RESUMEN
INTRODUCTION: The proton-coupled folate transporter (PCFT; SLC46A1) was discovered in 2006 as the principal mechanism by which folates are absorbed in the intestine and the causal basis for hereditary folate malabsorption (HFM). In 2011, it was found that PCFT is highly expressed in many tumors. This stimulated interest in using PCFT for cytotoxic drug targeting, taking advantage of the substantial levels of PCFT transport and acidic pH conditions commonly associated with tumors. AREAS COVERED: We summarize the literature from 2006 to 2022 that explores the role of PCFT in the intestinal absorption of dietary folates and its role in HFM and as a transporter of folates and antifolates such as pemetrexed (Alimta) in relation to cancer. We provide the rationale for the discovery of a new generation of targeted pyrrolo[2,3-d]pyrimidine antifolates with selective PCFT transport and inhibitory activity toward de novo purine biosynthesis in solid tumors. We summarize the benefits of this approach to cancer therapy and exciting new developments in the structural biology of PCFT and its potential to foster refinement of active structures of PCFT-targeted anti-cancer drugs. EXPERT OPINION: We summarize the promising future and potential challenges of implementing PCFT-targeted therapeutics for HFM and a variety of cancers.
Asunto(s)
Antineoplásicos , Antagonistas del Ácido Fólico , Neoplasias , Humanos , Transportador de Folato Acoplado a Protón/química , Antagonistas del Ácido Fólico/farmacología , Antagonistas del Ácido Fólico/uso terapéutico , Pemetrexed/farmacología , Pemetrexed/uso terapéutico , Antineoplásicos/farmacología , Antineoplásicos/uso terapéutico , Ácido Fólico/uso terapéutico , Neoplasias/tratamiento farmacológico , BiologíaRESUMEN
Introduction and importance: Hereditary Folate Malabsorption (HFM) is an extremely rare autosomal recessive disorder with in the existence of only 30 families world-wide. It presents with hematological, gastrointestinal, and neurological problems. Case presentation: Three-month-old-boy with a familial history of HFM presented to the clinic due to persistent fatigue, yellowish discoloration, feeding refusal, and pancytopenia. The patient received 3 packs of Red Blood Cells (RBCs). Five days after received 3 packs of RBCs, the patient presented with a fever of 38.3 Celsius with pancytopenia. The patient had low level of all immunoglobulins. He was started on broad-spectrum antibiotics. Testing for the HFM's SLC46A1 gene mutation, was positive. The patient was started on Leucovorin and Respirm. Clinical discussion: In this case, HFM presented as a neutropenic fever, hypoimmunoglobulinemia, low serum folate, elevated homocysteine, and a positive mutation on the SLC46A1. HFM has a wide-spectrum of presentations which includes hematological, neurological, immunological and gastrointestinal. Treatment involves the administration of folinic acid in either oral or intramuscular injections. Conclusion: HFM can present as neutropenic fever. High index of suspension is to be maintained when the presenting symptoms of the patients vary over a large number of systems. Genetic counseling is needed for parents when both are carrying an autosomal recessive allele.
RESUMEN
BACKGROUND: Hereditary folate malabsorption-a rare disorder caused by impairment of the folate transporter-can develop into severe folate deficiency manifesting as megaloblastic anemia and occasionally thrombocytopenia. Reportedly, megaloblastic anemia can manifest with hemorrhagic episodes, possibly due to ineffective platelet production and platelet dysfunction. However, life-threatening hemorrhage events in hereditary folate malabsorption have not been well investigated. CASE PRESENTATION: A 3-month-old Japanese boy was transferred to our hospital due to thrombocytopenia and severe megaloblastic anemia. During a thorough examination of hematopoietic abnormalities, the patient suddenly went into cardiac arrest due to pulmonary hemorrhage. Although intravenous folate supplementation was started soon after the identification of folate deficiency, the patient died of circulatory defect and multiple organ failure. The cause of pulmonary hemorrhage, such as respiratory infection, could not be confirmed. Genetic investigation revealed a mutation in the SLC46A1 gene to be the cause of the hereditary folate malabsorption. CONCLUSION: We report an infantile case of hereditary folate malabsorption that progressed to lethal pulmonary hemorrhage before folate deficiency was identified. Clinicians should consider that megaloblastic anemia could lead to severe bleeding without warning, and that nutrient supplementation should be initiated as soon as possible.
