FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W1997831277> ?p ?o ?g. }

• W1997831277 endingPage "17857" @default.
• W1997831277 startingPage "17846" @default.
• W1997831277 abstract "This report addresses the functional role of His residues in the proton-coupled folate transporter (PCFT; SLC46A1), which mediates intestinal folate absorption. Of ten His residues, only H247A and H281A mutations altered function. The folic acid influx Kt at pH 5.5 for H247A was ↓8.4-fold. Although wild type (WT)-PCFT Ki values varied among the folates, Ki values were much lower and comparable for H247-A, -R, -Q, or -E mutants. Homology modeling localized His247 to the large loop separating transmembrane domains 6 and 7 at the cytoplasmic entrance of the translocation pathway in hydrogen-bond distance to Ser172. The folic acid influx Kt for S172A-PCFT was decreased similar to H247A. His281 faces the extracellular region in the seventh transmembrane domain. H281A-PCFT results in loss-of-function due to ∼12-fold↑ in the folic acid influx Kt. When the pH was decreased from 5.5 to 4.5, the WT-PCFT folic acid influx Kt was unchanged, but the Kt decreased 4-fold for H281A. In electrophysiological studies in Xenopus oocytes, both WT-PCFT- and H281A-PCFT-mediated folic acid uptake produced current and acidification, and both exhibited a low level of folate-independent proton transport (slippage). Slippage was markedly increased for the H247A-PCFT mutant. The data suggest that disruption of the His247 to Ser172 interaction results in a PCFT conformational alteration causing a loss of selectivity, increased substrate access to a high affinity binding pocket, and proton transport in the absence of a folate gradient. The His281 residue is not essential for proton coupling but plays an important role in PCFT protonation, which, in turn, augments folate binding to the carrier. This report addresses the functional role of His residues in the proton-coupled folate transporter (PCFT; SLC46A1), which mediates intestinal folate absorption. Of ten His residues, only H247A and H281A mutations altered function. The folic acid influx Kt at pH 5.5 for H247A was ↓8.4-fold. Although wild type (WT)-PCFT Ki values varied among the folates, Ki values were much lower and comparable for H247-A, -R, -Q, or -E mutants. Homology modeling localized His247 to the large loop separating transmembrane domains 6 and 7 at the cytoplasmic entrance of the translocation pathway in hydrogen-bond distance to Ser172. The folic acid influx Kt for S172A-PCFT was decreased similar to H247A. His281 faces the extracellular region in the seventh transmembrane domain. H281A-PCFT results in loss-of-function due to ∼12-fold↑ in the folic acid influx Kt. When the pH was decreased from 5.5 to 4.5, the WT-PCFT folic acid influx Kt was unchanged, but the Kt decreased 4-fold for H281A. In electrophysiological studies in Xenopus oocytes, both WT-PCFT- and H281A-PCFT-mediated folic acid uptake produced current and acidification, and both exhibited a low level of folate-independent proton transport (slippage). Slippage was markedly increased for the H247A-PCFT mutant. The data suggest that disruption of the His247 to Ser172 interaction results in a PCFT conformational alteration causing a loss of selectivity, increased substrate access to a high affinity binding pocket, and proton transport in the absence of a folate gradient. The His281 residue is not essential for proton coupling but plays an important role in PCFT protonation, which, in turn, augments folate binding to the carrier. This laboratory recently identified SLC46A1 as a proton-coupled folate transporter (PCFT) 2The abbreviations used are: PCFTproton-coupled folate transporter (SLC46A1, SLC is the Human Genome Organization nomenclature for solute carrier genes, all capital names represent human genes, whereas lowercase designations represent orthologs from other species)5-formyl-THF5-formyltetrahydrofolate5-methylTHF5-methyltetrahydrofolateMTXmethotrexateGlpTglycerol-3-phosphate transporter. that mediates transport of folates across the apical (brush border) membrane of enterocytes within the acidic microclimate at the absorptive surface of the proximal jejunum (1Qiu A. Jansen M. Sakaris A. Min S.H. Chattopadhyay S. Tsai E. Sandoval C. Zhao R. Akabas M.H. Goldman I.D. Cell. 