Nav1.7, a peripheral neuron voltage-gated sodium channel, is essential for pain and olfaction in mice and humans. We examined the role of Nav1.7 as well as Nav1.3, Nav1.8, and Nav1.9 in different mouse models of chronic pain. Constriction-injury-dependent neuropathic pain is abolished when Nav1.7 is deleted in sensory neurons, unlike nerve-transection-related pain, which requires the deletion of Nav1.7 in sensory and sympathetic neurons for pain relief. Sympathetic sprouting that develops in parallel with nerve-transection pain depends on the presence of Nav1.7 in sympathetic neurons. Mechanical and cold allodynia required distinct sets of neurons and different repertoires of sodium channels depending on the nerve injury model. Surprisingly, pain induced by the chemotherapeutic agent oxaliplatin and cancer-induced bone pain do not require the presence of Nav1.7 sodium channels or Nav1.8-positive nociceptors. Thus, similar pain phenotypes arise through distinct cellular and molecular mechanisms. Therefore, rational analgesic drug therapy requires patient stratification in terms of mechanisms and not just phenotype.
•Phenotypically identical pain models have different underlying molecular mechanisms•Nav1.7 expression is required for sympathetic sprouting after neuronal damage•Oxaliplatin and cancer-induced bone pain are both Nav1.7-independent•Deleting Nav1.7 in adult mice reverses nerve damage-induced neuropathic pain
Wood and colleagues describe two pain syndromes that occur in the absence of Nav1.7, a sodium channel considered to be essential for pain perception and olfaction in humans. They provide evidence that pain phenotypes such as cold and mechanical allodynia can arise through distinct cell and molecular mechanisms after nerve injury in mouse peripheral sensory neurons. The existence of redundant mechanistically distinct peripheral pain mechanisms may help to explain recent difficulties with the development of new analgesic drugs.
Opiates are powerful drugs to treat severe pain, and act via mu opioid receptors distributed throughout the nervous system. Their clinical use is hampered by centrally-mediated adverse effects, including nausea or respiratory depression. Here we used a genetic approach to investigate the potential of peripheral mu opioid receptors as targets for pain treatment. We generated conditional knockout (cKO) mice in which mu opioid receptors are deleted specifically in primary afferent Nav1.8-positive neurons. Mutant animals were compared to controls for acute nociception, inflammatory pain, opiate-induced analgesia and constipation. There was a 76% decrease of mu receptor-positive neurons and a 60% reduction of mu-receptor mRNA in dorsal root ganglia of cKO mice. Mutant mice showed normal responses to heat, mechanical, visceral and chemical stimuli, as well as unchanged morphine antinociception and tolerance to antinociception in models of acute pain. Inflammatory pain developed similarly in cKO and controls mice after Complete Freund’s Adjuvant. In the inflammation model, however, opiate-induced (morphine, fentanyl and loperamide) analgesia was reduced in mutant mice as compared to controls, and abolished at low doses. Morphine-induced constipation remained intact in cKO mice. We therefore genetically demonstrate for the first time that mu opioid receptors partly mediate opiate analgesia at the level of Nav1.8-positive sensory neurons. In our study, this mechanism operates under conditions of inflammatory pain, but not nociception. Previous pharmacology suggests that peripheral opiates may be clinically useful, and our data further demonstrate that Nav1.8 neuron-associated mu opioid receptors are feasible targets to alleviate some forms of persistent pain.
Loss of function of the gene SCN9A, encoding the voltage-gated sodium channel Nav1.7, causes a congenital inability to experience pain in humans. Here we show that Nav1.7 is not only necessary for pain sensation but is also an essential requirement for odour perception in both mice and humans. We examined human patients with loss-of-function mutations in SCN9A and show that they are unable to sense odours. To establish the essential role of Nav1.7 in odour perception, we generated conditional null mice in which Nav1.7 was removed from all olfactory sensory neurons. In the absence of Nav1.7, these neurons still produce odour-evoked action potentials but fail to initiate synaptic signalling from their axon terminals at the first synapse in the olfactory system. The mutant mice no longer display vital, odour-guided behaviours such as innate odour recognition and avoidance, short-term odour learning, and maternal pup retrieval. Our study creates a mouse model of congenital general anosmia and provides new strategies to explore the genetic basis of the human sense of smell.
