DEG/ENaC but not TRP channels are the major mechanoelectrical transduction channels in a C. elegans nociceptor

Many nociceptors detect mechanical cues, but the ion channels responsible for mechanotransduction in these sensory neurons remain obscure. Using in?vivo recordings and genetic dissection, we identified the DEG/ENaC protein, DEG-1, as the major mechanotransduction channel in ASH, a polymodal nociceptor in Caenorhabditis elegans. But DEG-1 is not the only mechanotransduction channel in ASH: loss of deg-1 revealed a minor current whose properties differ from those expected of DEG/ENaC channels. This current was independent of two TRPV channels expressed in ASH. Although loss of these TRPV channels inhibits behavioral responses to noxious stimuli, we found that both mechanoreceptor currents and potentials were essentially wild-type in TRPV mutants. We propose that ASH nociceptors rely on two genetically distinct mechanotransduction channels and that TRPV channels contribute to encoding and transmitting information. Because mammalian and insect nociceptors also coexpress DEG/ENaCs and TRPVs, the cellular functions elaborated here for these ion channels may be conserved.     

Trigeminal-Rostral Ventromedial Medulla circuitry is involved in orofacial hyperalgesia contralateral to tissue injury

Background

Members of the degenerin/epithelial (DEG/ENaC) sodium channel family are mechanosensors in C elegans, and Nav1.7 and Nav1.8 voltage-gated sodium channel knockout mice have major deficits in mechanosensation. ?? and ??ENaC sodium channel subunits are present with acid sensing ion channels (ASICs) in mammalian sensory neurons of the dorsal root ganglia (DRG). The extent to which epithelial or voltage-gated sodium channels are involved in transduction of mechanical stimuli is unclear.

Results

Here we show that deleting ?? and ??ENaC sodium channels in sensory neurons does not result in mechanosensory behavioural deficits. We had shown previously that Nav1.7/Nav1.8 double knockout mice have major deficits in behavioural responses to noxious mechanical pressure. However, all classes of mechanically activated currents in DRG neurons are unaffected by deletion of the two sodium channels. In contrast, the ability of Nav1.7/Nav1.8 knockout DRG neurons to generate action potentials is compromised with 50% of the small diameter sensory neurons unable to respond to electrical stimulation in vitro.

Conclusion

Behavioural deficits in Nav1.7/Nav1.8 knockout mice reflects a failure of action potential propagation in a mechanosensitive set of sensory neurons rather than a loss of primary transduction currents. DEG/ENaC sodium channels are not mechanosensors in mouse sensory neurons.     

Behavioural and morphological evidence for the involvement of glial cell activation in delta opioid receptor function: implications for the development of opioid tolerance

The molecular identity and pharmacological properties of mechanically gated ion channels in sensory neurons are poorly understood. We show that FM1-43, a styryl dye used to fluorescently label cell membranes, permeates mechanosensitive ion channels in cultured dorsal root ganglion neurons, resulting in blockade of three previously defined subtypes of mechanically activated currents. Blockade and dye uptake is voltage dependent and regulated by external Ca²⁺. The structurally related larger dye FM3-25 inhibited mechanically activated currents to a lesser degree and did not permeate the channels. In vivo, FMI-43 decreases pain sensitivity in the Randall-Selitto test and increases the withdrawal threshold from von Frey hairs, together suggesting that the channels expressed at the cell body in culture mediate mechanosensation in the intact animal. These data give further insight into the mechanosensitive ion channels expressed by somatosensory neurons and suggest FM dyes are an interesting tool for studying them.     

Mechanotransduction in spider slit sensilla

Mechanoreception is a vital constituent of several sensory modalities and a wide range of internal regulatory processes, but fundamental mechanisms for neural detection of mechanical stimuli have been difficult to characterize because of the morphological properties of most mechanoreceptors and the nature of the stimulus itself. An invertebrate preparation, the VS-3 lyriform slit sense organ of the spider, Cupiennius salei, has proved useful because it possesses large mechanosensory neurons, whose cell bodies are close to the sites of sensory transduction, and accessible to intracellular recording during mechanotransduction. This has made it possible to observe and experiment with all the major stages of mechanosensation. Here, we describe several important findings from this preparation, including the estimated number, conductance and ionic selectivity of the ion channels responsible for mechanotransduction, the major voltage-activated ion channels responsible for action potential encoding and control of the dynamic properties of the neurons, the location of action potential initiation following mechanical stimulation, and the efferent control of mechanoreception. While many details of mechanosensation remain to be discovered, the VS-3 system continues to offer important opportunities to advance our understanding of this crucial physiological process.     

