Mechanically-activated Currents Research Articles

Amyloid β reduces mechanical sensation by attenuating the function of TRPV4 channels in rat small dorsal root ganglion neurons

Amyloid β (Aβ) is well known to be a key trigger for the progression of Alzheimer's disease and dementia. Aβ forms oligomers that develop into amyloid plaques, causing neuronal death and dysfunction. Aβ-induced neuronal dysfunction has been reported in peripheral neurons as well as in the brain. We previously reported that transient receptor potential vanilloid 4 (TRPV4) channels play an important role in mechanical sensation in sensory neurons innervating skeletal muscle. However, to date, it remains unclear whether Aβ modulates mechanosensation or TRPV4 channel activity in peripheral sensory neurons. As such, we hypothesized that Aβ acutely impairs the response of small dorsal root ganglion (DRG) neurons to mechanical stimuli by attenuating the function of TRPV4 channels. Purpose: The aim of this investigation was to determine the effects of Aβ1-40 and 1-42 on mechanosensation in small DRG neurons. Method: In in vitro whole-cell patch clamp recordings from cultured rat DRG neurons (Sprague-Dawley rat, n = 4, body weight: 316 ± 57 g), mechanically-activated (MA) current amplitudes or GSK1016790A (a selective agonist of TRPV4, 1 μM)-induced inward currents were measured before and 5 min after application of either HEPES buffered solution (control), Aβ1-40 (2.5 ng/μL), Aβ1-42 (2.5 ng/μL) or Aβ1-40/42 solution (2.5 ng/μL, respectively). Results: MA current amplitude was significantly decreased 5 min after application of both Aβ1-40 (Before vs. After: -71.7 ± 14.5 vs. -13.5 ± 4.8 pA. P < 0.01) and Aβ1-42 (Before vs. After: -63.7 ± 16.2 vs. -14.5 ± 5.9 pA. P < 0.05) while HEPES application did not alter MA currents (Before vs. After: -69.5 ± 17.9 ± -66.2 ± 13.9 pA. P = 0.36). Likewise, GSK1016790A-induced inward current was significantly suppressed by Aβ1-40/42 solution (Fold change in total charge transfer: HEPES vs. Aβ1-40/42: 0.92 ± 0.47 vs. 0.33 ± 0.06. P < 0.05). Conclusion: The data demonstrate that both Aβ1-40 and 1-42 suppress 1) the cellular response to mechanical stimulation and 2) TRPV4 currents in rat small DRG neurons. These findings suggest that both Aβ1-40 and 1-42 attenuate mechanical sensation in DRG neurons by inhibiting TRPV4 channels. Supported by NIH HL-151632 and JSPS 23KJ2207. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Caldendrin Is a Repressor of PIEZO2 Channels and Touch Sensation in Mice.

The sense of touch is crucial for cognitive, emotional, and social development and relies on mechanically activated (MA) ion channels that transduce force into an electrical signal. Despite advances in the molecular characterization of these channels, the physiological factors that control their activity are poorly understood. Here, we used behavioral assays, electrophysiological recordings, and various mouse strains (males and females analyzed separately) to investigate the role of the calmodulin-like Ca2+ sensor, caldendrin, as a key regulator of MA channels and their roles in touch sensation. In mice lacking caldendrin (Cabp1 KO), heightened responses to tactile stimuli correlate with enlarged MA currents with lower mechanical thresholds in dorsal root ganglion neurons (DRGNs). The expression pattern of caldendrin in the DRG parallels that of the major MA channel required for touch sensation, PIEZO2. In transfected cells, caldendrin interacts with and inhibits the activity of PIEZO2 in a manner that requires an alternatively spliced sequence in the N-terminal domain of caldendrin. Moreover, targeted genetic deletion of caldendrin in Piezo2-expressing DRGNs phenocopies the tactile hypersensitivity of complete Cabp1 KO mice. We conclude that caldendrin is an endogenous repressor of PIEZO2 channels and their contributions to touch sensation in DRGNs.

