Nav1.7 as a chondrocyte regulator and therapeutic target for osteoarthritis.

Osteoarthritis (OA) is the most common joint disease. Currently there are no effective methods that simultaneously prevent joint degeneration and reduce pain1. Although limited evidence suggests the existence of voltage-gated sodium channels (VGSCs) in chondrocytes2, their expression and function in chondrocytes and in OA remain essentially unknown. Here we identify Nav1.7 as an OA-associated VGSC and demonstrate that human OA chondrocytes express functional Nav1.7 channels, with a density of 0.1 to 0.15 channels per µm2 and 350 to 525 channels per cell. Serial genetic ablation of Nav1.7 in multiple mouse models demonstrates that Nav1.7 expressed in dorsal root ganglia neurons is involved in pain, whereas Nav1.7 in chondrocytes regulates OA progression. Pharmacological blockade of Nav1.7 with selective or clinically used pan-Nav channel blockers significantly ameliorates the progression of structural joint damage, and reduces OA pain behaviour. Mechanistically, Nav1.7 blockers regulate intracellular Ca2+ signalling and the chondrocyte secretome, which in turn affects chondrocyte biology and OA progression. Identification of Nav1.7 as a novel chondrocyte-expressed, OA-associated channel uncovers a dual target for the development of disease-modifying and non-opioid pain relief treatment for OA.

Ih current stabilizes excitability in rodent DRG neurons and reverses hyperexcitability in a nociceptive neuron model of inherited neuropathic pain.

We show here that hyperpolarization-activated current (Ih ) unexpectedly acts to inhibit the activity of dorsal root ganglion (DRG) neurons expressing WT Nav1.7, the largest inward current and primary driver of DRG neuronal firing, and hyperexcitable DRG neurons expressing a gain-of-function Nav1.7 mutation that causes inherited erythromelalgia (IEM), a human genetic model of neuropathic pain. In this study we created a kinetic model of Ih and used it, in combination with dynamic-clamp, to study Ih function in DRG neurons. We show, for the first time, that Ih increases rheobase and reduces the firing probability in small DRG neurons, and demonstrate that the amplitude of subthreshold oscillations is reduced by Ih . Our results show that Ih , due to slow gating, is not deactivated during action potentials (APs) and has a striking damping action, which reverses from depolarizing to hyperpolarizing, close to the threshold for AP generation. Moreover, we show that Ih reverses the hyperexcitability of DRG neurons expressing a gain-of-function Nav1.7 mutation that causes IEM. In the aggregate, our results show that Ih unexpectedly has strikingly different effects in DRG neurons as compared to previously- and well-studied cardiac cells. Within DRG neurons where Nav1.7 is present, Ih reduces depolarizing sodium current inflow due to enhancement of Nav1.7 channel fast inactivation and creates additional damping action by reversal of Ih direction from depolarizing to hyperpolarizing close to the threshold for AP generation. These actions of Ih limit the firing of DRG neurons expressing WT Nav1.7 and reverse the hyperexcitability of DRG neurons expressing a gain-of-function Nav1.7 mutation that causes IEM. KEY POINTS: Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels, the molecular determinants of hyperpolarization-activated current (Ih ) have been characterized as a 'pain pacemaker', and thus considered to be a potential molecular target for pain therapeutics. Dorsal root ganglion (DRG) neurons express Nav1.7, a channel that is not present in central neurons or cardiac tissue. Gain-of-function mutations (GOF) of Nav1.7 identified in inherited erythromelalgia (IEM), a human genetic model of neuropathic pain, produce DRG neuron hyperexcitability, which in turn produces severe pain. We found that Ih increases rheobase and reduces firing probability in small DRG neurons expressing WT Nav1.7, and demonstrate that the amplitude of subthreshold oscillations is reduced by Ih . We also demonstrate that Ih reverses the hyperexcitability of DRG neurons expressing a GOF Nav1.7 mutation (L858H) that causes IEM. Our results show that, in contrast to cardiac cells and CNS neurons, Ih acts to stabilize DRG neuron excitability and prevents excessive firing.

Dynamic-clamp analysis of wild-type human Nav1.7 and erythromelalgia mutant channel L858H.

The link between sodium channel Nav1.7 and pain has been strengthened by identification of gain-of-function mutations in patients with inherited erythromelalgia (IEM), a genetic model of neuropathic pain in humans. A firm mechanistic link to nociceptor dysfunction has been precluded because assessments of the effect of the mutations on nociceptor function have thus far depended on electrophysiological recordings from dorsal root ganglia (DRG) neurons transfected with wild-type (WT) or mutant Nav1.7 channels, which do not permit accurate calibration of the level of Nav1.7 channel expression. Here, we report an analysis of the function of WT Nav1.7 and IEM L858H mutation within small DRG neurons using dynamic-clamp. We describe the functional relationship between current threshold for action potential generation and the level of WT Nav1.7 conductance in primary nociceptive neurons and demonstrate the basis for hyperexcitability at physiologically relevant levels of L858H channel conductance. We demonstrate that the L858H mutation, when modeled using dynamic-clamp at physiological levels within DRG neurons, produces a dramatically enhanced persistent current, resulting in 27-fold amplification of net sodium influx during subthreshold depolarizations and even greater amplification during interspike intervals, which provide a mechanistic basis for reduced current threshold and enhanced action potential firing probability. These results show, for the first time, a linear correlation between the level of Nav1.7 conductance and current threshold in DRG neurons. Our observations demonstrate changes in sodium influx that provide a mechanistic link between the altered biophysical properties of a mutant Nav1.7 channel and nociceptor hyperexcitability underlying the pain phenotype in IEM.

