalling neurons.

The pentapeptide QYNAD does not block voltage-gated sodium channels.

BACKGROUND: An endogenous pentapeptide (Gln-Tyr-Asn-Ala-Asp; QYNAD) that is present at elevated levels in human CSF from patients with demyelinating diseases has been reported to block voltage-gated sodium channels at low (10 micro M) concentrations. Objective : Because of the potential importance of sodium channel blocking activity in demyelinating disorders, this study attempted to determine the sensitivity to QYNAD of different sodium channel subtypes, including Na(v)1.6, the major sodium channel at nodes of Ranvier, and Na(v)1.2, which is expressed in axons with abnormal myelin. METHODS: Sodium channel function was assayed using patch-clamp recordings, both in heterologous expression systems and in intact neurons. RESULTS: QYNAD synthesized in 10 different batches by four different facilities failed to block sodium currents, even at concentrations as high as 500 micro M (50-fold higher than the blocking concentration originally reported). QYNAD had no effect on the currents produced by recombinant Na(v)1.2, Na(v)1.4, Na(v)1.6, and Na(v)1.7 sodium channels or on the sodium currents that are produced by native channels in adult hippocampal or dorsal root ganglion neurons. QYNAD did not interfere with conduction in the optic nerve, a myelinated fiber tract that is often affected in MS. CONCLUSIONS: These experiments do not show any sodium channel blocking effect of QYNAD. The conclusion that QYNAD contributes to the pathophysiology of inflammatory neurologic disorders by blocking voltage-gated sodium channels should therefore be viewed with caution.

Na(v)1.5 underlies the 'third TTX-R sodium current' in rat small DRG neurons.

In addition to slow-inactivating and persistent TTX-R Na(+) currents produced by Na(v)1.8 and Na(v)1.9 Na(+) channels, respectively, a third TTX-R Na(+) current with fast activation and inactivation can be recorded in 80% of small neurons of dorsal root ganglia (DRG) from E15 rats, but in only 3% of adult small DRG neurons. The half-time for activation, the time constant for inactivation, and the midpoints of activation and inactivation of the third TTX-R Na(+) currents are significantly different from those of Na(v)1.8 and Na(v)1.9 Na(+) currents. The estimated TTX K(i) (2.11+/-0.34 microM) of the third TTX-R Na(+) current is significantly lower than those of Na(v)1.8 and Na(v)1.9 Na(+) currents. The Cd(2+) sensitivity of third TTX-R Na(+) current is closer to cardiac Na(+) currents. A concentration of 1 mM Cd(2+) is required to completely block this current, which is significantly lower than the 5 mM required to block Na(v)1.8 and Na(v)1.9 currents. The third TTX-R Na(+) channel is not co-expressed with Na(v)1.8 and Na(v)1.9 Na(+) channels in DRG neurons of E18 rats, at a time when all three currents show comparable densities. The physiological and pharmacological profiles of the third TTX-R Na(+) current are similar to those of the cardiac Na(+) channel Na(v)1.5 and RT-PCR and restriction enzyme polymorphism analysis, show a parallel pattern of expression of Na(v)1.5 in DRG during development. Taken together, these results demonstrate that Na(v)1.5 is expressed in a developmentally regulated manner in DRG neurons and suggest that Na(v)1.5 Na(+) channel produces the third TTX-R current.

Glycosylation alters steady-state inactivation of sodium channel Nav1.9/NaN in dorsal root ganglion neurons and is developmentally regulated.

Na channel NaN (Na(v)1.9) produces a persistent TTX-resistant (TTX-R) current in small-diameter neurons of dorsal root ganglia (DRG) and trigeminal ganglia. Na(v)1.9-specific antibodies react in immunoblot assays with a 210 kDa protein from the membrane fractions of adult DRG and trigeminal ganglia. The size of the immunoreactive protein is in close agreement with the predicted Na(v)1.9 theoretical molecular weight of 201 kDa, suggesting limited glycosylation of this channel in adult tissues. Neonatal rat DRG membrane fractions, however, contain an additional higher molecular weight immunoreactive protein. Reverse transcription-PCR analysis did not show additional longer transcripts that could encode the larger protein. Enzymatic deglycosylation of the membrane preparations converted both immunoreactive proteins into a single faster migrating band, consistent with two states of glycosylation of Na(v)1.9. The developmental change in the glycosylation state of Na(v)1.9 is paralleled by a developmental change in the gating of the persistent TTX-R Na(+) current attributable to Na(v)1.9 in native DRG neurons. Whole-cell patch-clamp analysis demonstrates that the midpoint of steady-state inactivation is shifted 7 mV in a hyperpolarized direction in neonatal (postnatal days 0-3) compared with adult DRG neurons, although there is no significant difference in activation. Pretreatment of neonatal DRG neurons with neuraminidase causes an 8 mV depolarizing shift in the midpoint of steady-state inactivation of Na(v)1.9, making it indistinguishable from that of adult DRG neurons. Our data show that extensive glycosylation of rat Na(v)1.9 is developmentally regulated and changes a critical property of this channel in native neurons.