Asunto(s)
Anemia Megaloblástica , Trombocitopenia , Anemia Megaloblástica/etiología , Ácido Fólico/uso terapéutico , Deficiencia de Ácido Fólico , Hemorragia/etiología , Humanos , Lactante , Síndromes de Malabsorción , Masculino , Transportador de Folato Acoplado a Protón/genética , Trombocitopenia/complicacionesRESUMEN
This study revealed the importance of serine 318 (S318) residue for proton-coupled folate transporter (PCFT, SLC46A1) functioning. Substitution of S318 with arginine or lysine impaired transport of methotrexate (MTX), but substitution with alanine (has a simple side chain structure), or cysteine (structurally similar to serine), had no significant effect on MTX transport. The initial uptake rate of MTX by S318A and S318C mutant at pH 5.0, followed by Michaelis-Menten kinetics with a Km value of approximately 2.3 µM (for S318A) and 2.9 µM (for S318C), was similar to that of the wild-type. The normalized Vmax value of the S318A mutant, calculated by dividing the Vmax value by the Western blot protein band's relative intensity, was approximately 2-fold greater than that of the wild-type. The normalized Vmax value of the S318C mutant was approximately 0.8-fold smaller than the wild-type. Results obtained showed that the substitution of S318 with basic amino acid residues results in the loss of transport activity, even though PCFT mutants are expressed at the cell membrane. Alternatively, the substitution of S318 with neutral amino acids did not significantly affect the transport function of PCFT.
Asunto(s)
Metotrexato , Transportador de Folato Acoplado a Protón , Ácido Fólico , Células HeLa , Humanos , Transportador de Folato Acoplado a Protón/genética , Serina/genéticaRESUMEN
Hereditary folate malabsorption (HFM) is a rare disorder of proton-coupled folate transporter deficiency. It is characterized by macrocytic anemia, recurrent infections, and epilepsy. A five-year-old girl presented with recurrent pneumonia, diarrhea, and mouth ulcers. On examination, pallor, microcephaly with spastic quadriparesis was noted. On investigations, leukopenia and thrombocytopenia with megaloblastic bone marrow picture and low folate levels was found. HFM was diagnosed at two years of age and the child was treated with folinic acid. Her diagnosis was confirmed by whole-exome sequencing which revealed a novel pathogenic homozygous frameshift insertion variation (c.620dupG) in the exon 2 of the SLC46A1 gene which was further confirmed by Sanger sequencing. The child improved significantly except for a partial improvement in neurological symptoms.
Asunto(s)
Epilepsia Refractaria , Deficiencia de Ácido Fólico , Pancitopenia , Niño , Preescolar , Femenino , Ácido Fólico/uso terapéutico , Deficiencia de Ácido Fólico/complicaciones , Deficiencia de Ácido Fólico/diagnóstico , Deficiencia de Ácido Fólico/tratamiento farmacológico , Humanos , Síndromes de Malabsorción , Pancitopenia/genética , Transportador de Folato Acoplado a Protón , ReinfecciónRESUMEN
The proton-coupled folate transporter (PCFT, SLC46A1) is required for folate intestinal absorption and transport across the choroid plexus. Recent work has identified a F392V mutation causing hereditary folate malabsorption. However, the residue properties responsible for this loss of function remains unknown. Using site-directed mutagenesis, we observed complete loss of function with charged (Lys, Asp, and Glu) and polar (Thr, Ser, and Gln) Phe-392 substitutions and minimal function with some neutral substitutions; however, F392M retained full function. Using the substituted-cysteine accessibility method (with N-biotinyl aminoethyl methanethiosulfonate labeling), Phe-392 mutations causing loss of function, although preserving membrane expression and trafficking, also resulted in loss of accessibility of the substituted cysteine in P314C-PCFT located within the aqueous translocation pathway. F392V function and accessibility of the P314C cysteine were restored by insertion of a G305L (suppressor) mutation. A S196L mutation localized in proximity to Gly-305 by homology modeling was inactive. However, when inserted into the inactive F392V scaffold, function was restored (mutually compensatory mutations), as was accessibility of the P314C cysteine residue. Reduced function, documented with F392H PCFT, was due to a 15-fold decrease in methotrexate influx Vmax, accompanied by a decreased influx Kt (4.5-fold) and Ki (3-fold). The data indicate that Phe-392 is required for rapid oscillation of the carrier among its conformational states and suggest that this is achieved by dampening affinity of the protein for its folate substrates. F392V and other inactivating Phe-392 PCFT mutations lock the protein in its inward-open conformation. Reach (length) and hydrophobicity of Phe-392 appear to be features required for full activity.
