Animal models of rheumatoid pain: experimental systems and insights.

Severe chronic pain is one of the hallmarks and most debilitating manifestations of inflammatory arthritis. It represents a significant problem in the clinical management of patients with common chronic inflammatory joint conditions such as rheumatoid arthritis, psoriatic arthritis and spondyloarthropathies. The functional links between peripheral inflammatory signals and the establishment of the neuroadaptive mechanisms acting in nociceptors and in the central nervous system in the establishment of chronic and neuropathic pain are still poorly understood, representing an area of intense study and translational priority. Several well-established inducible and spontaneous animal models are available to study the onset, progression and chronicization of inflammatory joint disease, and have been instrumental in elucidating its immunopathogenesis. However, quantitative assessment of pain in animal models is technically and conceptually challenging, and it is only in recent years that inflammatory arthritis models have begun to be utilized systematically in experimental pain studies using behavioral and neurophysiological approaches to characterize acute and chronic pain stages. This article aims primarily to provide clinical and experimental rheumatologists with an overview of current animal models of arthritis pain, and to summarize emerging findings, challenges and unanswered questions in the field.

Chronic exposure to tumor necrosis factor in vivo induces hyperalgesia, upregulates sodium channel gene expression and alters the cellular electrophysiology of dorsal root ganglion neurons.

The goal of these studies was to investigate the links between chronic exposure to the pro-inflammatory cytokine tumor necrosis factor (TNF), hyperalgesia and the excitability of dorsal root ganglion (DRG) sensory neurons. We employed transgenic mice that constitutively express TNF (TNFtg mice), a well-established model of chronic systemic inflammation. At 6 months of age, TNFtg mice demonstrated increased sensitivity to both mechanical and thermal heat stimulation relative to aged-matched wild-type controls. These increases in stimulus-evoked behaviors are consistent with nociceptor sensitization to normal physiological stimulation. The mechanisms underlying nociceptor sensitization were investigated using single-cell analysis to quantitatively compare gene expression in small-diameter (<30µm) DRG neurons. This analysis revealed the upregulation of mRNA encoding for tetrodotoxin-resistant (TTX-R) sodium (Na+) channels (Nav1.8, Nav1.9), Na+ channel ß subunits (ß1-ß3), TNF receptor 1 (TNFR1) and p38α mitogen-activated protein kinase in neurons of TNFtg mice. Whole-cell electrophysiology demonstrated a corresponding increase in TTX-R Na+ current density, hyperpolarizing shifts in activation and steady-state inactivation, and slower recovery from inactivation in the TNFtg neurons. Increased overlap of activation and inactivation in the TNFtg neurons produces inward Na+ currents at voltages near the resting membrane potential of sensory neurons (i.e. window currents). The combination of increased Na+ current amplitude, hyperpolarized shifts in Na+ channel activation and increased window current predicts a reduction in the action potential threshold and increased firing of small-diameter DRG neurons. Together, these data suggest that increases in the expression of Nav1.8 channels, regulatory ß1 subunits and TNFR1 contribute to increased nociceptor excitability and hyperalgesia in the TNFtg mice.

MTSET modification of D4S6 cysteines stabilize the fast inactivated state of Nav1.5 sodium channels.

The transmembrane S6 segments of Na(+) sodium channels form the cytoplasmic entrance of the channel and line the internal aspects of the aqueous pore. This region of the channel has been implicated in Na(+) channel permeation, gating, and pharmacology. In this study we utilized cysteine substitutions and methanethiosulfonate reagent (MTSET) to investigate the role of the S6 segment of homologous domain 4 (D4S6) in the gating of the cardiac (Nav1.5) channel. D4S6 cysteine mutants were heterologously expressed in tsA201 cells and currents recorded using whole-cell patch clamp. Internal MTSET reduced the peak Na(+) currents, induced hyperpolarizing shifts in steady-state inactivation and slowed the recovery of mutant channels with cysteines inserted near the middle (F1760C, V1763C) and C-terminus (Y1767C) of the D4S6. These findings suggested a link between the MTSET inhibition and fast inactivation. This was confirmed by expressing the V1763C and Y1767C mutations in non-inactivating Nav1.5 channels. Removing inactivation abolished the MTSET inhibition of the V1763C and Y1767C mutants. The data indicate that the MTSET-induced reduction in current primarily results from slower recovery from inactivation that produces hyperpolarizing shifts in fast inactivation and decreases the steady-state availability of the channels. This contrasted with a cysteine inserted near the C-terminus of the D4S6 (I1770C) where MTSET increased the persistent Na(+) current at depolarized voltages consistent with impaired fast inactivation. Covalent modification of D4S6 cysteines with MTSET adduct appears to reduce the mobility of the D4S6 segment and stabilize the channels in the fast inactivated state. These findings indicate that residues located near the middle and C-terminus of the D4S6 play an important role in fast inactivation.

