Functional and molecular analysis of D-serine transport in retinal Müller cells.
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 degrees 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 degrees MCs; immunohistochemistry confirmed these data in retinal sections and in cultures of 1 degrees 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 degrees MCs and suggest that D-serine, synthesized in Müller cells by SR, is effluxed via ASCT2 to regulate NMDA receptors in adjacent neurons.
Dun
Y
Y
Department of Cellular Biology and Anatomy, Medical College of Georgia, 1459 Laney-Walker Blvd., CB 2820, Augusta, GA 30912-2000, USA.
Mysona
B
B
Itagaki
S
S
Martin-Studdard
A
A
Ganapathy
V
V
Smith
S B
SB
D026962:Amino Acid Transport System ASC / Q000502:physiology; D026905:Amino Acid Transport Systems / Q000502:physiology; D000818:Animals; D001692:Biological Transport / Q000502:physiology; D002478:Cells, Cultured; D015870:Gene Expression; D051379:Mice; D015778:Minor Histocompatibility Antigens; D018377:Neurotransmitter Agents / Q000378:metabolism*; D012333:RNA, Messenger / Q000235:genetics; D019998:Racemases and Epimerases / Q000235:genetics / Q000378:metabolism; D051381:Rats; D012160:Retina / Q000166:cytology / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods; D012694:Serine / Q000378:metabolism*; D012964:Sodium / Q000502:physiology; D013379:Substrate Specificity; D000099300:Serine Racemase
D026962:Amino Acid Transport System ASC; D026905:Amino Acid Transport Systems; D015778:Minor Histocompatibility Antigens; D018377:Neurotransmitter Agents; D012333:RNA, Messenger; D019998:Racemases and Epimerases; D012694:Serine; D012964:Sodium; D000099300:Serine Racemase; C498850:Slc1a5 protein, mouse