TMEM63 proteins function as monomeric high-threshold mechanosensitive ion channels

OSCA/TMEM63s form mechanically activated (MA) ion channels in plants and animals, respectively. OSCAs and related TMEM16s and transmembrane channel-like (TMC) proteins form homodimers with two pores. Here, we uncover an unanticipated monomeric configuration of TMEM63 proteins. Structures of TMEM63A and TMEM63B (referred to as TMEM63s) revealed a single highly restricted pore. Functional analyses demonstrated that TMEM63s are bona fide mechanosensitive ion channels, characterized by small conductance and high thresholds. TMEM63s possess evolutionary variations in the intracellular linker IL2, which mediates dimerization in OSCAs. Replacement of OSCA1.2 IL2 with TMEM63A IL2 or mutations to key variable residues resulted in monomeric OSCA1.2 and MA currents with significantly higher thresholds. Structural analyses revealed substantial conformational differences in the mechano-sensing domain IL2 and gating helix TM6 between TMEM63s and OSCA1.2. Our studies reveal that mechanosensitivity in OSCA/TMEM63 channels is affected by oligomerization and suggest gating mechanisms that may be shared by OSCA/TMEM63, TMEM16, and TMC channels.

Potentiation of PIEZO2 mechanically-activated currents in sensory neurons mediates vincristine-induced mechanical hypersensitivity.

Vincristine, a widely used chemotherapeutic agent for treating different cancer, often induces severe peripheral neuropathic pain. A common symptom of vincristine-induced peripheral neuropathic pain is mechanical allodynia and hyperalgesia. However, mechanisms underlying vincristine-induced mechanical allodynia and hyperalgesia are not well understood. In the present study, we show with behavioral assessment in rats that vincristine induces mechanical allodynia and hyperalgesia in a PIEZO2 channel-dependent manner since gene knockdown or pharmacological inhibition of PIEZO2 channels alleviates vincristine-induced mechanical hypersensitivity. Electrophysiological results show that vincristine potentiates PIEZO2 rapidly adapting (RA) mechanically-activated (MA) currents in rat dorsal root ganglion (DRG) neurons. We have found that vincristine-induced potentiation of PIEZO2 MA currents is due to the enhancement of static plasma membrane tension (SPMT) of these cells following vincristine treatment. Reducing SPMT of DRG neurons by cytochalasin D (CD), a disruptor of the actin filament, abolishes vincristine-induced potentiation of PIEZO2 MA currents, and suppresses vincristine-induced mechanical hypersensitivity in rats. Collectively, enhancing SPMT and subsequently potentiating PIEZO2 MA currents in primary afferent neurons may be an underlying mechanism responsible for vincristine-induced mechanical allodynia and hyperalgesia in rats. Targeting to inhibit PIEZO2 channels may be an effective analgesic method to attenuate vincristine-induced mechanical hypersensitivity.

Deciphering mechanically activated ion channels at the single-channel level in dorsal root ganglion neurons.

Mechanically activated (MA) ion channels confer somatosensory neurons with the ability to sense a wide range of mechanical stimuli. MA ion channel activity in somatosensory neurons is best described by the electrophysiological recordings of MA currents in cultured dorsal root ganglion (DRG) neurons. Biophysical and pharmacological characterization of DRG MA currents has guided the field in screening/confirming channel candidates that induce the currents and facilitate the mechanosensory response. But studies on DRG MA currents have relied mostly on whole-cell macroscopic current properties obtained by membrane indentation, and little is known about the underlying MA ion channels at the single-channel level. Here, by acquiring indentation-induced macroscopic currents as well as stretch-activated single-channel currents from the same cell, we associate macroscopic current properties with single-channel conductance. This analysis reveals the nature of the MA channel responsible for the ensemble response. We observe four different conductances in DRG neurons with no association with a specific type of macroscopic current. Applying this methodology to a Piezo2 expressing DRG neuronal subpopulation allows us to identify PIEZO2-dependent stretch-activated currents and conductance. Moreover, we demonstrate that upon Piezo2 deletion, the remaining macroscopic responses are predominantly mediated by three different single-channel conductances. Collectively, our data predict that at least two other MA ion channels exist in DRG neurons that remain to be discovered.