Dysregulation of Kv3.4 channels in dorsal root ganglia following spinal cord injury.

Spinal cord injury (SCI) patients develop chronic pain involving poorly understood central and peripheral mechanisms. Because dysregulation of the voltage-gated Kv3.4 channel has been implicated in the hyperexcitable state of dorsal root ganglion (DRG) neurons following direct injury of sensory nerves, we asked whether such a dysregulation also plays a role in SCI. Kv3.4 channels are expressed in DRG neurons, where they help regulate action potential (AP) repolarization in a manner that depends on the modulation of inactivation by protein kinase C (PKC)-dependent phosphorylation of the channel's inactivation domain. Here, we report that, 2 weeks after cervical hemicontusion SCI, injured rats exhibit contralateral hypersensitivity to stimuli accompanied by accentuated repetitive spiking in putative DRG nociceptors. Also in these neurons at 1 week after laminectomy and SCI, Kv3.4 channel inactivation is impaired compared with naive nonsurgical controls. At 2-6 weeks after laminectomy, however, Kv3.4 channel inactivation returns to naive levels. Conversely, Kv3.4 currents at 2-6 weeks post-SCI are downregulated and remain slow-inactivating. Immunohistochemistry indicated that downregulation mainly resulted from decreased surface expression of the Kv3.4 channel, as whole-DRG-protein and single-cell mRNA transcript levels did not change. Furthermore, consistent with Kv3.4 channel dysregulation, PKC activation failed to shorten the AP duration of small-diameter DRG neurons. Finally, re-expressing synthetic Kv3.4 currents under dynamic clamp conditions dampened repetitive spiking in the neurons from SCI rats. These results suggest a novel peripheral mechanism of post-SCI pain sensitization implicating Kv3.4 channel dysregulation and potential Kv3.4-based therapeutic interventions.

Regulation/modulation of sensory neuron sodium channels.

The pseudounipolar sensory neurons of the dorsal root ganglia (DRG) give rise to peripheral branches that convert thermal, mechanical, and chemical stimuli into electrical signals that are transmitted via central branches to the spinal cord. These neurons express unique combinations of tetrodotoxin-sensitive (TTX-S) and tetrodotoxin-resistant (TTX-R) Na(+) channels that contribute to the resting membrane potential, action potential threshold, and regulate neuronal firing frequency. The small-diameter neurons (<25 µm) isolated from the DRG represent the cell bodies of C-fiber nociceptors that express both TTX-S and TTX-R Na(+) currents. The large-diameter neurons (>35 µm) are typically low-threshold A-fibers that predominately express TTX-S Na(+) currents. Peripheral nerve damage, inflammation, and metabolic diseases alter the expression and function of these Na(+) channels leading to increases in neuronal excitability and pain. The Na(+) channels expressed in these neurons are the target of intracellular signaling cascades that regulate the trafficking, cell surface expression, and gating properties of these channels. Post-translational regulation of Na(+) channels by protein kinases (PKA, PKC, MAPK) alter the expression and function of the channels. Injury-induced changes in these signaling pathways have been linked to sensory neuron hyperexcitability and pain. This review examines the signaling pathways and regulatory mechanisms that modulate the voltage-gated Na(+) channels of sensory neurons.

Differential expression of sodium channel ß subunits in dorsal root ganglion sensory neurons.