2006; 127: 917-928Abstract Full Text Full Text PDF PubMed Scopus (670) Google Scholar). The critical role that PCFT plays at this epithelium was confirmed with the demonstration by this laboratory that there are loss-of-function mutations in this gene in patients with the autosomal recessive disorder, hereditary folate malabsorption (Online Mendelian Inheritance in Man, 229050) (1Qiu A. Jansen M. Sakaris A. Min S.H. Chattopadhyay S. Tsai E. Sandoval C. Zhao R. Akabas M.H. Goldman I.D. Cell. 2006; 127: 917-928Abstract Full Text Full Text PDF PubMed Scopus (670) Google Scholar, 2Zhao R. Min S.H. Qiu A. Sakaris A. Goldberg G.L. Sandoval C. Malatack J.J. Rosenblatt D.S. Goldman I.D. Blood. 2007; 110: 1147-1152Crossref PubMed Scopus (136) Google Scholar). Hereditary folate malabsorption is characterized by impaired intestinal folate absorption and impaired transport of folates into the central nervous system (3Geller J. Kronn D. Jayabose S. Sandoval C. Medicine (Baltimore). 2002; 81: 51-68Crossref PubMed Scopus (109) Google Scholar), the latter likely due to a defect in transport across the blood-choroid plexus-cerebrospinal fluid barrier. While operating most efficiently at low pH, PCFT contributes to the transport of folates and the pharmacological activity of the new generation antifolate, pemetrexed, even at neutral pH (4Zhao R. Qiu A. Tsai E. Jansen M. Akabas M.H. Goldman I.D. Mol. Pharmacol. 2008; 74: 854-862Crossref PubMed Scopus (111) Google Scholar). PCFT may be even more important to the delivery of this, and possibly other antifolates, within the acidic interstitium of solid tumors (5Helmlinger G. Yuan F. Dellian M. Jain R.K. Nat. Med. 1997; 3: 177-182Crossref PubMed Scopus (1358) Google Scholar, 6Raghunand N. Altbach M.I. van Sluis R. Baggett B. Taylor C.W. Bhujwalla Z.M. Gillies R.J. Biochem. Pharmacol. 1999; 57: 309-312Crossref PubMed Scopus (84) Google Scholar). PCFT may also play a role in the export of folates from acidified endosomes (7Yang J. Chen H. Vlahov I.R. Cheng J.X. Low P.S. J. Pharmacol. Exp. Ther. 2007; 321: 462-468Crossref PubMed Scopus (102) Google Scholar) during folate receptor-mediated endocytosis (8Andrews N.C. Cell Metab. 2007; 5: 5-6Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar, 9Zhao R. Min S.H. Wang Y. Campanella E. Low P.S. Goldman I.D. J. Biol. Chem. 2009; 284: 4267-4274Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar). proton-coupled folate transporter (SLC46A1, SLC is the Human Genome Organization nomenclature for solute carrier genes, all capital names represent human genes, whereas lowercase designations represent orthologs from other species) 5-formyltetrahydrofolate 5-methyltetrahydrofolate methotrexate glycerol-3-phosphate transporter. Although facilitative carriers in lower organisms commonly use a proton gradient to drive uphill transport of substrates (10Pao S.S. Paulsen I.T. Saier Jr., M.H. Microbiol. Mol. Biol. Rev. 1998; 62: 1-34Crossref PubMed Google Scholar), there are only eight proton-coupled solute carrier families in humans; pcft is the latest to be identified (1Qiu A. Jansen M. Sakaris A. Min S.H. Chattopadhyay S. Tsai E. Sandoval C. Zhao R. Akabas M.H. Goldman I.D. Cell. 2006; 127: 917-928Abstract Full Text Full Text PDF PubMed Scopus (670) Google Scholar, 11Thwaites D.T. Anderson C.M. Exp. Physiol. 2007; 92: 603-619Crossref PubMed Scopus (169) Google Scholar). One or more His residues have been shown to be critical for function of some of these transporters (12Lam-Yuk-Tseung S. Govoni G. Forbes J. Gros P. Blood. 2003; 101: 3699-3707Crossref PubMed Scopus (93) Google Scholar, 13Metzner L. Natho K. Zebisch K. Dorn M. Bosse-Doenecke E. Ganapathy V. Brandsch M. Biochim. Biophys. Acta. 2008; 1778: 1042-1050Crossref PubMed Scopus (18) Google Scholar, 14Fei Y.J. Liu W. Prasad P.D. Kekuda R. Oblak T.G. Ganapathy V. Leibach F.H. Biochemistry. 1997; 36: 452-460Crossref PubMed Scopus (119) Google Scholar). Prior to the identification of PCFT, proton-coupled uphill folate transport was demonstrated in membrane vesicles derived from rat hepatocytes (15Horne D.W. Reed K.A. Arch. Biochem. Biophys. 1992; 298: 121-128Crossref PubMed Scopus (18) Google Scholar) and in rabbit jejunal brush border membrane vesicles (16Schron C.M. J. Membr. Biol. 1991; 120: 192-200Crossref PubMed Scopus (9) Google Scholar, 17Schron C.M. Washington Jr., C. Blitzer B.L. J. Clin. Invest. 