Andreas Leffler

Sensory neuron sodium channel Nav1.8 is essential for pain at low temperatures.

Sensory acuity and motor dexterity deteriorate when human limbs cool down, but pain perception persists and cold-induced pain can become excruciating. Evolutionary pressure to enforce protective behaviour requires that damage-sensing neurons (nociceptors) continue to function at low temperatures. Here we show that this goal is achieved by endowing superficial endings of slowly conducting nociceptive fibres with the tetrodotoxin-resistant voltage-gated sodium channel (VGSC) Nav1.8 (ref. 2). This channel is essential for sustained excitability of nociceptors when the skin is cooled. We show that cooling excitable membranes progressively enhances the voltage-dependent slow inactivation of tetrodotoxin-sensitive VGSCs. In contrast, the inactivation properties of Nav1.8 are entirely cold-resistant. Moreover, low temperatures decrease the activation threshold of the sodium currents and increase the membrane resistance, augmenting the voltage change caused by any membrane current. Thus, in the cold, Nav1.8 remains available as the sole electrical impulse generator in nociceptors that transmits nociceptive information to the central nervous system. Consistent with this concept is the observation that Nav1.8-null mutant mice show negligible responses to noxious cold and mechanical stimulation at low temperatures. Our data present strong evidence for a specialized role of Nav1.8 in nociceptors as the critical molecule for the perception of cold pain and pain in the cold.

The vanilloid receptor TRPV1 is activated and sensitized by local anesthetics in rodent sensory neurons

Local anesthetics (LAs) block the generation and propagation of action potentials by interacting with specific sites of voltage-gated Na(+) channels. LAs can also excite sensory neurons and be neurotoxic through mechanisms that are as yet undefined. Nonspecific cation channels of the transient receptor potential (TRP) channel family that are predominantly expressed by nociceptive sensory neurons render these neurons sensitive to a variety of insults. Here we demonstrated that the LA lidocaine activated TRP channel family receptors TRPV1 and, to a lesser extent, TRPA1 in rodent dorsal root ganglion sensory neurons as well as in HEK293t cells expressing TRPV1 or TRPA1. Lidocaine also induced a TRPV1-dependent release of calcitonin gene-related peptide (CGRP) from isolated skin and peripheral nerve. Lidocaine sensitivity of TRPV1 required segments of the putative vanilloid-binding domain within and adjacent to transmembrane domain 3, was diminished under phosphatidylinositol 4,5-bisphosphate depletion, and was abrogated by a point mutation at residue R701 in the proximal C-terminal TRP domain. These data identify TRPV1 and TRPA1 as putative key elements of LA-induced nociceptor excitation. This effect is sufficient to release CGRP, a key component of neurogenic inflammation, and warrants investigation into the role of TRPV1 and TRPA1 in LA-induced neurotoxicity.

Methylglyoxal Activates Nociceptors through Transient Receptor Potential Channel A1 (TRPA1) A POSSIBLE MECHANISM OF METABOLIC NEUROPATHIES

Background: Methylglyoxal is a reactive metabolite that modifies proteins and accumulates in diabetes and uremia. Results: Methylglyoxal excites nociceptors and releases neuropeptides via activation of TRPA1 channels by modifying their intracellular N-terminal cysteine and lysine residues. Conclusion: Methylglyoxal acting through TRPA1 is a possible cause of painful metabolic neuropathies. Significance: Methylglyoxal and its reaction with TRPA1 are promising targets for medicinal chemistry to fight neurotoxicity. Neuropathic pain can develop as an agonizing sequela of diabetes mellitus and chronic uremia. A chemical link between both conditions of altered metabolism is the highly reactive compound methylglyoxal (MG), which accumulates in all cells, in particular neurons, and leaks into plasma as an index of the severity of the disorder. The electrophilic structure of this cytotoxic ketoaldehyde suggests TRPA1, a receptor channel deeply involved in inflammatory and neuropathic pain, as a molecular target. We demonstrate that extracellularly applied MG accesses specific intracellular binding sites of TRPA1, activating inward currents and calcium influx in transfected cells and sensory neurons, slowing conduction velocity in unmyelinated peripheral nerve fibers, and stimulating release of proinflammatory neuropeptides from and action potential firing in cutaneous nociceptors. Using a model peptide of the N terminus of human TRPA1, we demonstrate the formation of disulfide bonds based on MG-induced modification of cysteines as a novel mechanism. In conclusion, MG is proposed to be a candidate metabolite that causes neuropathic pain in metabolic disorders and thus is a promising target for medicinal chemistry.

