The widely used antihistamine mepyramine causes topical pain relief through direct blockade of nociceptor sodium channels.

Mepyramine, a first-generation antihistamine targeting the histamine H(1) receptor, was extensively prescribed to patients suffering from allergic reactions and urticaria. Serious adverse effects, especially in case of overdose, were frequently reported, including drowsiness, impaired thinking, convulsion, and coma. Many of these side effects were associated with the blockade of histaminergic or cholinergic receptors. Here we show that mepyramine directly inhibits a variety of voltage-gated sodium channels, including the Tetrodotoxin-sensitive isoforms and the main isoforms (Nav1.7, Nav1.8, and Nav1.9) of nociceptors. Estimated IC50 were within the range of drug concentrations detected in poisoned patients. Mepyramine inhibited sodium channels through fast- or slow-inactivated state preference depending on the isoform. Moreover, mepyramine inhibited the firing responses of C- and Aß-type nerve fibers in ex vivo skin-nerve preparations. Locally applied mepyramine had analgesic effects on the scorpion toxin-induced excruciating pain and produced pain relief in acute, inflammatory, and chronic pain models. Collectively, these data provide evidence that mepyramine has the potential to be developed as a topical analgesic agent.

Membrane cholesterol depletion as a trigger of Nav1.9 channel-mediated inflammatory pain.

Cholesterol is a major lipid component of the mammalian plasma membrane. While much is known about its metabolism, its transport, and its role in atherosclerotic vascular disease, less is known about its role in neuronal pathophysiology. This study reveals an unexpected function of cholesterol in controlling pain transmission. We show that inflammation lowers cholesterol content in skin tissue and sensory DRG culture. Pharmacological depletion of cellular cholesterol entails sensitization of nociceptive neurons and promotes mechanical and thermal hyperalgesia through the activation of voltage-gated Nav1.9 channels. Inflammatory mediators enhance the production of reactive oxygen species and induce partitioning of Nav1.9 channels from cholesterol-rich lipid rafts to cholesterol-poor non-raft regions of the membrane. Low-cholesterol environment enhances voltage-dependent activation of Nav1.9 channels leading to enhanced neuronal excitability, whereas cholesterol replenishment reversed these effects. Consistently, we show that transcutaneous delivery of cholesterol alleviates hypersensitivity in animal models of acute and chronic inflammatory pain. In conclusion, our data establish that membrane cholesterol is a modulator of pain transmission and shed a new light on the relationship between cholesterol homeostasis, inflammation, and pain.

The Nav1.9 channel is a key determinant of cold pain sensation and cold allodynia.

Cold-triggered pain is essential to avoid prolonged exposure to harmfully low temperatures. However, the molecular basis of noxious cold sensing in mammals is still not completely understood. Here, we show that the voltage-gated Nav1.9 sodium channel is important for the perception of pain in response to noxious cold. Nav1.9 activity is upregulated in a subpopulation of damage-sensing sensory neurons responding to cooling, which allows the channel to amplify subthreshold depolarizations generated by the activation of cold transducers. Consequently, cold-triggered firing is impaired in Nav1.9(-/-) neurons, and Nav1.9 null mice and knockdown rats show increased cold pain thresholds. Disrupting Nav1.9 expression in rodents also alleviates cold pain hypersensitivity induced by the antineoplastic agent oxaliplatin. We conclude that Nav1.9 acts as a subthreshold amplifier in cold-sensitive nociceptive neurons and is required for the perception of cold pain under normal and pathological conditions.

The scorpion toxin Amm VIII induces pain hypersensitivity through gain-of-function of TTX-sensitive Na⁺ channels.