Painful channels in sensory neurons

Pain is an unpleasant sensation experienced when tissues are damaged. Thus, pain sensation in some way protects body from imminent threat or injury. Peripheral sensory nerves innervated to peripheral tissues initially respond to multiple forms of noxious or strong stimuli, such as heat, mechanical and chemical stimuli. In response to these stimuli, electrical signals for conducting the nociceptive neural signals through axons are generated. These action potentials are then conveyed to specific areas in the spinal cord and in the brain. Sensory afferent fibers are heterogeneous in many aspects. For example, sensory nerves are classified as Aa, -b, -d and C-fibers according to their diameter and degree of myelination. It is widely accepted that small sensory fibers tend to respond to vigorous or noxious stimuli and related to nociception. Thus these fibers are specifically called nociceptors. Most of nociceptors respond to noxious mechanical stimuli and heat. In addition, these sensory fibers also respond to chemical stimuli [Davis et al. (1993)] such as capsaicin. Thus, nociceptors are considered polymodal. Recent advance in research on ion channels in sensory neurons reveals molecular mechanisms underlying how various types of stimuli can be transduced to neural signals transmitted to the brain for pain perception. In particular, electrophysiological studies on ion channels characterize biophysical properties of ion channels in sensory neurons. Furthermore, molecular biology leads to identification of genetic structures as well as molecular properties of ion channels in sensory neurons. These ion channels are expressed in axon terminals as well as in cell soma. When these channels are activated, inward currents or outward currents are generated, which will lead to depolarization or hyperpolarization of the membrane causing increased or decreased excitability of sensory neurons. In order to depolarize the membrane of nerve terminals, either inward currents should be generated or outward currents should be inhibited. So far, many cationic channels that are responsible for the excitation of sensory neurons are introduced recently. Activation of these channels in sensory neurons is evidently critical to the generation of nociceptive signals. The main channels responsible for inward membrane currents in nociceptors are voltage-activated sodium and calcium channels, while outward current is carried mainly by potassium ions. In addition, activation of non-selective cation channels is also responsible for the excitation of sensory neurons. Thus, excitability of neurons can be controlled by regulating expression or by modulating activity of these channels.     

Neurosensory mechanotransduction through acid‐sensing ion channels

Acid‐sensing ion channels (ASICs) are voltage‐insensitive cation channels responding to extracellular acidification. ASIC proteins have two transmembrane domains and a large extracellular domain. The molecular topology of ASICs is similar to that of the mechanosensory abnormality 4‐ or 10‐proteins expressed in touch receptor neurons and involved in neurosensory mechanotransduction in nematodes. The ASIC proteins are involved in neurosensory mechanotransduction in mammals. The ASIC isoforms are expressed in Merkel cell–neurite complexes, periodontal Ruffini endings and specialized nerve terminals of skin and muscle spindles, so they might participate in mechanosensation. In knockout mouse models, lacking an ASIC isoform produces defects in neurosensory mechanotransduction of tissue such as skin, stomach, colon, aortic arch, venoatrial junction and cochlea. The ASICs are thus implicated in touch, pain, digestive function, baroreception, blood volume control and hearing. However, the role of ASICs in mechanotransduction is still controversial, because we lack evidence that the channels are mechanically sensitive when expressed in heterologous cells. Thus, ASIC channels alone are not sufficient to reconstruct the path of transducing molecules of mechanically activated channels. The mechanotransducers associated with ASICs need further elucidation. In this review, we discuss the expression of ASICs in sensory afferents of mechanoreceptors, findings of knockout studies, technical issues concerning studies of neurosensory mechanotransduction and possible missing links. Also we propose a molecular model and a new approach to disclose the molecular mechanism underlying the neurosensory mechanotransduction.     

TRPC3 and TRPC6 are essential for normal mechanotransduction in subsets of sensory neurons and cochlear hair cells

Transient receptor potential (TRP) channels TRPC3 and TRPC6 are expressed in both sensory neurons and cochlear hair cells. Deletion of TRPC3 or TRPC6 in mice caused no behavioural phenotype, although loss of TRPC3 caused a shift of rapidly adapting (RA) mechanosensitive currents to intermediate-adapting currents in dorsal root ganglion sensory neurons. Deletion of both TRPC3 and TRPC6 caused deficits in light touch and silenced half of small-diameter sensory neurons expressing mechanically activated RA currents. Double TRPC3/TRPC6 knock-out mice also showed hearing impairment, vestibular deficits and defective auditory brain stem responses to high-frequency sounds. Basal, but not apical, cochlear outer hair cells lost more than 75 per cent of their responses to mechanical stimulation. FM1-43-sensitive mechanically gated currents were induced when TRPC3 and TRPC6 were co-expressed in sensory neuron cell lines. TRPC3 and TRPC6 are thus required for the normal function of cells involved in touch and hearing, and are potential components of mechanotransducing complexes.     