Mendelian heritable pain disorders have provided insights into human pain mechanisms
and suggested new analgesic drug targets. Interestingly, many of the heritable
monogenic pain disorders have been mapped to mutations in genes encoding ion
channels. Studies in transgenic mice have also implicated many ion channels in damage
sensing and pain modulation. It seems likely that aberrant peripheral or central ion
channel activity underlies or initiates many pathological pain conditions.
Understanding the mechanistic basis of ion channel malfunction in terms of
trafficking, localization, biophysics, and consequences for neurotransmission is a
potential route to new pain therapies.
We identified and clinically investigated two patients with primary erythromelalgia mutations (PEM), which are the first reported to map to the fourth domain of Nav1.7 (DIV). The identified mutations (A1746G and W1538R) were cloned and transfected to cell cultures followed by electrophysiological analysis in whole-cell configuration. The investigated patients presented with PEM, while age of onset was very different (3 vs. 61 years of age). Electrophysiological characterization revealed that the early onset A1746G mutation leads to a marked hyperpolarizing shift in voltage dependence of steady-state activation, larger window currents, faster activation kinetics (time-to-peak current) and recovery from steady-state inactivation compared to wild-type Nav1.7, indicating a pronounced gain-of-function. Furthermore, we found a hyperpolarizing shift in voltage dependence of slow inactivation, which is another feature commonly found in Nav1.7 mutations associated with PEM. In silico neuron simulation revealed reduced firing thresholds and increased repetitive firing, both indicating hyperexcitability. The late-onset W1538R mutation also revealed gain-of-function properties, although to a lesser extent. Our findings demonstrate that mutations encoding for DIV of Nav1.7 can not only be linked to congenital insensitivity to pain or paroxysmal extreme pain disorder but can also be causative of PEM, if voltage dependency of channel activation is affected. This supports the view that the degree of biophysical property changes caused by a mutation may have an impact on age of clinical manifestation of PEM. In summary, these findings extent the genotype–phenotype correlation profile for SCN9A and highlight a new region of Nav1.7 that is implicated in PEM.
Electronic supplementary material
The online version of this article (doi:10.1007/s12017-012-8216-8) contains supplementary material, which is available to authorized users.
Erythromelalgia; Neuropathic pain; Voltage-gated sodium channels; Gain-of-function mutations; Nav1.7
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.
ion channel; genetics; pain; epilepsy; SCN1A
Nav1.8 is a tetrodotoxin-resistant sodium channel present in large subsets of peripheral sensory neurons, including both spinal and vagal afferents. In spinal afferents, Nav1.8 plays a key role in signaling different types of pain. Little is known, however, about the exact identity and role of Nav1.8-expressing vagal neurons. Here we generated mice with restricted expression of tdTomato fluorescent protein in all Nav1.8-expressing afferent neurons. As a result, intense fluorescence was visible in the cell bodies, central relays, and sensory endings of these neurons, revealing the full extent of their innervation sites in thoracic and abdominal viscera. For instance, vagal and spinal Nav1.8-expressing endings were seen clearly within the gastrointestinal mucosa and myenteric plexus, respectively. In the gastrointestinal muscle wall, labeled endings included a small subset of vagal tension receptors but not any stretch receptors. We also examined the detailed inner-vation of key metabolic tissues such as liver and pancreas and evaluated the anatomical relationship of Nav1.8-expressing vagal afferents with select enteroendocrine cells (i.e., ghrelin, glucagon, GLP-1). Specifically, our data revealed the presence of Nav1.8-expressing vagal afferents in several metabolic tissues and varying degrees of proximity between Nav1.8-expressing mucosal afferents and enteroendocrine cells, including apparent neuroendocrine apposition. In summary, this study demonstrates the power and versatility of the Cre-LoxP technology to trace identified visceral afferents, and our data suggest a previously unrecognized role for Nav1.8-expressing vagal neurons in gastrointestinal functions.
