Anthony M. Rush

Electrophysiological properties of two axonal sodium channels, Nav1.2 and Nav1.6, expressed in mouse spinal sensory neurones.

Sodium channels Nav1.2 and Nav1.6 are both normally expressed along premyelinated and myelinated axons at different stages of maturation and are also expressed in a subset of demyelinated axons, where coexpression of Nav1.6 together with the Na+/Ca2+ exchanger is associated with axonal injury. It has been difficult to distinguish the currents produced by Nav1.2 and Nav1.6 in native neurones, and previous studies have not compared these channels within neuronal expression systems. In this study, we have characterized and directly compared Nav1.2 and Nav1.6 in a mammalian neuronal cell background and demonstrate differences in their properties that may affect neuronal behaviour. The Nav1.2 channel displays more depolarized activation and availability properties that may permit conduction of action potentials, even with depolarization. However, Nav1.2 channels show a greater accumulation of inactivation at higher frequencies of stimulation (20–100 Hz) than Nav1.6 and thus are likely to generate lower frequencies of firing. Nav1.6 channels produce a larger persistent current that may play a role in triggering reverse Na+/Ca2+ exchange, which can injure demyelinated axons where Nav1.6 and the Na+/Ca2+ exchanger are colocalized, while selective expression of Nav1.2 may support action potential electrogenesis, at least at lower frequencies, while producing a smaller persistent current.

Sporadic onset of erythermalgia: a gain-of-function mutation in Nav1.7.

Inherited erythermalgia (erythromelalgia) is an autosomal dominant disorder in which patients experience severe burning pain in the extremities, in response to mild thermal stimuli and exercise. Although mutations in sodium channel Nav1.7 have been shown to underlie erythermalgia in several multigeneration families with the disease that have been investigated to date, the molecular basis of erythermalgia in sporadic cases is enigmatic. We investigated the role of Nav1.7 in a sporadic case of erythermalgia in a Chinese family.

PGE2 increases the tetrodotoxin-resistant Nav1.9 sodium current in mouse DRG neurons via G-proteins

Inflammation caused by tissue damage results in pain, reflecting an increase in excitability of the primary afferent neurons innervating the area. There is some evidence to suggest that altered function of voltage-gated sodium channels is responsible for the hyperexcitability produced by inflammatory agents, possibly acting through G-proteins, but the role of different channel subtypes has not been fully explored. The tetrodotoxin-resistant (TTX-R) sodium channel Na(v)1.9 is expressed selectively in C- and A-fibre nociceptive-type units and is upregulated by G-protein activation. In this study, we examined the effects of the inflammatory agent prostaglandin-E(2) (PGE(2)) on Na(v)1.9 current in both Na(v)1.8-null and wild-type (WT) mice and explored the role of specific G-proteins in modulation. PGE(2) caused a twofold increase in Na(v)1.9 current (p<0.05) in both systems. Steady-state activation was shifted in a hyperpolarizing direction by 6-8 mV and availability of channels by 12 mV. No differences in the activation and inactivation kinetics could be detected. The increase in current was blocked by pertussis toxin (PTX) but not cholera toxin (CTX), showing involvement of G(i/o) but not G(s) subunits. Our data indicate that Na(v)1.9 current can be increased during inflammation via a G-protein dependent mechanism and suggest that this could contribute to the regulation of electrogenesis in dorsal root ganglia (DRG) neurons.

Phosphorylation of Sodium Channel Nav1.8 by p38 Mitogen-Activated Protein Kinase Increases Current Density in Dorsal Root Ganglion Neurons

The sensory neuron-specific sodium channel Nav1.8 and p38 mitogen-activated protein kinase are potential therapeutic targets within nociceptive dorsal root ganglion (DRG) neurons in inflammatory, and possibly neuropathic, pain. Nav1.8 channels within nociceptive DRG neurons contribute most of the inward current underlying the depolarizing phase of action potentials. Nerve injury and inflammation of peripheral tissues cause p38 activation in DRG neurons, a process that may contribute to nociceptive neuron hyperexcitability, which is associated with pain. However, how substrates of activated p38 contribute to DRG neuron hyperexcitability is currently not well understood. We report here, for the first time, that Nav1.8 and p38 are colocalized in DRG neurons, that Nav1.8 within DRG neurons is a substrate for p38, and that direct phosphorylation of the Nav1.8 channel by p38 regulates its function in these neurons. We show that direct phosphorylation of Nav1.8 at two p38 phospho-acceptor serine residues on the L1 loop (S551 and S556) causes an increase in Nav1.8 current density that is not accompanied by changes in gating properties of the channel. Our study suggests a mechanism by which activated p38 contributes to inflammatory, and possibly neuropathic, pain through a p38-mediated increase of Nav1.8 current density.

