Expression of the type II voltage-dependent sodium channel gene is restricted to neurons by a silencer element active in nonneuronal cells. We have cloned cDNA coding for a transcription factor (REST) that binds to this silencer element. Expression of a recombinant REST protein confers the ability to silence type II reporter genes in neuronal cell types lacking the native REST protein, whereas expression of a dominant negative form of REST in nonneuronal cells relieves silencing mediated by the native protein. REST transcripts in developing mouse embryos are detected ubiquitously outside of the nervous system. We propose that expression of the type II sodium channel gene in neurons reflects a default pathway that is blocked in nonneuronal cells by the presence of REST.

The formalin model is widely used for evaluating the effects of analgesic compounds in laboratory animals. Injection of formalin into the hind paw induces a biphasic pain response; the first phase is thought to result from direct activation of primary afferent sensory neurons, whereas the second phase has been proposed to reflect the combined effects of afferent input and central sensitization in the dorsal horn. Here we show that formalin excites sensory neurons by directly activating TRPA1, a cation channel that plays an important role in inflammatory pain. Formalin induced robust calcium influx in cells expressing cloned or native TRPA1 channels, and these responses were attenuated by a previously undescribed TRPA1-selective antagonist. Moreover, sensory neurons from TRPA1-deficient mice lacked formalin sensitivity. At the behavioral level, pharmacologic blockade or genetic ablation of TRPA1 produced marked attenuation of the characteristic flinching, licking, and lifting responses resulting from intraplantar injection of formalin. Our results show that TRPA1 is the principal site of formalin's pain-producing action in vivo, and that activation of this excitatory channel underlies the physiological and behavioral responses associated with this model of pain hypersensitivity.analgesia ͉ inflammation ͉ trp channel ͉ formaldehyde T he formalin model was developed Ͼ30 years ago to assess pain and evaluate analgesic drugs in laboratory animals (1). In this test, a dilute (0.5-5%) formalin solution (in which formaldehyde is the active ingredient) is injected into the paw of a rodent, and pain-related behaviors are assessed over two temporally distinct phases, including an initial robust phase in which paw lifting, licking, and f linching are scored during the first 10 min, followed by a transient decline in these behaviors and a subsequent second phase of behavior lasting 30 -60 min (2, 3).Compounds that typically affect the first phase (Phase I) include local anesthetics, such as lidocaine (4). The second phase (Phase II) is proposed to result from activity-dependent sensitization of CNS neurons within the dorsal horn (3, 5, 6). Many analgesics, including intrathecal nonsteroidal antiinflammatory drugs (7), NMDA antagonists (8, 9), morphine (1, 10), and gabapentin (11, 12), inhibit only Phase II responses, but not Phase I.The formalin test has several advantages over other models, in that spontaneous pain-related responses can be observed in a freely moving unrestrained animal. Once injected, no additional stimulus is required to evoke nocifensive behaviors, and behaviors can be scored over a prolonged period such that the precise onset and duration of analgesics can be assessed (1). However, despite the utility and widespread use of the formalin model in pain research, the mechanism by which formalin triggers C-fiber activation remains unknown (13) and is often attributed to tissue injury (1,3,9).In this study, we show that formalin activates primary afferent sensory neurons through a specific and direct action o...

