Hereditary folate malabsorption due to a mutation in the external gate of the proton-coupled folate transporter SLC46A1

Abstract

Key Points• An N411K mutation in the external gate of the proton-coupled folate transporter within the aqueous channel results in impaired function.• The N411K mutation produces a substratespecific defect in transport, resulting in hereditary folate malabsorption.Hereditary folate malabsorption (HFM) is an autosomal recessive disorder characterized by impaired intestinal folate absorption and impaired folate transport across the choroid plexus due to loss of function of the proton-coupled folate transporter (PCFT-SLC46A1). We report a novel mutation, causing HFM, affecting a residue located in the 11th transmembrane helix within the external gate. The mutant N411K-PCFT was stable, trafficked to the cell membrane, and had sufficient residual activity to characterize the transport defect and the structural requirements at this site for gate function. The influx V max of the N411K mutant was markedly decreased, as was the affinity for most, but not all, folate/antifolate substrates.The greatest loss of activity was for 5-methyltetrahydrofolate. Substitutions with positive charged residues resulted in a loss of activity (arginine . lysine . histidine). Function was retained for the negative charged aspartate, but not the larger glutamate substitutions, whereas the bulky hydrophobic (leucine), or polar (glutamine) substitutions, were tolerated.Homology models of PCFT, in the inward and outward open conformations, based upon the mammalian Glut5 fructose transporter structures, localize Asn411 protruding into the aqueous pathway. This is most prominent when the carrier is in the inward open conformation when the external gate is closed. Mutations at this site likely result in highly specific steric and electrostatic interactions between the Asn411-substituted, and other, residues in the gate region that impede carrier function. The substrate specificity of the N411K mutant may be due to alterations of substrate flows through the external gate, downstream allosteric alterations in the folate-binding pocket, or both.

Upregulation of reduced folate carrier by vitamin D enhances brain folate uptake in mice lacking folate receptor alpha

“…The current therapeutic approach for neurological disorders resulting from folate transport defects is to achieve high 5-formylTHF blood levels in order to deliver sufficient folate to the brain to sustain normal neural development. However, despite achievement of high blood folate levels, CSF folate levels are still below the normal range and neurological signs, particularly seizures, may be difficult to control (8,32,45,46). Therefore, modulating folate transport at the BBB through RFC offers a therapeutic approach to improving brain folate delivery for the treatment of neurometabolic disorders caused by loss of FRα or PCFT function.…”

Successful Treatment of Hereditary Folate Malabsorption With Intramuscular Folinic Acid

“…This superiority is probably due to higher blood folate levels attained, which could accomplish transport over the defective PCFT transporter with some residual function or promote an alternative route via the vascular endothelial blood-brain barrier, or at the level of the choroid plexus via FRa. 1,14,16 A very recent report showed good results from IM treatment with levofolinic acid, the active L-isomer of 5-MTHF (folinic acid is the racemic mixture containing both L-and D-isomers of 5-MTHF). In one of the described patients, high doses of IM levofolinic acid resulted in even higher CSF 5-MTHF levels than treatment with IM folinic acid.…”

“…[5][6][7][9][10][11][12][13] Here we present our experience with the treatment of a patient with HFM; the functional impact of his mutation was previously published. 14 We compare biochemical outcomes on oral versus IM administration of folinic acid.…”

A proton-coupled folate transporter mutation causing hereditary folate malabsorption locks the protein in an inward-open conformation

“…Deciphering the functional role of PCFT residues mutated in HFM has contributed important insights into the properties of this transporter. A N411K mutation was localized within the external gate of the PCFT translocation pathway (14,17). A P425R mutation revealed a difference in the binding pocket for MTX versus pemetrexed (25).…”

“…The substituted cysteine accessibility method has been used to characterize the secondary structure, the accessibility of residues within transmembrane segments and defined elements of the aqueous translocation pathway. Residues that are involved in proton binding, protoncoupling, extracellular gating, folate substrate binding and carrier translocation rates have been identified (2,14,15). Homology models of PCFT in the inward-open (16,17), and more recently in both the inward-and outward-open (14,18,19), conformations, have been developed, the latter based upon the structures of the mammalian Glut 5 fructose transporter (20).…”