Fibroblast growth factor homologous factor 2B: association with Nav1.6 and selective colocalization at nodes of Ranvier of dorsal root axons.

Voltage-gated sodium channels interact with cytosolic proteins that regulate channel trafficking and/or modulate the biophysical properties of the channels. Nav1.6 is heavily expressed at the nodes of Ranvier along adult CNS and PNS axons and along unmyelinated fibers in the PNS. In an initial yeast two-hybrid screen using the C terminus of Nav1.6 as a bait, we identified FHF2B, a member of the FGF homologous factor (FHF) subfamily, as an interacting partner of Nav1.6. Members of the FHF subfamily share ∼70% sequence identity, and individual members demonstrate a cell- and tissue-specific expression pattern. FHF2 is abundantly expressed in the hippocampus and DRG neurons and colocalizes with Nav1.6 at mature nodes of Ranvier in myelinated sensory fibers in the dorsal root of the sciatic nerve. However, retinal ganglion cells and spinal ventral horn motor neurons show very low levels of FHF2 expression, and their axons exhibit no nodal FHF2 staining within the optic nerve and ventral root, respectively. Thus, FHF2 is selectively localized at nodes of dorsal root sensory but not ventral root motor axons. The coexpression of FHF2B and Nav1.6 in the DRG-derived cell line ND7/23 significantly increases the peak current amplitude and causes a 4 mV depolarizing shift of voltage-dependent inactivation of the channel. The preferential expression of FHF2B in sensory neurons may provide a basis for physiological differences in sodium currents that have been reported at the nodes of Ranvier in sensory versus motor axons.

Voltage-Gated Sodium Channel Nav1.6 Is Modulated by p38 Mitogen-Activated Protein Kinase

Nav1.6 is the major sodium channel isoform at nodes of Ranvier in myelinated axons and, additionally, is distributed along unmyelinated C-fibers of sensory neurons. Thus, modulation of the sodium current produced by Nav1.6 might significantly impact axonal conduction. Mitogen-activated protein kinases (MAPKs) are expressed in neurons and are activated after injury, for example, after sciatic nerve transection and hypoxia. Although the role of MAPK in signal transduction and in injury-induced regulation of gene expression is well established, the ability of these kinases to phosphorylate and modulate voltage-gated sodium channels has not been reported. Sequence analysis shows that Nav1.6 contains a putative MAP kinase-recognition module in the cytoplasmic loop (L1), which joins domains 1 and 2. We show in this study that sodium channels and p38 MAP kinase colocalize in rat brain tissue and that activated p38α phosphorylates L1 of Nav1.6, specifically at serine 553 (S553), in vitro. None of the other cytoplasmic loops and termini of the channel are phosphorylated by activated p38α in these assays. Activation of p38 in the neuronal ND7/23 cell line transfected with Nav1.6 leads to a significant reduction in the peak Nav1.6 current amplitude, without a detectable effect on gating properties. The substitution of S553 with alanine within L1 of the Nav1.6 channel prevents p38-mediated reduction of Nav1.6 current density. This is the first demonstration of MAPK phosphorylation and modulation of a voltage-gated sodium channel, and this modulation may represent an additional role for MAPK in regulating the neuronal response to injury.

Contactin regulates the current density and axonal expression of tetrodotoxin-resistant but not tetrodotoxin-sensitive sodium channels in DRG neurons

Contactin, a glycosyl‐phosphatidylinositol (GPI)‐anchored predominantly neuronal cell surface glycoprotein, associates with sodium channels Nav1.2, Nav1.3 and Nav1.9, and enhances the density of these channels on the plasma membrane in mammalian expression systems. However, a detailed functional analysis of these interactions and of untested putative interactions with other sodium channel isoforms in mammalian neuronal cells has not been carried out. We examined the expression and function of sodium channels in small‐diameter dorsal root ganglion (DRG) neurons from contactin‐deficient (CNTN–/–) mice, compared to CNTN+/+ litter mates. Nav1.9 is preferentially expressed in isolectin B4 (IB4)‐positive neurons and thus we used this marker to subdivide small‐diameter DRG neurons. Using whole‐cell patch‐clamp recording, we observed a greater than two‐fold reduction of tetrodotoxin‐resistant (TTX‐R) Nav1.8 and Nav1.9 current densities in IB4+ DRG neurons cultured from CNTN–/– vs. CNTN+/+ mice. Current densities for TTX‐sensitive (TTX‐S) sodium channels were unaffected. Contactins effect was selective for IB4+ neurons as current densities for both TTX‐R and TTX‐S channels were not significantly different in IB4– DRG neurons from the two genotypes. Consistent with these results, we have demonstrated a reduction in Nav1.8 and Nav1.9 immunostaining on peripherin‐positive unmyelinated axons in sciatic nerves from CNTN–/– mice but detected no changes in the expression for the two major TTX‐S channels Nav1.6 and Nav1.7. These data provide evidence of a role for contactin in selectively regulating the cell surface expression and current densities of TTX‐R but not TTX‐S Na+ channel isoforms in nociceptive DRG neurons; this regulation could modulate the membrane properties and excitability of these neurons.