vagotomy; autonomic nervous system; transgenic; visceral pain; obesity; connections
The molecular mechanisms determining magnitude and duration of inflammatory pain are still unclear. We assessed the contribution of G protein–coupled receptor kinase (GRK)-6 to inflammatory hyperalgesia in mice. We showed that GRK6 is a critical regulator of severity and duration of cytokine-induced hyperalgesia. In GRK6−/− mice, a significantly lower dose (100 times lower) of intraplantar interleukin (IL)-1β was sufficient to induce hyperalgesia compared with wild-type (WT) mice. In addition, IL-1β hyperalgesia lasted much longer in GRK6−/− mice than in WT mice (8 d in GRK6−/− versus 6 h in WT mice). Tumor necrosis factor (TNF)-α–induced hyperalgesia was also enhanced and prolonged in GRK6−/− mice. In vitro, IL-1β–induced p38 phosphorylation in GRK6−/− dorsal root ganglion (DRG) neurons was increased compared with WT neurons. In contrast, IL-1β only induced activation of the phosphatidylinositol (PI) 3-kinase/Akt pathway in WT neurons, but not in GRK6−/− neurons. In vivo, p38 inhibition attenuated IL-1β– and TNF-α–induced hyperalgesia in both genotypes. Notably, however, whereas PI 3-kinase inhibition enhanced and prolonged hyperalgesia in WT mice, it did not have any effect in GRK6-deficient mice. The capacity of GRK6 to regulate pain responses was also apparent in carrageenan-induced hyperalgesia, since thermal and mechanical hypersensitivity was significantly prolonged in GRK6−/− mice. Finally, GRK6 expression was reduced in DRGs of mice with chronic neuropathic or inflammatory pain. Collectively, these findings underline the potential role of GRK6 in pathological pain. We propose the novel concept that GRK6 acts as a kinase that constrains neuronal responsiveness to IL-1β and TNF-α and cytokine-induced hyperalgesia via biased cytokine-induced p38 and PI 3-kinase/Akt activation.
Tissue-specific gene deletion has proved informative in the analysis of pain pathways. Advillin has been shown to be a pan-neuronal marker of spinal and cranial sensory ganglia. We generated BAC transgenic mice using the Advillin promoter to drive a tamoxifen-inducible CreERT2 recombinase construct in order to be able to delete genes in adult animals. We used a floxed stop ROSA26LacZ reporter mouse to examine functional Cre expression, and analysed the behaviour of mice expressing Cre recombinase.
We used recombineering to introduce a CreERT2 cassette in place of exon 2 of the Advillin gene into a BAC clone (RPCI23-424F19) containing the 5' region of the Advillin gene. Transgenic mice were generated using pronuclear injection. The resulting AvCreERT2 transgenic mice showed a highly specific expression pattern of Cre activity after tamoxifen induction. Recombinase activity was confined to sensory neurons and no expression was found in other organs. Less than 1% of neurons showed Cre expression in the absence of tamoxifen treatment. Five-day intraperitoneal treatment with tamoxifen (2 mg per day) induced Cre recombination events in ≈90% of neurons in dorsal root and cranial ganglia. Cell counts of dorsal root ganglia (DRG) from transgenic animals with or without tamoxifen treatment showed no neuronal cell loss. Sensory neurons in culture showed ≈70% induction after 3 days treatment with tamoxifen. Behavioural tests showed no differences between wildtype, AvCreERT2 and tamoxifen-treated animals in terms of motor function, responses to light touch and noxious pressure, thermal thresholds as well as responses to inflammatory agents.
Our results suggest that the inducible pan-DRG AvCreERT2 deleter mouse strain is a useful tool for studying the role of individual genes in adult sensory neuron function. The pain phenotype of the Cre-induced animal is normal; therefore any alterations in pain processing can be unambiguously attributed to loss of the targeted gene.
AvCreERT2; pain and nociception; tamoxifen inducible; ROSA26 LacZ reporter; behaviour; DRG
Chronic pain associated with inflammation is a common clinical problem and the underlying mechanisms have only begun to be unravelled. GRK2 regulates cellular signalling by promoting G protein-coupled receptor (GPCR) desensitization and direct interaction with downstream kinases including p38. The aim of this study was to determine the contribution of GRK2 to regulation of inflammatory pain and to unravel the underlying mechanism. GRK2+/− mice with ~50% reduction in GRK2 developed increased and markedly prolonged thermal hyperalgesia and mechanical allodynia after carrageenan-induced paw inflammation or after intraplantar injection of the GPCR-binding chemokine CCL3. The effect of reduced GRK2 in specific cells was investigated using CRE-Lox technology. Carrageenan or CCL3-induced hyperalgesia was increased but not prolonged in mice with decreased GRK2 only in Nav1.8-nociceptors. In vitro, reduced neuronal GRK2 enhanced CCL3-induced TRPV1 sensitisation. In vivo, CCL3-induced acute hyperalgesia in GRK2+/− mice was mediated via TRPV1.