Piezo2 channel in nodose ganglia neurons is essential in controlling hypertension in a pathway regulated directly by Nedd4-2

Baroreflex plays a crucial role in regulation of arterial blood pressure (BP). Recently, Piezo1 and Piezo2, the mechanically-activated (MA) ion channels, have been identified as baroreceptors. However, the underlying molecular mechanism for regulating these baroreceptors in hypertension remains unknown. In this study, we used spontaneously hypertensive rats (SHR) and NG-Nitro-l-Arginine (L-NNA)- and Angiotensin II (Ang II)-induced hypertensive model rats to determine the role and mechanism of Piezo1 and Piezo2 in hypertension. We found that Piezo2 was dominantly expressed in baroreceptor nodose ganglia (NG) neurons and aortic nerve endings in Wistar-Kyoto (WKY) rats. The expression of Piezo2 not Piezo1 was significantly downregulated in these regions in SHR and hypertensive model rats. Electrophysiological results showed that the rapidly adapting mechanically-activated (RA-MA) currents and the responsive neuron numbers were significantly reduced in baroreceptor NG neurons in SHR. In WKY rats, the arterial BP was elevated by knocking down the expression of Piezo2 or inhibiting MA channel activity by GsMTx4 in NG. Knockdown of Piezo2 in NG also attenuated the baroreflex and increased serum norepinephrine (NE) concentration in WKY rats. Co-immunoprecipitation experiment suggested that Piezo2 interacted with Neural precursor cell-expressed developmentally downregulated gene 4 type 2 (Nedd4-2, also known as Nedd4L); Electrophysiological results showed that Nedd4-2 inhibited Piezo2 MA currents in co-expressed HEK293T cells. Additionally, Nedd4-2 was upregulated in NG baroreceptor neurons in SHR. Collectively, our results demonstrate that Piezo2 not Piezo1 may act as baroreceptor to regulate arterial BP in rats. Nedd4-2 induced downregulation of Piezo2 in baroreceptor NG neurons leads to hypertension in rats. Our findings provide a novel insight into the molecular mechanism for the regulation of baroreceptor Piezo2 and its critical role in the pathogenesis of hypertension.

Insulin potentiates the response to mechanical stimuli in small dorsal root ganglion neurons and thin fibre muscle afferents in vitro.

Insulin is known to activate the sympathetic nervous system centrally. A mechanical stimulus to tissues activates the sympathetic nervous system via thin fibre afferents. Evidence suggests that insulin modulates putative mechanosensitive channels in the dorsal root ganglion neurons of these afferents. In the present study, we report the novel finding that insulin augments the mechanical responsiveness of thin fibre afferents not only at dorsal root ganglion, but also at muscle tissue levels. Our data suggest that sympathoexcitation is mediated via the insulin-induced mechanical sensitization peripherally. The present study proposes a novel physiological role of insulin in the regulation of mechanical sensitivity in somatosensory thin fibre afferents. Insulin activates the sympathetic nervous system, although the mechanism underlying insulin-induced sympathoexcitation remains to be determined. A mechanical stimulus to tissues such as skin and/or skeletal muscle, no matter whether the stimulation is noxious or not, activates the sympathetic nervous system via thin fibre afferents. Evidence suggests that insulin modulates putative mechanosensitive channels in the dorsal root ganglion (DRG) neurons of these afferents. Accordingly, we investigated whether insulin augments whole-cell current responses to mechanical stimuli in small DRG neurons of normal healthy mice. We performed whole-cell patch clamp recordings using cultured DRG neurons and observed mechanically-activated (MA) currents induced by mechanical stimuli applied to the cell surface. Local application of vehicle solution did not change MA currents or mechanical threshold in cultured DRG neurons. Insulin (500mU mL-1 ) significantly augmented the amplitude of MA currents (P<0.05) and decreased the mechanical threshold (P<0.05). Importantly, pretreatment with the insulin receptor antagonist, GSK1838705, significantly suppressed the insulin-induced potentiation of the mechanical response. We further examined the impact of insulin on thin fibre muscle afferent activity in response to mechanical stimuli in normal healthy rats in vitro. Using a muscle-nerve preparation, we recorded single group IV fibre activity to a ramp-shaped mechanical stimulation. Insulin significantly decreased mechanical threshold (P<0.05), although it did not significantly increase the response magnitude to the mechanical stimulus. In conclusion, these data suggest that insulin augments the mechanical responsiveness of small DRG neurons and potentially sensitizes group IV afferents to mechanical stimuli at the muscle tissue level, possibly contributing to insulin-induced sympathoexcitation.

