David P. Corey

TRPA1 Contributes to Cold, Mechanical, and Chemical Nociception but Is Not Essential for Hair-Cell Transduction

TRPA1, a member of the transient receptor potential (TRP) family of ion channels, is expressed by dorsal root ganglion neurons and by cells of the inner ear, where it has proposed roles in sensing sound, painful cold, and irritating chemicals. To test the in vivo roles of TRPA1, we generated a mouse in which the essential exons required for proper function of the Trpa1 gene were deleted. Knockout mice display behavioral deficits in response to mustard oil, to cold ( approximately 0 degrees C), and to punctate mechanical stimuli. These mice have a normal startle reflex to loud noise, a normal sense of balance, a normal auditory brainstem response, and normal transduction currents in vestibular hair cells. TRPA1 is apparently not essential for hair-cell transduction but contributes to the transduction of mechanical, cold, and chemical stimuli in nociceptor sensory neurons.

The early-onset torsion dystonia gene (DYT1) encodes an ATP-binding protein

Early-onset torsion dystonia is a movement disorder, characterized by twisting muscle contractures, that begins in childhood. Symptoms are believed to result from altered neuronal communication in the basal ganglia. This study identifies the DYT1 gene on human chromosome 9q34 as being responsible for this dominant disease. Almost all cases of early-onset dystonia have a unique 3-bp deletion that appears to have arisen independently in different ethnic populations. This deletion results in loss of one of a pair of glutamic-acid residues in a conserved region of a novel ATP-binding protein, termed torsinA. This protein has homologues in nematode, rat, mouse and humans, with some resemblance to the family of heat-shock proteins and Clp proteases.

Sensitivity, polarity, and conductance change in the response of vertebrate hair cells to controlled mechanical stimuli

Hair cells, the primary receptors of the auditory, vestibular, and lateral-line sensory systems, produce electrical signals in response to mechanical stimulation of their apical hair bundles. We employed an in vitro preparation and intracellular recording to investigate the transduction mechanism of hair cells in the sacculus from the inner ear of the bullfrog (Rana catesbeiana). When stimulated directly by mechanical deflection of their hair bundles, these cells gave graded responses up to 15 mV in amplitude; the peak sensitivity was about 20 mV/micron deflection. The depolarizing component of the receptor potential corresponding to stimuli directed towards the kinocilium. Depolarizing responses were associated with a membrane resistance decrease, and hyperpolarizing responses with a resistance increase. Action potentials, possibly calcium spikes, were occasionally evoked in hair cells by mechanical or electrical stimulation.

TRPA1 is a candidate for the mechanosensitive transduction channel of vertebrate hair cells.

Mechanical deflection of the sensory hair bundles of receptor cells in the inner ear causes ion channels located at the tips of the bundle to open, thereby initiating the perception of sound. Although some protein constituents of the transduction apparatus are known, the mechanically gated transduction channels have not been identified in higher vertebrates. Here, we investigate TRP (transient receptor potential) ion channels as candidates and find one, TRPA1 (also known as ANKTM1), that meets criteria for the transduction channel. The appearance of TRPA1 messenger RNA expression in hair cell epithelia coincides developmentally with the onset of mechanosensitivity. Antibodies to TRPA1 label hair bundles, especially at their tips, and tip labelling disappears when the transduction apparatus is chemically disrupted. Inhibition of TRPA1 protein expression in zebrafish and mouse inner ears inhibits receptor cell function, as assessed with electrical recording and with accumulation of a channel-permeant fluorescent dye. TRPA1 is probably a component of the transduction channel itself.

TRP channels in mechanosensation: direct or indirect activation?

Ion channels of the transient receptor potential (TRP) superfamily are involved in a wide variety of neural signalling processes, most prominently in sensory receptor cells. They are essential for mechanosensation in systems ranging from fruitfly hearing, to nematode touch, to mouse mechanical pain. However, it is unclear in many instances whether a TRP channel directly transduces the mechanical stimulus or is part of a downstream signalling pathway. Here, we propose criteria for establishing direct mechanical activation of ion channels and review these criteria in a number of mechanosensory systems in which TRP channels are involved.

A chemical-genetic strategy implicates myosin-1c in adaptation by hair cells

Myosin-1c (also known as myosin-Ibeta) has been proposed to mediate the slow component of adaptation by hair cells, the sensory cells of the inner ear. To test this hypothesis, we mutated tyrosine-61 of myosin-1c to glycine, conferring susceptibility to inhibition by N(6)-modified ADP analogs. We expressed the mutant myosin-1c in utricular hair cells of transgenic mice, delivered an ADP analog through a whole-cell recording pipette, and found that the analog rapidly blocked adaptation to positive and negative deflections in transgenic cells but not in wild-type cells. The speed and specificity of inhibition suggests that myosin-1c participates in adaptation in hair cells.