Sodium channelopathies and pain

Chronic pain often represents a severe, debilitating condition. Up to 10% of the worldwide population are affected, and many patients are poorly responsive to current treatment strategies. Nociceptors detect noxious conditions to produce the sensation of pain, and this signal is conveyed to the CNS by means of action potentials. The fast upstroke of action potentials is mediated by voltage-gated sodium channels, of which nine pore-forming α-subunits (Nav1.1–1.9) have been identified. Heterogeneous functional properties and distinct expression patterns denote specialized functions of each subunit. The Nav1.7 and Nav1.8 subunits have emerged as key molecules involved in peripheral pain processing and in the development of an increased pain sensitivity associated with inflammation and tissue injury. Several mutations in the SCN9A gene encoding for Nav1.7 have been identified as important cellular substrates for different heritable pain syndromes. This review aims to cover recent progress on our understanding of how biophysical properties of mutant Nav1.7 translate into an aberrant electrogenesis of nociceptors. We also recapitulate the role of Nav1.8 for peripheral pain processing and of additional sodium channelopathies which have been linked to disorders with pain as a significant component.

Pharmacological properties of neuronal TTX-resistant sodium channels and the role of a critical serine pore residue

Voltage-gated sodium channels can be characterized by their sensitivity to inhibitors. Nav1.5 is sensitive to block by cadmium and extracellular QX-314, but relatively insensitive to tetrodotoxin and saxitoxin. Nav1.4 is tetrodotoxin- and saxitoxin-sensitive but resistant to cadmium and extracellular QX-314. Nav1.8 and Nav1.9 generate slowly inactivating (ITTXr-Slow) and persistent (ITTXr-Per) currents in sensory neurons that are tetrodotoxin-resistant. Tetrodotoxin sensitivity is largely determined by the identity of a single residue; tyrosine 401 in Nav1.4, cysteine 374 in Nav1.5 and serine 356 and 355 in Nav1.8 and Nav1.9. We asked whether Nav1.8 and Nav1.9 share other pharmacological properties as a result of this serine residue. ITTXr-Slow and ITTXr-Per were saxitoxin-resistant and resistant to internal QX-314. ITTXr-Slow was also resistant to external QX-314 and displayed a approximately fourfold higher sensitivity than ITTXr-Per to cadmium. The impact of the serine residue was investigated by replacing tyrosine 401 in Nav1.4 with serine (Y401S) or cysteine (Y401C). Both mutants were resistant to tetrodotoxin and saxitoxin. Whereas Nav1.4-Y401C displayed an increased sensitivity to cadmium and extracellular QX-314, the serine substitution did not alter the sensitivity of Nav1.4 to cadmium or QX-314. Our data indicates that while the serine residue determines the sensitivity of ITTXr-Slow and ITTXr-Per to tetrodotoxin and saxitoxin, it does not determine their insensitivity to QX-314 or their differential sensitivities to cadmium.