Reduced GRK2 in microglia/monocytes only was required and sufficient to transform acute carrageenan- or CCL3-induced hyperalgesia into chronic hyperalgesia. Chronic hyperalgesia in GRK2+/− mice was associated with ongoing microglial activation and increased phospho-p38 and TNF-α in the spinal cord. Inhibition of spinal cord microglial, p38, or TNF-α activity by intrathecal administration of specific inhibitors reversed ongoing hyperalgesia in GRK2+/− mice. Microglia/macrophage GRK2 expression was reduced in the lumbar ipsilateral spinal cord during neuropathic pain, underlining the patho-physiological relevance of microglial GRK2.
Thus, we identified completely novel cell-specific roles of GRK2 in regulating acute and chronic inflammatory hyperalgesia.
inflammatory hyperalgesia; G protein-coupled receptor kinase; microglia; p38; TNF; TRPV1; CCL3
The natural response to itch sensation is to scratch, which relieves the itch through an unknown mechanism. Interaction between pain and itch has been frequently demonstrated, and the selectivity hypothesis of itch, based on data from electrophysiological and behavioral experiments, postulates the existence of primary pain afferents capable of repressing itch. Here, we demonstrate that deletion of vesicular glutamate transporter (VGLUT) 2 in a subpopulation of neurons partly overlapping with the vanilloid receptor (TRPV1) primary afferents resulted in a dramatic increase in itch behavior accompanied by a reduced responsiveness to thermal pain. The increased itch behavior was reduced by administration of antihistaminergic drugs and by genetic deletion of the gastrin-releasing peptide receptor, demonstrating a dependence on VGLUT2 to maintain normal levels of both histaminergic and nonhistaminergic itch. This study establishes that VGLUT2 is a major player in TRPV1 thermal nociception and also serves to regulate a normal itch response.
Small unmyelinated sensory neurons classified as nociceptors are divided into two subpopulations based on phenotypic differences including expression of neurotrophic factor receptors. Approximately half of unmyelinated nociceptors express the NGF receptor TrkA and half express the GDNF Family Ligand (GFL) receptor Ret. The function of NGF/TrkA signaling in the TrkA population of nociceptors has been extensively studied and NGF/TrkA signaling is a well established mediator of pain. The GFLs are analgesic in models of neuropathic pain emphasizing the importance of understanding the physiological function of GFL/Ret signaling in nociceptors. However, perinatal lethality of Ret-null mice has precluded the study of the physiological role of GFL/Ret signaling in the survival, maintenance and function of nociceptors in viable mice. We deleted Ret exclusively in nociceptors by crossing nociceptor-specific Nav1.8 Cre and Ret conditional mice to produce Ret-Nav1.8 conditional knock out (CKO) mice. Loss of Ret exclusively in nociceptors results in a reduction in nociceptor number and size indicating Ret signaling is important for the survival and trophic support of these cells. Ret-Nav1.8 CKO mice exhibit reduced epidermal innervation, but normal central projections. In addition, Ret-Nav1.8 CKO mice have increased sensitivity to cold and increased formalin-induced pain, demonstrating that Ret signaling modulates the function of nociceptors in vivo. Enhanced inflammation-induced pain may be mediated by decreased Prostatic Acid Phosphatase (PAP) as PAP levels are markedly reduced in Ret-Nav1.8 CKO mice. The results of this study identify the physiological role of endogenous Ret signaling in the survival and function of nociceptors.
Ret; neurotrophic factor; GDNF; pain; inflammation; nociceptor
The voltage-gated sodium channel NaV1.8 is expressed exclusively in nociceptive sensory neurons and plays an important role in pain pathways. NaV1.8 cannot be functionally expressed in non-neuronal cells even in the presence of β-subunits. We have previously identified Pdzd2, a multi PDZ-domain protein, as a potential interactor for NaV1.8. Here we report that Pdzd2 binds directly to the intracellular loops of NaV1.8 and NaV1.7. The endogenous NaV1.8 current in sensory neurons is inhibited by antisense- and siRNA-mediated downregulation of Pdzd2. However, no marked change in pain behaviours is observed in Pdzd2-decificent mice. This may be due to compensatory upregulation of p11, another regulatory factor for NaV1.8, in dorsal root ganglia of Pdzd2-deficient mice. These findings reveal that Pdzd2 and p11 play collaborative roles in regulation of NaV1.8 expression in sensory neurons.