Voltage-gated Na(+) channels (Nav) are the targets of a variety of scorpion toxins. Here, we investigated the effects of Amm VIII, a toxin isolated from the venom of the scorpion Androctonus mauretanicus mauretanicus, on pain-related behaviours in mice. The effects of Amm VIII were compared with the classic scorpion α-toxin AaH II from Androctonus australis. Contrary to AaH II, intraplantar injection of Amm VIII at relatively high concentrations caused little nocifensive behaviours. However, Amm VIII induced rapid mechanical and thermal pain hypersensitivities. We evaluated the toxins' effects on Nav currents in nociceptive dorsal root ganglion (DRG) neurons and immortalized DRG neuron-derived F11 cells. Amm VIII and AaH II enhanced tetrodotoxin-sensitive (TTX-S) Nav currents in DRG and F11 cells. Both toxins impaired fast inactivation and negatively shifted activation. AaH II was more potent than Amm VIII at modulating TTX-S Nav currents with EC50 of 5 nM and 1 µM, respectively. AaH II and Amm VIII also impaired fast inactivation of Nav1.7, with EC50 of 6.8 nM and 1.76 µM, respectively. Neither Nav1.8 nor Nav1.9 was affected by the toxins. AaH II and Amm VIII reduced first spike latency and lowered action potential threshold. Amm VIII was less efficient than AaH II in increasing the gain of the firing frequency-stimulation relationship. In conclusion, our data show that Amm VIII, although less potent than AaH II, acts as a gating-modifier peptide reminiscent of classic α-toxins, and suggest that its hyperalgesic effects can be ascribed to gain-of-function of TTX-S Na(+) channels in nociceptors.

Kv1.1 channels act as mechanical brake in the senses of touch and pain.

Molecular determinants of threshold sensitivity of mammalian mechanoreceptors are unknown. Here, we identify a mechanosensitive (MS) K(+) current (IKmech) that governs mechanical threshold and adaptation of distinct populations of mechanoreceptors. Toxin profiling and transgenic mouse studies indicate that IKmech is carried by Kv1.1-Kv1.2 heteromers. Mechanosensitivity is attributed to Kv1.1 subunits, through facilitation of voltage-dependent open probability. IKmech is expressed in high-threshold C-mechano-nociceptors (C-HTMRs) and Aß-mechanoreceptors, but not in low-threshold C-mechanoreceptors. IKmech opposes depolarization induced by slow/ultraslow MS cation currents in C-HTMRs, thereby shifting mechanical threshold for firing to higher values. However, due to kinetics mismatch with rapidly-adapting MS cation currents, IKmech tunes firing adaptation but not mechanical threshold in Aß-mechanoreceptors. Expression of Kv1.1 dominant negative or inhibition of Kv1.1/IKmech caused severe mechanical allodynia but not heat hyperalgesia. By balancing the activity of excitatory mechanotransducers, Kv1.1 acts as a mechanosensitive brake that regulates mechanical sensitivity of fibers associated with mechanical perception.

Nav1.9 channel contributes to mechanical and heat pain hypersensitivity induced by subacute and chronic inflammation.

Inflammation is known to be responsible for the sensitization of peripheral sensory neurons, leading to spontaneous pain and invalidating pain hypersensitivity. Given its role in regulating neuronal excitability, the voltage-gated Nav1.9 channel is a potential target for the treatment of pathological pain, but its implication in inflammatory pain is yet not fully described. In the present study, we examined the role of the Nav1.9 channel in acute, subacute and chronic inflammatory pain using Nav1.9-null mice and Nav1.9 knock-down rats. In mice we found that, although the Nav1.9 channel does not contribute to basal pain thresholds, it plays an important role in heat pain hypersensitivity induced by subacute paw inflammation (intraplantar carrageenan) and chronic ankle inflammation (complete Freund's adjuvant-induced monoarthritis). We showed for the first time that Nav1.9 also contributes to mechanical hypersensitivity in both models, as assessed using von Frey and dynamic weight bearing tests. Consistently, antisense-based Nav1.9 gene silencing in rats reduced carrageenan-induced heat and mechanical pain hypersensitivity. While no changes in Nav1.9 mRNA levels were detected in dorsal root ganglia (DRGs) during subacute and chronic inflammation, a significant increase in Nav1.9 immunoreactivity was observed in ipsilateral DRGs 24 hours following carrageenan injection. This was correlated with an increase in Nav1.9 immunolabeling in nerve fibers surrounding the inflamed area. No change in Nav1.9 current density could be detected in the soma of retrolabeled DRG neurons innervating inflamed tissues, suggesting that newly produced channels may be non-functional at this level and rather contribute to the observed increase in axonal transport. Our results provide evidence that Nav1.9 plays a crucial role in the generation of heat and mechanical pain hypersensitivity, both in subacute and chronic inflammatory pain models, and bring new elements for the understanding of its regulation in those models.