From stress and strain to spikes: mechanotransduction in spider slit sensilla

This review focuses on the structure and function of a single mechanoreceptor organ in the cuticle of spiders. Knowledge emerging from the study of this organ promises to yield general principles that can be applied to mechanosensation in a wide range of animal systems. The lyriform slit sense organ on the antero-lateral leg patella of the spider Cupiennius salei is unusual in possessing large sensory neurons, whose cell bodies are close to the sites of sensory transduction, and accessible to intracellular recording during mechanotransduction. This situation, combined with recent technical developments, has made it possible to observe and experiment with all the major stages of mechanosensation. Important findings include the approximate size, number and ionic selectivity of the ion channels responsible for mechanotransduction, the types of voltage-activated ion channels responsible for action potential encoding, and the mechanisms controlling the dynamic properties of transduction and encoding. Most recently, a complex efferent system for peripheral modulation of mechanosensation has been discovered and partially characterized. Much remains to be learned about mechanosensation, but the lyriform slit sense organ system continues to offer important opportunities to advance our understanding of this crucial sense.     

The DRASIC cation channel contributes to the detection of cutaneous touch and acid stimuli in mice.

Cation channels in the DEG/ENaC family are proposed to detect cutaneous stimuli in mammals. We localized one such channel, DRASIC, in several different specialized sensory nerve endings of skin, suggesting it might participate in mechanosensation and/or acid-evoked nociception. Disrupting the mouse DRASIC gene altered sensory transduction in specific and distinct ways. Loss of DRASIC increased the sensitivity of mechanoreceptors detecting light touch, but it reduced the sensitivity of a mechanoreceptor responding to noxious pinch and decreased the response of acid- and noxious heat-sensitive nociceptors. The data suggest that DRASIC subunits participate in heteromultimeric channel complexes in sensory neurons. Moreover, in different cellular contexts, DRASIC may respond to mechanical stimuli or to low pH to mediate normal touch and pain sensation.     

A TRP channel that senses cold stimuli and menthol

A distinct subset of sensory neurons are thought to directly sense changes in thermal energy through their termini in the skin. Very little is known about the molecules that mediate thermoreception by these neurons. Vanilloid Receptor 1 (VR1), a member of the TRP family of channels, is activated by noxious heat. Here we describe the cloning and characterization of TRPM8, a distant relative of VR1. TRPM8 is specifically expressed in a subset of pain- and temperature-sensing neurons. Cells overexpressing the TRPM8 channel can be activated by cold temperatures and by a cooling agent, menthol. Our identification of a cold-sensing TRP channel in a distinct subpopulation of sensory neurons implicates an expanded role for this family of ion channels in somatic sensory detection.     

Impairment of proprioceptive movement and mechanical nociception in Drosophila melanogaster larvae lacking Ppk30, a Drosophila member of the Degenerin/Epithelial Sodium Channel family

The mechanosensory neurons of Drosophila larvae are demonstrably activated by diverse mechanical stimuli, but the mechanisms underlying this function are not completely understood. Here we report a genetic, immunohistochemical, and electrophysiological analysis of the Ppk30 ion channel, a member of the Drosophila pickpocket (ppk) family, counterpart of the mammalian Degenerin/Epithelial Na⁺ Channel family. Ppk30 mutant larvae displayed deficits in proprioceptive movement and mechanical nociception, which are detected by class IV sensory (mdIV) neurons. The same neurons also detect heat nociception, which was not impaired in ppk30 mutant larvae. Similarly, Ppk30 mutation did not alter gentle touch mechanosensation, a distinct mechanosensation detected by other neurons, suggesting that Ppk30 has a functional role in mechanosensation in mdIV neurons. Consistently, Ppk30 was expressed in class IV neurons, but was not detectable in other larval skin sensory neurons. Mutant phenotypes were rescued by expressing Ppk30 in mdIV neurons. Electrophysiological analysis of heterologous cells expressing Ppk30 did not detect mechanosensitive channel activities, but did detect acid‐induced currents. These data show that Ppk30 has a role in mechanosensation, but not in thermosensation, in class IV neurons, and possibly has other functions related to acid response.     