FGF14 N-terminal splice variants differentially modulate Nav1.2 and Nav1.6-encoded sodium channels.

The Intracellular Fibroblast Growth Factor (iFGF) subfamily includes four members (FGFs 11-14) of the structurally related FGF superfamily. Previous studies showed that the iFGFs interact directly with the pore-forming (alpha) subunits of voltage-gated sodium (Nav) channels and regulate the functional properties of sodium channel currents. Sequence heterogeneity among the iFGFs is thought to confer specificity to this regulation. Here, we demonstrate that the two N-terminal alternatively spliced FGF14 variants, FGF14-1a and FGF14-1b, differentially regulate currents produced by Nav1.2 and Nav1.6 channels. FGF14-1b, but not FGF14-1a, attenuates both Nav1.2 and Nav1.6 current densities. In contrast, co-expression of an FGF14 mutant, lacking the N-terminus, increased Nav1.6 current densities. In neurons, both FGF14-1a and FGF14-1b localized at the axonal initial segment, and deletion of the N-terminus abolished this localization. Thus, the FGF14 N-terminus is required for targeting and functional regulation of Nav channels, suggesting an important function for FGF14 alternative splicing in regulating neuronal excitability.

Differential modulation of sodium channel Nav1.6 by two members of the fibroblast growth factor homologous factor 2 subfamily

FHF2A and FHF2B are two members of the fibroblast growth factor homologous factor 2 (FHF2) subfamily with distinct N termini. Using a generic antibody and electrophysiological methods, we previously showed that FHF2 is expressed in hippocampus and dorsal root ganglion (DRG) neurons and is colocalized with sodium channel Nav1.6 at sensory but not motor nodes of Ranvier, and that FHF2B associates with Nav1.6, causing an increase in current density and a small depolarizing shift in availability of channels. Using immunolabeling of adult rat tissue, we demonstrate that FHF2A is present within DRG but not in hippocampal or cerebellar neurons or at nodes of Ranvier in sciatic nerve, and that Nav1.6 and FHF2A are colocalized in nonmyelinated fibers. We also show that FHF2A binds directly to Nav1.6, and that the two proteins coimmunoprecipitate from transfected HEK293 cells. Because Nav1.6 has been associated with rapid firing rates, we examined the possible effects of FHF2B and the sister isoform, FHF2A, on electrophysiological properties of this channel in the DRG‐derived ND7/23 cell line. We show that FHF2B inhibits accumulation of inactivation in response to trains of stimulation at high frequencies. In marked contrast, FHF2A causes an accumulation of inactivated channels at all frequencies tested due to a slowing of recovery from inactivation. Thus different FHF2 subfamily members have different functional effects on Nav1.6 and are differentially distributed in DRG neurons and their axons. This suggests that FHF2A and FHF2B may selectively alter firing behaviour of specific neuronal compartments via differential modulation of Nav1.6.

Contactin Associates with Sodium Channel Nav1.3 in Native Tissues and Increases Channel Density at the Cell Surface

The upregulation of voltage-gated sodium channel Nav1.3 has been linked to hyperexcitability of axotomized dorsal root ganglion (DRG) neurons, which underlies neuropathic pain. However, factors that regulate delivery of Nav1.3 to the cell surface are not known. Contactin/F3, a cell adhesion molecule, has been shown to interact with and enhance surface expression of sodium channels Nav1.2 and Nav1.9. In this study we show that contactin coimmunoprecipitates with Nav1.3 from postnatal day 0 rat brain where this channel is abundant, and from human embryonic kidney (HEK) 293 cells stably transfected with Nav1.3 (HEK-Nav1.3). Purified GST fusion proteins of the N and C termini of Nav1.3 pull down contactin from lysates of transfected HEK 293 cells. Transfection of HEK-Nav1.3 cells with contactin increases the amplitude of the current threefold without changing the biophysical properties of the channel. Enzymatic removal of contactin from the cell surface of cotransfected cells does not reduce the elevated levels of the Nav1.3 current. Finally, we show that, similar to Nav1.3, contactin is upregulated in axotomized DRG neurons and accumulates within the neuroma of transected sciatic nerve. We propose that the upregulation of contactin and its colocalization with Nav1.3 in axotomized DRG neurons may contribute to the hyper-excitablity of the injured neurons.