Figure 1.
Inhibition of hTAS2R16, hTAS2R38, and hTAS2R43 by probenecid.
HEK-293T cells were transiently transfected with Gα16gust44 and the indicated TAS2R receptors in a 384-well microplate. 22 hours post-transfection, calcium influx was measured in cells challenged with the indicated ligands in the presence (closed triangles) or absence (open diamonds) of probenecid (1 mM; 1 hour pre-incubation). Probenecid treatment completely attenuated (A) salicin-dependent (3 mM) calcium influx by the hTAS2R16 receptor and (B) PTC- (100 µM) and (C) PROP-dependent (30 µM) calcium influx by the hTAS2R38 receptor. (D) Probenecid treatment similarly attenuated aloin-induced (3 mM) hTAS2R43 signaling. (E) Probenecid treatment did not inhibit saccharin induced signaling of hTAS2R31. (F) hTAS2R38 transfected cells challenged with probenecid or buffer alone (1 mM) did not result in calcium influx, but do flux with the PTC control.
Figure 2.
Structures of TAS2R agonists, probenecid, MRP1 inhibitors, and probenecid analogs used.
Figure 3.
Non-Gustatory GPCRs are not inhibited by probenecid.
HEK-293T cells were transiently transfected with Gα16gust44 and the indicated GPCR receptors. In the case of the endogenously expressed βAR receptor, only Gα16gust44 was transfected. 22 hours post-transfection, calcium influx was measured for cells that were challenged with the indicated ligands in the presence (closed triangles) or absence (open diamonds) of probenecid (1 mM; 1 hour pre-incubation). Probenecid treatment did not attenuate calcium influx upon challenge of (A) CCR5 with 10 nM RANTES, (B) CXCR4 with 10 nM SDF-1α, or (C) βAR with 10 µM isoproterenol.
Figure 4.
Probenecid inhibition of hTAS2R16 occurs rapidly and is not dependent on the MRP1 transporter.
(A) HEK-293T cells were transiently transfected with hTAS2R16 and Gα16gust44. 22 hours post-transfection, calcium influx was measured for cells that were challenged with 3 mM salicin in the presence of 1 mM probenecid pretreatment for the indicated amount of time (0 min indicates co-injection of salicin with probenecid). hTAS2R16 was completely inactivated by 5 minutes of probenecid pretreatment. (B) HEK-293T cells were transiently transfected with hTAS2R16 and Gα16gust44 followed by challenge with 3 mM salicin in the presence or absence of the indicated compounds (1 mM, pretreatment for 60 minutes). The MRP1 transporter inhibitor indomethacin did not inhibit hTAS2R16 function. Error bars represent standard errors (n = 3).
Figure 5.
Pharmacological mechanism of action of probenecid inhibition.
(A) HEK-293T cells were transiently transfected with Gα16gust44 and hTAS2R16 or hTAS2R38. 22 hours post-transfection, cells were pre-treated with increasing amounts of probenecid for 1 hour followed by challenge with 3 mM salicin or 300 µM PTC. (B) HEK-293T cells were transiently transfected with Gα16gust44 and hTAS2R16, pre-treated with increasing amounts of probenecid for 1 hour, and then challenged with different concentrations of salicin. Error bars represent standard errors (n = 4). (C) HEK-293T cells were transiently transfected with Gα16gust44 and hTAS2R38, pre-treated with increasing amounts of probenecid for 1 hour, and then challenged with different concentrations of PTC. Error bars represent standard errors (n = 4).
Figure 6.
Differential effect of probenecid analogs on the activation of hTAS2R16 and hTAS2R38 receptors.
HEK-293T cells were transiently transfected with Gα16gust44 and the indicated TAS2R receptor. 22 hours post-transfection, calcium influx was measured after challenge with (A) 3 mM salicin or (B) 100 µM PTC in the presence or absence of the indicated compounds (1 mM, pretreatment for 60 minutes). Error bars represent standard deviations (n = 6 for hTAS2R16; n = 12 for hTAS2R38).
Figure 7.
Identification of hTAS2R16 residues required for probenecid inhibition.
(A) HEK-293T cells were transiently transfected with wild type hTAS2R16 and Gα16gust44. 22 hours post-transfection, calcium flux was measured for cells that were challenged with 3 mM salicin in the presence (closed triangles) or absence (open diamonds) of probenecid (1 mM; 1 hour pre-incubation). Salicin response to mock transfected (vector alone) HEK-293T cells is shown for comparison. (B, C) HEK-293T cells were similarly transfected with hTAS2R16 variants containing the mutations N96T or P44T/H113R, and challenged with 3 mM salicin in the presence or absence of probenecid (1 mM; 1 hour pre-incubation). N96T and P44T/H113R mutants showed decreased sensitivity to probenecid. A separate clone containing the single point mutant H113R was also tested to rule out this residue (Figure S1). Error bars represent standard deviations (n = 4).
Figure 8.
Suppression of human bitterness perception of salicin by probenecid.
(A) 15 human subjects were asked to rate the bitterness intensity of 10 mM salicin before (pre) and after (post) treatment with 10 mM probenecid or control treatment with 8.1 µM quinine HCl (QHCl) on a general labeled magnitude scale (gLMS). Treatment with probenecid (black bars) significantly inhibited the perceived bitterness of salicin (p<0.05), whereas a bitter taste control treatment with QHCl had no affect (white bars). (B) 9 human subjects were asked to rate the bitterness intensity of 10 mM salicin or 250 mM saccharin before (pre) and after (post) treatment with 10 mM probenecid or control treatment with 8.1 µM quinine HCl (QHCl). Treatment with probenecid (black bars) significantly inhibited the perceived bitterness of salicin (p<0.05), whereas a control treatment with QHCl had no affect (white bars). Probenecid failed to inhibit the bitterness of saccharin (in the presence of the sweet taste inhibitor lactisole (lac) to enable subjects to focus exclusively on the bitterness of saccharin). The inhibitory effect of probenecid on salicin was also observed in the presence of lactisole, demonstrating that lactisole did not interfere with probenecid's inhibition of perceived bitterness. At the concentrations used, salicin and saccharin did not differ in their overall perceived bitterness. Error bars represent standard errors (SE).