Serum response factor (SRF) is a prototypic transcription factor that mediates stimulus-dependent gene expression. Here, we show that SRF mediates NGF signaling, axonal growth, branching, and target innervation by embryonic DRG sensory neurons. Conditional deletion of the murine SRF gene in DRGs results in no deficits in neuronal viability or differentiation but causes defects in extension and arborization of peripheral axonal projections in the target field in vivo, similar to the target innervation defects observed in mice lacking NGF. Moreover, SRF is both necessary and sufficient for NGF-dependent axonal outgrowth in vitro, and NGF regulates SRF-dependent gene expression and axonal outgrowth through activation of both MEK/ERK and MAL signaling pathways. These findings show that SRF is a major effector of both MEK/ERK and MAL signaling by NGF and that SRF is a key mediator of NGF-dependent target innervation by embryonic sensory neurons.
Neuropathic pain may arise following peripheral nerve injury though the molecular mechanisms associated with this are unclear. We used proteomic profiling to examine changes in protein expression associated with the formation of hyper-excitable neuromas derived from rodent saphenous nerves. A two-dimensional difference gel electrophoresis (2D-DIGE) profiling strategy was employed to examine protein expression changes between developing neuromas and normal nerves in whole tissue lysates. We found around 200 proteins which displayed a >1.75-fold change in expression between neuroma and normal nerve and identified 55 of these proteins using mass spectrometry. We also used immunoblotting to examine the expression of low-abundance ion channels Nav1.3, Nav1.8 and calcium channel α2δ-1 subunit in this model, since they have previously been implicated in neuronal hyperexcitability associated with neuropathic pain. Finally, S35methionine in vitro labelling of neuroma and control samples was used to demonstrate local protein synthesis of neuron-specific genes. A number of cytoskeletal proteins, enzymes and proteins associated with oxidative stress were up-regulated in neuromas, whilst overall levels of voltage-gated ion channel proteins were unaffected. We conclude that altered mRNA levels reported in the somata of damaged DRG neurons do not necessarily reflect levels of altered proteins in hyper-excitable damaged nerve endings. An altered repertoire of protein expression, local protein synthesis and topological re-arrangements of ion channels may all play important roles in neuroma hyper-excitability.
Pain, which afflicts up to 20% of the population at any time, provides
both a massive therapeutic challenge and a route to understanding mechanisms in
the nervous system. Specialised sensory neurons (nociceptors) signal the
existence of tissue damage to the central nervous system (CNS), where pain is
represented in a complex matrix involving many CNS structures. Genetic
approaches to investigating pain pathways using model organisms have identified
the molecular nature of the transducers, regulatory mechanisms involved in
changing neuronal activity, as well as the critical role of immune system cells
in driving pain pathways. In man, mapping of human pain mutants as well as twin
studies and association studies of altered pain behaviour have identified
important regulators of the pain system. In turn, new drug targets for chronic
pain treatment have been validated in transgenic mouse studies. Thus, genetic
studies of pain pathways have complemented the traditional neuroscience
approaches of electrophysiology and pharmacology to give us fresh insights into
the molecular basis of pain perception.
The molecular identity and pharmacological properties of mechanically gated ion channels in sensory neurons are poorly understood. We show that FM1-43, a styryl dye used to fluorescently label cell membranes, permeates mechanosensitive ion channels in cultured dorsal root ganglion neurons, resulting in blockade of three previously defined subtypes of mechanically activated currents. Blockade and dye uptake is voltage dependent and regulated by external Ca2+. The structurally related larger dye FM3-25 inhibited mechanically activated currents to a lesser degree and did not permeate the channels. In vivo, FMI-43 decreases pain sensitivity in the Randall-Selitto test and increases the withdrawal threshold from von Frey hairs, together suggesting that the channels expressed at the cell body in culture mediate mechanosensation in the intact animal. These data give further insight into the mechanosensitive ion channels expressed by somatosensory neurons and suggest FM dyes are an interesting tool for studying them.
