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1Department of Physiology and Biophysics, and 2Neuroscience Program, University of Miami School of Medicine, Miami, Florida 33136
Submitted 19 March 2004; accepted in final form 7 May 2004
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ABSTRACT |
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17 mM), which acts on TREK-1 and TASK-1 channels, blocked acid taste responses. Agents diagnostic for other 2-pore domain and voltage-gated potassium channels (anandamide, 10 µM; Gd3+, 1 mM; arachidonic acid, 100 µM; quinidine, 200 µM; quinine, 100 mM; 4-AP, 10 mM; and TEA, 1 mM) did not affect acid responses. The expression of 2-pore domain channels and our pharmacological characterization suggest that a matrix of ion channels, including one or more acid-sensitive 2-pore domain K channels, could play a role in sour taste transduction. However, our results do not unambiguously identify any one channel as the acid taste transducer. |
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INTRODUCTION |
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; Richter et al. 2003a) and Ca2+ influx in a select subset of the acidified taste cells (Richter et al. 2003a). Ca2+ influx presumably leads to neurotransmitter release at synapses between sensory afferent neurons and taste cells. A complete description of the mechanisms by which cytoplasmic acidification stimulates Ca2+ influx in the acid-sensitive taste receptor cells remains to be elucidated. Our previous findings indicate that intracellular acidification opens voltage-gated Ca channels in a fraction of taste cells (Richter et al. 2003a), presumably attributable to depolarizing receptor potentials generated by acid taste stimuli. Thus a key question of sour taste transduction is how intracellular acidification evokes depolarizing receptor potentials.
Protons are postulated to act on ion channels in the membranes of acid-sensitive taste receptor cells and generate inward current. Taste cells express several proton-gated or proton-permeant cation channels that have been proposed to be sour taste transducers, including cation channels (ENaCs, MDEG1, ASIC-2b: Gilbertson and Gilbertson 1994; Gilbertson et al. 1992; Lin et al. 2002; Liu and Simon 2001; Ugawa et al. 1998), hyperpolarization-activated cyclic nucleotide-gated channels (HCNs: Stevens et al. 2001), and proton-sensitive chloride channels (Miyamoto et al. 2000). However, even though most of these ion channels are expressed in subsets of taste cells, it is not known whether these cells are the same cells that mediate acid taste. Thus a direct link has not been firmly established between these various channels and sour taste transduction.
Potassium leak channels establish the membrane potential of all cell types and play especially important roles in excitable cells. The ion channels that produce leak conductances have an unusual structure, with each subunit possessing 4 transmembrane helices and 2 pore-lining domains (Lesage et al. 1996). As many as 15 mammalian genes, constituting the K2P family, encode these potassium channels (Talley et al. 2003). The K2P channels are widely expressed and regulate the resting potential and membrane excitability in neurons and cardiac cells by their sensitivity to physiological signals such as Ca2+, cyclic nucleotides, or pH (for review, see Goldstein et al. 2001; Talley et al. 2003). Certain members of this family are strongly modulated by intracellular acidification, that is, TWIK-1 and -2 and TREK-1 and -2 (Chavez et al. 1999; Lesage et al. 1996, 2000; Maingret et al. 1999). Other K2P channels, that is, TASK-1, -2, and -3 (Kim et al. 1999, 2000) and the distantly related TRESK (Sano et al. 2003), are modulated strongly by extracellular acid and only weakly (if at all) by intracellular acidification. Acid taste stimuli give rise to intracellular acidification and this is postulated to be the proximate stimulus for acid (sour) taste (Lyall et al. 2001). In the present study, we investigated whether 2-pore domain K channels sensitive to intracellular acidification are expressed in mouse taste buds and whether they might be relevant to acid taste transduction. We also tested the effects of a panel of pharmacological agents that have been reported to affect these channels. The results are consistent with the notion that one or more acid-sensitive 2-pore domain K channels may play a role in acid taste transduction. Some of these data were previously presented in abstract form (Richter et al. 2003b).
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METHODS |
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All experiments were carried out according to the National Institutes of Health Guidelines for the Care and Use of Animals and protocols were approved by the University of Miami Animal Care and Use Committee. Adult C57/BL mice (The Jackson Laboratory, Bar Harbor, ME) were killed by exposure to CO2 followed by cervical dislocation. Tongues were dissected free and transferred to Tyrode' s buffer.