Direct interaction with contactin targets voltage-gated sodium channel Na(v)1.9/NaN to the cell membrane.

The mechanisms that target various sodium channels within different regions of the neuronal membrane, which they endow with different physiological properties, are not yet understood. To examine this issue we studied the voltage-gated sodium channel Na(v)1.9/NaN, which is preferentially expressed in small sensory neurons of dorsal root ganglia and trigeminal ganglia and the nonmyelinated axons that arise from them. Our results show that the cell adhesion molecule contactin binds directly to Na(v)1.9/NaN and recruits tenascin to the protein complex in vitro. Na(v)1.9/NaN and contactin co-immunoprecipitate from dorsal root ganglia and transfected Chinese hamster ovary cell line, and co-localize in the C-type neuron soma and along nonmyelinated C-fibers and at nerve endings in the skin. Co-transfection of Chinese hamster ovary cells with Na(v)1.9/NaN and contactin enhances the surface expression of the sodium channel over that of Na(v)1.9/NaN alone. Thus contactin binds directly to Na(v)1.9/NaN and participates in the surface localization of this channel along nonmyelinated axons.

Nav1.3 sodium channels: rapid repriming and slow closed-state inactivation display quantitative differences after expression in a mammalian cell line and in spinal sensory neurons.

Although rat brain Nav1.3 voltage-gated sodium channels have been expressed and studied in Xenopus oocytes, these channels have not been studied after their expression in mammalian cells. We characterized the properties of the rat brain Nav1.3 sodium channels expressed in human embryonic kidney (HEK) 293 cells. Nav1.3 channels generated fast-activating and fast-inactivating currents. Recovery from inactivation was relatively rapid at negative potentials (<-80 mV) but was slow at more positive potentials. Development of closed-state inactivation was slow, and, as predicted on this basis, Nav1.3 channels generated large ramp currents in response to slow depolarizations. Coexpression of beta3 subunits had small but significant effects on the kinetic and voltage-dependent properties of Nav1.3 currents in HEK 293 cells, but coexpression of beta1 and beta2 subunits had little or no effect on Nav1.3 properties. Nav1.3 channels, mutated to be tetrodotoxin-resistant (TTX-R), were expressed in SNS-null dorsal root ganglion (DRG) neurons via biolistics and were compared with the same construct expressed in HEK 293 cells. The voltage dependence of steady-state inactivation was approximately 7 mV more depolarized in SNS-null DRG neurons, demonstrating the importance of background cell type in determining physiological properties. Moreover, consistent with the idea that cellular factors can modulate the properties of Nav1.3, the repriming kinetics were twofold faster in the neurons than in the HEK 293 cells. The rapid repriming of Nav1.3 suggests that it contributes to the acceleration of repriming of TTX-sensitive (TTX-S) sodium currents that are seen after peripheral axotomy of DRG neurons. The relatively rapid recovery from inactivation and the slow closed-state inactivation kinetics of Nav1.3 channels suggest that neurons expressing Nav1.3 may exhibit a reduced threshold and/or a relatively high frequency of firing.

Beta1 adducin gene expression in DRG is developmentally regulated and is upregulated by glial-derived neurotrophic factor and nerve growth factor.

Differential display technique has proven to be effective in identifying differentially regulated genes under a variety of experimental conditions. We identified beta1 adducin as a target in primary rat dorsal root ganglia (DRG) cultures that is upregulated by exposure to nerve growth factor (NGF) and glial-derived neurotrophic factor (GDNF). We used real-time reverse-transcription polymerase chain reaction (RT-PCR) for quantitative measurement of beta1 adducin gene expression both in DRG cultures and in vivo. Significant increase in beta1 adducin expression level was observed in DRG cultures treated with either GDNF or NGF, compared to untreated cultures. The expression of beta1 adducin in rat tissues was highest in the brain and high in the cerebellum, superior cervical ganglion and DRG tissues. By contrast, low expression levels of beta1 adducin are detected in sciatic nerve and in non-neural tissues. Our study also showed that expression of beta1 adducin gene is developmentally regulated in rat DRG and trigeminal ganglia, with a peak around P0 and significant attenuation by P21. The level of expression of beta1 adducin in adult rat DRG and trigeminal ganglia may be maintained by the action of neurotrophic factors that are produced in innervated targets like skin and muscle.