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Transportador de Folato Acoplado a Protón/metabolismo , Secuencia de Aminoácidos , Animales , Transporte Biológico , Cisteína/química , Cisteína/metabolismo , Deficiencia de Ácido Fólico/patología , Células HeLa , Humanos , Cinética , Síndromes de Malabsorción/patología , Metotrexato/metabolismo , Mutagénesis Sitio-Dirigida , Estructura Terciaria de Proteína , Transportador de Folato Acoplado a Protón/química , Transportador de Folato Acoplado a Protón/genéticaAsunto(s)
Deficiencia de Ácido Fólico , Hipertensión Pulmonar , Síndromes de Malabsorción , Humanos , LactanteRESUMEN
BACKGROUND: Hereditary folate malabsorption is a multisystem disease owing to biallelic variants in the gene encoding the proton-coupled folate transporter. Hereditary folate malabsorption is treated with folinic acid, aimed to restore blood and cerebrospinal fluid folate levels. Little is known as to whether oral or intramuscular supplementation of folinic acid is most effective. METHODS: Here we describe a one-year-old boy with hereditary folate malabsorption presenting with the typical features including failure to thrive, aphthous stomatitis, macrocytic anemia along with severe developmental impairment and epilepsy, as well as a magnetic resonance imaging of the brain showing bilateral occipital, cortical calcifications characteristic of hereditary folate malabsorption. We compared the effect of treatment with oral folinic acid versus intramuscular folinic acid supplementation by measuring plasma and cerebrospinal fluid folate levels. RESULTS: Compared with oral administration, intramuscular treatment resulted in higher folate levels in blood and, most importantly, normalization of folate levels in cerebrospinal fluid. Clinically, nearly all systemic and neurological symptoms resolved. CONCLUSION: Normal cerebrospinal fluid folate levels can be achieved in individuals with hereditary folate malabsorption with intramuscular (but not with oral) administration of folinic acid.
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Deficiencia de Ácido Fólico/tratamiento farmacológico , Leucovorina/farmacología , Síndromes de Malabsorción/tratamiento farmacológico , Complejo Vitamínico B/farmacología , Humanos , Lactante , Inyecciones Intramusculares , Leucovorina/administración & dosificación , Masculino , Complejo Vitamínico B/administración & dosificaciónRESUMEN
Hereditary folate malabsorption (HFM) is an autosomal recessive disease caused by mutations in SLC46A1 encoding the proton-coupled folate transporter (PCFT). HFM patients present with various clinical features including megaloblastic anemia, thrombocytopenia, combined immunodeficiency and neurodevelopmental disorders. In this study, we report the same deep intronic mutation of c.1166-285â¯Tâ¯>â¯G shared by four unrelated Japanese patients with HFM. This mutation was shown to generate a cryptic splice donor site for a 168-bp insertion of intron 3 sequences, leading to premature termination in the middle of this insertion. This mutation could be a founder mutation in the Japanese population, but also could be a hot-spot and could be present in undiagnosed HFM patients worldwide because of the difficulty to detect this mutation.
Asunto(s)
Deficiencia de Ácido Fólico/genética , Síndromes de Malabsorción/genética , Transportador de Folato Acoplado a Protón/genética , Pueblo Asiatico/genética , Femenino , Humanos , Lactante , Masculino , MutaciónRESUMEN
Hereditary folate malabsorption is a rare autosomal recessive disorder caused by impaired active folate transport across membranes and into the central nervous system due to loss-of-function mutations in proton-coupled folate transporter (PCFT). Newborns with this condition have initially normal folate stores, but as they are unable to absorb dietary folate and use rapidly their stores because of their growth demands, symptoms appear in the early infancy. Significant neurological morbidity usually follows the initial non-specific clinical presentation and delayed initiation of treatment. High dose oral and parenteral folinic acid treatment have been previously reported in literature to improve the clinical outcome without achieving optimal cerebrospinal fluid (CSF) folate levels though. The active isomer of 5-formyltetrahydrofolate, also known as levofolinic acid, is available for administration. We report our experience in achieving normal (age dependent) CSF 5-Methyltetrahydrofolate (5-MTHF) levels following daily intramuscular administration of levofolinic acid in three patients with HFM. Follow-up assessment with repeated lumbar punctures has shown a stabilization of 5-MTHF levels within normal range. Clinical features and brain MRI findings had as well either improvement or stabilization. To the best of our knowledge, we provide as well for the first time data in regard to the im levofolinate treatment dosage.