RT-PCR
Taste epithelium was delaminated by subepithelial injection of proteases as described previously (Gilbertson et al. 1993) and nontaste epithelium surrounding the papilla was trimmed away. The preparation was composed predominantly (but not exclusively) of taste buds. Total RNA was isolated from these taste buds or from adjacent nonsensory lingual epithelium using the Absolutely RNA Nanoprep kit (Stratagene, La Jolla, CA). Taste buds from a single papilla were dispersed in 100 µl lysis buffer containing guanidine thiocyanate and 
-mercaptoethanol; RNA from the lysate was captured on a silica-based matrix, treated with DNase I, washed, and then eluted in 10 µl 10 mM Tris-HCl (pH 7.5). Purified taste RNA was denatured and first-strand cDNA was synthesized at 42°C for 60 min using SuperScript II Reverse Transcriptase in a 20 µl final volume. After removing template RNA with RNase H, 1 µl cDNA was used as template in a 25 µl PCR. Each cDNA preparation was used to test for expression of the entire set of channels described in RESULTS. All reagents were purchased from Invitrogen (Carlsbad, CA).
We designed PCR primers using published full-length cDNA sequences from mouse for TWIK-1 and -2, TREK-1, and TASK-1 and -2 (Table 1). Because no published mouse cDNA sequences were available for TASK-3 or TREK-2, we used full-length cDNAs from rat to identify putative orthologs (>96% identity) in the mouse genome. We confirmed that intron locations and exon sizes were identical between the rat genes and the presumed orthologs in mouse. Primers for TASK-3 and TREK-2 were then designed in identified mouse exons. We used a similar strategy starting from published human TRESK full-length cDNA sequence to design primers for mouse TRESK. In all cases, each primer pair spanned at least one intron. Conditions for PCR were: 94°C for 2 min; 25–40 cycles at 94°C for 30 s, 56–65°C for 30 s, 72°C for 45 s; and a final extension at 72°C for 5 min. Annealing temperatures were 56°C for TWIK-1 and -2, TREK-1, TASK-3, and TRESK; 57°C for TASK-2; 59°C for TREK-2; and 65°C for TASK-1. We also amplified 2 control mRNAs, -actin (5'-caaccgtgaaaagatgacc-3', and 5'-ctggaaaagagcctcagg-3', 449 bp product) and the taste cell-specific G protein, gustducin (5'-gcaaccacctccattgttct-3', and 5'-agaagagcccacagtctttgag-3', 286 bp product), using primer pairs located in separate exons.
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We used circumvallate taste buds, which have been shown by us and others to contain acid-responsive taste cells (Caicedo et al. 2002; Richter et al. 2003; Ugawa et al. 2003). We imaged changes of Ca2+, pH, and membrane potential in taste cells using one of two preparations: 1) 100-µm-thick slices of circumvallate taste papilla ("lingual epithelium slice"; Richter et al. 2003) or 2) isolated circumvallate taste buds and cells. For imaging, the functional indicator dyes listed in Table 2 were obtained from Molecular Probes (Eugene, OR) and were stored as stock solutions at –20°C.
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; Caicedo et al. 2002). To record changes in pH in lingual epithelial slices, we loaded taste cells using the same method for the pH-sensitive fluorescent indicator dye, BCECF (Richter et al. 2003a). It should be noted that we measured only relative changes in [Ca2+]i, pHi, and membrane potential with this method and were not able to determine the absolute values of [Ca2+], pH, and mV reached during the experiments. For recordings on taste cells removed from their epithelial environment, taste buds were gently aspirated from the delaminated lingual epithelium (see above, RT-PCR) and plated onto coverslips coated with Cell-Tak adhesive (BD Biosciences, Bedford, MA). Such preparations, which typically included elongate taste receptor cells dissociated from the taste buds, were loaded with membrane-permeant indicator dyes (AM esters) by incubating them in the dark at 25°C. Dyes and incubation times were: for [Ca2+], 2 µM OG-AM for 25 min; for pHi, 100 µM HPTS for 10 min; and for membrane potential, 100 µM ANEPPS for 15 min (see Table 2).