Feeling the pressure in mammalian somatosensation

Mechanoreceptor cells of the somatosensory system initiate the perception of touch and pain. Molecules required for mechanosensation have been identified from invertebrate neurons, and recent functional studies indicate that ion channels of the transient receptor potential and degenerin/epithelial Na+ channel families are likely to be transduction channels. The expression of related channels in mammalian somatosensory neurons has fueled the notion that these channels mediate mechanotransduction in vertebrates; however, genetic disruption and heterologous expression have not yet revealed a direct role for any of these candidates in somatosensory mechanotransduction. Thus, new systems are needed to define the function of these ion channels in somatosensation and to pinpoint molecules or signaling pathways that underlie mechanotransduction in vertebrates.     

Recording of mechanosensitive currents using piezoelectrically driven mechanostimulator

Mechanotransduction constitutes the basis of a variety of physiological processes, such as the senses of touch, balance, proprioception and hearing. In vertebrates, mechanosensation is mediated by mechanosensory receptors. The aptitude of these mechanoreceptors for detecting mechanical information relies on the presence of mechanosensitive channels that transform mechanical forces into electrical signals. However, advances in understanding mechanical transduction processes have proven difficult because sensory nerve endings have historically been inaccessible to patch-clamp recording. We report here an in vitro model of mechanotransduction that allows the application of focal force on sensory neuron membrane during whole-cell patch clamping. This technique, called mechano-clamp, provides an opportunity to explore the properties and identities of mechanotransducer channels in mammalian sensory neurons. The protocol-from tissue dissociation to patch-clamp recording-can be completed in 7 h.     

Molecular determinants of mechanosensation in the muscle spindle

The muscle spindle (MS) provides essential sensory information for motor control and proprioception. The Group Ia and II MS afferents are low threshold slowly-adapting mechanoreceptors and report both static muscle length and dynamic muscle movement information. The exact molecular mechanism by which MS afferents transduce muscle movement into action potentials is incompletely understood. This short review will discuss recent evidence suggesting that PIEZO2 is an essential mechanically sensitive ion channel in MS afferents and that vesicle-released glutamate contributes to maintaining afferent excitability during the static phase of stretch. Other mechanically gated ion channels, voltage-gated sodium channels, other ion channels, regulatory proteins, and interactions with the intrafusal fibers are also important for MS afferent mechanosensation. Future studies are needed to fully understand mechanosensation in the MS and whether different complements of molecular mediators contribute to the different response properties of Group Ia and II afferents.     

Neuropeptide FF and FMRFamide potentiate acid-evoked currents from sensory neurons and proton-gated DEG/ENaC channels

Acidosis is associated with inflammation and ischemia and activates cation channels in sensory neurons. Inflammation also induces expression of FMRFamidelike neuropeptides, which modulate pain. We found that neuropeptide FF (Phe-Leu-Phe-Gln-Pro-Gln-Arg-Phe amide) and FMRFamide (Phe-Met-Arg-Phe amide) generated no current on their own but potentiated H+-gated currents from cultured sensory neurons and heterologously expressed ASIC and DRASIC channels. The neuropeptides slowed inactivation and induced sustained currents during acidification. The effects were specific; different channels showed distinct responses to the various peptides. These results suggest that acid-sensing ion channels may integrate multiple extracellular signals to modify sensory perception.     

Activation of mu opioid receptors inhibits transient high- and low-threshold Ca2+ currents, but spares a sustained current.

Opioids and opiates decrease the duration of action potentials and the amount of neurotransmitter released from sensory neurons. The mu-type opioid receptor, the binding site for morphine, is thought to act exclusively on K+ channels. Here, we show that activation of the mu receptor inhibits Ca2+ channels in rat sensory neurons; the effect is blocked by a mu antagonist and is not mimicked by kappa or delta receptor agonists. Both low-threshold (T-type) and high-threshold Ca2+ currents are partially suppressed. omega-Conotoxin-sensitive and omega-conotoxin-insensitive, high-threshold Ca2+ currents are inhibited. The kinetic effect on high-threshold current is like that caused by diminished rest potential: the transient component is selectively lost, whereas the sustained component is spared.     