The voltage gated sodium channel Nav 1.8 has a highly restricted expression pattern to predominantly nociceptive peripheral sensory neurones. Behaviourally Nav 1.8-null mice show an increased acute pain threshold to noxious mechanical pressure and also deficits in inflammatory and visceral, but not neuropathic pain. Here we have made in vivo electrophysiology recordings of dorsal horn neurones in intact anaesthetised Nav 1.8-null mice, in response to a wide range of stimuli to further the understanding of the functional roles of Nav 1.8 in pain transmission from the periphery to the spinal cord.
Nav 1.8-null mice showed marked deficits in the coding by dorsal horn neurones to mechanical, but not thermal, -evoked responses over the non-noxious and noxious range compared to littermate controls. Additionally, responses evoked to other stimulus modalities were also significantly reduced in Nav 1.8-null mice where the reduction observed to pinch > brush. The occurrence of ongoing spontaneous neuronal activity was significantly less in mice lacking Nav 1.8 compared to control. No difference was observed between groups in the evoked activity to electrical activity of the peripheral receptive field.
This study demonstrates that deletion of the sodium channel Nav 1.8 results in stimulus-dependent deficits in the dorsal horn neuronal coding to mechanical, but not thermal stimuli applied to the neuronal peripheral receptive field. This implies that Nav 1.8 is either responsible for, or associated with proteins involved in mechanosensation.
Two voltage gated sodium channel α-subunits, Nav1.7 and Nav1.8, are expressed at high levels in nociceptor terminals and have been implicated in the development of inflammatory pain. Mis-expression of voltage-gated sodium channels by damaged sensory neurons has also been implicated in the development of neuropathic pain, but the role of Nav1.7 and Nav1.8 is uncertain. Here we show that deleting Nav1.7 has no effect on the development of neuropathic pain. Double knockouts of both Nav1.7 and Nav1.8 also develop normal levels of neuropathic pain, despite a lack of inflammatory pain symptoms and altered mechanical and thermal acute pain thresholds. These studies demonstrate that, in contrast to the highly significant role for Nav1.7 in determining inflammatory pain thresholds, the development of neuropathic pain does not require the presence of either Nav1.7 or Nav1.8 alone or in combination.
Human acute and inflammatory pain requires the expression of voltage-gated sodium channel Nav1.7 but its significance for neuropathic pain is unknown. Here we show that Nav1.7 expression in different sets of mouse sensory and sympathetic neurons underlies distinct types of pain sensation. Ablating Nav1.7 gene (SCN9A) expression in all sensory neurons using Advillin-Cre abolishes mechanical pain, inflammatory pain and reflex withdrawal responses to heat. In contrast, heat-evoked pain is retained when SCN9A is deleted only in Nav1.8-positive nociceptors. Surprisingly, responses to the hotplate test, as well as neuropathic pain, are unaffected when SCN9A is deleted in all sensory neurons. However, deleting SCN9A in both sensory and sympathetic neurons abolishes these pain sensations and recapitulates the pain-free phenotype seen in humans with SCN9A loss-of-function mutations. These observations demonstrate an important role for Nav1.7 in sympathetic neurons in neuropathic pain, and provide possible insights into the mechanisms that underlie gain-of-function Nav1.7-dependent pain conditions.
Sodium channel Nav1.7 is essential for acute human pain but its role in chronic neuropathic pain is unclear. Minett and colleagues show that Nav1.7 expression specifically in sympathetic neurons, rather than sensory neurons, is required for the development of chronic neuropathic pain after injury.
Genes encoding the α subunits of neuronal sodium channels have evolutionarily conserved sites of alternative splicing but no functional differences have been attributed to the splice variants. Here, using NaV1.7 as an exemplar, we show that the sodium channel isoforms are functionally distinct when co-expressed with β subunits. The gene, SCN9A, encodes the α subunit of the NaV1.7 channel, and contains both sites of alternative splicing that are highly conserved. In conditions where the intrinsic properties of the NaV1.7 splice variants were similar when expressed alone, co-expression of β1 subunits had different effects on channel availability that were determined by splicing at either site in the α subunit. While the identity of exon 5 determined the degree to which β1 subunits altered voltage-dependence of activation (P = 0.027), the length of exon 11 regulated how far β1 subunits depolarised voltage-dependence of inactivation (P = 0.00012). The results could have a significant impact on channel availability, for example with the long version of exon 11, the co-expression of β1 subunits could lead to nearly twice as large an increase in channel availability compared to channels containing the short version. Our data suggest that splicing can change the way that NaV channels interact with β subunits. Because splicing is conserved, its unexpected role in regulating the functional impact of β subunits may apply to multiple voltage-gated sodium channels, and the full repertoire of β subunit function may depend on splicing in α subunits.