1985; 76: 2030-2033Crossref PubMed Scopus (83) Google Scholar). In the latter case, His residues were implicated, because this process was blocked by diethyl pyrocarbonate (18Said H.M. Mohammadkhani R. Biochem. J. 1993; 290: 237-240Crossref PubMed Scopus (21) Google Scholar). However, diethyl pyrocarbonate effects are nonspecific; this reagent can also react with tyrosine, lysine, and cysteine residues (19Miles E.W. Methods Enzymol. 1977; 47: 431-442Crossref PubMed Scopus (814) Google Scholar, 20Cohen L.A. Annu. Rev. Biochem. 1968; 37: 695-726Crossref PubMed Scopus (104) Google Scholar). The current study was designed to assess directly, by site-directed mutagenesis, the potential role that His residues might play in folate transport mediated by the human PCFT. This study also addresses, for the first time, the direct assessment of cellular acidification, and the relationship between folate and proton transport in wild-type and His mutants, which accompanies PCFT-mediated transport in Xenopus oocytes. Tritiated folic acid (diammonium salt, 3′,5′,7,9-[3H], cat. no. MT-783) and methotrexate (MTX-disodium salt, 3′,5′,7-[3H](N), cat. no. MT701) were obtained from Moravek Biochemicals Inc. (Brea, CA), purified by liquid chromatography, and maintained as previously described (21Fry D.W. Yalowich J.C. Goldman I.D. J. Biol. Chem. 1982; 257: 1890-1896Abstract Full Text PDF PubMed Google Scholar). Unlabeled folic acid and tunicamycin (T7765) were purchased from Sigma-Aldrich. MTX, (6S)5-formylTHF, and (6S)5-methylTHF were obtained from Schircks Laboratories (Jona, Switzerland). Protease inhibitor mixture (cat. no. 11836170001) was obtained from Roche Applied Science. All other reagents were of the highest purity available from commercial sources. HeLa cells, originally obtained from the American Type Tissue Collection (Manassas, VA), have been maintained in this laboratory in RPMI 1640 medium supplemented with 5% fetal bovine serum, 100 units/ml penicillin, and 100 μg/ml streptomycin at 37 °C in a humidified atmosphere of 5% CO2. HeLa R1-11 cells (4Zhao R. Qiu A. Tsai E. Jansen M. Akabas M.H. Goldman I.D. Mol. Pharmacol. 2008; 74: 854-862Crossref PubMed Scopus (111) Google Scholar) is a more stable subclone of the HeLa R1 cell line (22Zhao R. Chattopadhyay S. Hanscom M. Goldman I.D. Clin. Cancer Res. 2004; 10: 8735-8742Crossref PubMed Scopus (48) Google Scholar) that lacks both PCFT and reduced folate carrier expression, the later due to a genomic deletion. Lipofectamine 2000 (Invitrogen) was used according to the manufacturer's protocol for transient transfection of plasmid DNAs into these cells. R1-11 or HeLa cells were transfected with empty pcDNA3.1(+) vector (mock), or the same vector containing wild-type (WT), or mutant pcft cDNAs. Tunicamycin was dissolved in DMSO and added to growth medium to achieve a concentration of 1 μg/ml, a level previously shown to block the N-linked glycosylation of PCFT with minimal loss of cell viability (23Unal E.S. Zhao R. Qiu A. Goldman I.D. Biochim. Biophys. Acta. 2008; 1778: 1407-1414Crossref PubMed Scopus (72) Google Scholar). Cells were exposed to tunicamycin in growth medium for 48 h following which they were prepared for Western blot analysis. Site-directed mutagenesis was carried out according to the QuikChange II XL protocol from Stratagene (La Jolla, CA). WT-pcft cDNA, cloned into the BamHI site of the mammalian expression vector pcDNA3.1(+), was used as the template (1Qiu A. Jansen M. Sakaris A. Min S.H. Chattopadhyay S. Tsai E. Sandoval C. Zhao R. Akabas M.H. Goldman I.D. Cell. 2006; 127: 917-928Abstract Full Text Full Text PDF PubMed Scopus (670) Google Scholar). Briefly, complementary forward and reverse primers, which carry the targeted nucleotide changes in the middle region, were designed individually for introducing Ala into human pcft primary sequence at positions indicated in supplemental Table S1. Initial DNA denaturation, number of plasmid amplification cycles, and annealing and extension conditions were the same as previously defined (23Unal E.S. Zhao R. Qiu A. Goldman I.D. Biochim. Biophys. Acta. 2008; 1778: 1407-1414Crossref PubMed Scopus (72) Google Scholar). DpnI (10 units/μl) restriction enzyme was used to digest the parental double strand DNA. The mixture (3 μl) was transformed into DH5α-competent cells (Invitrogen). Plasmids carrying the desired mutations were identified by DNA-automated sequencing in the Albert Einstein Cancer Center Genomics Shared Resource. The entire coding region of pcft was sequenced to confirm the mutation and the fidelity of DNA. A hemagglutinin (HA) peptide epitope (YPYDVPDYA) was fused to the C terminus of WT and the various pcft constructs by PCR-based site-directed mutagenesis as previously described (23Unal E.S. Zhao R. Qiu A. Goldman I.D. Biochim. Biophys. Acta. 2008; 1778: 1407-1414Crossref PubMed Scopus (72) Google Scholar). The C-HA-pcft vector was used as a template for the amino acid substitutions at His247, His281, and Ser172. The forward primers used to introduce these mutations are shown in supplemental Table S1. To express WT and mutant transporters in Xenopus oocytes, WT-pcft was initially cloned into the pGEMHE vector at the EcoRI and XbaI restriction sites (24Liman E.R. Tytgat J. Hess P. Neuron. 1992; 9: 861-871Abstract Full Text PDF PubMed Scopus (979) Google Scholar). The pGEMHE vector carrying WT-pcft was employed as a template for site-directed mutagenesis to create the H281A construct using the same primers as described above (supplemental Table S1). Influx measurements were performed by rapid determination of membrane transport parameters as previously described (23Unal E.S. Zhao R. Qiu A. Goldman I.D. Biochim. Biophys. Acta. 2008; 1778: 1407-1414Crossref PubMed Scopus (72) Google Scholar). R1-11 cells were seeded in 17-mm glass scintillation vials at 4 × 105 cells/ml density. Influx was assessed at mid-log phase growth ∼72 h after transfection. Most transport studies were performed in MES-buffered saline (20 mm MES, 140 mm NaCl, 5 mm KCl, 2 mm MgCl2, 5 mm dextrose), at pH 5.5. This buffer was titrated to achieve pH levels of 4.5, 5.0, 6.0, and 6.5. For pH levels ≥ 7.0, MES-buffered saline was replaced with HBS and titrated to the appropriate pH. Cells were incubated in HEPES buffer (HBS) (20 mm HEPES, 140 mm NaCl, 5 mm KCl, 2 mm MgCl2, 5 mm dextrose, at pH 7.4) at 37 °C for 20 min before transfer into the prewarmed uptake buffer containing tritiated folate/antifolate substrate. Uptake was halted after 1 min by the injection of 8 ml of HBS (pH 7.4) at 0 °C, an interval over which the relationship between cell folate and time was linear and the ordinate intercept at zero time was near the point of origin. Cells were washed twice in the 0 °C HBS buffer after which radioactivity and protein levels were determined as reported previously (23Unal E.S. Zhao R. Qiu A. Goldman I.D. Biochim. Biophys. Acta. 2008; 1778: 1407-1414Crossref PubMed Scopus (72) Google Scholar). Influx is expressed as picomoles of [3H]MTX or [3H]folic acid/mg of protein/min. [3H]Folic acid and [3H]MTX influx kinetic parameters (Kt and Vmax) were obtained from a non-linear regression of influx as a function of extracellular folate/antifolate concentration according to the Michaelis-Menten equation. Influx Ki values for (6S)5-formylTHF, (6S)5-methylTHF, and folic acid were based upon inhibition of [3H]MTX influx according to the formula: Vinhibited = VmaxS/(1 + i/Ki)Kt + S, where i and S are the inhibitor and substrate concentrations, respectively, and Kt and Vmax are the parameters determined for WT-PCFT. The [3H]MTX concentrations used were comparable to the Kt for each PCFT construct, and the concentrations of inhibitors were adjusted so that the level of inhibition achieved was in the 35–65% range. As reported previously (23Unal E.S. Zhao R. Qiu A. Goldman I.D. Biochim. Biophys. Acta. 2008; 1778: 1407-1414Crossref PubMed Scopus (72) Google Scholar), a rabbit anti-HA antibody (#H6908) obtained from Sigma was used to detect HA-tagged proteins in cell plasma membrane protein fractions prepared from HeLa cells transiently transfected with empty vector (mock), WT-pcft, or mutant constructs at equal amounts. Briefly, to obtain membrane fractions, 200 μl (106 cells) were incubated on ice for 30 min in hypotonic buffer (0.5 mm Na2HPO4, 0.1 mm EDTA at pH 7.4) containing protease inhibitor. After centrifugation at 14,000 rpm for 2 min, the pellet was dissolved in 20 mm Tris base, 150 mm NaCl, 1% Triton X-100, 0.1% SDS, 1 mm EDTA buffer. Proteins were dissolved in SDS-PAGE loading buffer (0.225 m Tris.Cl (pH 