The small-diameter (<25 µm) and large-diameter (>30 µm) sensory neurons of the dorsal root ganglion (DRG) express distinct combinations of tetrodotoxin sensitive and tetrodotoxin-resistant Na(+) channels that underlie the unique electrical properties of these neurons. In vivo, these Na(+) channels are formed as complexes of pore-forming α and auxiliary ß subunits. The goal of this study was to investigate the expression of ß subunits in DRG sensory neurons. Quantitative single-cell RT-PCR revealed that ß subunit mRNA is differentially expressed in small (ß(2) and ß(3)) and large (ß(1) and ß(2)) DRG neurons. This raises the possibility that ß subunit availability and Na(+) channel composition and functional regulation may differ in these subpopulations of sensory neurons. To further explore these possibilities, we quantitatively compared the mRNA expression of the ß subunit with that of Na(v)1.7, a TTX-sensitive Na(+) channel widely expressed in both small and large DRG neurons. Na(v)1.7 and ß subunit mRNAs were significantly correlated in small (ß(2) and ß(3)) and large (ß(1) and ß(2)) DRG neurons, indicating that these subunits are coexpressed in the same populations. Co-immunoprecipitation and immunocytochemistry indicated that Na(v)1.7 formed stable complexes with the ß(1)-ß(3) subunits in vivo and that Na(v)1.7 and ß(3) co-localized within the plasma membranes of small DRG neurons. Heterologous expression studies showed that ß(3) induced a hyperpolarizing shift in Na(v)1.7 activation, whereas ß(1) produced a depolarizing shift in inactivation and faster recovery. The data indicate that ß(3) and ß(1) subunits are preferentially expressed in small and large DRG neurons, respectively, and that these auxiliary subunits differentially regulate the gating properties of Na(v)1.7 channels.

Modulation of Kv3.4 channel N-type inactivation by protein kinase C shapes the action potential in dorsal root ganglion neurons.

Fast inactivation of heterologously expressed Kv3.4 channels is dramatically slowed upon phosphorylation of the channel's N-terminal (N-type) inactivation gate by protein kinase C (PKC). However, the presence and physiological importance of this exquisite modulation in excitable tissues were unknown. Here, we employed minimally invasive cell-attached patch-clamping, single-cell qPCR and specific siRNAs to unambiguously demonstrate that fast-inactivating Kv3.4 channels underlie a robust high voltage-activated A-type K(+) current (I(AHV)) in nociceptive dorsal root ganglion neurons from 7-day-old rats. We also show that PKC activation with phorbol 12,13-dibutyrate (PDBu) causes a 4-fold slowing of Kv3.4 channel inactivation and, consequently, accelerates the repolarization of the action potential (AP) by 22%, which shortens the AP duration by 14%. G-protein coupled receptor (GPCR) agonists eliminate I(AHV) fast inactivation in a membrane-delimited manner, suggesting a Kv3.4 channel signalling complex. Preincubation of the neurons with the PKC inhibitor bisindolylmaleimide II inhibits the effect of GPCR agonists and PDBu. Furthermore, activation of PKC via GPCR agonists recapitulates the effects of PDBu on the AP. Finally, transfection of the neurons with Kv3.4 siRNA prolongs the AP by 25% and abolishes the GPCR agonist-induced acceleration of the AP repolarization. These results show that Kv3.4 channels help shape the repolarization of the nociceptor AP, and that modulation of Kv3.4 channel N-type inactivation by PKC regulates AP repolarization and duration. We propose that the dramatic modulation of I(AHV) fast inactivation by PKC represents a novel mechanism of neural plasticity with potentially significant implications in the transition from acute to chronic pain.

Regulatory Role of Voltage-Gated Na Channel ß Subunits in Sensory Neurons.

