In the present study, physiological responses to amiloride were examined in defined subsets of mouse fungiform taste cells. The principal finding is that amiloride-sensitive Na+
channels, thought to be required for amiloride-sensitive NaCl transduction, appear to be functionally expressed in taste cells lacking voltage-dependent inward currents. These cells are likely to be the Type I taste cells, thought previously to have only a support function in the taste bud [3
]. However, it is also possible that cells lacking inward currents are developing taste cells that have not yet reached the taste pore. Developing taste cells have slowly activating outward currents compared to mature receptor cells [48
]. Indeed, we recorded from a small number of taste cells that had slowly activating outward currents typical of developing taste cells, but none of these exhibited an amiloride effect. Interestingly, only 7.7% of the total cells recorded from were amiloride-sensitive. The proportion of cells responding to amiloride seems low compared to previous studies showing that about 65% of the cells responded to amiloride in mouse taste cells maintained in an intact taste bud [44
]. However, it is now well-know that taste cells communicate between each other [49
] and that the information contained in one cell can be transferred to adjacent cell(s). Hence several cells can respond to NaCl even if they do not possess the ENaC. Nevertheless, using in situ hybridization, Shigemura et al. (2005) showed that, in mouse, only 2 to 4 cells express the different ENaC subunits in a fungiform taste bud. This observation correlates with our findings and suggests that only a small proportion of taste cells express amiloride-sensitive channels in mouse. Further, rat and mouse show numerous morphological differences [50
] as well as physiological differences. Indeed, responses to amiloride occurred in rat taste cells with both inward and outward voltage-gated currents (Type II cells) but not in cells showing a Ca2+
current (Type III cells) [42
]. However, these authors did not record from cells with only outward voltage-gated currents (likely Type I cells). It would be predictable that many rat type I cells would be sensitive to amiloride since Doolin and Gilbertson showed that 75% of rat fungiform taste cells expressing only outward currents are sensitive to amiloride. Thus, our results disclose a remarkable difference between species and could explain the different detection threshold measured in rat and in mouse, i.e: between 0.001 M and 0.002 M in Wistar rat [51
] compared to 0.065 M in C57BL/6J mouse [28
Because the cells were isolated in this study, and amiloride was bath-applied, it was not possible to tell whether the amiloride-sensitive Na+
channels were localized to the apical membrane. However, amiloride-sensitive cells responded to the lowest concentration of amiloride (0.2 μM), which corresponds to the defined Ki for apical ENaCs [40
]. Basolateral amiloride-sensitive Na+
channels are less sensitive to amiloride (Ki = 0.56 μM) [52
]. One could propose that the enzymatic treatment used to isolate the taste bud cells as well as the high concentration of Na+
ions in the bath solution could degrade or desensitize the amiloride-sensitive channels [40
]. However, the use of amiloride in the enzymatic treatment solution or the bathing medium did not enhance the occurrence of amiloride responses, suggesting that the channels are functional. Moreover, it seems unlikely that the enzymatic treatment degrades the channels in a particular type of cell and not in another type of cell.
The observation of responses to amiloride in cells with only voltage-gated K+
channels raises the question of how amiloride-sensitive taste information is transmitted to the nervous system. According to morphological studies, type I cells do not possess synaptic contacts [1
] or subsurface-cisternae, which have been proposed to be involved in activation of afferent nerve fibers [8
]. Besides, the absence of voltage-gated Na+
channels in the amiloride-sensitive cells would appear to eliminate the cell's ability to produce action potentials, however, the cells should depolarize in response to NaCl. Recently, Huang et al.
] showed that Pannexin 1 (Px1) hemichannel mRNA was detected in about half of the cells expressing NTPDaseII, a marker for glial-like cells (Type I cells). Since the presence of Px1 channels is believed to underlie ATP release in the taste bud, which is required for salt taste transduction [10
], Type I cells may release ATP to signal to the nervous system. Further studies will be needed to determine if Px1 channels are required for transmitting salt taste information to the nervous system, either directly, or via other cells in the taste bud.
Another interesting finding in this study is that fungiform taste buds appear to have a different complement of taste cell types than circumvallate papillae. TRPM5-GFP mice were used to identify the cells expressing the TRPM5 channel, presumably found in all type II cells, however, GFP fluorescence was observed only in a few cells in each taste bud of fungiform papillae, while it was observed in many cells of circumvallate taste buds. Further, many of the unlabeled cells exhibited physiological criteria used to characterize cells as a Type II cell (i.e): presence of voltage-gated Na+
channels and lack of voltage-gated Ca2+
]. Unlike circumvallate taste cells, PLCβ2 was expressed in some non-TRPM5-GFP fungiform cells, although the number of these taste cells is not sufficient to account for the large percentage of unlabeled taste cells expressing voltage-dependent Na+
currents. These observations suggest that, in mouse fungiform taste buds, only a subset of cells with Type II cell membrane properties expresses PLC signalling components. The function of the PLCβ2-independent cells with voltage-gated Na+
channels is unclear. Many cells in intact fungiform taste buds are electrically excitable, and generate action potentials to apically-applied stimuli representing all the taste qualities, including salt [54
]. It is possible that these PLCβ2-independent cells integrate signals transduced by other cells in the taste bud, including Type I cells.
Fungiform taste cells also have a different complement of Type III taste cells than circumvallate taste buds. The proportion of SNAP-25 labeled cells is higher in circumvallate taste buds than in fungiform taste buds. This correlates with the small number of synapses observed ultrastructurally in mouse fungiform taste buds compared to foliate and vallate taste buds [55
]. These observations also correspond with our inability to randomly find taste cells expressing voltage-gated Ca2+
currents in the unlabeled taste cells of the TRPM5-GFP mice.
This study, taken together with previous studies, suggests that at least in fungiform taste buds, separate subsets of taste cells are specialized for transducing different taste qualities. Subsets of Type II cells with PLC signaling components mediate the transduction of sweet, umami, or bitter compounds, while a different subset, likely of Type III cells, mediates sour transduction [56
]. We now show that a subset of Type I cells expresses functional ENaC channels, involved in the transduction of amiloride-sensitive, Na+
specific salt taste. In addition, we show that a large subset of taste cells with Type II cell membrane properties lacks expression of PLC signaling components. Whether these cells are involved in the transduction of tastants, or in signal processing in the taste bud awaits further investigation.