Fibroblast growth factor homologous factor 1B binds to the C terminus of the tetrodotoxin-resistant sodium channel rNav1.9a (NaN).

In this study we demonstrate a direct interaction between a cytosolic fibroblast growth factor family member and a sodium channel. A yeast two-hybrid screen for proteins that associate with the cytoplasmic domains of the tetrodotoxin-resistant sodium channel rNa(v)1.9a (NaN) led to the identification of fibroblast growth factor homologous factor 1B (FHF1B), a member of the fibroblast growth factor family, as an interacting partner of rNa(v)1.9a. FHF1B selectively interacts with the C-terminal region but not the other four intracellular segments of rNa(v)1.9a. FHF1B binds directly to the C-terminal polypeptide of rNa(v)1.9a both in vitro and in mammalian cell lines. The N-terminal 5-77 amino acid residues of FHF1B are essential for binding to rNa(v)1.9a. FHF1B did not interact with C termini of two other sodium channels hNa(v)1.7a (hNaNE) and rNa(v)1.8a (SNS), which share 50% similarity to the C-terminal polypeptide of rNa(v)1.9a. FHF1B is the first growth factor found to bind specifically to a sodium channel. Although the functional significance of this interaction is not clear, FHF1B may affect the rNa(v)1.9a channel directly or by recruiting other proteins to the channel complex. Alternatively, it is possible that rNa(v)1.9a may help deliver this factor to the cell membrane, where it exerts its function.

Sodium channels and their genes: dynamic expression in the normal nervous system, dysregulation in disease states(1).

Although classical neurophysiological doctrine rested on the concept of the sodium channel, it is now clear that there are nearly a dozen sodium channel genes, each encoding a molecularly distinct channel. Different repertoires of channels endow different types of neurons with distinct transduction and encoding properties. Sodium channel expression is highly dynamic, exhibiting plasticity at both the transcriptional and post-transcriptional levels. In some types of neurons within the normal nervous system, e.g. hypothalamic magnocellular neurosecretory neurons, changes in sodium channel gene expression occur in association with the transition from a quiescent to a bursting state; these changes are accompanied by the insertion of a different set of sodium channel subtypes in the cell membrane, a form of molecular plasticity which results in altered electrogenic properties. Dysregulation of sodium channel genes has been observed in a number of disease states. For example, transection of the peripheral axons of spinal sensory neurons triggers down-regulation of some sodium channel genes, and up-regulation of other sodium channel genes; the resultant changes in sodium channel expression contribute to hyperexcitability that can lead to chronic pain. There is also evidence, in experimental models of demyelination and in post-mortem tissue from patients with multiple sclerosis, for dysregulation of sodium channel gene expression in the cell bodies of some neurons whose axons have been demyelinated, suggesting that an acquired channelopathy may contribute to the pathophysiology of demyelinating diseases such as multiple sclerosis. The dynamic nature of sodium channel gene expression makes it a complex topic for investigation, but it also introduces therapeutic opportunities, since subtype-specific sodium channel modulating drugs may soon be available.

Sensory neuron-specific sodium channel SNS is abnormally expressed in the brains of mice with experimental allergic encephalomyelitis and humans with multiple sclerosis.

Clinical abnormalities in multiple sclerosis (MS) have classically been considered to be caused by demyelination and/or axonal degeneration; the possibility of molecular changes in neurons, such as the deployment of abnormal repertoires of ion channels that would alter neuronal electrogenic properties, has not been considered. Sensory Neuron-Specific sodium channel SNS displays a depolarized voltage dependence, slower activation and inactivation kinetics, and more rapid recovery from inactivation than classical "fast" sodium channels. SNS is selectively expressed in spinal sensory and trigeminal ganglion neurons within the peripheral nervous system and is not expressed within the normal brain. Here we show that sodium channel SNS mRNA and protein, which are not present within the cerebellum of control mice, are expressed within cerebellar Purkinje cells in a mouse model of MS, chronic relapsing experimental allergic encephalomyelitis. We also demonstrate SNS mRNA and protein expression within Purkinje cells from tissue obtained postmortem from patients with MS, but not in control subjects with no neurological disease. These results demonstrate a change in sodium channel expression in neurons within the brain in an animal model of MS and in humans with MS and suggest that abnormal patterns of neuronal ion channel expression may contribute to clinical abnormalities such as ataxia in these disorders.