RESUMEN
The proton-coupled folate transporter (PCFT-SLC46A1) is the mechanism by which folates are absorbed across the brush-border membrane of the small intestine. The transporter is also expressed in the choroid plexus and is required for transport of folates into the cerebrospinal fluid. Loss of PCFT function, as occurs in the autosomal recessive disorder "hereditary folate malabsorption" (HFM), results in a syndrome characterized by severe systemic and cerebral folate deficiency. Folate-receptor alpha (FRα) is expressed in the choroid plexus, and loss of function of this protein, as also occurs in an autosomal recessive disorder, results solely in "cerebral folate deficiency" (CFD), the designation for this disorder. This paper reviews the current understanding of the functional and structural properties and regulation of PCFT, an electrogenic proton symporter, and contrasts PCFT properties with those of the reduced folate carrier (RFC), an organic anion antiporter, that is the major route of folate transport to systemic tissues. The clinical characteristics of HFM and its treatment, based upon the thirty-seven known cases with the clinical syndrome, of which thirty have been verified by genotype, are presented. The ways in which PCFT and FRα might interact at the level of the choroid plexus such that each is required for folate transport from blood to cerebrospinal fluid are considered along with the different clinical presentations of HFM and CFD.
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Deficiencia de Ácido Fólico/metabolismo , Síndromes de Malabsorción/metabolismo , Transportador de Folato Acoplado a Protón/metabolismo , Animales , Transporte Biológico , Deficiencia de Ácido Fólico/líquido cefalorraquídeo , Humanos , Absorción Intestinal , Síndromes de Malabsorción/líquido cefalorraquídeo , Modelos Moleculares , Transportador de Folato Acoplado a Protón/químicaRESUMEN
The proton-coupled folate transporter (PCFT) mediates folate absorption across the brush-border membrane of the proximal small intestine and is required for folate transport across the choroid plexus into the cerebrospinal fluid. In this study, the functional role and accessibility of the seven PCFT Trp residues were assessed by the substituted-cysteine accessibility method. Six Trp residues at a lipid-aqueous interface tolerated Cys substitution in terms of protein stability and function. W85C, W202C, and W213C were accessible to N-biotinyl aminoethylmethanethiosulfonate; W48C and W299C were accessible only after treatment with dithiotreitol (DTT), consistent with modification of these residues by an endogenous thiol-reacting molecule and their extracellular location. Neither W107C nor W333C was accessible (even after DTT) consistent with their cytoplasmic orientation. Biotinylation was blocked by pemetrexed only for the W48C (after DTT), W85C, W202C residues. Function was impaired only for the W299C PCFT mutant located in the 4th external loop between the 7th and 8th transmembrane helices. Despite its aqueous location, function could only be fully preserved with Phe and, to a lesser extent, Ala substitutions. There was a 6.5-fold decrease in the pemetrexed influx Vmax and a 3.5- and 6-fold decrease in the influx Kt and Ki, respectively, for the W299S PCFT. The data indicate that the hydrophobicity of the W299 residue is important for function suggesting that during the transport cycle this residue interacts with the lipid membrane thereby impacting on the oscillation of the carrier and, indirectly, on the folate binding pocket.