6.8), 50% glycerol, 5% SDS, 0.05% bromphenol blue) with 0.25 m dithiothreitol). A 12% SDS-polyacrylamide gel was used to resolve the proteins, which were then transferred to polyvinylidene difluoride Transfer Membranes (Amersham Biosciences). After antibody treatment, membranes were processed by the ECL Plus Western Blotting Detection System (Amersham Biosciences). Rabbit β-actin antibody (#4967L, Cell Signaling Technology) was used to determine the loading amounts. Xenopus oocytes were employed to assess the functional roles of the WT and mutant pcfts based upon currents generated and intracellular acidification. cRNA (23 ng in 50 nl) or water (50 nl) were injected into stage V/VI oocytes, and electrophysiological measurements were made 3–7 days later. Voltage electrodes were made from fiber-capillary borosilicate and filled with 3 m KCl; pH electrodes were pulled similarly, silanized, filled with hydrogen ionophore 1 mixture B (Fluka), and back-filled with phosphate buffer (pH 7.0). These procedures and the electrical configuration were the same as described previously (25Chang M.H. DiPiero J. Sönnichsen F.D. Romero M.F. J. Biol. Chem. 2008; 283: 18402-18410Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar, 26Gunshin H. Mackenzie B. Berger U.V. Gunshin Y. Romero M.F. Boron W.F. Nussberger S. Gollan J.L. Hediger M.A. Nature. 1997; 388: 482-488Crossref PubMed Scopus (2667) Google Scholar). For these experiments, as with the divalent metal transporter, DMT1, oocytes were voltage clamped to −90 mV to maximize folate-induced currents (26Gunshin H. Mackenzie B. Berger U.V. Gunshin Y. Romero M.F. Boron W.F. Nussberger S. Gollan J.L. Hediger M.A. Nature. 1997; 388: 482-488Crossref PubMed Scopus (2667) Google Scholar, 27Mackenzie B. Ujwal M.L. Chang M.H. Romero M.F. Hediger M.A. Pflugers Arch. 2006; 451: 544-558Crossref PubMed Scopus (112) Google Scholar). ND90 solution (96 mm NaCl, 1.8 mm CaCl2, 1 mm MgCl2) was pH-adjusted using Tris (for pH 7.4) or MES (for pH 4.5–5.5). During these experiments, oocytes were continuously superfused with ND90 solutions (with and without folates as indicated) at 5 ml/min. Related, experimentally solved three-dimensional structures were identified based upon the sequence of human PCFT with the PSIPRED fold recognition program (28McGuffin L.J. Bryson K. Jones D.T. Bioinformatics. 2000; 16: 404-405Crossref PubMed Scopus (2740) Google Scholar). The most suitable template was that of glycerol-3-phosphate (GlpT) transporter from Escherichia coli (Protein Data Bank code 1pw4) sharing 14% sequence identity to PCFT (29Huang Y. Lemieux M.J. Song J. Auer M. Wang D.N. Science. 2003; 301: 616-620Crossref PubMed Scopus (854) Google Scholar). A three-dimensional comparative protein structure model for PCFT was built using the Multiple Mapping Method (30Rai B.K. Madrid-Aliste C.J. Fajardo J.E. Fiser A. Bioinformatics. 2006; 22: 2691-2692Crossref PubMed Scopus (31) Google Scholar, 31Rai B.K. Fiser A. Proteins. 2006; 63: 644-661Crossref PubMed Scopus (58) Google Scholar). This method employs an alignment optimization algorithm to produce accurate input alignment between the target and template for the MODELLER program (32Sali A. Blundell T.L. J. Mol. Biol. 1993; 234: 779-815Crossref PubMed Scopus (10562) Google Scholar, 33Fiser A. Sali A. Methods Enzymol. 2003; 374: 461-491Crossref PubMed Scopus (1355) Google Scholar) that generates a comparative model. The quality of the resulting model was checked with PROSA energy function (34Sippl M.J. Proteins. 1993; 17: 355-362Crossref PubMed Scopus (1777) Google Scholar). Electrostatic potential around the molecule was calculated with the DelPhi macromolecular electrostatics modeling package (35Honig B. Nicholls A. Science. 1995; 268: 1144-1149Crossref PubMed Scopus (2537) Google Scholar) and visualized in GRASP2 (36Petrey D. Honig B. Methods Enzymol. 