TTX-resistant Voltage-gated Sodium Channels Research Articles

A-type FHFs mediate resurgent currents through TTX-resistant voltage-gated sodium channels.

Resurgent currents (INaR) produced by voltage-gated sodium channels are required for many neurons to maintain high-frequency firing and contribute to neuronal hyperexcitability and disease pathophysiology. Here, we show, for the first time, that INaR can be reconstituted in a heterologous system by coexpression of sodium channel α-subunits and A-type fibroblast growth factor homologous factors (FHFs). Specifically, A-type FHFs induces INaR from Nav1.8, Nav1.9 tetrodotoxin (TTX)-resistant neuronal channels, and, to a lesser extent, neuronal Nav1.7 and cardiac Nav1.5 channels. Moreover, we identified the N-terminus of FHF as the critical molecule responsible for A-type FHFs-mediated INaR. Among the FHFs, FHF4A is the most important isoform for mediating Nav1.8 and Nav1.9 INaR. In nociceptive sensory neurons, FHF4A knockdown significantly reduces INaR amplitude and the percentage of neurons that generate INaR, substantially suppressing excitability. Thus, our work reveals a novel molecular mechanism underlying TTX-resistant INaR generation and provides important potential targets for pain treatment.

Is neuronal inhibition or excitability controlled by Na+K+2CL– transporters?

Is neuronal inhibition or excitability controlled by Na⁺K⁺2CL^– transporters?

MrgprX1 Mediates Neuronal Excitability and Itch Through Tetrodotoxin-Resistant Sodium Channels.

In this study, we sought to elucidate the molecular mechanism underlying human Mas-related G protein-coupled receptor X1 (MrgprX1) mediated itch sensation. We found that activation of MrgprX1 by BAM8-22 triggered robust action potential discharges in dorsal root ganglion (DRG) neurons. This neuronal excitability is not mediated by Transient receptor potential (TRP) cation channels, M-type potassium channels, or chloride channels. Instead, activation of MrgprX1 lowers the activation threshold of TTX-resistant sodium channels and induces inward sodium currents. These MrgprX1-elicited action potential discharges can be blocked by Pertussis toxin (PTX) and a Gβγ inhibitor - Gallein. Behavioral results showed that Nav1.9 knockout but not Trpa1 knockout significantly reduced BAM8-22 evoked scratching behavior. Collectively, these data suggest that activation of MrgprX1 triggers itch sensation by increasing the activity of TTX-resistant voltage-gated sodium channels.

(400) An Ex-Vivo Human Model of Inflammatory Pain for Enabling Translational Research and Drug Discovery

The incomplete understanding of chronic neuropathic and inflammatory pain mechanisms poses a major challenge for the development of new, non-addictive pain medications. Cross-species differences in pharmacological responses and analgesic effects lead to poor translation of preclinical animal models to clinical studies in humans. We have developed methodologies for the isolation of human sensory neurons, from organ donors, which allow for ex-vivo studies of sensory neurons physiology and pharmacology. The cells remain phenotypically stable for up to 14 days in culture and can be used to assess pharmacological responses by employing imaging- or electrophysiology-based methods. Here we present a novel preclinical discovery strategy, which utilizes human sensory neurons to establish cellular models of pathological states. In this model, we recapitulate inflammatory conditions by incubating human sensory neurons with pro-inflammatory agents (PGE2 and bradykinin) for 72 hrs. This treatment induced changes in neuronal excitability and in the nociceptive profile of human sensory neurons. Indeed, sensory neurons showed increased excitability, heightened responsiveness to TRPV1 agonists and increased expression of TTX-resistant voltage gated sodium channels. These changes are consistent with phenotypes of inflammatory pain observed in the clinic. Furthermore, we demonstrated that this cell-based model can be used to differentiate the effects of analgesic drugs that have demonstrated varying degree of success in clinical trials with patient populations affected by different pain indications. This approach demonstrates high potential for ranking drug candidates based on potential analgesic efficacy and for predicting the most appropriate pain indication for clinical trial development. The research presented was sponsored by AnaBios Corporation.

Historical Contingency in a Multigene Family Facilitates Adaptive Evolution of Toxin Resistance

Novel adaptations must originate and function within an already established genome [1]. As a result, the ability of a species to adapt to new environmental challenges is predicted to be highly contingent on the evolutionary history of its lineage [2-6]. Despite a growing appreciation of the importance of historical contingency in the adaptive evolution of single proteins [7-11], we know surprisingly little about its role in shaping complex adaptations that require evolutionary change in multiple genes. One such adaptation, extreme resistance to tetrodotoxin (TTX), has arisen in several species of snakes through coevolutionary arms races with toxic amphibian prey, which select for TTX-resistant voltage-gated sodium channels (Nav) [12-16]. Here, we show that the relatively recent origins of extreme toxin resistance, which involve the skeletal muscle channel Nav1.4, were facilitated by ancient evolutionary changes in two other members of the same gene family. A substitution conferring TTX resistance to Nav1.7, a channel found in small peripheral neurons, arose in lizards ∼170 million years ago (mya) and was present in the common ancestor of all snakes. A second channel found in larger myelinated neurons, Nav1.6, subsequently evolved resistance in four different snake lineages beginning ∼38 mya. Extreme TTX resistance has evolved at least five times within the past 12 million years via changes in Nav1.4, but only within lineages that previously evolved resistant Nav1.6 and Nav1.7. Our results show that adaptive protein evolution may be contingent upon enabling substitutions elsewhere in the genome, in this case, in paralogs of the same gene family.