The G1662S NaV1.8 mutation in small fibre neuropathy: impaired inactivation underlying DRG neuron hyperexcitability.

OBJECTIVE: Painful small fibre neuropathy (SFN) represents a significant public health problem, with no cause apparent in one-half of cases (termed idiopathic, I-SFN). Gain-of-function mutations of sodium channel NaV1.7 have recently been identified in nearly 30% of patients with biopsy-confirmed I-SFN. More recently, gain-of-function mutations of NaV1.8 have been found in patients with I-SFN. These NaV1.8 mutations accelerate recovery from inactivation, enhance the response to slow depolarisations, and enhance activation at the channel level, thereby producing hyperexcitability of small dorsal root ganglion (DRG) neurons, which include nociceptors, at the cellular level. Identification and functional profiling of additional NaV1.8 variants are necessary to determine the spectrum of changes in channel properties that underlie DRG neuron hyperexcitability in these patients. METHODS: Two patients with painful SFN were evaluated by skin biopsy, quantitative sensory testing, nerve conduction studies, screening of genomic DNA for mutations in SCN9A and SCN10A and electrophysiological functional analysis. RESULTS: A novel sodium channel NaV1.8 mutation G1662S was identified in both patients. Voltage-clamp analysis revealed that the NaV1.8/G1662S substitution impairs fast-inactivation, depolarising the midpoint (V1/2) by approximately 7 mV. Expression of G1662S mutant channels within DRG neurons rendered these cells hyperexcitable. CONCLUSIONS: We report for the first time a mutation of NaV1.8 which impairs inactivation, in patients with painful I-SFN. Together with our earlier results, our observations indicate that an array of NaV1.8 mutations, which affect channel function in multiple ways, can contribute to the pathophysiology of painful peripheral neuropathy.

Multistate structural modeling and voltage-clamp analysis of epilepsy/autism mutation Kv10.2-R327H demonstrate the role of this residue in stabilizing the channel closed state.

Voltage-gated potassium channel Kv10.2 (KCNH5) is expressed in the nervous system, but its functions and involvement in human disease are poorly understood. We studied a human Kv10.2 channel mutation (R327H) recently identified in a child with epileptic encephalopathy and autistic features. Using multistate structural modeling, we demonstrate that the Arg327 residue in the S4 helix of voltage-sensing domain has strong ionic interactions with negatively charged residues within the S1-S3 helices in the resting (closed) and early-activation state but not in the late-activation and fully-activated (open) state. The R327H mutation weakens ionic interactions between residue 327 and these negatively charged residues, thus favoring channel opening. Voltage-clamp analysis showed a strong hyperpolarizing (∼70 mV) shift of voltage dependence of activation and an acceleration of activation. Our results demonstrate the critical role of the Arg327 residue in stabilizing the channel closed state and explicate for the first time the structural and functional change of a Kv10.2 channel mutation associated with neurological disease.

Differential effect of D623N variant and wild-type Na(v)1.7 sodium channels on resting potential and interspike membrane potential of dorsal root ganglion neurons.

Sodium channel NaV1.7 is preferentially expressed in dorsal root ganglion (DRG) and sympathetic ganglion neurons. Gain-of-function NaV1.7 mutations/variants have been identified in the painful disorders inherited erythromelalgia and small-fiber neuropathy (SFN). DRG neurons transfected with these channel variants display depolarized resting potential, reduced current-threshold, increased firing-frequency and spontaneous firing. Whether the depolarizing shift in resting potential and enhanced spontaneous firing are due to persistent activity of variant channels, or to compensatory changes in other conductance(s) in response to expression of the variant channel, as shown in model systems, has not been studied. We examined the effect of wild-type NaV1.7 and a NaV1.7 mutant channel, D623N, associated with SFN, on resting potential and membrane potential during interspike intervals in DRG neurons. Resting potential in DRG neurons expressing D623N was depolarized compared to neurons expressing WT-NaV1.7. Exposure to TTX hyperpolarized resting potential by 7mV, increased current-threshold, decreased firing-frequency, and reduced NMDG-induced-hyperpolarization in DRG neurons expressing D623N. To assess the contribution of depolarized resting potential to DRG neuron excitability, we mimicked the mutant channel's depolarizing effect by current injection to produce equivalent depolarization; the depolarization decreased current threshold and increased firing-frequency. Voltage-clamp using ramp or repetitive action potentials as commands showed that D623N channels enhance the TTX-sensitive inward current, persistent at subthreshold membrane voltages, as predicted by a Hodgkin-Huxley model. Our results demonstrate that a variant of NaV1.7 associated with painful neuropathy depolarizes resting membrane potential and produces an enhanced inward current during interspike intervals, thereby contributing to DRG neuron hyperexcitability.