Activation of TRPA1 by membrane permeable local anesthetics

BackgroundLow concentrations of local anesthetics (LAs) suppress cellular excitability by inhibiting voltage-gated Na+ channels. In contrast, LAs at high concentrations can be excitatory and neurotoxic. We recently demonstrated that LA-evoked activation of sensory neurons is mediated by the capsaicin receptor TRPV1, and, to a lesser extent by the irritant receptor TRPA1. LA-induced activation and sensitization of TRPV1 involves a domain that is similar, but not identical to the vanilloid-binding domain. Additionally, activation of TRPV1 by LAs involves PLC and PI(4,5)P2-signalling. In the present study we aimed to characterize essential structural determinants for LA-evoked activation of TRPA1.ResultsRecombinant rodent and human TRPA1 were expressed in HEK293t cells and investigated by means of whole-cell patch clamp recordings. The LA lidocaine activates TRPA1 in a concentration-dependent manner. The membrane impermeable lidocaine-derivative QX-314 is inactive when applied extracellularly. Lidocaine-activated TRPA1-currents are blocked by the TRPA1-antagonist HC-030031. Lidocaine is also an inhibitor of TRPA1, an effect that is more obvious in rodent than in human TRPA1. This species-specific difference is linked to the pore region (transmembrane domain 5 and 6) as described for activation of TRPA1 by menthol. Unlike menthol-sensitivity however, lidocaine-sensitivity is not similarly determined by serine- and threonine-residues within TM5. Instead, intracellular cysteine residues known to be covalently bound by reactive TRPA1-agonists seem to mediate activation of TRPA1 by LAs.ConclusionsThe structural determinants involved in activation of TRPA1 by LAs are disparate from those involved in activation by menthol or those involved in activation of TRPV1 by LAs.

The Molecular Basis for Species-specific Activation of Human TRPA1 Protein by Protons Involves Poorly Conserved Residues within Transmembrane Domains 5 and 6

Background: Extracellular acidosis mediates pain and inflammation by activating sensory afferent neurons. Results: Protons activate and sensitize human TRPA1 in a strongly species-specific manner encoded by transmembrane domains 5 and 6. Conclusion: Our data identify TRPA1 as an ion channel likely to mediate acid-induced pain in humans. Significance: Protons are the first known endogenous agonists of TRPA1 with species-specificity for human TRPA1. The surveillance of acid-base homeostasis is concerted by diverse mechanisms, including an activation of sensory afferents. Proton-evoked activation of rodent sensory neurons is mainly mediated by the capsaicin receptor TRPV1 and acid-sensing ion channels. In this study, we demonstrate that extracellular acidosis activates and sensitizes the human irritant receptor TRPA1 (hTRPA1). Proton-evoked membrane currents and calcium influx through hTRPA1 occurred at physiological acidic pH values, were concentration-dependent, and were blocked by the selective TRPA1 antagonist HC030031. Both rodent and rhesus monkey TRPA1 failed to respond to extracellular acidosis, and protons even inhibited rodent TRPA1. Accordingly, mouse dorsal root ganglion neurons lacking TRPV1 only responded to protons when hTRPA1 was expressed heterologously. This species-specific activation of hTRPA1 by protons was reversed in both mouse and rhesus monkey TRPA1 by exchange of distinct residues within transmembrane domains 5 and 6. Furthermore, protons seem to interact with an extracellular interaction site to gate TRPA1 and not via a modification of intracellular N-terminal cysteines known as important interaction sites for electrophilic TRPA1 agonists. Our data suggest that hTRPA1 acts as a sensor for extracellular acidosis in human sensory neurons and should thus be taken into account as a yet unrecognized transduction molecule for proton-evoked pain and inflammation. The species specificity of this property is unique among known endogenous TRPA1 agonists, possibly indicating that evolutionary pressure enforced TRPA1 to inherit the role as an acid sensor in human sensory neurons.

The general anesthetic propofol excites nociceptors by activating TRPV1 and TRPA1 rather than GABAA receptors.

Anesthetic agents can induce a paradox activation and sensitization of nociceptive sensory neurons and, thus, potentially facilitate pain processing. Here we identify distinct molecular mechanisms that mediate an activation of sensory neurons by 2,6-diisopropylphenol (propofol), a commonly used intravenous anesthetic known to elicit intense pain upon injection. Clinically relevant concentrations of propofol activated the recombinant transient receptor potential (TRP) receptors TRPA1 and TRPV1 heterologously expressed in HEK293t cells. In dorsal root ganglion (DRG) neurons, propofol-induced activation correlated better to expression of TRPA1 than of TRPV1. However, pretreatment with the protein kinase C activator 4β-phorbol 12-myristate 13-acetate (PMA) resulted in a significantly sensitized propofol-induced activation of TRPV1 in DRG neurons as well as in HEK293t cells. Pharmacological and genetic silencing of both TRPA1 and TRPV1 only partially abrogated propofol-induced responses in DRG neurons. The remaining propofol-induced activation was abolished by the selective γ-aminobutyric acid, type A (GABAA) receptor antagonist picrotoxin. Propofol but not GABA evokes a release of calcitonin gene-related peptide, a key component of neurogenic inflammation, from isolated peripheral nerves of wild-type but not TRPV1 and TRPA1-deficient mice. Moreover, propofol but not GABA induced an intense pain upon intracutaneous injection. As both the release of calcitonin gene-related peptide and injection pain by propofol seem to be independent of GABAA receptors, our data identify TRPV1 and TRPA1 as key molecules for propofol-induced excitation of sensory neurons. This study warrants further investigations into the role of anesthetics to induce nociceptor sensitization and to foster postoperative pain.

