Abstract
Human proton-coupled folate transporter (hPCFT/SLC46A1) has recently been found to be inhibited by myricetin by a sustained mechanism, raising a concern that the inhibition might lead to malabsorption of folates in the intestine, where hPCFT works for their epithelial uptake. However, rat PCFT (rPCFT) has more recently been found not to be inhibited by myricetin. Prompted by this finding, we attempted to determine the amino acid residue involved in that by analyses comparing between hPCFT and rPCFT. In the initial analysis, chimeric constructs prepared from hPCFT and rPCFT were examined for myricetin sensitivity to determine the hPCFT segment involved in the sensitivity. Focusing on the thereby determined segment from 83rd to 186th amino acid residue, hPCFT mutants having a designated amino acid residue replaced with its counterpart in rPCFT were prepared for the subsequent analysis. Among them, only G158N-substituted hPCFT was found to be transformed to be insensitive to myricetin and, accordingly, oppositely N158G-substituted rPCFT was transformed to be sensitive to myricetin. These results indicate the critical role of Gly158 in the myricetin sensitivity of hPCFT. This finding would help advance the elucidation of the mechanism of the myricetin-induced inhibition of hPCFT and manage the potential risk arising from that.
Highlights
Human proton-coupled folate transporter has recently been found to be inhibited by myricetin by a sustained mechanism, raising a concern that the inhibition might lead to malabsorption of folates in the intestine, where hPCFT works for their epithelial uptake
The myricetin insensitivity of rat PCFT (rPCFT) can be taken advantage of to determine the amino acid residue involved in the myricetin-induced inhibition in hPCFT, as its myricetin sensitivity should be derived from the amino acid residues that are different from their counterparts in rPCFT
We first attempted to identify the hPCFT segment involved in the myricetin sensitivity to narrow down the candidate amino acid residues by a comparative analysis of the effects www.nature.com/scientificreports of myricetin on chimeric constructs derived from hPCFT and rPCFT (Fig. 2)
Summary
Human proton-coupled folate transporter (hPCFT/SLC46A1) has recently been found to be inhibited by myricetin by a sustained mechanism, raising a concern that the inhibition might lead to malabsorption of folates in the intestine, where hPCFT works for their epithelial uptake. 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 (Vmax) was reduced[19,20]. We here report our successful attempt to pursue that by analyses comparing between the two PCFTs. The acquired knowledge of the responsible amino acid residue would help advance the elucidation of the mechanism of the myricetin sensitivity of hPCFT and manage the potential risk of the myricetin-induced impairment of the intestinal absorption of folate and analogues
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