Membrane properties and electrogenesis in the distal axons of small dorsal root ganglion neurons in vitro.

Although it is generally thought that sensory transduction occurs at or close to peripheral nerve endings, with action potentials subsequently propagating along the axons of dorsal root ganglia (DRG) neurons toward the central nervous system, the small diameter of nociceptive axons and their endings have made it difficult to estimate their membrane properties and electrogenic characteristics. Even the resting potentials of nociceptive axons are unknown. In this study, we developed the capability to record directly with patch-clamp electrodes from the small-diameter distal axons of DRG neurons in vitro. We showed using current-clamp recordings that 1) these sensory axons have a resting potential of -60.2 ± 1 mV; 2) both tetrodotoxin (TTX)-sensitive (TTX-S) and TTX-resistant (TTX-R) Na(+) channels are present and available for activation at resting potential, at densities that can support action potential electrogenesis in these axons; 3) TTX-sensitive channels contribute to the amplification of small depolarizations that are subthreshold with respect to the action potential in these axons; 4) TTX-R channels can support the production of action potentials in these axons; and 5) these TTX-R channels can produce repetitive firing, even at depolarized membrane potentials where TTX-S channels are inactivated. Finally, using voltage-clamp recordings with an action potential as the command, we confirmed the presence of both TTX-S and TTX-R channels, which are activated sequentially during action potential in these axons. These results provide direct evidence for the presence of TTX-S and TTX-R Na(+) channels that are functionally available at resting potential and contribute to electrogenesis in small-diameter afferent axons.

Amiloride is neuroprotective in an MPTP model of Parkinson's disease.

The diuretic amiloride has recently proven neuroprotective in models of cerebral ischemia, a property attributable to the drug's inhibition of central acid-sensing ion channels (ASICs). Given that Parkinson's disease (PD), like ischemia, is associated with cerebral lactic acidosis, we tested amiloride in the MPTP-treated mouse, a model of PD also manifesting lactic acidosis. Amiloride was found to protect substantia nigra (SNc) neurons from MPTP-induced degeneration, as determined by attenuated reductions in striatal tyrosine hydroxylase (TH) and dopamine transporter (DAT) immunohistochemistry, as well as smaller declines in striatal DAT radioligand binding and dopamine levels. More significantly, amiloride also preserved dopaminergic cell bodies in the SNc. Administration of psalmotoxin venom (PcTX), an ASIC1a blocker, resulted in a much more modest effect, attenuating only the deficits in striatal DAT binding and dopamine. These findings represent the first experimental evidence of a potential role for ASICs in the pathogenesis of Parkinson's disease.

The LXR agonist TO901317 selectively lowers hippocampal Abeta42 and improves memory in the Tg2576 mouse model of Alzheimer's disease.

Recent studies show that intracellular cholesterol levels can modulate the processing of amyloid precursor protein to Abeta peptide. Moreover, cholesterol-rich apoE-containing lipoproteins may also promote Abeta clearance. Agonists of the liver X receptor (LXR) transcriptionally induce genes involved in intracellular lipid efflux and transport, including apoE. Thus, LXR agonists have the potential to both inhibit APP processing and promote Abeta clearance. Here we show that LXR agonist, TO901317, increased hippocampal ABCA1 and apoE and decreased Abeta42 levels in APP transgenic mice. TO901317 had no significant effects on levels of Abeta40, full length APP, or the APP processing products. Next, we examined the effects of TO901317 in the contextual fear conditioning paradigm; TO901317 completely reversed the contextual memory deficit in these mice. These data demonstrate that LXR agonists do not directly inhibit APP processing but rather facilitate the clearance of Abeta42 and may represent a novel therapeutic approach to Alzheimer's disease.

The effect of mGlu5 receptor positive allosteric modulators on signaling molecules in brain slices.

Positive allosteric modulators of metabotropic glutamate receptor subtype 5 (mGlu5) have promising therapeutic potential. The effects of selective mGlu5 receptor positive allosteric modulators on signaling molecules in brain slices have not been previously reported. The current study demonstrated that the selective mGlu5 receptor positive allosteric modulator, N-{4-chloro-2-[(1,3-dioxo-1,3-dihydro-2H-isoindol-2yl)-methyl]phenyl}-2-hydrobenzamide (CPPHA) potentiated the response to a subthreshold concentration of 3,5-dihydroxy-phenylglycine (DHPG) on extracellular signal-regulated protein kinase (ERK) and cyclic-AMP responsive element-binding protein (CREB) activity, as well as N-methyl d-aspartate (NMDA) receptor subunit NR1 phosphorylation in cortical and hippocampal slices. These results suggest that allosteric modulators of mGlu5 receptor could have physiologically significant effects by potentiating the actions of glutamate.