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

Umapathy N Siddaramappa, Jaya Pranava Gnana-Prakasam, Pamela Moore Martin, Barbara A Mysona, Ying Dun, Sylvia B Smith, Vadivel Ganapathy, Puttur D Prasad

Research output: Contribution to journalArticle

43 Citations (Scopus)

Abstract

PURPOSE. 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 Kt values of 1.2 ± 0.1, 4.6 ± 0.5, and 3.5 ± 0.8 μM, 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.

Original languageEnglish (US)
Pages (from-to)5299-5305
Number of pages7
JournalInvestigative Ophthalmology and Visual Science
Volume48
Issue number11
DOIs
StatePublished - Nov 1 2007

Fingerprint

Proton-Coupled Folate Transporter
Retina
Organism Cloning
Folic Acid
Xenopus laevis
Ganglia
Messenger RNA
Oocytes
In Situ Hybridization
Polymerase Chain Reaction
Methotrexate
Intestines
Protons

ASJC Scopus subject areas

  • Ophthalmology
  • Sensory Systems
  • Cellular and Molecular Neuroscience

Cite this

@article{db829a3bb8a34fe1a0557453457d87d2,
title = "Cloning and functional characterization of the proton-coupled electrogenic folate transporter and analysis of its expression in retinal cell types",
abstract = "PURPOSE. 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, M{\"u}ller, and RPE cells of mouse retina and in RPE, ganglion, and M{\"u}ller 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 Kt values of 1.2 ± 0.1, 4.6 ± 0.5, and 3.5 ± 0.8 μM, 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.",
author = "Siddaramappa, {Umapathy N} and Gnana-Prakasam, {Jaya Pranava} and Martin, {Pamela Moore} and Mysona, {Barbara A} and Ying Dun and Smith, {Sylvia B} and Vadivel Ganapathy and Prasad, {Puttur D}",
year = "2007",
month = "11",
day = "1",
doi = "10.1167/iovs.07-0288",
language = "English (US)",
volume = "48",
pages = "5299--5305",
journal = "Investigative Ophthalmology and Visual Science",
issn = "0146-0404",
publisher = "Association for Research in Vision and Ophthalmology Inc.",
number = "11",

}

TY - JOUR

T1 - Cloning and functional characterization of the proton-coupled electrogenic folate transporter and analysis of its expression in retinal cell types

AU - Siddaramappa, Umapathy N

AU - Gnana-Prakasam, Jaya Pranava

AU - Martin, Pamela Moore

AU - Mysona, Barbara A

AU - Dun, Ying

AU - Smith, Sylvia B

AU - Ganapathy, Vadivel

AU - Prasad, Puttur D

PY - 2007/11/1

Y1 - 2007/11/1

N2 - PURPOSE. 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 Kt values of 1.2 ± 0.1, 4.6 ± 0.5, and 3.5 ± 0.8 μM, 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.

AB - PURPOSE. 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 Kt values of 1.2 ± 0.1, 4.6 ± 0.5, and 3.5 ± 0.8 μM, 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.

UR - http://www.scopus.com/inward/record.url?scp=38449116048&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=38449116048&partnerID=8YFLogxK

U2 - 10.1167/iovs.07-0288

DO - 10.1167/iovs.07-0288

M3 - Article

C2 - 17962486

AN - SCOPUS:38449116048

VL - 48

SP - 5299

EP - 5305

JO - Investigative Ophthalmology and Visual Science

JF - Investigative Ophthalmology and Visual Science

SN - 0146-0404

IS - 11

ER -