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Y. Dun, B.A. Mysona, L.P. Amarnath, V. Ganapathy, S.B. Smith; Functional Analysis of D–Serine Transport in Retinal Müller Cells . Invest. Ophthalmol. Vis. Sci. 2006;47(13):131.
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© ARVO (1962-2015); The Authors (2016-present)
Recent data suggest that D–serine is the endogenous coagonist of the NMDA receptor in vertebrate retina and may modulate ganglion and amacrine cell sensitivity. Studies from Miller’s lab showed that D–serine and its synthesizing enzyme, serine racemase (SR), are present in retinal glial cells (Müller cells, astrocytes). The uptake of D–serine in whole retina preparations using capillary electrophoresis suggested that the Na+–dependent ASC (alanine–serine–cysteine)–type of transporter mediates D–serine uptake. The purpose of this study was to determine at the functional and molecular level the transport mechanisms responsible for D–serine uptake in isolated retinal Müller cells.
Uptake of [3H]–D–serine was measured in cultured rMC–1 and primary mouse Müller cells. The ion–dependence and relative contributions of various transport systems to D–serine uptake were determined based on differential substrate specificities and kinetics analysis. RT–PCR was used to analyze the expression of transporter–specific mRNAs (ASCT–1, ASCT–2, ATB0,+) in these cells. To analyze SR expression, RT–PCR, western blot, and immunohistochemistry were used.
D–serine transport was obligatorily dependent on Na+ (thus eliminating the Na+–independent asc–1 as the transporter for D–serine in Müller cells). The transport had a Na+:substrate stoichiometry of 1:1. Substrate specificity experiments showed that D–serine transport in Müller cells was inhibited by Ala, Ser, Cys, Gln and Asn, consistent with ASCT–2, but not ASCT–1–mediated transport. D–serine transport was not inhibited by Arg or Lys, ruling out ATB0,+ as the mediator of D–serine uptake. RT–PCR analysis of RNA isolated from Müller cells confirmed the expression of ASCT–2. For SR analysis, RT–PCR of RNA isolated from Müller cells amplified the expected 415 bp product using primers specific for rat SR. Western blot and immunocytochemistry confirmed that SR was expressed in rMC–1 cells and primary Müller cells, but not in primary mouse ganglion cells nor in the ganglion cell line, RGC–5.
These data provide functional and molecular evidence that ASCT–2 mediates the transport of D–serine in Müller cells and that D–serine is synthesized in these cells by the racemization of L–serine. Since ASCT–2 is an obligatory amino acid exchanger, we hypothesize that the transporter is involved in the release of D–Serine from Müller cells for subsequent modulation of NMDA receptors in retinal neurons.
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