Formation of a new receptor-operated channel by heteromeric assembly of TRPP2 and TRPC1 subunits.

Although several protein-protein interactions have been reported between transient receptor potential (TRP) channels, they are all known to occur exclusively between members of the same group. The only intergroup interaction described so far is that of TRPP2 and TRPC1; however, the significance of this interaction is unknown. Here, we show that TRPP2 and TRPC1 assemble to form a channel with a unique constellation of new and TRPP2/TRPC1-specific properties. TRPP2/TRPC1 is activated in response to G-protein-coupled receptor activation and shows a pattern of single-channel conductance, amiloride sensitivity and ion permeability distinct from that of TRPP2 or TRPC1 alone. Native TRPP2/TRPC1 activity is shown in kidney cells by complementary gain-of-function and loss-of-function experiments, and its existence under physiological conditions is supported by colocalization at the primary cilium and by co-immunoprecipitation from kidney membranes. Identification of the heteromultimeric TRPP2/TRPC1 channel has implications in mechanosensation and cilium-based Ca(2+) signalling.

Inflammatory mediators increase Nav1.9 current and excitability in nociceptors through a coincident detection mechanism.

Altered function of Na+ channels is responsible for increased hyperexcitability of primary afferent neurons that may underlie pathological pain states. Recent evidence suggests that the Nav1.9 subunit is implicated in inflammatory but not acute pain. However, the contribution of Nav1.9 channels to the cellular events underlying nociceptor hyperexcitability is still unknown, and there remains much uncertainty as to the biophysical properties of Nav1.9 current and its modulation by inflammatory mediators. Here, we use gene targeting strategy and computer modeling to identify Nav1.9 channel current signature and its impact on nociceptors' firing patterns. Recordings using internal fluoride in small DRG neurons from wild-type and Nav1.9-null mutant mice demonstrated that Nav1.9 subunits carry the TTX-resistant "persistent" Na+ current called NaN. Nav1.9(-/-) nociceptors showed no significant change in the properties of the slowly inactivating TTX-resistant SNS/Nav1.8 current. The loss in Nav1.9-mediated Na+ currents was associated with the inability of small DRG neurons to generate a large variety of electrophysiological behaviors, including subthreshold regenerative depolarizations, plateau potentials, active hyperpolarizing responses, oscillatory bursting discharges, and bistable membrane behaviors. We further investigated, using CsCl- and KCl-based pipette solutions, whether G-protein signaling pathways and inflammatory mediators upregulate the NaN/Nav1.9 current. Bradykinin, ATP, histamine, prostaglandin-E2, and norepinephrine, applied separately at maximal concentrations, all failed to modulate the Nav1.9 current. However, when applied conjointly as a soup of inflammatory mediators they rapidly potentiated Nav1.9 channel activity, generating subthreshold amplification and increased excitability. We conclude that Nav1.9 channel, the molecular correlate of the NaN current, is potentiated by the concerted action of inflammatory mediators that may contribute to nociceptors' hyperexcitability during peripheral inflammation.

Expression and localization of the Nav1.9 sodium channel in enteric neurons and in trigeminal sensory endings: implication for intestinal reflex function and orofacial pain.

The Nav1.9 sodium channel is expressed in nociceptive DRG neurons where it contributes to spontaneous pain behavior after peripheral inflammation. Here, we used a newly developed antibody to investigate the distribution of Nav1.9 in rat and mouse trigeminal ganglion (TG) nerve endings and in enteric nervous system (ENS). In TGs, Nav1.9 was expressed in the soma of small- and medium-sized, peripherin-positive neurons. Nav1.9 was present along trigeminal afferent fibers and at terminals in lip skin and dental pulp. In the ENS, Nav1.9 was detected within the soma and proximal axons of sensory, Dogiel type II, myenteric and submucosal neurons. Immunological data were correlated with the detection of persistent TTX-resistant Na(+) currents sharing similar properties in DRG, TG and myenteric neurons. Collectively, our data support a potential role of Nav1.9 in the transmission of trigeminal pain and the regulation of intestinal reflexes. Nav1.9 might therefore constitute a molecular target for therapeutic treatments of orofacial pain and gastrointestinal syndromes.

The versatile nature of the calcium-permeable cation channel TRPP2.

