Functional and molecular analysis of d-serine transport in retinal Müller cells

Y. Dun, Barbara A Mysona, S. Itagaki, A. Martin-Studdard, V. Ganapathy, Sylvia B Smith

Research output: Contribution to journalArticle

38 Citations (Scopus)

Abstract

d-serine, an endogenous co-agonist of NMDA receptors in vertebrate retina, may modulate glutamate sensitivity of retinal neurons. This study determined at the functional and molecular level the transport process responsible for d-serine in retinal Müller cells. RT-PCR and immunoblotting showed that serine racemase (SR), the synthesizing enzyme for d-serine, is expressed in the rMC-1 Müller cell line and primary cultures of mouse Müller cells (1°MCs). The relative contributions of different amino acid transport systems to d-serine uptake were determined based on differential substrate specificities and ion dependencies. d-serine uptake was obligatorily dependent on Na+, eliminating Na+-independent transporters (asc-1 and system L) for d-serine in Müller cells. The Na+:substrate stoichiometry for the transport process was 1:1. d-serine transport was inhibited by alanine, serine, cysteine, glutamine, and asparagine, but not anionic amino acids or cationic amino acids, suggesting that d-serine transport in Müller cells occurs via ASCT2 rather than ASCT1 or ATB0,+. The expression of mRNAs specific for ASCT1, ASCT2, and ATB0,+ was analyzed by RT-PCR confirming the expression of ASCT2 (and ASCT1) mRNA, but not ATB0,+, in Müller cells. Immunoblotting detected ASCT2 in neural retina and in 1°MCs; immunohistochemistry confirmed these data in retinal sections and in cultures of 1°MCs. The efflux of d-serine via ASCT2 by ASCT2 substrates was demonstrable using the Xenopus laevis oocyte heterologous expression system. These data provide the first molecular evidence for SR and ASCT2 expression in a Müller cell line and in 1°MCs and suggest that d-serine, synthesized in Müller cells by SR, is effluxed via ASCT2 to regulate NMDA receptors in adjacent neurons.

Original languageEnglish (US)
Pages (from-to)191-199
Number of pages9
JournalExperimental eye research
Volume84
Issue number1
DOIs
StatePublished - Jan 1 2007

Fingerprint

Serine
N-Methyl-D-Aspartate Receptors
Immunoblotting
Retina
Retinal Neurons
Amino Acid Transport Systems
Amino Acids
Cell Line
Polymerase Chain Reaction
Messenger RNA
Asparagine
Xenopus laevis
Substrate Specificity
Glutamine
Alanine
Oocytes
Cysteine
Vertebrates
Glutamic Acid
Immunohistochemistry

Keywords

  • d-serine
  • neurotransmitter
  • retinal Müller cells
  • retinal transport
  • serine racemase

ASJC Scopus subject areas

  • Ophthalmology
  • Sensory Systems
  • Cellular and Molecular Neuroscience

Cite this

Functional and molecular analysis of d-serine transport in retinal Müller cells. / Dun, Y.; Mysona, Barbara A; Itagaki, S.; Martin-Studdard, A.; Ganapathy, V.; Smith, Sylvia B.

In: Experimental eye research, Vol. 84, No. 1, 01.01.2007, p. 191-199.

Research output: Contribution to journalArticle

Dun, Y. ; Mysona, Barbara A ; Itagaki, S. ; Martin-Studdard, A. ; Ganapathy, V. ; Smith, Sylvia B. / Functional and molecular analysis of d-serine transport in retinal Müller cells. In: Experimental eye research. 2007 ; Vol. 84, No. 1. pp. 191-199.
@article{0d7daa05ba474e2e8124b66303cf36f9,
title = "Functional and molecular analysis of d-serine transport in retinal M{\"u}ller cells",
abstract = "d-serine, an endogenous co-agonist of NMDA receptors in vertebrate retina, may modulate glutamate sensitivity of retinal neurons. This study determined at the functional and molecular level the transport process responsible for d-serine in retinal M{\"u}ller cells. RT-PCR and immunoblotting showed that serine racemase (SR), the synthesizing enzyme for d-serine, is expressed in the rMC-1 M{\"u}ller cell line and primary cultures of mouse M{\"u}ller cells (1°MCs). The relative contributions of different amino acid transport systems to d-serine uptake were determined based on differential substrate specificities and ion dependencies. d-serine uptake was obligatorily dependent on Na+, eliminating Na+-independent transporters (asc-1 and system L) for d-serine in M{\"u}ller cells. The Na+:substrate stoichiometry for the transport process was 1:1. d-serine transport was inhibited by alanine, serine, cysteine, glutamine, and asparagine, but not anionic amino acids or cationic amino acids, suggesting that d-serine transport in M{\"u}ller cells occurs via ASCT2 rather than ASCT1 or ATB0,+. The expression of mRNAs specific for ASCT1, ASCT2, and ATB0,+ was analyzed by RT-PCR confirming the expression of ASCT2 (and ASCT1) mRNA, but not ATB0,+, in M{\"u}ller cells. Immunoblotting detected ASCT2 in neural retina and in 1°MCs; immunohistochemistry confirmed these data in retinal sections and in cultures of 1°MCs. The efflux of d-serine via ASCT2 by ASCT2 substrates was demonstrable using the Xenopus laevis oocyte heterologous expression system. These data provide the first molecular evidence for SR and ASCT2 expression in a M{\"u}ller cell line and in 1°MCs and suggest that d-serine, synthesized in M{\"u}ller cells by SR, is effluxed via ASCT2 to regulate NMDA receptors in adjacent neurons.",
keywords = "d-serine, neurotransmitter, retinal M{\"u}ller cells, retinal transport, serine racemase",
author = "Y. Dun and Mysona, {Barbara A} and S. Itagaki and A. Martin-Studdard and V. Ganapathy and Smith, {Sylvia B}",
year = "2007",
month = "1",
day = "1",
doi = "10.1016/j.exer.2006.09.015",
language = "English (US)",
volume = "84",
pages = "191--199",
journal = "Experimental Eye Research",
issn = "0014-4835",
publisher = "Academic Press Inc.",
number = "1",