Taste stimuli
Citric acid (100 mM in Tyrode's buffer, pH 3), a potent acid taste stimulus in rodents and humans and which, like other weak organic acids, is a more effective tastant than HCl (e.g., Beatty and Craig 1935), was focally applied with pressure ejection from a puffer micropipette. We measured the concentration of citric acid delivered to the taste cells by including a fluorescent tracer dye in the stimulus solutions (200 µM Lucifer yellow CH; Molecular Probes). For some experiments as indicated, we used a bath application of KCl or acidified Tyrode's buffer (pH adjusted to 1.5 with HCl).
Microscopy, data analysis, and statistics
Cell responses in the lingual slice preparation were recorded using scanning confocal microscopy with argon laser excitation (488 nm) combined with an FITC filter set (510 LP) to view cells loaded with CGD (Caicedo et al. 2000). Data were captured by scanning a field (10 µm optical slice) containing 1 or more taste buds every 0.5 to 5.0 s. Data were stored for off-line analysis using Fluoview v. 2.1 (Olympus). Throughout this analysis, we included only data from taste cells that exhibited Ca2+ responses to citric acid with 
F/F >0.1 and that could be elicited 
3 times in succession.
When testing the effects of pharmacological agents on taste cell responses (i.e., [Ca2+]) to citric acid, we included data from cells for which we could collect 3 control responses (before drug application) and 3 or more responses during drug treatment. Data from cells that did not recover their control response by 30 min after washout were not included. We used the same selection criteria for pHi responses of taste cells.
All recordings for individual cells are presented as tracings of F/F. Responses were measured as the peak F/F in a record. Histograms comparing taste cell responses before and after a drug treatment were obtained by taking the mean of 3 or more responses (F/F) before applying a drug and normalizing all responses to that mean. To compare the effects of pharmacological treatments against controls, we used a paired Student's t-test. For all statistics, P < 0.05 was considered significant. Curves were fitted using Prism (GraphPad Software, San Diego, CA).
Reagents and solutions
Unless stated otherwise, all reagents for physiological experiments were obtained from Sigma (St. Louis, MO). Solutions were prepared freshly for each experiment by dissolving in Tyrode's buffer (in mM: 135 NaCl, 5 KCl, 2 CaCl2, 1 MgCl2, 5 NaHCO3, 10 Hepes, 10 glucose, and 10 sodium pyruvate; pH 7.4). The following pharmacological agents were used at concentrations that are 10 times the IC50 for 2-pore domain K+ channels: anandamide (10 µM), Gd3+ (1 mM), arachidonic acid (AA, 100 µM), quinidine (200 µM), quinine (100 mM), and riluzole (500 µM). We also tested the effect of 4-aminopyridine (4-AP, 10 mM) and tetraethylammonium (TEA, 1 mM) because, although 2-pore domain channels are resistant to these treatments (Lesage 2003), most other potassium channels are not. All test solutions were prepared freshly immediately before experimentation by dissolving in Tyrode's buffer. Riluzole was prepared as a 100 mM stock solution in DMSO and stored at –80°C. The stock solution was diluted in Tyrode's buffer to a final concentration of 500 µM (0.1% final DMSO concentration). Halothane was made up as a saturated solution (17 mM, pH 7.4, cf. Patel et al. 1999). To minimize loss of this volatile anesthetic from the perfusion system, the halothane solution was prepared immediately before experimentation and was delivered by gravity from 10-ml syringes sealed with plastic stoppers.
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RESULTS |
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), applying citric acid focally to the taste pore of vallate taste buds produces an intracellular acidification throughout the entire taste bud (Fig. 1A). In a subset of taste cells, this acidification elicits an increase in [Ca2+]i (Fig. 1B).