TRPP2 is a member of the transient receptor potential (TRP) superfamily of cation channels, which is mutated in autosomal dominant polycystic kidney disease (ADPKD). TRPP2 is thought to function with polycystin 1-a large integral protein-as part of a multiprotein complex involved in transducing Ca(2+)-dependent information. TRPP2 has been implicated in various biological functions including cell proliferation, sperm fertilization, mating behaviour, mechanosensation and asymmetric gene expression. Although its function as a Ca(2+)-permeable cation channel is well established, its precise role in the plasma membrane, the endoplasmic reticulum and the cilium is controversial. Recent studies suggest that TRPP2 function is highly dependent on the subcellular compartment of expression, and is regulated by many interactions with adaptor proteins. This review summarizes the most pertinent evidence about the properties of TRPP2 channels, focusing on the compartment-specific functions of mammalian TRPP2.

Differential targeting and functional specialization of sodium channels in cultured cerebellar granule cells.

The ion channel dynamics that underlie the complex firing patterns of cerebellar granule (CG) cells are still largely unknown. Here, we have characterized the subcellular localization and functional properties of Na+ channels that regulate the excitability of CG cells in culture. As evidenced by RT-PCR and immunocytochemical analysis, morphologically differentiated CG cells expressed Nav1.2 and Nav1.6, though both subunits appeared to be differentially regulated. Nav1.2 was localized at most axon initial segments (AIS) of CG cells from 8 days in vitro DIV 8 to DIV 15. At DIV 8, Nav1.6 was found uniformly throughout somata, dendrites and axons with occasional clustering in a subset of AIS. Accumulation of Nav1.6 at most AIS was evident by DIV 13-14, suggesting it is developmentally regulated at AIS. The specific contribution of these differentially distributed Na+ channels has been assessed using a combination of methods that allowed discrimination between functionally compartmentalized Na+ currents. In agreement with immunolocalization, we found that fast activating-fully inactivating Na+ currents predominate at the AIS membrane and in the somatic plasma membrane.

A novel function for cadherin-11 in the regulation of motor axon elongation and fasciculation.

We previously observed that cadherin-11, a type II cadherin, is expressed in growing motor and sensory axons in the mouse embryo. Here, we assessed its functional involvement in the regulation of axon elongation and fasciculation by evaluating the activity of a specific cadherin-11 homophilic ligand, cad11-Fc (cadherin-11 extracellular region fused to Fc fragment of IgG), on the length and organization of motor axons outgrowing from embryonic ventral spinal cord explants. Cad11-Fc substrate enhanced axon growth and prevented interactions occurring between growing axons, providing evidences for a role of cadherin-11 in the control of growth cone progression. Comparison of cadherin-11 with N-cadherin, a type I cadherin concomitantly expressed by motor axons, revealed similarities in their functional properties, including the ability to reorganize the actin cytoskeleton through interactions with catenins, but differences in their axon growth-promoting activity, arguing for subtle differences in their contributions to peripheral nerve elongation.

Migrating and myelinating potential of neural precursors engineered to overexpress PSA-NCAM.

Polysialic acid (PSA) on NCAM is an important modulator of cell-cell interactions during development and regeneration. Here we investigated whether PSA overexpression influences neural cell migration and myelination. We stably expressed a GFP-tagged polysialytransferase, PSTGFP, in mouse neurospheres and induced prolonged PSA synthesis. Using a chick xenograft assay for migration, we show that PSA can instruct precursor migration along the ventral pathway. PSA persistence did not change neural precursor multipotentiality in vitro but induced a delay in oligodendrocyte differentiation. PSTGFP+ precursors showed widespread engraftment in shiverer brain, closely similar to that observed with control precursors expressing a fluorescent protein. Initially, myelination by oligodendrocytes was delayed but, eventually, down-regulation of PSTGFP occurred, allowing myelination to proceed. Thus down-regulation of polysialyltransferases takes place even in cells where its RNA is under the control of a heterologous promoter and engineering PSA overexpression in neural precursors does not cause irreversible unphysiological effects.

Polycystins, calcium signaling, and human diseases.

