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


METHODS
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.


RESULTS
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.


CONCLUSIONS
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.
Umapathy
Nagavedi S
NS
Department of Biochemistry and Molecular Biology, Medical College of Georgia, Augusta, GA 30912, USA.
Gnana-Prakasam
Jaya P
JP
Martin
Pamela M
PM
Mysona
Barbara
B
Dun
Ying
Y
Smith
Sylvia B
SB
Ganapathy
Vadivel
V
Prasad
Puttur D
PD
D000818:Animals; D002460:Cell Line; D003001:Cloning, Molecular; D005260:Female; D005492:Folic Acid / Q000378:metabolism / Q000494:pharmacology; D015870:Gene Expression; D006801:Humans; D017403:In Situ Hybridization; D026901:Membrane Transport Proteins / Q000096:biosynthesis / Q000235:genetics*; D008727:Methotrexate / Q000494:pharmacology; D051379:Mice; D008807:Mice, Inbred BALB C; D008810:Mice, Inbred C57BL; D009457:Neuroglia / Q000378:metabolism*; D009865:Oocytes / Q000378:metabolism; D010857:Pigment Epithelium of Eye / Q000378:metabolism*; D058979:Proton-Coupled Folate Transporter; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D012160:Retina / Q000166:cytology / Q000378:metabolism*; D012165:Retinal Ganglion Cells / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013763:Tetrahydrofolates / Q000494:pharmacology; D014982:Xenopus laevis
D026901:Membrane Transport Proteins; D058979:Proton-Coupled Folate Transporter; D012333:RNA, Messenger; C516877:Slc46a1 protein, mouse; D013763:Tetrahydrofolates; D005492:Folic Acid; C005984:5-methyltetrahydrofolate; D008727:Methotrexate