Changes in expression of two tetrodotoxin-resistant sodium channels and their currents in dorsal root ganglion neurons after sciatic nerve injury but not rhizotomy.

Two TTX-resistant sodium channels, SNS and NaN, are preferentially expressed in c-type dorsal root ganglion (DRG) neurons and have been shown recently to have distinct electrophysiological signatures, SNS producing a slowly inactivating and NaN producing a persistent sodium current with a relatively hyperpolarized voltage-dependence. An attenuation of SNS and NaN transcripts has been demonstrated in small DRG neurons after transection of the sciatic nerve. However, it is not known whether changes in the currents associated with SNS and NaN or in the expression of SNS and NaN channel protein occur after axotomy of the peripheral projections of DRG neurons or whether similar changes occur after transection of the central (dorsal root) projections of DRG neurons. Peripheral and central projections of L4/5 DRG neurons in adult rats were axotomized by transection of the sciatic nerve and the L4 and L5 dorsal roots, respectively. DRG neurons were examined using immunocytochemical and patch-clamp methods 9-12 d after sciatic nerve or dorsal root lesion. Levels of SNS and NaN protein in the two types of injuries were paralleled by their respective TTX-resistant currents. There was a significant decrease in SNS and NaN signal intensity in small DRG neurons after peripheral, but not central, axotomy compared with control neurons. Likewise, there was a significant reduction in slowly inactivating and persistent TTX-resistant currents in these neurons after peripheral, but not central, axotomy compared with control neurons. These results indicate that peripheral, but not central, axotomy results in a reduction in expression of functional SNS and NaN channels in c-type DRG neurons and suggest a basis for the altered electrical properties that are observed after peripheral nerve injury.

Localization of the tetrodotoxin-resistant sodium channel NaN in nociceptors.

Tetrodotoxin-resistant sodium currents contribute to the somal and axonal sodium currents of small diameter primary sensory neurons, many of which are nociceptive. NaN is a recently described tetrodotoxin-resistant sodium channel expressed preferentially in IB4-labeled dorsal root ganglion (DRG) neurons. We employed an antibody raised to a NaN specific peptide to show that NaN is preferentially localized along axons of IB4-positive unmyelinated fibers in the sciatic nerve and in axon terminals in the cornea. NaN immunoreactivity was also found at some nodes of Ranvier of thinly myelinated axons of the sciatic nerve, where it was juxtaposed to Kv1.2 potassium channel immunoreactivity. This distribution of NaN is consistent with a role for NaN sodium channels in nociceptive transmission.

Glial-derived neurotrophic factor upregulates expression of functional SNS and NaN sodium channels and their currents in axotomized dorsal root ganglion neurons.

Dorsal root ganglion (DRG) neurons produce multiple sodium currents, including several different TTX-sensitive (TTX-S) currents and TTX-resistant (TTX-R) currents, which are produced by distinct sodium channels. We previously demonstrated that, after sciatic nerve transection, the levels of SNS and NaN sodium channel alpha-subunit transcripts and protein in small (18-30 micrometer diameter) DRG neurons are reduced, as are the amplitudes and densities of the slowly inactivating and persistent TTX-R currents produced by these two channels. In this study, we asked whether glial-derived neurotrophic factor (GDNF), which has been shown to prevent some axotomy-induced changes such as the loss of somatostatin expression in DRG neurons, can ameliorate the axotomy-induced downregulation of SNS and NaN TTX-R sodium channels. We show here that exposure to GDNF can significantly increase both slowly inactivating and persistent TTX-R sodium currents, which are paralleled by increases in SNS and NaN mRNA and protein levels, in axotomized DRG neurons in vitro. We also show that intrathecally administered GDNF increases the amplitudes of the slowly inactivating and persistent TTX-R currents, and SNS and NaN protein levels, in peripherally axotomized DRG neurons in vivo. Finally, we demonstrate that GDNF upregulates the persistent TTX-R current in SNS-null mice, thus demonstrating that the upregulated persistent sodium current is not produced by SNS. Because TTX-R sodium channels have been shown to be important in nociception, the effects of GDNF on axotomized DRG neurons may have important implications for the regulation of nociceptive signaling by these cells.