Autosomal dominant polycystic kidney disease (ADPKD) is a major, inherited nephropathy affecting over 1:1000 of the worldwide population. It is a systemic condition with frequent hepatic and cardiovascular manifestations in addition to the progressive development of fluid-filled cysts from the tubules and collecting ducts of affected kidneys. The pathogenesis of cyst formation is currently thought to involve increased proliferation of epithelial cells, mild dedifferentiation, and fluid accumulation. In the past decade, study of ADPKD led to the discovery of a unique family of highly complex proteins, the polycystins. Loss-of-function mutations in either of two polycystin proteins, polycystin-1 or polycystin-2, give rise to ADPKD. These proteins are thought to function together as part of a multiprotein complex that may initiate Ca2+ signals, directing attention to the regulation of intracellular Ca2+ as a possible misstep that participates in cyst formation. Here we review what is known about the Ca2+ signaling functions of polycystin proteins and focus on findings that have significantly advanced our physiological insight. Special attention is paid to the recently discovered role of these proteins in the mechanotransduction of the renal primary cilium and the model it suggests.

Gating and modulation of presumptive NaV1.9 channels in enteric and spinal sensory neurons.

The NaV1.9 subunit is expressed in nociceptive dorsal root ganglion (DRG) neurons and sensory myenteric neurons in which it generates 'persistent' tetrodotoxin-resistant (TTX-R) Na+ currents of yet unknown physiological functions. Here, we have analyzed these currents in details by combining single-channel and whole-cell recordings from cultured rat DRG and myenteric neurons. Comparison of single-channel with whole-cell data indicates that recording using internal CsCl best reflects the basic electrical features of NaV1.9 currents. Inclusion of fluoride in the pipette solution caused a negative shift in the activation and inactivation gates of NaV1.9 but not NaV1.8. Fluoride acts by promoting entry of NaV1.9 channels into a preopen closed state, which causes a strong bias towards opening and enhances the ability of sensory neurons to sustain spiking. Thus, the modulation of the resting-closed states of NaV1.9 channels strongly influences nociceptor excitability and may provide a mechanism by which inflammatory mediators alter pain threshold.

FGFR4 signaling is a necessary step in limb muscle differentiation.

In chick embryos, most if not all, replicating myoblasts present within the skeletal muscle masses express high levels of the FGF receptor FREK/FGFR4, suggesting an important role for this molecule during myogenesis. We examined FGFR4 function during myogenesis, and we demonstrate that inhibition of FGFR4, but not FGFR1 signaling, leads to a dramatic loss of limb muscles. All muscle markers analyzed (such as Myf5, MyoD and the embryonic myosin heavy chain) are affected. We show that inhibition of FGFR4 signal results in an arrest of muscle progenitor differentiation, which can be rapidly reverted by the addition of exogenous FGF, rather than a modification in their proliferative capacities. Conversely, over-expression of FGF8 in somites promotes FGFR4 expression and muscle differentiation in this tissue. Together, these results demonstrate that in vivo, myogenic differentiation is positively controlled by FGF signaling, a notion that contrasts with the general view that FGF promotes myoblast proliferation and represses myogenic differentiation. Our data assign a novel role to FGF8 during chick myogenesis and demonstrate that FGFR4 signaling is a crucial step in the cascade of molecular events leading to terminal muscle differentiation.

Complementary expression and regulation of cadherins 6 and 11 during specific steps of motoneuron differentiation.

Cadherins constitute one of the major classes of adhesion receptors participating in the architectural organization of embryonic tissues. We previously identified type II cadherins 6 and 11, whose mRNA expression was tightly regulated during mouse neuromuscular development. Here we determine the regulation of expression and the localization of the corresponding proteins in relation to spinal neuron differentiation and peripheral nerve outgrowth. Cadherin-11 expression initially appeared in motor columns before extending to the whole spinal cord, dorsal root ganglia, and sensory-motor peripheral nerves. Then, its expression decreased in nervous tissues and became predominant in mesenchymes. Cadherin-6 was exclusively expressed in floor and roof plates, motor columns, and motor peripheral nerves, including motor axons and Schwann cell precursors. Compared to cadherin-11, its expression in motor columns was delayed and restricted to certain subpopulations of motoneurons. These results strongly implicate cadherins 6 and 11 in the control of spinal motoneuron differentiation and segregation and in axoaxonal, axoglial, and glio-glial interactions during sensory-motor nerve progression.