}

TY - JOUR

T1 - Functional and molecular analysis of d-serine transport in retinal Müller cells

AU - Dun, Y.

AU - Mysona, Barbara A

AU - Itagaki, S.

AU - Martin-Studdard, A.

AU - Ganapathy, V.

AU - Smith, Sylvia B

PY - 2007/1/1

Y1 - 2007/1/1

N2 - d-serine, an endogenous co-agonist of NMDA receptors in vertebrate retina, may modulate glutamate sensitivity of retinal neurons. This study determined at the functional and molecular level the transport process responsible for d-serine in retinal Müller cells. RT-PCR and immunoblotting showed that serine racemase (SR), the synthesizing enzyme for d-serine, is expressed in the rMC-1 Müller cell line and primary cultures of mouse Müller cells (1°MCs). The relative contributions of different amino acid transport systems to d-serine uptake were determined based on differential substrate specificities and ion dependencies. d-serine uptake was obligatorily dependent on Na+, eliminating Na+-independent transporters (asc-1 and system L) for d-serine in Müller cells. The Na+:substrate stoichiometry for the transport process was 1:1. d-serine transport was inhibited by alanine, serine, cysteine, glutamine, and asparagine, but not anionic amino acids or cationic amino acids, suggesting that d-serine transport in Müller cells occurs via ASCT2 rather than ASCT1 or ATB0,+. The expression of mRNAs specific for ASCT1, ASCT2, and ATB0,+ was analyzed by RT-PCR confirming the expression of ASCT2 (and ASCT1) mRNA, but not ATB0,+, in Müller cells. Immunoblotting detected ASCT2 in neural retina and in 1°MCs; immunohistochemistry confirmed these data in retinal sections and in cultures of 1°MCs. The efflux of d-serine via ASCT2 by ASCT2 substrates was demonstrable using the Xenopus laevis oocyte heterologous expression system. These data provide the first molecular evidence for SR and ASCT2 expression in a Müller cell line and in 1°MCs and suggest that d-serine, synthesized in Müller cells by SR, is effluxed via ASCT2 to regulate NMDA receptors in adjacent neurons.

AB - d-serine, an endogenous co-agonist of NMDA receptors in vertebrate retina, may modulate glutamate sensitivity of retinal neurons. This study determined at the functional and molecular level the transport process responsible for d-serine in retinal Müller cells. RT-PCR and immunoblotting showed that serine racemase (SR), the synthesizing enzyme for d-serine, is expressed in the rMC-1 Müller cell line and primary cultures of mouse Müller cells (1°MCs). The relative contributions of different amino acid transport systems to d-serine uptake were determined based on differential substrate specificities and ion dependencies. d-serine uptake was obligatorily dependent on Na+, eliminating Na+-independent transporters (asc-1 and system L) for d-serine in Müller cells. The Na+:substrate stoichiometry for the transport process was 1:1. d-serine transport was inhibited by alanine, serine, cysteine, glutamine, and asparagine, but not anionic amino acids or cationic amino acids, suggesting that d-serine transport in Müller cells occurs via ASCT2 rather than ASCT1 or ATB0,+. The expression of mRNAs specific for ASCT1, ASCT2, and ATB0,+ was analyzed by RT-PCR confirming the expression of ASCT2 (and ASCT1) mRNA, but not ATB0,+, in Müller cells. Immunoblotting detected ASCT2 in neural retina and in 1°MCs; immunohistochemistry confirmed these data in retinal sections and in cultures of 1°MCs. The efflux of d-serine via ASCT2 by ASCT2 substrates was demonstrable using the Xenopus laevis oocyte heterologous expression system. These data provide the first molecular evidence for SR and ASCT2 expression in a Müller cell line and in 1°MCs and suggest that d-serine, synthesized in Müller cells by SR, is effluxed via ASCT2 to regulate NMDA receptors in adjacent neurons.

KW - d-serine

KW - neurotransmitter

KW - retinal Müller cells

KW - retinal transport

KW - serine racemase

UR - http://www.scopus.com/inward/record.url?scp=33751242713&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=33751242713&partnerID=8YFLogxK

U2 - 10.1016/j.exer.2006.09.015

DO - 10.1016/j.exer.2006.09.015

M3 - Article

C2 - 17094966

AN - SCOPUS:33751242713

VL - 84

SP - 191

EP - 199

JO - Experimental Eye Research

JF - Experimental Eye Research

SN - 0014-4835

IS - 1

ER -