ATP is known to depolarize sensory neurons, and may play a role in nociceptor activation when released from damaged tissue. Here we report the molecular cloning and characterization of a new member of the P2X receptor family, P2X3, expressed by these cells. The channel transcript was present in a subset of rat dorsal-root-ganglion sensory neurons, some of which express nociceptor-associated markers; it was absent in other tissues that were tested, including sympathetic, enteric and central nervous system neurons. Moreover, when expressed in Xenopus oocytes, the channel showed an ATP-dependent cation flux. P2X3 is the only ligand-gated channel known to be expressed exclusively by a subset of sensory neurons. The remarkable selectivity of expression of the channel coupled with its sensory neuron-like pharmacology suggests that this channel may transduce ATP-evoked nociceptor activation.

Proton-gated channels expressed by sensory neurons are of particular interest because low pH causes pain. Two proton-gated channels, acid-sensing ionic channel (ASIC) and dorsal root ASIC (DRASIC), that are members of the amiloride-sensitive ENaC͞Degenerin family are known to be expressed by sensory neurons. Here, we describe the cloning and characterization of an ASIC splice variant, ASIC-␤, which contains a unique N-terminal 172 aa, as well as unique 5 and 3 untranslated sequences. ASIC-␤, unlike ASIC and DRASIC, is found only in a subset of small and large diameter sensory neurons and is absent from sympathetic neurons or the central nervous system. The patterns of expression of ASIC and ASIC-␤ transcripts in rat dorsal root ganglion neurons are distinct. When expressed in COS-7 cells, ASIC-␤ forms a functional channel with electrophysiological properties distinct from ASIC and DRASIC. The pH dependency and sensitivity to amiloride of ASIC-␤ is similar to that described for ASIC, but unlike ASIC, the channel is not permeable to calcium, nor are ASIC-␤-mediated currents inhibited by extracellular calcium. The unique distribution of ASIC-␤ suggests that it may play a specialized role in sensory neuron function.

Acid-sensing ion channel 3 (ASIC3), a proton-gated ion channel of the degenerins͞epithelial sodium channel (DEG͞ENaC) receptor family is expressed predominantly in sensory neurons including nociceptive neurons responding to protons. To study the role of ASIC3 in pain signaling, we generated ASIC3 knockout mice. Mutant animals were healthy and responded normally to most sensory stimuli. However, in behavioral assays for pain responses, ASIC3 null mutant mice displayed a reduced latency to the onset of pain responses, or more pain-related behaviors, when stimuli of moderate to high intensity were used. This unexpected effect seemed independent of the modality of the stimulus and was observed in the acetic acid-induced writhing test (0.6 vs. 0.1-0.5%), in the hot-plate test (52.5 and 55 vs. 50°C), and in tests for mechanically induced pain (tail-pinch vs. von Frey filaments). We postulate that ASIC3 is involved in modulating moderate-to high-intensity pain sensation. C ation channels of the degenerin͞epithelial sodium channel family (DEG͞ENaC) have been proposed as transducers of somatosensory stimuli in several species (1). The structural hallmarks of these proteins are two hydrophobic transmembrane domains, with short N and C termini and a large extracellular loop. In vertebrates, the DEG͞ENaC family includes several related subunits of Na ϩ -selective (P NA ͞P K , 8-40) acid-sensing ion channels (ASIC1a, ASIC1b, ASIC2a, ASC2b, ASIC3, and ASIC4; previously named ASIC-␣͞BNC2, ASIC-␤, MDEG1͞ BNC1, MDEG2, DRASIC, and SPASIC, respectively) (2-4). ASIC proteins associate as homo-or heteromultimers to form functional receptors. Because these receptors are gated by protons, it has been suggested that they might be involved in the perception of pain during tissue acidosis (5). However, there is evidence also that they are involved in mechanosensation; many DEG͞ENaC proteins are localized to mechanosensitive cells in Caenorhabditis elegans, Drosophila melanogaster, rat, and mouse (1, 6, 7). In C. elegans, mutations in DEG͞ENaC proteins such as MEC-4 and MEC-10 lead to impaired touch responses (8,9), and the targeted deletion of ASIC2 in mice resulted in a reduced sensitivity of low-threshold mechanoreceptors (7).One member of the ASIC family, ASIC3, seems to be a particularly good candidate for the transduction of proton and mechanical stimuli, because ASIC3 is expressed predominantly in dorsal root ganglia neurons (10) including large-diameter mechanoreceptors and unmyelinated small-diameter peptidergic nociceptors (3, 6, 11). In addition, ASIC3 protein was found to be present in sensory nerve terminals in Meissner corpuscles lanceolate fibers, which correspond to rapidly adapting lowthreshold mechanoreceptors, as well as in free nerve endings, which may correspond to nociceptors (6).Previous studies have shown that ASIC3 can be activated by protons and generate biphasic inward currents when it was expressed in heterologous cells (12). A transient inward current can be induced when extracellular pH falls to 7.0, which is...

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