Some but not all TSCs generated synergistic response to the L-amino acid+IMP MIXes.

<p>Ca²⁺ responses of WT TSCs to the four L-amino acid sets are shown. Each L-amino acid set consisted of one of the four different L-amino acids (MPG (10mM), Ser (20mM), Arg (10mM), or Gln (10mM), respectively), IMP (1mM), and the MIX of L-amino acid+IMP. [A, D, G, J] Representative TSC responses where the magnitudes of MIX responses were greater than the summation of individual L-amino acid and IMP responses, i.e., the MIX responses were synergistic. [B, E, H, I] Representative TSC responses where the magnitudes of MIX responses were greater than or equal to the responses of the L-amino acid or IMP individually, but not greater than the summation of individual L-amino acid and IMP responses, i.e., the MIX responses were not synergistic. [C, F, I, L] A Mean±SEM response for the L-amino acid sets exhibiting synergistic and non-synergistic responses. For each L-amino acid set, the MIX responses generated by synergistic cells were significantly greater than the calculated sum (Sum) of L-amino acid and IMP responses. For non-synergistic cells, MIX responses were significantly smaller than the calculated sums of responses to L-amino acid and IMP. MIX responses of synergistic cells were also significantly greater than the MIX responses of non-synergistic cells. Numbers in parenthesis are the number of cells. One-way ANOVA followed by Bonferroni post hoc tests were used for statistical comparisons. ***P<0.0001, **P<0.001.</p

Representative Ca²⁺ responses of TSCs from WT mice.

<p>Stimuli tested were IMP (1mM), 4 L-amino acids from different side-chain groups (MPG (10mM), Ser (20mM), Arg (10mM), and Gln (10mM)), and L-amino acids with IMP. [A] Ca²⁺ responses of 3 sample TSCs. Each cell was tested with all 9 stimuli. The responses of the three cells are examples of some of the different response patterns to the array of stimuli. The bar above each stimulus trace represents stimulus application time (30 sec). [B] Mean±SEM amplitude of Ca²⁺ increase above baseline for responsive cells only, i.e. cells with a change in Ca²⁺ ≥5%. Numbers in parenthesis are the number of cells.</p

Representative Ca²⁺ responses elicited by TSCs during stimulation with 4 different stimuli.

<p>Cells were stimulated with 4 different stimuli, L-amino acid MIX (AA-MIX) (MPG (10mM), Ser (20mM), Arg (10mM), and Gln (10 mM)), sweet (SC45647 (100μM)), bitter (denatonium (2mM) or cycloheximide (20μM)+denatonium (2mM)), and high-K⁺ (65mM) solution. The bar above each stimulus trace represents the 30 s stimulus application period. [A] A TSC that responded only to AA-MIX. [B] A TSC that responded to bitter and sweet stimuli. [C] A TSC that responded to AA-MIX, sweet, and high-K⁺ solution. AA-MIX elicited a very small increase in cytosolic Ca²⁺ compared to sweet stimulus. The high-K⁺ solution elicited very large increase in cytosolic Ca²⁺, suggesting the presence to voltage-gated calcium channels. Note: A different Y axis scale was used for the high-K⁺ response as the high-K⁺ elicited response was much larger compared to AA-MIX or sweet responses. [D] This TSC responded to both AA-MIX and bitter stimuli. AA-MIX elicited a very small increase in cytosolic Ca²⁺ compared to bitter stimulus. [E] This TSC responded only to bitter stimuli. [F] This TSC responded only to sweet stimuli.</p

Ca²⁺ responses to L-amino acids and IMP in MIX-responsive and non-MIX-responsive cells.

<p>Bars represent Mean±SEM Ca²⁺ responses. Average Ca²⁺ responses to the individual stimulus components of the MIX were compared for two groups of MIX-responsive cells: 1) non-synergistic and 2) synergistic cells. The increases in intracellular Ca²⁺ in response to IMP and the L-amino acids were significantly smaller for synergistic cells (gray bars) compared to non-synergistic (black bars) MIX-responsive cells. Cells that did not respond to the MIX but responded to an L-amino acid (non-MIX-responsive cells; white bars) presented alone also generated Ca²⁺ responses with a similar magnitude as those of MIX-responsive non-synergistic cells (gray bars). To eliminate bias by cells that may not have a receptor, only those cells with Ca²⁺ responses (ΔF≥5%) to L-amino acids were analyzed (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0130088#sec009" target="_blank">results</a>). For these cells, Ca²⁺ responses to individual L-amino acids were not significantly different from Ca²⁺ responses of MIX-responsive, non-synergistic cells (patterned bars). Unpaired t-test (for IMP), and One-way ANOVA followed by Bonferroni post hoc t-test (for L-amino acids) were used for statistical comparison. ***P<0.0001, **P<0.001, *P<0.05.</p

Representative Ca²⁺ responses of TSCs from T1r3 KO mice.

