Protons can modulate the activity of a number of receptors and ion channels expressed in nociceptors (1). Among such entities, they can directly activate vanilloid receptor subtype-1 (VR1) and acid-sensing ion channel ASICs (2, 3). VR1 is selectively expressed in polymodal nociceptors, which are responsive to noxiously thermal, mechanical, and chemical stimuli, and is broadly regarded as a major detector of multiple pain-producing stimuli. The contribution of VR1 to the pH sensitivity of nociceptors has been established in vitro by gene knockout experiments (4). However, the activation of the VR1 channel requires extremely severe acidification to pH less than 6.0 (4, 5), raising the possibility that another signal sensor that is more sensitive to protons than VR1 may be present in nociceptors, because, for example, skin nociceptors have activation thresholds as high as pH 6.9 (6). In muscle and cardiac ischemia, an extracellular pH drop from 7.4 to 7.0 is sufficient to induce persistent activation of a subset of nociceptors (7-9). Recent electrophysiological experiments have strongly suggested the involvement of ASICs (amiloride-blockable proton-gated channel subunits expressed in mammalian central and peripheral nervous systems) (10) in nociception linked to acidoses. Sensory neurons from mice lacking ASIC-3 (nomenclature as in ref. 3) are severely deficient in their responses to acidic stimuli in vitro (11). The heterologously expressed ASIC-2b/ASIC-3 channel generates a biphasic inward current that is similar to the native proton-activated current in dorsal root ganglion (DRG) neurons (12). The ASIC-3 channel is capable of reproducing the features of acid-evoked currents in cardiac nociceptors (13). Despite these observations, there is still controversy about the functional roles of ASICs in mammals, because proton detection through ASICs has not yet been demonstrated in vivo. In this report, we evaluated the efficacy of amiloride (an inhibitor of ASICs) and capsazepine (an inhibitor of VR1) on acid-evoked pain in humans using a psychophysical method. To confirm the specificities of both drugs, we investigated their effects on capsaicinevoked pain using a similar psychophysical approach. Our results indicate the involvement of ASICs and VR1 in proton-induced pain in humans and show their relative importance in the nociception. Methods Psychophysical experiments. The following experiments were approved by the Ethics Committee of the Nagoya City University Medical School and conducted in accordance with the Declaration of Helsinki. A total of 56 healthy men, 21-41 years of age, participated in the study. All subjects stated that they had not used drugs of any kind within one week preceding the experiments.
Bitter taste perception is a conserved chemical sense against the ingestion of poisonous substances in mammals. A multigene family of G-protein-coupled receptors, T2R (so-called TAS2R or TRB) receptors and a G-protein alpha subunit (Galpha), gustducin, are believed to be key molecules for its perception, but little is known about the molecular basis for its interaction. Here, we use a heterologous expression system to determine a specific domain of gustducin necessary for T2R coupling. Two chimeric Galpha16 proteins harboring 37 and 44 gustducin-specific sequences at their C termini (G16/gust37 and G16/gust44) responded to different T2R receptors with known ligands, but G16/gust 23, G16/gust11, and G16/gust5 did not. The former two chimeras contained a predicted beta6 sheet, an alpha5 helix, and an extreme C terminus of gustducin, and all the domains were indispensable to the expression of T2R activity. We also expressed G16 protein chimeras with the corresponding domain from other Galpha(i) proteins, cone-transducin (Galpha(t2)), Galpha(i2), and Galpha(z) (G16/t2, G16/i2, and G16/z). As a result, G16/t2 and G16/i2 produced specific responses of T2Rs, but G16/z did not. Because Galpha(t2) and Galpha(i2) are expressed in the taste receptor cells, these G-protein alpha(i) subunits may also be involved in bitter taste perception via T2R receptors. The present Galpha16-based chimeras could be useful tools to analyze the functions of many orphan G-protein-coupled taste receptors.
Acid-sensing ion channel-2a (ASIC2a) is an amiloride-blockable proton-gated cation channel, probably contributing to sour-taste detection in rat taste cells. To isolate another subtype of the sour-taste receptor, we screened a rat circumvallate papilla cDNA library and identified ASIC2b, an N-terminal splice variant of ASIC2a. Reverse transcription-PCR analyses confirmed the expression of ASIC2b transcripts in the circumvallate papilla and, moreover, demonstrated its expression in the foliate and fungiform papillae. Immunohistochemical analyses revealed that ASIC2b, as well as ASIC2a, was expressed in a subpopulation of taste cells in the circumvallate, foliate, and fungiform papillae, and some of the cells displayed both ASIC2a and ASIC2b immunoreactivities. Subsequent coimmunoprecipitation studies with circumvallate papillae extracts indicated that ASIC2b associated with ASIC2a to form assemblies and, together with our immunohistochemical findings, strongly suggested that both ASIC2 subunits formed heteromeric channels in taste cells in the circumvallate, foliate, and fungiform papillae. Oocyte electrophysiology demonstrated that the ASIC2a/ASIC2b channel generated maximal inward currents at a pH of < or =2.0, which is in agreement with the in vivo pH sensitivity of rat taste cells, and that the amiloride sensitivity of the heteromer decreased with decreasing pH and was almost completely abolished at a pH of 2.0. These findings provide persuasive explanations for the amiloride insensitivity of acid-induced responses of rat taste cells.
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