Voltage-gated sodium Na(+) channels are membrane-bound proteins incorporating aqueous conduction pores that are highly selective for sodium Na(+) ions. The opening of these channels results in the rapid influx of Na(+) ions that depolarize the cell and drive the rapid upstroke of nerve and muscle action potentials. While the concept of a Na(+)-selective ion channel had been formulated in the 1940s, it was not until the 1980s that the biochemical properties of the 260-kDa and 36-kDa auxiliary ß subunits (ß(1), ß(2)) were first described. Subsequent cloning and heterologous expression studies revealed that the α subunit forms the core of the channel and is responsible for both voltage-dependent gating and ionic selectivity. To date, 10 isoforms of the Na(+) channel α subunit have been identified that vary in their primary structures, tissue distribution, biophysical properties, and sensitivity to neurotoxins. Four ß subunits (ß(1)-ß(4)) and two splice variants (ß(1A), ß(1B)) have been identified that modulate the subcellular distribution, cell surface expression, and functional properties of the α subunits. The purpose of this review is to provide a broad overview of ß subunit expression and function in peripheral sensory neurons and examine their contributions to neuropathic pain.

Regulation of Nav1.6 and Nav1.8 peripheral nerve Na+ channels by auxiliary ß-subunits.

Voltage-gated Na(+) (Na(v)) channels are composed of a pore-forming α-subunit and one or more auxiliary ß-subunits. The present study investigated the regulation by the ß-subunit of two Na(+) channels (Na(v)1.6 and Na(v)1.8) expressed in dorsal root ganglion (DRG) neurons. Single cell RT-PCR was used to show that Na(v)1.8, Na(v)1.6, and ß(1)-ß(3) subunits were widely expressed in individually harvested small-diameter DRG neurons. Coexpression experiments were used to assess the regulation of Na(v)1.6 and Na(v)1.8 by ß-subunits. The ß(1)-subunit induced a 2.3-fold increase in Na(+) current density and hyperpolarizing shifts in the activation (-4 mV) and steady-state inactivation (-4.7 mV) of heterologously expressed Na(v)1.8 channels. The ß(4)-subunit caused more pronounced shifts in activation (-16.7 mV) and inactivation (-9.3 mV) but did not alter the current density of cells expressing Na(v)1.8 channels. The ß(3)-subunit did not alter Na(v)1.8 gating but significantly reduced the current density by 31%. This contrasted with Na(v)1.6, where the ß-subunits were relatively weak regulators of channel function. One notable exception was the ß(4)-subunit, which induced a hyperpolarizing shift in activation (-7.6 mV) but no change in the inactivation or current density of Na(v)1.6. The ß-subunits differentially regulated the expression and gating of Na(v)1.8 and Na(v)1.6. To further investigate the underlying regulatory mechanism, ß-subunit chimeras containing portions of the strongly regulating ß(1)-subunit and the weakly regulating ß(2)-subunit were generated. Chimeras retaining the COOH-terminal domain of the ß(1)-subunit produced hyperpolarizing shifts in gating and increased the current density of Na(v)1.8, similar to that observed for wild-type ß(1)-subunits. The intracellular COOH-terminal domain of the ß(1)-subunit appeared to play an essential role in the regulation of Na(v)1.8 expression and gating.

Single-cell analysis of sodium channel expression in dorsal root ganglion neurons.

Sensory neurons of the dorsal root ganglia (DRG) express multiple voltage-gated sodium (Na) channels that substantially differ in gating kinetics and pharmacology. Small-diameter (<25 µm) neurons isolated from the rat DRG express a combination of fast tetrodotoxin-sensitive (TTX-S) and slow TTX-resistant (TTX-R) Na currents while large-diameter neurons (>30 µm) predominately express fast TTX-S Na current. Na channel expression was further investigated using single-cell RT-PCR to measure the transcripts present in individually harvested DRG neurons. Consistent with cellular electrophysiology, the small neurons expressed transcripts encoding for both TTX-S (Nav1.1, Nav1.2, Nav1.6, and Nav1.7) and TTX-R (Nav1.8 and Nav1.9) Na channels. Nav1.7, Nav1.8 and Nav1.9 were the predominant Na channels expressed in the small neurons. The large neurons highly expressed TTX-S isoforms (Nav1.1, Nav1.6, and Nav1.7) while TTX-R channels were present at comparatively low levels. A unique subpopulation of the large neurons was identified that expressed TTX-R Na current and high levels of Nav1.8 transcript. DRG neurons also displayed substantial differences in the expression of neurofilaments (NF200, peripherin) and Necl-1, a neuronal adhesion molecule involved in myelination. The preferential expression of NF200 and Necl-1 suggests that large-diameter neurons give rise to thick myelinated axons. Small-diameter neurons expressed peripherin, but reduced levels of NF200 and Necl-1, a pattern more consistent with thin unmyelinated axons. Single-cell analysis of Na channel transcripts indicates that TTX-S and TTX-R Na channels are differentially expressed in large myelinated (Nav1.1, Nav1.6, and Nav1.7) and small unmyelinated (Nav1.7, Nav1.8, and Nav1.9) sensory neurons.