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; Kinnamon and Roper 1988). We thus postulated that the link between cytoplasmic acidification and the rise in [Ca2+]i in acid-responsive taste cells is membrane depolarization followed by activation of voltage-gated Ca channels. Thus we tested whether we could measure depolarizing receptor potentials in acid-sensitive taste cells using the voltage-sensitive fluorescent dye, ANEPPS (Hayashi et al. 1996). We first determined whether changes in ANEPPS fluorescence (F/FANEPPS) reliably reflected changes in membrane potential in isolated taste buds. To depolarize cells, we applied Tyrode's buffer with elevated concentrations of KCl (20, 50, and 100 mM: substituted for equimolar NaCl) to isolated taste buds preloaded with ANEPPS. As expected, KCl application resulted in a concentration-dependent increase in F/FANEPPS, consistent with membrane depolarization of taste cells (Fig. 1C). To estimate the incidence of taste cells responding to acid with membrane depolarization, we measured responses from single cells loaded with ANEPPS. Applying citric acid (100 mM) to isolated taste cells resulted in a pronounced increase in ANEPPS fluorescence in about 40% of the isolated cells, consistent with depolarization in a subset of acid-sensitive taste cells (Fig. 1D). Collectively, these findings suggest the model for acid taste transduction shown in Fig. 2.
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Using RT-PCR, we asked whether any of the seven K2P channels (Sano et al. 2003), which are modulated by intracellular acidification, were expressed in taste buds and might play a role in acid taste. Expression of five of these acid-sensitive channels was detected in cDNA prepared from mouse circumvallate taste buds, that is, TWIK-1 and -2, TREK-1 and -2, and TASK-1. Expression of TASK-3 and TRESK mRNAs was not apparent (Fig. 3). We found an identical pattern of expression in circumvallate and foliate taste buds for each of these 5 channels (not shown). TASK-1 appeared to be expressed selectively in taste buds when compared with nontaste lingual epithelium. The taste-selective expression pattern of TASK-1 was confirmed in five independent preparations of RNA from mouse circumvallate and foliate taste buds and three preparations from nontaste lingual epithelium. By contrast, RT-PCR products for TWIK-2, TREK-1 and -2, and TASK-2 were readily detected in nontaste lingual epithelium. TWIK-1 appeared to be expressed at a higher level in taste buds compared with nonsensory lingual tissue, although we have not attempted to quantify this differential expression. We also tested for the expression of a related channel, TASK-2, that is activated by external (but not cytoplasmic) acidification. We detected TASK-2 expression at similar levels in taste and nontaste lingual RNAs. PCR for -actin and the taste cell-specific G protein, 
-gustducin, served to confirm the overall quality and inclusion of taste buds, respectively, in each cDNA preparation.
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40 cycles to be consistently detected. This suggests that the corresponding mRNAs for TWIK-1 and -2 may be present at relatively high concentrations in taste buds and nonsensory lingual epithelium. Pharmacological characterization of acid responses
A number of pharmacological agents known to affect the above acid-modulated 2-pore domain K channels were tested for their effect on acid taste responses in mouse taste cells. For example, both quinine and quinidine selectively inhibit TWIK-1 and TASK-2 (Lesage et al. 1996). Continuous perfusion of lingual slices with quinidine (200 µM) had no effect on citric acid–induced responses (Fig. 4A). Similarly, 100 mM quinine did not alter citric acid-induced Ca2+ responses (not shown). Parenthetically, other (bitter-sensitive) taste cells did respond to quinine, even at 1 mM, as we have shown previously (Caicedo et al. 2002). The following agents, which act on various 2-pore domain K channels, did not affect citric acid-induced taste responses (Fig. 4): 10 µM anandamide (an inhibitor of TASK-1; Maingret et al. 2001), 1 mM Gd3+ (an inhibitor of TREK-1; Maingret et al. 2000), 100 µM arachidonic acid (an enhancer of TREK-1 and -2; Lesage et al. 2000). We also tested 4-AP and TEA, which are potent inhibitors of voltage-gated and other types of K channels but are relatively ineffective at 2-pore domain K channels (Lesage 2003). Neither 10 mM 4-AP nor 1 mM TEA had an effect on citric acid taste responses (Fig. 4, E and F).
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; Lesage 2003). In the lingual slice preparation, bath-applied, riluzole (500 µM) increased citric acid taste responses by a small, but statistically significant extent (Fig. 5). This effect is consistent with block of a resting (leak) K+ conductance.