A mechanically activated ion channel is functionally expressed in the MrgprB4 positive sensory neurons, which detect stroking of hairy skin in mice

Mas-related G-protein coupled receptor B4 (MrgprB4) has been reported to be expressed in the dorsal root ganglion (DRG) neurons which detect stroking of hairy skin of mice. However, the mechanisms by which the MrgprB4 positive (+) neurons respond to adequate stimulus remain unsolved as it was also reported that electrophysiological analysis of cultured MrgprB4+ neurons did not reveal responses to mechanical stimuli. Contrary to the observation, however, in this study we show that the MrgprB4+ neurons functionally express a mechanically activated channel using DRG neurons dissociated from genetically-modified mice whose MrgprB4+ neurons express a red fluorescent protein. Hypotonicity-induced cell swelling increased intracellular Ca2+ concentrations ([Ca2+]i) of MrgprB4+ neurons. The [Ca2+]i increases were prevented by extracellular Ca2+ removal and by applications of nonselective Piezo channel blockers. Patch clamp analysis revealed that the MrgprB4+ neurons exhibited rapidly-adapting mechanically-activated currents. The MrgprB4+ neurons were stained with anti-Piezo2 antibody. These results raise the possibility that the MrgprB4+ neurons directly detect the stroking-like stimuli of hairy skin.

Endogenous Piezo1 Can Confound Mechanically Activated Channel Identification and Characterization

A gold standard for characterizing mechanically activated (MA) currents is via heterologous expression ofcandidate channels in naive cells. Two recent studies described MA channels using this paradigm. TMEM150c was proposed to be a component of an MA channel partly based on a heterologous expression approach (Hong etal., 2016). In another study, Piezo1's N-terminal "propeller" domain was proposed to constitute an intrinsic mechanosensitive module based on expression of a chimera between a pore-forming domain of the mechanically insensitive ASIC1 channel and Piezo1 (Zhao etal., 2016). When we attempted to replicate these results, we found each construct conferred modest MA currents in a small fraction of naive HEK cells similar to thepublished work. Strikingly, these MA currents were not detected in cells in which endogenous Piezo1 was CRISPR/Cas9 inactivated. These results highlight the importance of choosing cells lacking endogenous MA channels to assay the mechanotransduction properties of various proteins. This Matters Arising paper is in response to Hong etal. (2016) and Zhao etal. (2016) in Neuron. See also the response papers by Hong etal. (2017) and Zhao etal. (2017) published concurrently with this Matters Arising.

Mechanical sensitivity and electrophysiological properties of acutely dissociated dorsal root ganglion neurons of rats

Primary afferent fibers use mechanically activated (MA) currents to transduce innocuous and noxious mechanical stimuli. However, it is largely unknown about the differences in MA currents between the afferents for sensing innocuous and noxious stimuli. In the present study, we used dorsal root ganglion (DRG) neurons acutely dissociated from rats and studied their MA currents and also their intrinsic membrane properties. Recorded from small-sized DRG neurons, we found that most of these neurons were mechanically sensitive (MS) showing MA currents. The MS neurons could be classified into nociceptive-like mechanically sensitive (Noci-MS) and non-nociceptive-like mechanically sensitive (nonNoci-MS) neurons based on their action potential shapes. Noci-MS neurons responded to mechanical stimulation with three types of MA currents, rapidly adapting (RA), intermediately adapting (IA), and slowly adapting (SA) currents. In contrast, almost all nonNoci-MS neurons showed RA current type in response to mechanical stimulation. Mechanical thresholds had a broad range for both nonNoci-MS and Noci-MS neurons, and the thresholds were not significantly different between them. However, MA current densities were significantly smaller in Noci-MS than in nonNoci-MS neurons. Noci-MS and nonNoci-MS neurons also showed significant differences in their electrophysiological properties including action potential (AP) thresholds and AP firing patterns. These differences may contribute to the differential sensory encoding for innocuous and noxious mechanical stimuli.