Transient receptor potential (TRP) channels TRPC3 and TRPC6 are expressed in both sensory neurons and cochlear hair cells. Deletion of TRPC3 or TRPC6 in mice caused no behavioural phenotype, although loss of TRPC3 caused a shift of rapidly adapting (RA) mechanosensitive currents to intermediate-adapting currents in dorsal root ganglion sensory neurons. Deletion of both TRPC3 and TRPC6 caused deficits in light touch and silenced half of small-diameter sensory neurons expressing mechanically activated RA currents. Double TRPC3/TRPC6 knock-out mice also showed hearing impairment, vestibular deficits and defective auditory brain stem responses to high-frequency sounds. Basal, but not apical, cochlear outer hair cells lost more than 75 per cent of their responses to mechanical stimulation. FM1-43-sensitive mechanically gated currents were induced when TRPC3 and TRPC6 were co-expressed in sensory neuron cell lines. TRPC3 and TRPC6 are thus required for the normal function of cells involved in touch and hearing, and are potential components of mechanotransducing complexes.
mechanosensation; touch; hearing
Members of the degenerin/epithelial (DEG/ENaC) sodium channel family are mechanosensors in C elegans, and Nav1.7 and Nav1.8 voltage-gated sodium channel knockout mice have major deficits in mechanosensation. β and γENaC sodium channel subunits are present with acid sensing ion channels (ASICs) in mammalian sensory neurons of the dorsal root ganglia (DRG). The extent to which epithelial or voltage-gated sodium channels are involved in transduction of mechanical stimuli is unclear.
Here we show that deleting β and γENaC sodium channels in sensory neurons does not result in mechanosensory behavioural deficits. We had shown previously that Nav1.7/Nav1.8 double knockout mice have major deficits in behavioural responses to noxious mechanical pressure. However, all classes of mechanically activated currents in DRG neurons are unaffected by deletion of the two sodium channels. In contrast, the ability of Nav1.7/Nav1.8 knockout DRG neurons to generate action potentials is compromised with 50% of the small diameter sensory neurons unable to respond to electrical stimulation in vitro.
Behavioural deficits in Nav1.7/Nav1.8 knockout mice reflects a failure of action potential propagation in a mechanosensitive set of sensory neurons rather than a loss of primary transduction currents. DEG/ENaC sodium channels are not mechanosensors in mouse sensory neurons.
Mechanotransduction; Sodium channels; Pain; Nav1.7; Nav1.8; ENaCs
EphB receptors and their ephrin-B ligands play an important role in nervous system development, as well as synapse formation and plasticity in the adult brain. Recent studies show that intrathecal treatment with EphB-receptor activator ephrinB2-Fc induced thermal hyperalgesia and mechanical allodynia in rat, indicating that ephrin-B2 in small dorsal root ganglia (DRG) neurons and EphB receptors in the spinal cord modulate pain processing. To examine the role of ephrin-B2 in peripheral pain pathways, we deleted ephrin-B2 in Nav1.8+ nociceptive sensory neurons with the Cre-loxP system. Sensory neuron numbers and terminals were examined using neuronal makers. Pain behavior in acute, inflammatory and neuropathic pain models was assessed in the ephrin-B2 conditional knockout (CKO) mice. We also investigated the c-Fos expression and NMDA receptor NR2B phosphorylation in ephrin-B2 CKO mice and littermate controls.
The ephrin-B2 CKO mice were healthy with no sensory neuron loss. However, pain-related behavior was substantially altered. Although acute pain behavior and motor co-ordination were normal, inflammatory pain was attenuated in ephrin-B2 mutant mice. Complete Freund's adjuvant (CFA)-induced mechanical hyperalgesia was halved. Formalin-induced pain behavior was attenuated in the second phase, and this correlated with diminished tyrosine phosphorylation of N-methyl-D-aspartic acid (NMDA) receptor subunit NR2B in the dorsal horn. Thermal hyperalgesia and mechanical allodynia were significantly reduced in the Seltzer model of neuropathic pain.
Presynaptic ephrin-B2 expression thus plays an important role in regulating inflammatory pain through the regulation of synaptic plasticity in the dorsal horn and is also involved in the pathogenesis of some types of neuropathic pain.