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; Patel and Honore 2001; Patel et al. 1999; Terrenoire et al. 2001), significantly reduced acid-evoked Ca2+ transients in taste cells (P < 0.05, n = 8; Fig. 6, C and D). Nevertheless, halothane did not modify acid-evoked intracellular pH changes in taste cells (P > 0.05, n = 10; Fig. 6, A and B). This result suggests that halothane affects acid taste responses at a step after cytoplasmic acidification but before Ca2+ influx. Halothane did not alter responses to a bitter stimulus, cycloheximide (P > 0.05, n = 5, Fig. 6, E and F), demonstrating that this anesthetic does not globally affect taste cell responsivity.
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). Importantly, halothane also prevented acid-evoked depolarization of isolated taste cells (Fig. 7D). Halothane thus appears to block the Ca2+ influx in response to acid stimulation by stabilizing the membrane potential of taste cells.
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DISCUSSION |
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; Lin et al. 2002), although the findings in the rat generally concur with our results in mouse. Burks et al. (2003) reported TASK-3 in vallate and foliate (but not fungiform) taste buds and apparently did not detect TWIK-2. The origin and significance of the differences in expression between rat and mouse taste buds regarding TWIK-2 and TASK-3 expression is unclear. Indeed, there is precedence for such species difference in channel expression. For example, the proton-gated cation channel, ASIC-2, is expressed in rat but not mouse taste buds (Richter et al. 2004).
Several acid-sensitive ion channels have been proposed to mediate sour taste perception in mammals, including the cation channels ENaC, MDEG1, ASIC-2, and HCN1 and HCN4 (Gilbertson and Gilbertson 1994; Gilbertson et al. 1992; Lin et al. 2002 Liu and Simon 2001; Stevens et al. 2001; Ugawa et al. 1998, 2003), and chloride channels (Miyamoto et al. 2000). Downstream of the acid transduction channel(s), a basolateral N+-H+ exchanger, NHE-1, is believed to play a significant role in the adaptation of the acid taste response (Lyall et al. 2004). Despite the apparent variety of acid-sensitive ion channels expressed by taste cells, none of the aforementioned ion channels has been directly and unequivocally demonstrated to mediate sour taste. For example, HCN1 and HCN4 are expressed in a subset of taste cells and ionic currents produced by the cloned channel resemble those in some taste cells (Stevens et al. 2001). Similarly, ASIC-2 is expressed in a subset of rat taste cells and ASIC-like currents were recorded in rat taste cells (Ugawa et al. 1998, 2003). In both instances, it is not known whether the cells that express HCN and ASIC2 are those that respond to acid taste stimuli. Further, ASIC-2 is not significantly expressed in mouse taste cells (Richter et al. 2004). Genetic ablation of ASIC-2 in mice does not affect behavioral or Ca2+ responses to acid taste stimuli (Kinnamon et al. 2000; Richter et al. 2004). In short, none of the proposed cation or chloride channels has been demonstrated to be a predominantly compelling sour taste transducer in mammalian taste buds.
The absence of an unequivocal sour taste receptor was in part the rationale for investigating a role for K2P channels in acid taste. These potassium leak channels are expressed in many tissues. K2P channels are constitutively active and play a major role in physiological functions including establishing membrane potential and regulating neuronal and muscular excitability in response to neurotransmitters and hormones (Lesage and Lazdunski 2000). Some members of the K2P family are regulated by pH changes in the extracellular milieu or in the cytoplasm or both, whereas others are relatively pH insensitive. We have tested those that are affected by intracellular acidification, which is believed to be the proximate stimulus for acid taste (Lyall et al. 2001). One such channel, TASK-1, is a proposed acid sensor in the carotid body (Buckler et al. 2000). It may be significant that we found TASK-1 is expressed in taste buds and not in surrounding nonsensory tissue. TASK-1 is blocked by both intra- and extracellular acidification (Kim et al. 1999). Thus acid taste stimulation should depolarize taste cells that express this channel. Indeed, citric acid stimulation was shown to result in depolarizing receptor potentials that originate from a blocked resting conductance (Cummings and Kinnamom 1992). In short, TASK-1 might be a good candidate for an acid taste transducer in mouse taste buds. Opposing this conclusion, however, is the lack of action of anandamide (a TASK-1 blocker) on acid taste responses in our experiments (Fig. 4B). Further studies using in situ hybridization, immunohistochemistry, and more detailed functional analyses would be necessary to definitively establish or refute a role for TASK-1 in sour taste.
