Folates are one-carbon donors required for de novo nucleotide synthesis and methylation reactions. Folate requirements are met entirely from dietary sources and, and while several transporters were known to mediate transport of folates into systemic tissues, the mechanism of intestinal folate absorption was only recently clarified. This laboratory identified SLC46A1 as a Proton-Coupled Folate Transporter (PCFT) that mediates folate transport across the apical (brush border) membrane of enterocytes in the acidic microclimate at the absorptive surface of the proximal small intestine. The role that PCFT plays at this epithelium was confirmed by the demonstration that there are loss-of-function mutations in PCFT in patients with the autosomal recessive disorder, hereditary folate malabsorption (HFM, OMIM 229050). HFM is characterized by impaired intestinal folate absorption and impaired transport of folates into central nervous system, the latter likely due to a defect in transport across the blood:choroid plexus:cerebrospinal fluid barrier. PCFT also facilitates export of folates from acidified endosomes during folate receptor (FR) – mediated endocytosis. Both proteins co-localize to the endosomal compartment. PCFT-mediated transport is highly specific for, and among, folates and antifolates with influx K_ts of ~ 0.2 – 2 μM. PCFT does not transport folate polyglutamates. PCFT-mediated folate transport in Xenopus oocytes evokes current accompanied by cellular acidification. A low level of current and acidification is detected in the absence of folate substrate in the presence of a high proton gradient suggesting that PCFT can also act as a proton channel. Folates are also transported by PCFT in the absence of an inwardly directed proton gradient. The “working” PCFT secondary structure model at this point is a protein with a large intracellular loop separating six transmembrane domains (TMDs) on either side. The N- and C- termini are located in the cytoplasm as established by immunocytochemistry. The extracellular location of the first loop was confirmed based upon glycosylation ot the two predicted sites in this region (N58, N68). Studies are exploring PCFT structure-function informed, in part, by point mutations located within, or adjacent to, predicted TMDs detected in patients with HFM. Other studies utilize site-directed mutagenesis to identify functionally important residues with an initial focus on histidine. Of the 10 His residues, mutation of only two to Ala (H247 and H281 – both conserved) results in functional changes. H247 resides in the mid-region of the large central intracellular loop. Mutation of H247 to residues of different size, polarity and charge results in a marked increase in affinity for its folate substrates and a loss of selectivity. Using a homology model based upon the GlpT crystal structure, H247 is located in hydrogen bond distance to S172 in the 2^nd intracellular loop, (also fully conserved), at the cytoplasmic opening of the translocation pathway. S172A-PCFT has a transport phenotype similar to that of H247A. When the H247A mutant is expressed in Xenopus oocytes, the proton slippage observed with wild-type PCFT-mediated transport in the absence of folate is markedly increased, especially at high extracellular proton concentrations. These observations suggest that disruption of the H147-S172 interaction, which may play a “tethering or filtering” role, results in a shift in the PCFT conformation state(s) that (i) increases the accessibility of folate substrates to a high-affinity binding pocket, (ii) accounts for the loss of selectivity and increase in substrate affinity, and (iii) allows folate-independent proton transport in a channel-like mode. The H281A mutation, on the other hand, results in a marked increase in the folate influx K_t but the carrier remains electrogenic and acidifies oocytes indicating that this residue is not required for proton-coupling. When the buffer pH is decreased below 5.5, there is a fall in the folic acid influx K_t for the H281A-PCFT with little change in the wild-type PCFT influx K_t. The homology model places H281 in the 7^th TMD at the extracellular opening of the translocation pathway. These observations suggest that protonation of H281 results in an increased affinity of carrier for its folate substrates that is impaired in the H281A-PCFT mutant. Enhanced folate binding to the H281A mutant, observed with decreasing pH, appears to be due to the increase in proton concentration required to induce a conformation of the binding pocket that is inherent to wild-type PCFT at higher pH. Ongoing studies are directed to further confirm the PCFT secondary structure and to identify residues that play a role in proton-coupling and substrate selectivity.