Voltage changes across the cell membrane control the gating of many cation-selective ion channels. Conserved from bacteria to humans, the voltage-gated-ligand superfamily of ion channels are encoded as polypeptide chains of six transmembrane-spanning segments (S1-S6). S1-S4 functions as a self-contained voltage-sensing domain (VSD), in essence a positively charged lever that moves in response to voltage changes. The VSD 'ligand' transmits force via a linker to the S5-S6 pore domain 'receptor', thereby opening or closing the channel. The ascidian VSD protein Ci-VSP gates a phosphatase activity rather than a channel pore, indicating that VSDs function independently of ion channels. Here we describe a mammalian VSD protein (H(V)1) that lacks a discernible pore domain but is sufficient for expression of a voltage-sensitive proton-selective ion channel activity. H(v)1 currents are activated at depolarizing voltages, sensitive to the transmembrane pH gradient, H+-selective, and Zn2+-sensitive. Mutagenesis of H(v)1 identified three arginine residues in S4 that regulate channel gating and two histidine residues that are required for extracellular inhibition of H(v)1 by Zn2+. H(v)1 is expressed in immune tissues and manifests the characteristic properties of native proton conductances (G(vH+)). In phagocytic leukocytes, G(vH+) are required to support the oxidative burst that underlies microbial killing by the innate immune system. The data presented here identify H(v)1 as a long-sought voltage-gated H+ channel and establish H(v)1 as the founding member of a family of mammalian VSD proteins.

Transient receptor potential (TRP) proteins are cation-selective channels that function in processes as diverse as sensation and vasoregulation. Mammalian TRP channels that are gated by heat and capsaicin (>43 degrees C; TRPV1 (ref. 1)), noxious heat (>52 degrees C; TRPV2 (ref. 2)), and cooling (< 22 degrees C; TRPM8 (refs 3, 4)) have been cloned; however, little is known about the molecular determinants of temperature sensing in the range between approximately 22 degrees C and 40 degrees C. Here we have identified a member of the vanilloid channel family, human TRPV3 (hTRPV3) that is expressed in skin, tongue, dorsal root ganglion, trigeminal ganglion, spinal cord and brain. Increasing temperature from 22 degrees C to 40 degrees C in mammalian cells transfected with hTRPV3 elevated intracellular calcium by activating a nonselective cationic conductance. As in published recordings from sensory neurons, the current was steeply dependent on temperature, sensitized with repeated heating, and displayed a marked hysteresis on heating and cooling. On the basis of these properties, we propose that hTRPV3 is thermosensitive in the physiological range of temperatures between TRPM8 and TRPV1.

Mammalian spermatozoa become motile at ejaculation, but before they can fertilize the egg, they must acquire more thrust to penetrate the cumulus and zona pellucida. The forceful asymmetric motion of hyperactivated spermatozoa requires Ca 2؉ entry into the sperm tail by an alkalinization-activated voltage-sensitive Ca 2؉ -selective current (ICatSper). Hyperactivation requires CatSper1 and CatSper2 putative ion channel genes, but the function of two other related genes (CatSper3 and CatSper4) is not known. Here we show that targeted disruption of murine CatSper3 or CatSper4 also abrogated I CatSper , sperm cell hyperactivated motility and male fertility but did not affect spermatogenesis or initial motility. Direct protein interactions among CatSpers, the sperm specificity of these proteins, and loss of I CatSper in each of the four CatSper ؊/؊ mice indicate that CatSpers are highly specialized flagellar proteins.calcium ͉ contraception ͉ flagella S permatozoa first acquire the potential for motility in the epididymis. They are capacitated in the female reproductive tract (1), where they acquire hyperactivated motility and other attributes that enable fertilization (2). During hyperactivation, the sperm tail motion changes from symmetric, fast, and low amplitude (sinusoidal) to asymmetric, slow, and large amplitude (whip-like; refs. 3-5). Hyperactivation is required for fertilization, providing the force needed to free the sperm cell from the oviductal reservoir and to penetrate the cumulus and zona pellucida surrounding the egg (1, 6, 7).Sperm cells become motile and progress directionally once they enter the female reproductive tract. Ca 2ϩ -independent flagellar dynein and ATP orchestrate the low-amplitude sinusoidal-activated motility of the tail. As the sperm cells encounter a more alkaline environment in the higher female reproductive tract, they hyperactivate, a process that requires Ca 2ϩ entry (3,8,9). Studies with antibodies or nucleotide probes have labeled several Ca 2ϩ -permeant channels, including voltage-sensitive Ca 2ϩ -selective channels (CatSpers and CaVs), cyclic nucleotide-gated channels, and transient receptor potential channels, in spermatocytes or spermatozoa (10-18). However, recent patch-clamp recordings of mouse epididymal spermatozoa (19) show that the predominant Ca 2ϩ -carrying current requires the CatSper1 gene that encodes a six-transmembrane-spanning protein of the voltage-gated ligand ion channel superfamily (20). In both whole-cell and perforatedpatch configurations, the Ca 2ϩ -selective current (I CatSper ) originated from the principal piece of the sperm tail and was absent in spermatozoa from CatSper1 Ϫ/Ϫ mice. CatSper1 Ϫ/Ϫ and CatSper2 Ϫ/Ϫ male mice are infertile (11,21), and sperm cells from CatSper1 Ϫ/Ϫ and CatSper2 Ϫ/Ϫ mice are unable to hyperactivate (4, 21). I CatSper was dramatically potentiated by a rise in intracellular pH, suggesting that the alkalinization that occurs during sperm capacitation activates I CatSper to increase intracellular [Ca 2ϩ ] and induce hypera...