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Membrana Celular/metabolismo , Ácido Fólico/metabolismo , Transportador de Folato Acoplado a Protón/metabolismo , Sitios de Unión , Biotinilación , Membrana Celular/efectos de los fármacos , Cisteína , Antagonistas del Ácido Fólico/farmacología , Genotipo , Células HeLa , Humanos , Concentración de Iones de Hidrógeno , Interacciones Hidrofóbicas e Hidrofílicas , Cinética , Mutagénesis Sitio-Dirigida , Mutación , Pemetrexed/farmacología , Fenotipo , Unión Proteica , Conformación Proteica en Hélice alfa , Dominios Proteicos , Estabilidad Proteica , Transportador de Folato Acoplado a Protón/química , Transportador de Folato Acoplado a Protón/efectos de los fármacos , Transportador de Folato Acoplado a Protón/genética , Relación Estructura-Actividad , Transfección , TriptófanoRESUMEN
The proton-coupled folate transporter (PCFT) mediates intestinal folate absorption and transport of folates across the choroid plexus. This study focuses on the role of Tyr residues in PCFT function. The substituted Cys-accessibility method identified four Tyr residues (Y291, Y362, Y315, and Y414) that are accessible to the extracellular compartment; three of these (Y291, Y362, and Y315) are located within or near the folate binding pocket. When the Tyr residues were replaced with Cys or Ala, these mutants showed similar (up to 6-fold) increases in influx Vmax and Kt/Ki for [(3)H]methotrexate and [(3)H]pemetrexed. When the Tyr residues were replaced with Phe, these changes were moderated or absent. When Y315A PCFT was used as representative of the mutants and [(3)H]pemetrexed as the transport substrate, this substitution did not increase the efflux rate constant. Furthermore, neither influx nor efflux mediated by Y315A PCFT was transstimulated by the presence of substrate in the opposite compartment; however, substantial bidirectional transstimulation of transport was mediated by wild-type PCFT. This resulted in a threefold greater efflux rate constant for cells that express wild-type PCFT than for cells that express Y315 PCFT under exchange conditions. These data suggest that these Tyr residues, possibly through their rigid side chains, secure the carrier in a high-affinity state for its folate substrates. However, this may be achieved at the expense of constraining the carrier's mobility, thereby decreasing the rate at which the protein oscillates between its conformational states. The Vmax generated by these Tyr mutants may be so rapid that further augmentation during transstimulation may not be possible.
Asunto(s)
Ácido Fólico/metabolismo , Absorción Intestinal/fisiología , Transportador de Folato Acoplado a Protón/metabolismo , Tirosina/química , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Sitios de Unión/genética , Transporte Biológico/genética , Línea Celular Tumoral , Glutamatos/metabolismo , Guanina/análogos & derivados , Guanina/metabolismo , Células HeLa , Humanos , Concentración de Iones de Hidrógeno , Absorción Intestinal/genética , Metotrexato/metabolismo , Modelos Moleculares , Pemetrexed , Estructura Terciaria de Proteína , Transportador de Folato Acoplado a Protón/genéticaRESUMEN
BACKGROUND: Hereditary folate malabsorption is a rare, autosomal recessive disorder of proton-coupled folate transporter deficiency resulting in folate deficiency. Left untreated, the condition can cause severe brain damage and megaloblastic anemia, leading to progressive psychomotor retardation, seizures and other neurological problems. Early diagnosis and treatment are crucial. No case has been documented yet in Mainland China until now. METHODS: A Chinese girl affected by hereditary folate malabsorption was studied. The girl presented with recurrent megaloblastic anemia from the age of 7 months. Paroxysmal limbs trembling and seizures were presented from the age of three years. Intracranial calcification was noted by CT. At her age of 5 years, mental regression, lower-extremity weakness and sleeping problems were observed. Her plasma folate decreased to 4.49 nmol/L (normal control>6.8nmol/L). Plasma total homocysteine elevated to 28.11 µmol/L (normal control<15 µmol/L). Folate and 5-methylterahydrofolate in cerebrospinal fluid were significantly decreased to undetectable level. RESULTS: On SLC46A1 gene, a novel mutation, c.1A>T (M1L), and a reported mutation c.194-195 insG (p.Cys66LeufsX99) were identified, supported the diagnosis of hereditary folate malabsorption. Each parent carries one of two mutations. Folinic calcium supplement resulted in rapid clinical improvement. She is currently 6 years old with normal development and routine blood features. CONCLUSION: Hereditary folate malabsorption is one of the few easily-treatable inherited metabolic diseases. Measurements of folate and 5-methyltetrahydrofolate in cerebrospinal fluid are keys for the diagnosis of the patients.