Y1767C, a novel SCN5A mutation, induces a persistent Na+ current and potentiates ranolazine inhibition of Nav1.5 channels.

Long QT syndrome type 3 (LQT3) has been traced to mutations of the cardiac Na(+) channel (Na(v)1.5) that produce persistent Na(+) currents leading to delayed ventricular repolarization and torsades de pointes. We performed mutational analyses of patients suffering from LQTS and characterized the biophysical properties of the mutations that we uncovered. One LQT3 patient carried a mutation in the SCN5A gene in which the cysteine was substituted for a highly conserved tyrosine (Y1767C) located near the cytoplasmic entrance of the Na(v)1.5 channel pore. The wild-type and mutant channels were transiently expressed in tsA201 cells, and Na(+) currents were recorded using the patch-clamp technique. The Y1767C channel produced a persistent Na(+) current, more rapid inactivation, faster recovery from inactivation, and an increased window current. The persistent Na(+) current of the Y1767C channel was blocked by ranolazine but not by many class I antiarrhythmic drugs. The incomplete inactivation, along with the persistent activation of Na(+) channels caused by an overlap of voltage-dependent activation and inactivation, known as window currents, appeared to contribute to the LQTS phenotype in this patient. The blocking effect of ranolazine on the persistent Na(+) current suggested that ranolazine may be an effective therapeutic treatment for patients with this mutation. Our data also revealed the unique role for the Y1767 residue in inactivating and forming the intracellular pore of the Na(v)1.5 channel.

Role of voltage-gated sodium, potassium and calcium channels in the development of cocaine-associated cardiac arrhythmias.

Cocaine is a highly active stimulant that alters dopamine metabolism in the central nervous system resulting in a feeling of euphoria that with time can lead to addictive behaviours. Cocaine has numerous deleterious effects in humans including seizures, vasoconstriction, ischaemia, increased heart rate and blood pressure, cardiac arrhythmias and sudden death. The cardiotoxic effects of cocaine are indirectly mediated by an increase in sympathomimetic stimulation to the heart and coronary vasculature and by a direct effect on the ion channels responsible for maintaining the electrical excitability of the heart. The direct and indirect effects of cocaine work in tandem to disrupt the co-ordinated electrical activity of the heart and have been associated with life-threatening cardiac arrhythmias. This review focuses on the direct effects of cocaine on cardiac ion channels, with particular focus on sodium, potassium and calcium channels, and on the contributions of these channels to cocaine-induced arrhythmias. Companion articles in this edition of the journal examine the epidemiology of cocaine use (Wood & Dargan) and the treatment of cocaine-associated arrhythmias (Hoffmann).

Lidocaine promotes the trafficking and functional expression of Na(v)1.8 sodium channels in mammalian cells.

Nociceptive neurons of the dorsal root ganglion (DRG) express a combination of rapidly gating TTX-sensitive and slowly gating TTX-resistant Na currents, and the channels that produce these currents have been cloned. The Na(v)1.7 and Na(v)1.8 channels encode for the rapidly inactivating TTX-sensitive and slowly inactivating TTX-resistant Na currents, respectively. Although the Na(v)1.7 channel expresses well in cultured mammalian cell lines, attempts to express the Na(v)1.8 channel using similar approaches has been met with limited success. The inability to heterologously express Na(v)1.8 has hampered detailed characterization of the biophysical properties and pharmacology of these channels. In this study, we investigated the determinants of Na(v)1.8 expression in tsA201 cells, a transformed variant of HEK293 cells, using a combination of biochemistry, immunochemistry, and electrophysiology. Our data indicate that the unusually low expression levels of Na(v)1.8 in tsA201 cells results from a trafficking defect that traps the channel protein in the endoplasmic reticulum. Incubating the cultured cells with the local anesthetic lidocaine dramatically enhanced the cell surface expression of functional Na(v)1.8 channels. The biophysical properties of the heterologously expressed Na(v)1.8 channel are similar but not identical to those of the TTX-resistant Na current of native DRG neurons, recorded under similar conditions. Our data indicate that the lidocaine acts as a molecular chaperone that promotes efficient trafficking and increased cell surface expression of Na(v)1.8 channels.