TRPA1 is differentially modulated by the amphipathic molecules trinitrophenol and chlorpromazine

TRPA1, a poorly selective Ca(2+)-permeable cation channel, is expressed in peripheral sensory neurons, where it is considered to contribute to a variety of sensory processes such as the detection of painful stimuli. Furthermore, TRPA1 was also identified in hair cells of the inner ear, but its involvement in sensing mechanical forces is still being controversially discussed. Amphipathic molecules such as trinitrophenol and chlorpromazine have been shown to provide useful tools to study mechanosensitive channels. Depending on their charge, they partition in the inner or outer sheets of the lipid bilayer, causing a curvature of the membrane, which has been demonstrated to activate or inhibit mechanosensitive ion channels. In the present study, we investigated the effect of these molecules on TRPA1 gating. TRPA1 was robustly activated by the anionic amphipathic molecule trinitrophenol. The whole-cell and single channel properties resemble those previously described for TRPA1. Moreover, we could show that the toxin GsMTx-4 acts on TRPA1. In addition to its recently described role as an inhibitor of stretch-activated ion channels, it serves as a potent activator of TRPA1 channels. On the other hand, the positively charged drug chlorpromazine modulates activated TRPA1 currents in a voltage-dependent way. The exposure of activated TRPA1 channels to chlorpromazine led to a block at positive potentials and an increased open probability at negative potentials. The variability in the shape of the I-V curve gives a first indication that native mechanically activated TRPA1 currents must not necessarily exhibit the same biophysical properties as ligand-activated TRPA1 currents.     

Understanding Sensory Nerve Mechanotransduction through Localized Elastomeric Matrix Control

Background

While neural systems are known to respond to chemical and electrical stimulation, the effect of mechanics on these highly sensitive cells is still not well understood. The ability to examine the effects of mechanics on these cells is limited by existing approaches, although their overall response is intimately tied to cell-matrix interactions. Here, we offer a novel method, which we used to investigate stretch-activated mechanotransduction on nerve terminals of sensory neurons through an elastomeric interface.

Methodology/Principal Findings

To apply mechanical force on neurites, we cultured dorsal root ganglion neurons on an elastic substrate, polydimethylsiloxane (PDMS), coated with extracellular matrices (ECM). We then implemented a controlled indentation scheme using a glass pipette to mechanically stimulate individual neurites that were adjacent to the pipette. We used whole-cell patch clamping to record the stretch-activated action potentials on the soma of the single neurites to determine the mechanotransduction-based response. When we imposed specific mechanical force through the ECM, we noted a significant neuronal action potential response. Furthermore, because the mechanotransduction cascade is known to be directly affected by the cytoskeleton, we investigated the cell structure and its effects. When we disrupted microtubules and actin filaments with nocodozale or cytochalasin-D, respectively, the mechanically induced action potential was abrogated. In contrast, when using blockers of channels such as TRP, ASIC, and stretch-activated channels while mechanically stimulating the cells, we observed almost no change in action potential signalling when compared with mechanical activation of unmodified cells.

Conclusions/Significance

These results suggest that sensory nerve terminals have a specific mechanosensitive response that is related to cell architecture.     

Evidence for a protein tether involved in somatic touch

The gating of ion channels by mechanical force underlies the sense of touch and pain. The mode of gating of mechanosensitive ion channels in vertebrate touch receptors is unknown. Here we show that the presence of a protein link is necessary for the gating of mechanosensitive currents in all low‐threshold mechanoreceptors and some nociceptors of the dorsal root ganglia (DRG). Using TEM, we demonstrate that a protein filament with of length ～100 nm is synthesized by sensory neurons and may link mechanosensitive ion channels in sensory neurons to the extracellular matrix. Brief treatment of sensory neurons with non‐specific and site‐specific endopeptidases destroys the protein tether and abolishes mechanosensitive currents in sensory neurons without affecting electrical excitability. Protease‐sensitive tethers are also required for touch‐receptor function in vivo. Thus, unlike the majority of nociceptors, cutaneous mechanoreceptors require a distinct protein tether to transduce mechanical stimuli.     

Inflammatory signals enhance piezo2-mediated mechanosensitive currents

Heightened nociceptor function caused by inflammatory mediators such as bradykinin (BK) contributes to increased pain sensitivity (hyperalgesia) to noxious mechanical and thermal stimuli. Although it is known that sensitization of the heat transducer TRPV1 largely subserves thermal hyperalgesia, the cellular mechanisms underlying mechanical hyperalgesia have been elusive. The role of the mechanically activated (MA) channel piezo2 (known as FAM38B) present in mammalian sensory neurons is unknown. We test the hypothesis that piezo2 activity is enhanced by BK, an algogenic peptide that induces mechanical hyperalgesia within minutes. Piezo2 current amplitude is increased and inactivation is slowed by bradykinin receptor beta 2 (BDKRB2) activation in heterologous expression systems. Protein kinase A (PKA) and protein kinase C (PKC) agonists enhance piezo2 activity. BDKRB2-mediated effects are abolished by PKA and PKC inhibitors. Finally, piezo2-dependent MA currents in a class of native sensory neurons are enhanced 8-fold by BK via PKA and PKC. Thus, piezo2 sensitization may contribute to PKA- and PKC-mediated mechanical hyperalgesia.