2003; 374: 492-509Crossref PubMed Scopus (198) Google Scholar). pKa values for charged residues were obtained from multiconformation continuum electrostatics calculations (37Alexov E.G. Gunner M.R. Biophys. J. 1997; 72: 2075-2093Abstract Full Text PDF PubMed Scopus (340) Google Scholar, 38Georgescu R.E. Alexov E.G. Gunner M.R. Biophys. J. 2002; 83: 1731-1748Abstract Full Text Full Text PDF PubMed Scopus (398) Google Scholar). Multiconformation continuum electrostatics combines continuum electrostatics and molecular mechanics force fields in Monte Carlo sampling to simultaneously calculate side-chain ionization and conformation. Data presented are the means ± S.E. from at least three independent experiments. Statistical comparisons were performed by the two-tailed Student's paired t test. All statistical analyses utilized GraphPad Prism (version 3.0 for Windows, GraphPad Software). The human PCFT protein (SLC46A1, NP_542400) consists of 459 amino acids with 10 His residues in the primary sequence. Transmembrane topology prediction programs identified 12 integral transmembrane helices with N and C termini located within the cytoplasm (23Unal E.S. Zhao R. Qiu A. Goldman I.D. Biochim. Biophys. Acta. 2008; 1778: 1407-1414Crossref PubMed Scopus (72) Google Scholar, 39Qiu A. Min S.H. Jansen M. Malhotra U. Tsai E. Cabelof D.C. Matherly L.H. Zhao R. Akabas M.H. Goldman I.D. Am. J. Physiol. Cell Physiol. 2007; 293: C1669-C1678Crossref PubMed Scopus (111) Google Scholar). Localization of the termini was subsequently confirmed experimentally (23Unal E.S. Zhao R. Qiu A. Goldman I.D. Biochim. Biophys. Acta. 2008; 1778: 1407-1414Crossref PubMed Scopus (72) Google Scholar, 39Qiu A. Min S.H. Jansen M. Malhotra U. Tsai E. Cabelof D.C. Matherly L.H. Zhao R. Akabas M.H. Goldman I.D. Am. J. Physiol. Cell Physiol. 2007; 293: C1669-C1678Crossref PubMed Scopus (111) Google Scholar). Accordingly, His140 is located in the second extracellular loop, His247 and His248 in the large intracellular loop between transmembrane domains (TMDs) VI and VII, His281 in TMD VII, and His312 in TMD VIII. Five His residues (His49, His84, His201, His266, and His449) are at membrane-aqueous interfaces (Fig. 1). Although all His residues are partially conserved, His84, His247, and His281 are identical in all species (supplemental Table S2). All ten His residues were individually replaced with Ala. The mutant pcft cDNA constructs, along with WT-pcft and the empty vector (mock), were transiently transfected, individually, into HeLa-R1-11 cells. As indicated in Fig. 2, influx of 0.5 μm [3H]MTX over 1 min at pH 5.5 in cells transiently transfected with the H49A, H84A, H140A, and H201A mutants was equal to that of the WT carrier with only a modest reduction in function of the H248A and H449A mutants. There were substantial reductions in function for the H247A and H281A mutants, both fully conserved, and, to a much lesser extent, the H312A mutant that is not fully conserved. The pattern of transport changes was the same when influx of 0.5 μm [3H]MTX was assessed in the absence of a proton driving force at pH 7.0, although the overall rate was substantially lower (∼1/25th the rate at pH 5.5, data not shown). These results indicated that His247 and His281 are residues that play important roles in PCFT function. Western blot analyses of crude cell plasma membrane protein fractions were evaluated to assess levels of protein expression of the two fully conserved mutants along with the H312A mutant. Immunoblots of crude membrane protein fractions from HeLa cells transiently transfected with empty vector (mock) or C-HA-tagged WT-pcft, H247A, H281A, and H312A-pcft mutants are shown in Fig. 3A. At equivalent protein loading, H281A and H312A expression was similar to WT-pcft. The H247A mutant protein migrated at ∼47 kDa as a narrower band, faster than the other proteins that ran as a broad band at ∼55 kDa. To determine the role of glycosylation in this pattern along with the structural integrity of the H247A mutant, HeLa cells were transiently transfected with WT-PCFT or the H247A mutant and incubated with 1.0 μg/ml tunicamycin for 48 h. Cell membrane protein fractions were prepared and subjected to Western blot analysis along with membranes from cells transfected with the N58Q/N68Q double mutant (deglycosylated PCFT) (23Unal E.S. Zhao R. Qiu A. Goldman I.D. Biochim. Biophys. Acta. 2008; 1778: 1407-1414Crossref PubMed Scopus (72) Google Scholar). As indicated in Fig. 3B, high molecular weight broad PCFT bands disappeared for the WT carrier and the protein migrated at the same size (∼35 kDa) as the deglycosylated (C-HA-N58Q/N68Q) carrier. Similarly, the H247A mutant protein showed a size shift from ∼47 kDa to ∼35 kDa in cells exposed to tunicamycin. Hence, H247A PCFT is expressed to the