Neuronal excitability relies on coordinated action of functionally distinction channels. Voltage-gated sodium (Na V ) and potassium (K V ) channels have distinct but complementary roles in firing action potentials: Na V channels provide depolarizing current while K V channels provide hyperpolarizing current. Mutations and dysfunction of multiple Na V and K V channels underlie disorders of excitability, including pain and epilepsy. Modulating ion channel trafficking may offer a potential therapeutic strategy for these diseases. A fundamental question, however, is whether these channels with distinct functional roles are transported independently or packaged together in the same vesicles in sensory axons. We have used Optical Pulse-Chase Axonal Long-distance imaging to investigate trafficking of Na V and K V channels and other axonal proteins from distinct functional classes in live rodent sensory neurons (from male and female rats). We show that, similar to Na V 1.7 channels, Na V 1.8 and K V 7.2 channels are transported in Rab6a-positive vesicles, and that each of the Na V channel isoforms expressed in healthy, mature sensory neurons (Na V 1.6, Na V 1.7, Na V 1.8, and Na V 1.9) is cotransported in the same vesicles. Further, we show that multiple axonal membrane proteins with different physiological functions (Na V 1.7, K V 7.2, and TNFR1) are cotransported in the same vesicles. However, vesicular packaging of axonal membrane proteins is not indiscriminate, since another axonal membrane protein (NCX2) is transported in separate vesicles. These results shed new light on the development and organization of sensory neuron membranes, revealing complex sorting of axonal proteins with diverse physiological functions into specific transport vesicles.
Small fiber neuropathy (SFN) is a common condition affecting thinly myelinated Aδ and unmyelinated C fibers, often resulting in excruciating pain and dysautonomia. SFN has been associated with several conditions, but a significant number of cases have no discernible cause. Recent genetic studies have identified potentially pathogenic gain-of-function mutations in several the pore-forming voltage-gated sodium channel α subunits (NaVs) in a subset of patients with SFN, but the auxiliary sodium channel β subunits have been less implicated in the development of the disease. β subunits modulate NaV trafficking and gating, and several mutations have been linked to epilepsy and cardiac dysfunction. Recently, we provided the first evidence for the contribution of a mutation in the β2-subunit to pain in human painful diabetic neuropathy. Here, we provide the first evidence for the involvement of a sodium channel β subunit mutation in the pathogenesis of SFN with no other known causes. We show, through current-clamp analysis, that the newly-identified Y69H variant of the β2 subunit induces neuronal hyperexcitability in dorsal root ganglion neurons, lowering the threshold for action potential firing and allowing for increased repetitive action potential spiking. Underlying the hyperexcitability induced by the β2-Y69H variant, we demonstrate an upregulation in tetrodotoxin-sensitive, but not tetrodotoxin-resistant sodium currents. This provides the first evidence for the involvement of β2 subunits in SFN and strengthens the link between sodium channel β subunits and the development of neuropathic pain in humans.
Fibroblast Growth Factor Homologous Factors (FHFs) are cytosolic members of the of the FGF proteins. Four members of this subfamily (FHF1-4) are differentially expressed in multiple tissues in an isoform-dependent manner. Mutations in FHF proteins have been associated with multiple neurological disorders. FHF proteins bind to the C-terminus of voltage-gated sodium (Nav) channels and regulate current amplitude and gating properties of these channels. FHF2, which is expressed in DRG neurons, has two main splicing isoforms, FHF2A and FHF2B, which differ in the length and sequence of their N-termini, have been shown to differentially regulate gating properties of Nav1.7, a channel that is a major driver of DRG neuron firing. FHF2 expression levels are downregulated following peripheral nerve axotomy, which suggests that they may regulate neuronal excitability via an action on Nav channels after injury. We have previously shown that knockdown of FHF2 leads to gain-of-function changes in Nav1.7 gating properties: enhanced repriming, increased current density and hyperpolarized activation. From this we posited that knockdown of FHF2 might also lead to DRG hyperexcitability. Here we show that knockdown of either FHF2A alone or all isoforms of FHF2 results in increased DRG neuron excitability. In addition, we demonstrate that supplementation of FHF2A and FHF2B reduces DRG neuron excitability. Overexpression of FHF2A or FHF2B also reduced excitability of DRG neurons treated with a cocktail of inflammatory mediators, a model of inflammatory pain. Our data suggest that increased neuronal excitability after nerve injury might be triggered, in part, via a loss of FHF2-Nav1.7 interaction.
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