Sustained inhibition of proton-coupled folate transporter by myricetin

Identification of the amino acid residue responsible for the myricetin sensitivity of human proton-coupled folate transporter

“…Moreover, for both of the myricetin-sensitive constructs of B and C, myricetin induced a greater extent of inhibition at the high folate concentration than at the low folate concentration (Fig. 3B), consistent with the characteristic of the myricetin-induced hPCFT inhibition described in our previous study in the aspect of apparent kinetics and indicating a reduction in both V max and K m 19–21 . These results suggest that the amino acid residue responsible for the sensitivity of hPCFT to myricetin is present in the hPCFT-derived segment commonly included in the two constructs, which is the segment spanning 83rd to 186th amino acid residues.…”

“…In an additional set of experiments using the N158G mutant tagged with GFP for detection by western blotting and fluorescent microscopic observation, there was not any appreciable myricetin-induced alteration in the expression and localization of the mutant at the plasma membrane (Supplementary Figs. S1 and S2), suggesting that a decrease in V max indicated by the reduced folate uptake at the high folate concentration implies a decrease in the velocity of substrate translocation, at least in agreement with the characteristic of myricetin-induced alteration in the V max of hPCFT 19 . The saturable characteristic of transport by carrier-mediated mechanism was also confirmed for the N158G mutant (Fig.…”

“…All the other mutants were found to retain the myricetin sensitive characteristic similar to that observed for the wild type of hPCFT (Fig. 3) in the aspect of apparent kinetics 19–21 , their mediated folate uptake being reduced by myricetin to a greater extent at the high folate concentration than at the low folate concentration. These results suggest that Gly 158 is the amino acid residue responsible for the myricetin sensitivity of hPCFT.…”

“…hPCFT has recently been found to be extensively inhibited by myricetin by a sustaining mechanism that persists after its removal 19–21 . The extensive and sustained inhibition of hPCFT by this flavonoid, which is widely present in vegetables and fruits 22 , raises a concern that it could potentially lead to harmful impairment of the intestinal absorption of those PCFT substrates.…”

“…Although the sustaining mechanism of the myricetin-induced inhibition is yet to be fully clarified, kinetic analyses of the myricetin-induced inhibition of the uptake of folate in stably hPCFT-transfected Madin-Darby canine kidney II (MDCKII) cells and in the Caco-2 cell line, which is a well recognized intestinal epithelial cell model, consistently indicated that the maximum transport rate (V max ) was reduced 19,20 . It is notable, however, that the Michaelis constant (K m ) was reduced at the same time, attenuating the inhibition at lower folate concentrations.…”