same extent as WT-PCFT, and the protein is not degraded. Rather, the alteration in protein mobility for the H247A mutant appears to be due to a change in the extent of glycosylation or a protein conformational change. There were profound and distinct changes in influx kinetics mediated by the H247A mutant as compared with WT-PCFT (Fig. 4A). The influx Kt for folic acid decreased by a factor of ∼8.4 (from 1.43 ± 0.64 to 0.17 ± 0.03 μm). This was associated with a ∼11-fold decrease in influx Vmax (from 247 ± 37 to 23 ± 1.5 pmol/mg/min). Hence, this amino acid substitution resulted in a marked increase in the affinity of carrier for folic acid. The change in folic acid influx kinetics for the H281A mutant was quite different (Fig. 4B). The influx Kt for folic acid increased ∼12-fold (from 1.88 ± 0.58 to 21.4 ± 2.8 μm). The Vmax values for the WT-PCFT and H281A mutant were similar (743 ± 84 and 715 ± 49 pmol/mg/min, respectively, p > 0.05). Hence, in the case of this mutant, the major change was a marked decrease in carrier affinity for folic acid. The inter-experimental differences between Vmax values are due to differences in transient transfection efficiency which affect Vmax, but not Kt, determinations. The loss of function for the H312A mutant was small, and this was, as expected, associated with small changes in folic acid influx kinetics. The folic acid influx Kt for H312A was only 2-fold higher than WT-PCFT (2.43 ± 0.3 versus 1.22 ± 0.12 μm, respectively). This was accompanied by a 2-fold decrease in influx Vmax of 127 ± 4.5 versus 256 ± 5.7 pmol/mg/min, respectively (data not shown). Substrate specificity of the three His mutants was evaluated. The pcft cDNA constructs were transiently transfected into HeLa-R1-11 cells and inhibition of 0.5 μm [3H]MTX influx by several other folates (at 2 μm) was assessed. As indicated in Fig. 5A, the pattern of [3H]MTX influx inhibition was 5-methyl-THF > 5-formylTHF > folic acid for WT-PCFT. To facilitate comparison of the degree of inhibition by folates, the data were plotted as a percentage of control influx in the absence of inhibitors for each pcft construct (Fig. 5B). The pattern of inhibition of MTX influx into H281A and H312A transfectants by the various folates was similar to that of WT-pcft transfectants. However, the magnitude and pattern of inhibition was remarkably different for the H247A mutant. The degree of inhibition was markedly increased and was comparable for all the folates. This was more evident when the inhibitor concentration was decreased to a level (0.25 μm) so low that there was no inhibition at all of MTX influx mediated by WT-PCFT. Under these conditions there still was marked (∼40%), and comparable, inhibition of MTX influx into the H247A transfectant by the three folate substrates (supplemental Fig. S1). Ki values were computed based upon inhibition of 0.5 μm [3H]MTX influx by 5-methylTHF, 5-formylTHF, or folic acid, each at 2 μm for the WT carrier, and each at 0.25 μm for the H247A mutant (Table 1). Influx Ki values for the H247A mutant were similar for all the substrates and were decreased by factors of ∼3.4, ∼7, and ∼8 for 5-methylTHF, 5-formylTHF, and folic acid, respectively, as compared with the WT-PCFT. Hence, the H247A mutation resulted in both increased affinity for all its folate substrates and a loss of substrate selectivity.TABLE 1Ki values for 5-methylTHF, 5-formylTHF, and folic acid based upon inhibition of 0.5 μm [3H]MTX influxki5-formylTHF5-methylTHFFolic acidμmWT-PCFT1.17 ± 0.120.67 ± 0.211.63 ± 0.18H247A0.17 ± 0.020.20 ± 0.010.20 ± 0.02 Open table in a new tab To further explore the role of the His247 residue in PCFT function, other mutants were constructed with a variety of amino acid substitutions. When His was replaced with Arg (positively charged) there was a marked loss of function (Fig. 6A). On the other hand, substitution with the uncharged Gln produced transport activity higher than that of H247A; substitution with the negatively charged Glu preserved t" @default.
• W1997831277 created "2016-06-24" @default.
• W1997831277 creator A5017974771 @default.
• W1997831277 creator A5040379692 @default.
• W1997831277 creator A5041914018 @default.
• W1997831277 creator A5050833235 @default.
• W1997831277 creator A5071038254 @default.
• W1997831277 creator A5071736781 @default.