Ion channel expression by white matter glia: The O-2A glial progenitor cell

We describe electrophysiological properties of the O-2A glial progenitor cell in a new serum-free culture system. O-2A progenitors have many properties characteristic of neurons: they have glutamate-activated ion channels, express the neuronal form of the sodium channel, fire single regenerative potentials, and synthesize the neurotransmitter GABA by an alternative synthetic pathway. Nearly identical properties were observed in acutely isolated O-2A progenitors, indicating that this phenotype is not an artifact of culture. The O-2A did not express a simple subset of channel types found in its descendant cells, the type-2 astrocyte and oligodendrocyte, studied in the same culture system. During development, these electrophysiological properties may contribute to O-2A function in vivo.

An Ion Channel Essential for Sensing Chemical Damage

Tissue damage and its downstream consequences are experimentally assayed by formaldehyde application, which indiscriminately modifies proteins and is presumed to cause pain through broadly acting mechanisms. Here we show that formaldehyde activates the ion channel TRPA1 and that TRPA1-deficient mice exhibit dramatically reduced formaldehyde-induced pain responses. 4-Hydroxynonenal, a reactive chemical produced endogenously during oxidative stress, and other related aldehydes also activate TRPA1 in vitro. Furthermore, painful responses to iodoacetamide, a nonspecific cysteine-alkylating compound, are abolished in TRPA1-deficient mice. Therefore, although these reactive chemicals modify many proteins, the associated pain appears mainly dependent on a single ion channel.

TRPA1 Modulates Mechanotransduction in Cutaneous Sensory Neurons

Transient receptor potential ankyrin 1 (TRPA1) is expressed by nociceptive neurons of the dorsal root ganglia (DRGs) and trigeminal ganglia, but its roles in cold and mechanotransduction are controversial. To determine the contribution of TRPA1 to cold and mechanotransduction in cutaneous primary afferent terminals, we used the ex vivo skin–nerve preparation from Trpa1+/+, Trpa1+/−, and Trpa1−/− adult mouse littermates. Cutaneous fibers from TRPA1-deficient mice showed no deficits in acute cold sensitivity, but they displayed striking deficits in mechanical response properties. C-fiber nociceptors from Trpa1−/− mice exhibited action potential firing rates 50% lower than those in wild-type C-fibers across a wide range of force intensities. Aδ-fiber mechanonociceptors also had reduced firing, but only at high intensity forces (>100 mN). Surprisingly, the firing rates of low-threshold Aβ and D-hair mechanoreceptive fibers were also altered. TRPA1 protein and mRNA expression was assessed in DRG neurons and cutaneous innervation by using Trpa1 in situ hybridization, an antibody for TRPA1, and an antibody for placental alkaline phosphatase (PLAP) in mice in which PLAP was substituted for Trpa1. DRG neurons of all sizes expressed Trpa1 mRNA or PLAP immunoreactivity. TRPA1 or PLAP immunolabeling was detected not only on many thin-caliber axons and intraepidermal endings but also on many large-caliber axons as well as lanceolate and Meissner endings. Epidermal and hair follicle keratinocytes also express TRPA1 message and protein. We propose that TRPA1 modulates mechanotransduction via a cell-autonomous mechanism in nociceptor terminals and possibly through a modulatory role in keratinocytes, which may interact with sensory terminals to modify their mechanical firing properties.

The ion channel TRPA1 is required for normal mechanosensation and is modulated by algesic stimuli

BACKGROUND & AIMS The transient receptor potential (TRP) channel family includes transducers of mechanical and chemical stimuli for visceral sensory neurons. TRP ankyrin 1 (TRPA1) is implicated in inflammatory pain; it interacts with G-protein-coupled receptors, but little is known about its role in the gastrointestinal (GI) tract. Sensory information from the GI tract is conducted via 5 afferent subtypes along 3 pathways. METHODS Nodose and dorsal root ganglia whose neurons innnervate 3 different regions of the GI tract were analyzed from wild-type and TRPA1(-/-) mice using quantitative reverse-transcription polymerase chain reaction, retrograde labeling, and in situ hybridization. Distal colon sections were analyzed by immunohistochemistry. In vitro electrophysiology and pharmacology studies were performed, and colorectal distension and visceromotor responses were measured. Colitis was induced by administration of trinitrobenzene sulphonic acid. RESULTS TRPA1 is required for normal mechano- and chemosensory function in specific subsets of vagal, splanchnic, and pelvic afferents. The behavioral responses to noxious colonic distension were substantially reduced in TRPA1(-/-) mice. TRPA1 agonists caused mechanical hypersensitivity, which increased in mice with colitis. Colonic afferents were activated by bradykinin and capsaicin, which mimic effects of tissue damage; wild-type and TRPA1(-/-) mice had similar direct responses to these 2 stimuli. After activation by bradykinin, wild-type afferents had increased mechanosensitivity, whereas, after capsaicin exposure, mechanosensitivity was reduced: these changes were absent in TRPA1(-/-) mice. No interaction between protease-activated receptor-2 and TRPA1 was evident. CONCLUSIONS These findings demonstrate a previously unrecognized role for TRPA1 in normal and inflamed mechanosensory function and nociception within the viscera.