Tentonin 3/TMEM150c Confers Distinct Mechanosensitive Currents in Dorsal-Root Ganglion Neurons with Proprioceptive Function

Touch sensation or proprioception requires the transduction of mechanical stimuli into electrical signals by mechanoreceptors in the periphery. These mechanoreceptors are equipped with various transducer channels. Although Piezo1 and 2 are mechanically activated (MA) channels with rapid inactivation, MA molecules with other inactivation kinetics have not been identified. Here we report that heterologously expressed Tentonin3 (TTN3)/TMEM150C isactivated by mechanical stimuli with distinctly slow inactivation kinetics. Genetic ablation of Ttn3/Tmem150c markedly reduced slowly adapting neurons in dorsal-root ganglion neurons. The MA TTN3 currents were inhibited by known blockers of mechanosensitive ion channels. Moreover, TTN3 was localized in muscle spindle afferents. Ttn3-deficient mice exhibited the loss of coordinated movements and abnormal gait. Thus, TTN3 appears to be a component of a mechanosensitive channel with a slow inactivation rate and contributes to motor coordination. Identification of this gene advances our understanding of the various types of mechanosensations, including proprioception.

A critical role for Piezo2 channels in the mechanotransduction of mouse proprioceptive neurons

Proprioceptors are responsible for the conscious sensation of limb position and movement, muscle tension or force, and balance. Recent evidence suggests that Piezo2 is a low threshold mechanosensory receptor in the peripheral nervous system, acting as a transducer for touch sensation and proprioception. Thus, we characterized proprioceptive neurons in the mesencephalic trigeminal nucleus that are involved in processing proprioceptive information from the face and oral cavity. This is a specific population of neurons that produce rapidly adapting mechanically-activated currents that are fully dependent on Piezo2. As such, we analyzed the deficits in balance and coordination caused by the selective deletion of the channel in proprioceptors (conditional knockout). The data clearly shows that Piezo2 fulfills a critical role in a defined homogeneous population of proprioceptor neurons that innervate the head muscles, demonstrating that this ion channel is essential for mammalian proprioceptive mechanotransduction.

Effect of protons on the mechanical response of rat muscle nociceptive fibers and neurons in vitro

Strong exercise makes muscle acidic, and painful. The stimulus that activates muscle nociceptors in such instance may be protons. Reportedly, however, not many afferents are excited by protons alone. We, therefore, posited that protons sensitize muscular nociceptors to mechanical stimuli. We examined effects of protons on mechanical sensitivity of muscle nociceptors by single-fiber recording from rat muscle-nerve preparations in vitro and by whole cell patch-clamp recording of mechanically activated (MA) currents from cultured rat dorsal root ganglion neurons. We recorded 38 Aδ- and C-fibers. Their response magnitude was increased by both pH 6.2 and pH 6.8; in addition the mechanical threshold was lowered by pH 6.2. Decrease in the threshold by pH6.2 was also observed in MA currents. Presently observed sensitization by protons could be involved in several types of ischemic muscle pain, and may also be involved in cardiovascular and respiratory controls during exercise.