• W1997831277 date "2009-06-01" @default.
• W1997831277 modified "2023-10-18" @default.
• W1997831277 title "The Functional Roles of the His247 and His281 Residues in Folate and Proton Translocation Mediated by the Human Proton-coupled Folate Transporter SLC46A1" @default.
• W1997831277 cites W1524947972 @default.
• W1997831277 cites W1540897124 @default.
• W1997831277 cites W1568628619 @default.
• W1997831277 cites W1576609740 @default.
• W1997831277 cites W1584359630 @default.
• W1997831277 cites W1595347157 @default.
• W1997831277 cites W1598655624 @default.
• W1997831277 cites W1627569217 @default.
• W1997831277 cites W1677997440 @default.
• W1997831277 cites W1933777541 @default.
• W1997831277 cites W1965314140 @default.
• W1997831277 cites W1971456879 @default.
• W1997831277 cites W1972178394 @default.
• W1997831277 cites W1973501824 @default.
• W1997831277 cites W1975591144 @default.
• W1997831277 cites W1976450208 @default.
• W1997831277 cites W1985592310 @default.
• W1997831277 cites W1985629967 @default.
• W1997831277 cites W1989643384 @default.
• W1997831277 cites W1994710858 @default.
• W1997831277 cites W1997432723 @default.
• W1997831277 cites W1998486888 @default.
• W1997831277 cites W1999334756 @default.
• W1997831277 cites W2002096718 @default.
• W1997831277 cites W2004145462 @default.
• W1997831277 cites W2004316399 @default.
• W1997831277 cites W2010003901 @default.
• W1997831277 cites W2011018782 @default.
• W1997831277 cites W2012419473 @default.
• W1997831277 cites W2012538550 @default.
• W1997831277 cites W2021534482 @default.
• W1997831277 cites W2021676916 @default.
• W1997831277 cites W2023052348 @default.
• W1997831277 cites W2024343185 @default.
• W1997831277 cites W2029667189 @default.
• W1997831277 cites W2033461453 @default.
• W1997831277 cites W2033835940 @default.
• W1997831277 cites W2034496929 @default.
• W1997831277 cites W2036427272 @default.
• W1997831277 cites W2037728003 @default.
• W1997831277 cites W2052116640 @default.
• W1997831277 cites W2052242603 @default.
• W1997831277 cites W2064085430 @default.
• W1997831277 cites W2065283382 @default.
• W1997831277 cites W2070002280 @default.
• W1997831277 cites W2073326152 @default.
• W1997831277 cites W2073886406 @default.
• W1997831277 cites W2085134575 @default.
• W1997831277 cites W2089150909 @default.
• W1997831277 cites W2090857098 @default.
• W1997831277 cites W2091745271 @default.
• W1997831277 cites W2094673798 @default.
• W1997831277 cites W2099384461 @default.
• W1997831277 cites W2099551469 @default.
• W1997831277 cites W2101625991 @default.
• W1997831277 cites W2103566186 @default.
• W1997831277 cites W2104212924 @default.
• W1997831277 cites W2106154817 @default.
• W1997831277 cites W2110476441 @default.
• W1997831277 cites W2110666633 @default.
• W1997831277 cites W2117545594 @default.
• W1997831277 cites W2119226590 @default.
• W1997831277 cites W2122092859 @default.
• W1997831277 cites W2128841415 @default.
• W1997831277 cites W2130350228 @default.
• W1997831277 cites W2130381741 @default.
• W1997831277 cites W2142529984 @default.
• W1997831277 cites W2147241123 @default.
• W1997831277 cites W2153306103 @default.
• W1997831277 cites W2329765761 @default.
• W1997831277 cites W74057563 @default.
• W1997831277 cites W955725593 @default.
• W1997831277 doi "https://doi.org/10.1074/jbc.m109.008060" @default.
• W1997831277 hasPubMedCentralId "https://www.ncbi.nlm.nih.gov/pmc/articles/2719423" @default.
• W1997831277 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/19389703" @default.
• W1997831277 hasPublicationYear "2009" @default.
• W1997831277 type Work @default.
• W1997831277 sameAs 1997831277 @default.
• W1997831277 citedByCount "58" @default.
• W1997831277 countsByYear W19978312772012 @default.
• W1997831277 countsByYear W19978312772013 @default.
• W1997831277 countsByYear W19978312772014 @default.
• W1997831277 countsByYear W19978312772015 @default.
• W1997831277 countsByYear W19978312772016 @default.
• W1997831277 countsByYear W19978312772017 @default.
• W1997831277 countsByYear W19978312772018 @default.
• W1997831277 countsByYear W19978312772019 @default.

• next