Asunto(s)
Deficiencia de Ácido Fólico/genética , Síndromes de Malabsorción/genética , Transportador de Folato Acoplado a Protón/genética , Edad de Inicio , Pueblo Asiatico , Preescolar , Femenino , Ácido Fólico/uso terapéutico , Deficiencia de Ácido Fólico/complicaciones , Deficiencia de Ácido Fólico/patología , Humanos , Síndromes de Malabsorción/complicaciones , Síndromes de Malabsorción/patología , Mutación Puntual , Convulsiones/etiologíaRESUMEN
Hereditary folate malabsorption (HFM) is a rare autosomal recessive disorder. Severe folate deficiency in HFM can result in immunodeficiency. We describe a female infant with HFM who acquired severe Pneumocystis pneumonia. The objective of the present study was to elucidate her immunological phenotype and to examine the time course of immune recovery following parenteral folate therapy. The patient demonstrated a combined immunodeficiency with an impaired T cell proliferation response, pan-hypogammaglobulinemia, and an imbalanced pro-inflammatory cytokine profile. She had normal white blood cell count, normal lymphocyte subsets, and normal complement levels. Two novel mutations were identified within the SLC46A1 gene to produce a compound heterozygote. We confirmed full recovery of her immunological and neurophysiological status with parenteral folate replacement. The time course of recovery of her immunological profile varied widely, however. HFM should be recognized as a unique form of immunodeficiency.
Asunto(s)
Deficiencia de Ácido Fólico/complicaciones , Ácido Fólico/uso terapéutico , Síndromes de Inmunodeficiencia/tratamiento farmacológico , Síndromes de Inmunodeficiencia/etiología , Síndromes de Malabsorción/complicaciones , Recuento de Células Sanguíneas , Citocinas/sangre , Femenino , Heterocigoto , Humanos , Isotipos de Inmunoglobulinas/sangre , Isotipos de Inmunoglobulinas/inmunología , Síndromes de Inmunodeficiencia/diagnóstico , Lactante , Activación de Linfocitos/inmunología , Subgrupos Linfocitarios/inmunología , Subgrupos Linfocitarios/metabolismo , Mutación , Transportador de Folato Acoplado a Protón/genéticaRESUMEN
Hereditary folate malabsorption (OMIM 229050) is a rare autosomal recessive disorder caused by loss-of-function mutations in the proton-coupled folate transporter gene (pcft/SLC46A1) resulting in impaired folate transport across the intestine and into the central nervous system. We report a novel, homozygous, deletion mutation in a child of Nicaraguan descent in exon 2 (c.558-588 del, ss778190447) at amino acid position I188 resulting in a frameshift with a premature stop.
Asunto(s)
Ácido Fólico/metabolismo , Síndromes de Malabsorción/genética , Transportador de Folato Acoplado a Protón/genética , Eliminación de Secuencia , Humanos , Lactante , Masculino , NicaraguaRESUMEN
The proton-coupled folate transporter (PCFT, SLC46A1) mediates folate transport across the apical brush-border membrane of the proximal small intestine and the basolateral membrane of choroid plexus ependymal cells. Two loss-of-function mutations in PCFT, which are the basis for hereditary folate malabsorption, have been identified within the fourth transmembrane domain (TMD4) in subjects with this disorder. We have employed the substituted Cys accessibility method (SCAM) to study the accessibilities of all residues in TMD4 and their roles in folate substrate binding to the carrier. When residues 146-167 were replaced by Cys, all except R148C were expressed at the cell surface. Modification of five of these substituted Cys residues (positions 147, 152, 157, 158, and 161) by methanethiosulfonate (MTS) reagents led to reduction of PCFT function. All five residues could be labeled with N-biotinylaminoethyl-MTS, and this could be blocked by the high-affinity PCFT substrate pemetrexed. Pemetrexed also protected PCFT mutant function from inhibitory modification of the substituted Cys at positions 157, 158, and 161 by a MTS. The findings indicate that these five residues in TMD4 are accessible to the aqueous translocation pathway, play a role in folate substrate binding, and are likely located within or near the folate binding pocket. A homology model of PCFT places three of these residues, Phe¹57, Gly¹58, and Leu¹6¹, within a breakpoint in the midportion of TMD4, a region that likely participates in alterations in the PCFT conformational state during carrier cycling.