Asthma is an inflammatory disorder caused by airway exposures to allergens and chemical irritants. Studies focusing on immune, smooth muscle, and airway epithelial function revealed many aspects of the disease mechanism of asthma. However, the limited efficacies of immune-directed therapies suggest the involvement of additional mechanisms in asthmatic airway inflammation. TRPA1 is an irritant-sensing ion channel expressed in airway chemosensory nerves. TRPA1-activating stimuli such as cigarette smoke, chlorine, aldehydes, and scents are among the most prevalent triggers of asthma. Endogenous TRPA1 agonists, including reactive oxygen species and lipid peroxidation products, are potent drivers of allergen-induced airway inflammation in asthma. Here, we examined the role of TRPA1 in allergic asthma in the murine ovalbumin model. Strikingly, genetic ablation of TRPA1 inhibited allergen-induced leukocyte infiltration in the airways, reduced cytokine and mucus production, and almost completely abolished airway hyperreactivity to contractile stimuli. This phenotype is recapitulated by treatment of wild-type mice with HC-030031, a TRPA1 antagonist. HC-030031, when administered during airway allergen challenge, inhibited eosinophil infiltration and prevented the development of airway hyperreactivity. Trpa1 ؊/؊ mice displayed deficiencies in chemically and allergen-induced neuropeptide release in the airways, providing a potential explanation for the impaired inflammatory response. Our data suggest that TRPA1 is a key integrator of interactions between the immune and nervous systems in the airways, driving asthmatic airway inflammation following inhaled allergen challenge. TRPA1 may represent a promising pharmacological target for the treatment of asthma and other allergic inflammatory conditions. airway hyperreactivity ͉ TRP channel ͉ TRPA1

TRPA1 is a nonselective cation channel expressed by nociceptors. Although it is widely accepted that TRPA1 serves as a broad irritancy receptor for a variety of reactive chemicals, its role in cold sensation remains controversial. Here, we demonstrate that mild cooling markedly increases agonist-evoked rat TRPA1 currents. In the absence of an agonist, even noxious cold only increases current amplitude slightly. These results suggest that TRPA1 is a key mediator of cold hypersensitivity in pathological conditions in which reactive oxygen species and proinflammatory activators of the channel are present, but likely plays a comparatively minor role in acute cold sensation. Supporting this, cold hypersensitivity can be induced in wild-type but not Trpa1 Ϫ/Ϫ mice by subcutaneous administration of a TRPA1 agonist. Furthermore, the selective TRPA1 antagonist HC-030031 [2-(1,3-dimethyl-2,6-dioxo-1,2,3,6-tetrahydro-7H-purin-7-yl)-N-(4-isopropylphenyl)acetamide] reduces cold hypersensitivity in rodent models of inflammatory and neuropathic pain.

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