The results from the pharmacological testing did not allow us to identify unambiguously any one acid-sensitive K2P channel as uniquely associated with acid taste responses. Expression alone would suggest TWIK-1 and TASK-1 are the most relevant channels because they are preferentially expressed in taste cells. However, this conclusion was not supported by the pharmacological profile of acid responses in our experiments.
The effects of riluzole on acid responses are consistent with an involvement of TREK-1 and -2, both of which are blocked by riluzole. However, because intracellular acidification opens TREK-1 and -2 channels to hyperpolarize cells (Lesage and Lazdunski 2000), neither of these channels is a candidate for a primary transducer for acid taste. One would anticipate that pharmacological block of TREK-1 and -2 leak conductances would enhance any acid-evoked depolarizing currents, in line with what we observed for taste cells (Fig. 5). This would suggest that TREK-1 and/or TREK-2 might act as modulatory channels in acid-sensing taste cells, serving to oppose acid-evoked responses and thereby keeping depolarizing receptor potentials in check, or assisting in recovery from depolarization. A major caveat is that, although riluzole has been widely used to diagnose K2P channels in numerous studies, it has been shown to affect other ion channels as well (Cao et al. 2002). Similarly, the ability of halothane to depress acid taste responses may result from its known ability to activate TREK-1 and TASK-1 channels. By opening these leak conductances, halothane hyperpolarizes membranes and shunts inward currents, thus preventing depolarizing receptor potentials. These outcomes readily explain the results in taste cells (Fig. 7, C and D). Yet, as with riluzole, the actions of halothane are not completely specific to K2P channels. For instance, halothane also blocks voltage-gated Ca channels (Kamatchi et al. 1999), an action that would cooperate with its hyperpolarizing effect on membrane potential to further reduce any acid-evoked Ca2+ influx (Fig. 6, C and D).
In aggregate, our findings are generally consistent with—but do not prove—a role for K2P channels in taste in general, and acid taste in particular. Certain of the acid-sensitive K2P channels may be more likely than others to be involved in sour taste because of their preferential expression in taste buds versus nontaste tissue (i.e., TWIK-1 and TASK-1) and because riluzole and halothane affected acid taste responses (i.e., TREK-1 and -2 and TASK-1). It is possible that acid taste transduction is the result of the effect of intracellular acidification on a matrix of acid-sensitive ion channels in taste cells, some of which would tend to depolarize (e.g., ASIC2, HCN-1 and -4, TASK-1) and others, like TREK-1 and -2 channels, to stabilize the membrane potential, with a net depolarizing receptor potential. If this interpretation is correct, it may be difficult to use knockout mice to isolate any one contributor to sour taste.
As a footnote, throughout this report the assumption has been that intracellular acidification in the taste bud is the proximate stimulus for acid taste responses, as proposed by others (Lyall et al. 2001) and as we have also observed (Richter et al. 2003a). Yet it should be noted that applying acid taste stimuli to the lingual sensory surface will acidify intra- and extracellular compartments alike. Measurements of pH alterations selectively in the narrow intercellular spaces within taste buds after acid taste stimulation have not been possible to date. This may be important because extracellular, rather than intracellular, protons affect different K2P leak channels. It might also be noted that ASIC-2, a candidate sour taste transducer in rat taste cells (Ugawa et al. 1998, 2003) is gated by extracellular protons. Further, ENaC channels would require a proton gradient across the membrane (i.e., extracellular > intracellular concentration) to generate inward current carried by H+. Ambiguity about the actual site of action of protons released from sour tastants only adds to the present uncertainties about sour taste transduction.
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GRANTS |
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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Address for reprint requests and other correspondence: S. Roper, Dept. of Physiology and Biophysics, University of Miami School of Medicine, 1600 NW 10th Ave., Miami, FL 33136 (E-mail: Roper{at}miami.edu).
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) indicate focal application of citric acid (CA, 100 mM). Data show responses recorded before (left traces) and during (right traces, horizontal bars) continuous perfusion with the agent indicated. Pharmacological agents were applied for 5 to 40 min at 10 times the IC50 reported for K2P channels. All calibrations are as labeled in F.
). C: average response (mean 