Local anesthetic-like inhibition of voltage-gated Na(+) channels by the partial μ-opioid receptor agonist buprenorphine.

Background: Opioids induce analgesia mainly by inhibiting synaptic transmission via G protein-coupled opioid receptors. In addition to analgesia, buprenorphine induces a pronounced antihyperalgesia and is an effective adjuvant to local anesthetics. These properties only partially apply to other opioids, and thus targets other than opioid receptors are likely to be employed. Here we asked if buprenorphine inhibits voltage-gated Na+ channels. Methods: Na+ currents were examined by whole cell patch clamp recordings on different recombinant Na+ channel &agr;-subunits. The effect of buprenorphine on unmyelinated mouse C-fibers was examined with the skin-nerve preparation. Data are presented as mean ± SEM. Results: Buprenorphine induced a concentration-dependent tonic (IC50 33 ± 2 &mgr;M) and use-dependent block of endogenous Na+ channels in ND7/23 cells. This block was state-dependent and displayed slow on and off characteristics. The effect of buprenorphine was reduced on local anesthetic insensitive Nav1.4-mutant constructs and was more pronounced on the inactivation-deficient Nav1.4-WCW mutant. Neuronal (Nav1.3, Nav1.7, and Nav1.8), cardiac (Nav1.5), and skeletal muscle (Nav1.4) &agr;-subunits displayed small differences in tonic block, but similar degrees of use-dependent block. According to our patch clamp data, buprenorphine blocked electrically evoked action potentials in C-fiber nerve terminals. Buprenorphine was more potent than other opioids, including morphine (IC50 378 ± 20 &mgr;M), fentanyl (IC50 95 ± 5 &mgr;M), sufentanil (IC50 111 ± 6 &mgr;M), remifenatil (IC50 612 ± 17 &mgr;M), and tramadol (IC50 194 ± 9 &mgr;M). Conclusions: Buprenorphine is a potent local anesthetic and blocks voltage-gated Na+ channels via the local anesthetic binding site. This property is likely to be relevant when buprenorphine is used for pain treatment and for local anesthesia.

Alternative splicing may contribute to time-dependent manifestation of inherited erythromelalgia.

The Na(v)1.7 sodium channel is preferentially expressed in nocioceptive dorsal root ganglion and sympathetic ganglion neurons. Gain-of-function mutations in Na(v)1.7 produce the nocioceptor hyperexcitability underlying inherited erythromelalgia, characterized in most kindreds by early-age onset of severe pain. Here we describe a mutation (Na(v)1.7-G616R) in a pedigree with adult-onset of pain in some family members. The mutation shifts the voltage-dependence of channel fast-inactivation in a depolarizing direction in the adult-long, but not in the neonatal-short splicing isoform of Na(v)1.7 in dorsal root ganglion neurons. Altered inactivation does not depend on the age of the dorsal root ganglion neurons in which the mutant is expressed. Expression of the mutant adult-long, but not the mutant neonatal-short, isoform of Na(v)1.7 renders dorsal root ganglion neurons hyperexcitable, reducing the current threshold for generation of action potentials, increasing spontaneous activity and increasing the frequency of firing in response to graded suprathreshold stimuli. This study shows that a change in relative expression of splice isoforms can contribute to time-dependent manifestation of the functional phenotype of a sodium channelopathy.