Publications

• Cloning and functional characterization of the proton-coupled electrogenic folate transporter and analysis of its expression in retinal cell types.

  Umapathy N.S., Gnana-Prakasam J.P., Martin P.M., Mysona B., Dun Y., Smith S.B., Ganapathy V., Prasad P.D.

  <h4>Purpose</h4>In a prior study the cellular uptake of folate was investigated in the retina. Recently, a new proton-coupled folate transporter (PCFT) in human intestine was reported. In the present study, the expression of this novel transporter in the retina was determined, the mouse orthologue ... >> More

  <h4>Purpose</h4>In a prior study the cellular uptake of folate was investigated in the retina. Recently, a new proton-coupled folate transporter (PCFT) in human intestine was reported. In the present study, the expression of this novel transporter in the retina was determined, the mouse orthologue was cloned from retinal tissue, and its transport function was characterized.<h4>Methods</h4>RT-PCR and folate uptake measurements were used to detect the expression of PCFT in mouse retina and in retinal cell types. The expression of PCFT mRNA in intact retina was investigated by in situ hybridization. Mouse PCFT cDNA was cloned, and its transport characteristics were analyzed by electrophysiological methods after expression of the cloned transporter in Xenopus laevis oocytes.<h4>Results</h4>RT-PCR showed expression of PCFT mRNA in both neural retina and RPE eye cup. In situ hybridization detected PCFT mRNA in all retinal cell layers. Proton-coupled folate uptake was detectable in primary cultures of ganglion, Müller, and RPE cells of mouse retina and in RPE, ganglion, and Müller cells of human or rat origin. In X. laevis oocytes expressing the cloned mouse PCFT, folate and its derivatives methotrexate and 5-methyltetrahydrofolate induced H(+)-coupled inward currents with K(t) values of 1.2 +/- 0.1, 4.6 +/-0.5, and 3.5 +/-0.8 microM, respectively. The transport process showed an H(+)-folate stoichiometry of 1:1, suggesting that PCFT transports the zwitterionic form of folate.<h4>Conclusions</h4>This is the first report on the expression of PCFT in the retina. All cell layers of the retina express this transporter. Mouse PCFT, cloned from retina, mediates H(+)-coupled electrogenic transport of folate and its derivatives. << Less

  Invest. Ophthalmol. Vis. Sci. 48:5299-5305(2007) [PubMed] [EuropePMC]

  This publication is cited by 2 other entries.

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

  Zhan H.Q., Najmi M., Lin K., Aluri S., Fiser A., Goldman I.D., Zhao R.

  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 rem ... >> More

  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 <i>N</i>-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 <i>V</i>_max, accompanied by a decreased influx K_t (4.5-fold) and <i>K_i</i> (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. << Less

  J. Biol. Chem. 295:15650-15661(2020) [PubMed] [EuropePMC]