State-dependent trapping of flecainide in the cardiac sodium channel.

Flecainide is a Class I antiarrhythmic drug and a potent inhibitor of the cardiac (Nav1.5) sodium channel. Although the flecainide inhibition of Nav1.5 is typically enhanced by depolarization, the contributions of the open and inactivated states to flecainide binding and inhibition remain controversial. We further investigated the state-dependent binding of flecainide by examining its inhibition of rapidly inactivating and non-inactivating mutants of Nav1.5 expressed in Xenopus oocytes. Applying flecainide while briefly depolarizing from a relatively negative holding potential resulted in a low-affinity inhibition of the channel (IC(50) = 345 microM). Increasing the frequency of stimulation potentiated the flecainide inhibition (IC(50) = 7.4 microM), which progressively increased over the range of voltages where Nav1.5 channels activated. This contrasts with sustained depolarizations that effectively stabilize the channels in inactivated states, which failed to promote significant flecainide inhibition. The voltage sensitivity and strong dependence of the flecainide inhibition on repetitive depolarization suggests that flecainide binding is facilitated by channel opening and that the drug does not directly bind to closed or inactivated channels. The binding of flecainide to open channels was further investigated in a non-inactivating mutant of Nav1.5. Flecainide produced a time-dependent decay in the current of the non-inactivating mutant that displayed kinetics consistent with a simple pore blocking mechanism (K(D) = 11 microM). At hyperpolarized voltages, flecainide slowed the recovery of both the rapidly inactivating (tau = 81 +/- 3 s) and non-inactivating (tau = 42 +/- 3 s) channels. Mutation of a conserved isoleucine of the D4S6 segment (I1756C) creates an alternative pathway that permits the rapid diffusion of anaesthetics out of the Nav1.5 channel. The I1756C mutation accelerated the recovery of both the rapidly inactivating (tau = 12.6 +/- 0.4 s) and non-inactivating (tau = 7.4 +/- 0.1 s) channels, suggesting that flecainide is trapped and not tightly bound within the pore when the channels are closed or inactivated. The data indicate that flecainide rapidly gains access to its binding site when the channel is open and inhibits Na(+) current by a pore blocking mechanism. Closing of either the activation or the inactivation gate traps flecainide within the pore resulting in the slow recovery of the drug-modified channels at hyperpolarized voltages.

Inhibition of HERG potassium channels by cocaethylene: a metabolite of cocaine and ethanol.

OBJECTIVE: To investigate the mechanism by which cocaethylene, a metabolite of cocaine and alcohol inhibits a cardiac delayed rectifier potassium channel. METHODS: The cDNA of the HERG potassium channel that underlies I(Kr) in humans was transiently expressed in tsA201 cells and currents recorded using the patch clamp technique. RESULTS: The cocaethylene inhibition of HERG is concentration-dependent with an IC(50) of 4.0 microM. The inhibition increases over the range of voltages where the channels activate suggesting that cocaethylene binding may be linked to the activation or opening of the channels. Cocaethylene slows the deactivation of the tail current indicating that drug-modified channels are stabilized in the open conformation. Cocaethylene also accelerates inactivation but has no effect of the recovery from inactivation. CONCLUSIONS: Cocaethylene inhibits HERG by binding to the activated or open channels and by modulating the kinetics of inactivation. The cocaethylene inhibition of the channels occurs within the range of concentrations detected in the plasma of humans following the ingestion of cocaine and alcohol and is likely to contribute to the potent cardiotoxicity of this drug combination.