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 present in mammalian sensory neurons is unknown. We tested the hypothesis that piezo2 activity is enhanced by BK, an algogenic peptide that induces mechanical hyperalgesia within minutes. We elicited piezo2-mediated currents by poking transiently transfected HEK293T cells with a blunt glass probe. Piezo2 current amplitude increases and inactivation is slowed by activation of exogenous bradykinin receptor B2. The area under the curve was enhanced 6.5-fold by BK compared with vehicle. Inclusion of GDPbetaS in the pipette occluded both effects on amplitude and inactivation. The protein kinase A (PKA) and protein kinase C (PKC) agonists 8-BrcAMP and PMA, respectively, enhanced piezo2 activity. No enhancement of piezo2-mediated currents was observed during exposure to the PLC activator m-3M3FBS (25 microM), suggesting phosphatidylinositol-PLC pathways do not increase piezo2 activity; inhibition of store release by thapsigargin had no effect on BK-induced changes. The PKA inhibitor H-89 and PKC inhibitor BIM I applied together effectively abrogated the BK-induced changes. Finally, piezo2-dependent MA currents in a class of native sensory neurons are enhanced 8-fold by BK via a PKA and PKC dependent mechanism, similarly to that observed in HEK cells. Thus, piezo2 sensitization may contribute to PKA- and PKC-mediated mechanical hyperalgesia. The mechanism described here differs from another model of mechanical hyperalgesia (epinephrine-induced PKCe-mediated) in which the downstream effector of cAMP is EPAC rather than PKA, since the BK effects observed here require PKA activity, and neither PI-PLC nor phospholipase D appears to be involved.

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.

Piezo1 and Piezo2 are essential components of distinct mechanically activated cation channels.

Mechanical stimuli drive many physiological processes, including touch and pain sensation, hearing, and blood pressure regulation. Mechanically activated (MA) cation channel activities have been recorded in many cells, but the responsible molecules have not been identified. We characterized a rapidly adapting MA current in a mouse neuroblastoma cell line. Expression profiling and RNA interference knockdown of candidate genes identified Piezo1 (Fam38A) to be required for MA currents in these cells. Piezo1 and related Piezo2 (Fam38B) are vertebrate multipass transmembrane proteins with homologs in invertebrates, plants, and protozoa. Overexpression of mouse Piezo1 or Piezo2 induced two kinetically distinct MA currents. Piezos are expressed in several tissues, and knockdown of Piezo2 in dorsal root ganglia neurons specifically reduced rapidly adapting MA currents. We propose that Piezos are components of MA cation channels.

TRPA1 Mediates Mechanical Currents in the Plasma Membrane of Mouse Sensory Neurons

Mechanosensitive channels serve as essential sensors for cells to interact with their environment. The identity of mechanosensitive channels that underlie somatosensory touch transduction is still a mystery. One promising mechanotransduction candidate is the Transient Receptor Potential Ankyrin 1 (TRPA1) ion channel. To determine the role of TRPA1 in the generation of mechanically-sensitive currents, we used dorsal root ganglion (DRG) neuron cultures from adult mice and applied rapid focal mechanical stimulation (indentation) to the soma membrane. Small neurons (diameter <27 µm) were studied because TRPA1 is functionally present in these neurons which largely give rise to C-fiber afferents in vivo. Small neurons were classified by isolectin B4 binding.Mechanically-activated inward currents were classified into two subtypes: Slowly Adapting and Transient. First, significantly more IB4 negative neurons (84%) responded to mechanical stimulation than IB4 positive neurons (54%). Second, 89% of Slowly Adapting currents were present in IB4 negative neurons whereas only 11% were found in IB4 positive neurons. Third, Slowly Adapting currents were completely absent in IB4 negative neurons from TRPA1−/− mice. Consistent with this, Slowly Adapting currents were abolished in wild type IB4 negative neurons stimulated in the presence of a TRPA1 antagonist, HC-030031. In addition, the amplitude of Transient mechanically-activated currents in IB4 positive neurons from TRPA1−/− mice was reduced by over 60% compared to TRPA1+/+ controls; however, a similar reduction did not occur in wild-type neurons treated with HC-030031. Transfection of TRPA1 in HEK293 cells did not significantly alter the proportion or magnitude of mechanically-activated currents in HEK293 cells, indicating that TRPA1 alone is not sufficient to confer mechanical sensitivity.These parallel genetic and pharmacological data demonstrate that TRPA1 mediates the Slowly Adapting mechanically-activated currents in small-diameter IB4 negative neurons from adult mice. The TRPA1 protein may also contribute to a complex that mediates Transient mechanically-activated currents in small IB4 positive C fiber type neurons.