Roles of ASIC3, TRPV1, and NaV1.8 in the transition from acute to chronic pain in a mouse model of fibromyalgia.

BackgroundTissue acidosis is effective in causing chronic muscle pain. However, how muscle nociceptors contribute to the transition from acute to chronic pain is largely unknown.ResultsHere we showed that a single intramuscular acid injection induced a priming effect on muscle nociceptors of mice. The primed muscle nociceptors were plastic and permitted the development of long-lasting chronic hyperalgesia induced by a second acid insult. The plastic changes of muscle nociceptors were modality-specific and required the activation of acid-sensing ion channel 3 (ASIC3) or transient receptor potential cation channel V1 (TRPV1). Activation of ASIC3 was associated with increased activity of tetrodotoxin (TTX)-sensitive voltage-gated sodium channels but not protein kinase Cϵ (PKCϵ) in isolectin B4 (IB4)-negative muscle nociceptors. In contrast, increased activity of TTX-resistant voltage-gated sodium channels with ASIC3 or TRPV1 activation in NaV1.8-positive muscle nociceptors was required for the development of chronic hyperalgesia. Accordingly, compared to wild type mice, NaV1.8-null mice showed briefer acid-induced hyperalgesia (5 days vs. >27 days).ConclusionASIC3 activation may manifest a new type of nociceptor priming in IB4-negative muscle nociceptors. The activation of ASIC3 and TRPV1 as well as enhanced NaV1.8 activity are essential for the development of long-lasting hyperalgesia in acid-induced, chronic, widespread muscle pain.

Membrane Properties of Neuron-Like Cells Generated from Adult Human Bone-Marrow-Derived Mesenchymal Stem Cells

Adult mesenchymal stem cells (MeSCs) isolated from human bone marrow are capable of generating neural stem cell (NSC)-like cells that can be subsequently differentiated into cells expressing molecular markers for neurons. Here we report that these neuron-like cells had functional properties similar to those of brain-derived neurons. Whole-cell patch-clamp recordings and calcium imaging experiments were performed on neuron-like cells differentiated from bone-marrow-derived NSC-like cells. The neuron-like cells were subjected to current pulses to determine if they were capable of generating depolarization-induced action potentials. We found that nearly all of the cells with neuron-like morphology exhibited active membrane properties in response to the depolarizing pulses. The most common response was a single spike-like event with an overshoot and brief afterhyperpolarization. Cells that did not generate overshooting spike-like events usually displayed rectifying current-voltage relationships. The prevalence of these active membrane properties in response to the depolarizing current pulses suggested that the human MeSCs (hMeSCs) were capable of converting to a neural lineage under defined culture conditions. The spike-like events were blocked by the voltage-gated sodium channel inhibitor lidocaine, but unaffected by another sodium channel inhibitor tetrodotoxin (TTX). In calcium imaging experiments, the neuron-like cells responded to potassium chloride depolarization and l-glutamate application with increases in the cytoplasmic calcium levels. Thus, the neuron-like cells appeared to express TTX-resistant voltage-gated sodium channels, voltage-gated calcium channels, and functional l-glutamate receptors. These results demonstrate that hMeSCs were capable of generating cells with characteristics typical of functional neurons that may prove useful for neuroreplacement therapies.

Tetrodotoxin-resistant conductivity and spinal effects of cutaneous C-fibre afferents in the rat.

The effect of the sodium channel blocking agent tetrodotoxin (TTX) on signal processing in afferent fibres of dorsal roots was tested in Sprague-Dawley rats. TTX applied to the dorsal roots L4-L6 blocked the fast afferent volleys from the sural nerve, which was stimulated electrically with supramaximal strength for A-fibres. Afferent C-fibre compound action potentials (CAPs) elicited by electrical stimulation of the dorsal root L5 peripherally from the TTX block or by electrical stimulation of the sural nerve likewise disappeared from the recording. Cord dorsum potentials (CDPs) recorded at the dorsal root entry zone of L4 were blocked completely if elicited by A-fibre volleys. In contrast, CDPs elicited by C-fibre stimulation persisted with longer latency and reduced amplitude in the first part of the CDP. During TTX block, C-fibre potentials could also be recorded from dorsal root filaments after stimulation of the sural nerve or the dorsal root L5 peripherally of the TTX-block. The results suggest that in the axonal membrane of cutaneous C-afferents, both TTX sensitive and TTX-resistant voltage gated sodium channels exist, the latter being responsible for the propagation of signals in a portion of C-fibres after TTX application. The TTX-resistant portion of the afferent potential does not seem to contribute much to the afferent C-fibre CAP before TTX application, but its central effects seem to be overproportionally strong.