<p>Stimuli tested were IMP (1mM), 4 different L-amino acids (MPG (10mM), Ser (20mM), Arg (10mM), and Gln (10mM)), and L-amino acids with IMP. [A] Ca²⁺ responses of 2 sample TSCs. Each cell was tested with all 9 stimuli. The responses of these cells are examples of some of the different response patterns elicited by the array of stimuli. The bar above each stimulus trace represents the stimulus application time (30 sec). [B] Mean±SEM amplitude of Ca²⁺ increase (ΔF%) above baseline for responsive cells only, i.e. only cells with a change in baseline Ca²⁺≥5%. Numbers in parenthesis are the number of cells.</p

TSCs from T1r3 KO mice can also elicit synergistic responses.

<p>Ca²⁺ responses of T1r3 KO TSCs to the four L-amino acid sets are shown. Each L-amino acid set consisted of one of the four L-amino acids (MPG (10mM), Ser (20mM), Arg (10mM), or Gln (10mM), respectively), IMP (1mM), and the MIX of L-amino acid+IMP. [A, D, G, J] Representative TSC responses when the magnitudes of MIX responses were greater than the summation of individual L-amino acid and IMP responses, i.e., the MIX responses were synergistic. [B, E, H, I] Representative TSC responses where the magnitudes of MIX responses were greater than or equal to the response of the L-amino acid or IMP individually, but not greater than the summation of the individual L-amino acid and IMP responses, i.e., the MIX responses were not synergistic. [C, F, I, L] Mean±SEM response for the L-amino acid sets exhibiting synergistic and non-synergistic responses. We were unable to conclude anything about Gln set as only 2 cells responded synergistically to Gln+IMP. Numbers in parenthesis are the number of cells. Mann Whitney test was used for statistical testing. ***P<0.001, **P<0.01, *P<0.05.</p

Summary of T1r3 KO MIX-responsive synergistic and non-synergistic TSCs.

<p>Values are number of cells. Values in parenthesis are percentages. +, response; -, no response to the stimulus.</p><p>Summary of T1r3 KO MIX-responsive synergistic and non-synergistic TSCs.</p

Representative Ca²⁺ responses of adjacent WT TSCs in clusters.

<p>Cells were stimulated with 9 different stimuli: IMP (1mM) 4 different L-amino acids (MPG (10mM), Ser (20mM), Arg (10mM), and Gln (10mM)), and L-amino acid+IMP. The bar above each stimulus trace represents the stimulus application time (30 sec). [A] Cells 1 and 2, adjacent TSCs in a cluster, generated Ca²⁺ responses similar in temporal and amplitude to each stimulus, including synergistic responses to all the L-amino acid+IMP MIXes. [B] These cells were adjacent TSCs in a cluster, exhibited similar non-synergistic Ca²⁺ responses to all the L-amino acid+IMP MIXes, but the response amplitudes of cell 2 was 50% of cell 1. [C] Cells 1 and 2 were adjacent TSCs in a cluster that responded to the same stimuli. However, one cell responded synergistically to the MIXes whereas the other cell responded non-synergistically. [D] Cells 1 and 2 were adjacent TSCs. Each cell exhibited different response patterns to the array of test stimuli.</p

Comparison of amplitude of synergistic responses between WT and T1r3 KO mice.

<p>Bars represent Mean±SEM for synergistic responses (i.e., responses to the MIX that were greater than the sum of individual responses) of TSCs from WT and T1r3 KO mice. The amplitude of responses elicited by MPG+IMP and Arg+IMP was significantly smaller in T1r3 KO cells than those of WT cells (Unpaired t-test). *P<0.05.</p

Summary of WT MIX-responsive synergistic and non-synergistic TSCs.

<p>MIX-responsive TSCs responded differently to the individual components of each L-amino acid set. Values are number of cells. Values in parenthesis are percentages. +, response;-, no response to the stimulus. Note: For 2 cells in the Arg set, and 2 cells in the Gln set, MIX responses were not synergistic even though IMP nor the L-amino acid alone elicited a response (their increases in Ca²⁺ were <5% of baseline). For these cells, the increases in Ca²⁺ in response to IMP, and L-amino acids individually were between 2.5% and 3% above baseline, and thus were not considered to be responses. However, the MIX elicited very small responses that were just above 5% of baseline Ca²⁺, but not larger than the added sum of individual responses. Thus, these MIX responses were not synergistic. Since only around 2% of the cells showed this type of response, we did not include these cells in our discussion of synergy.</p><p>Summary of WT MIX-responsive synergistic and non-synergistic TSCs.</p