Abstract
Abstract: :
Purpose: In the retina, Müller glial cells play a pivotal role in the modulation of synaptic transmission and express functional N–methyl–D–aspartate (NMDA) glutamate receptors, which may regulate glial activity by establishing feedback loops between glia and neurons. Glycine acts as a co–agonist at these receptors through interactions with strychnine–insensitive sites. N–methyl–D–aspartate receptors (NMDARs) differ in the retina and the brain regarding properties of glycine coagonist–binding site, which confer distinct functional characteristics to the receptor. We present the pharmacological characterization of the glycine binding site and the molecular analysis of the subunit expression as well as the alternative splicing–generated NR1 isoform composition of NMDA receptors in Müller cells. Methods: Specific binding of radiolabeled ligands in the presence of cold competitors to membrane fractions from Müller glia and retinal neuron primary cultures was determined. Radioligand binding data were analyzed via nonlinear regression using PRISM computer program. The molecular composition of NMDA receptor was analyzed by RT–PCR and Western blot. Results: Glycine binds specifically to NMDA receptors in these cells in a saturable, pH–dependent, temperature–independent fashion. Importantly, we report cell–specific differences in properties of the strychnine–insensitive glycine binding site and NR1 splice variant expression in neurons and Müller glia. Glycine–binding to cultured Müller cells shows lower affinity (Kd=632±198 nM) and higher density (Bmax=43.58±7.02 pmol/mg protein) than in neurons. D–serine, postulated as the endogenous glutamate coagonist at NMDARs competitively inhibits glycine binding with up to 60 fold lower affinity than glycine. Spermine has no effect on the glial coagonist site, in contrast with the stimulation by this polyamine of neuronal glycine–binding and NMDAR activity. Molecular analysis of NR2 subunit expression and of N–terminal and C–terminal NR1 variants in MC further confirms NMDAR structural differences in neurons and glia which could underlie a differential regulation of glutamate function in the retina. Conclusions: The observed structural differences between glial and neuronal cells in the retina could account for cell–specific pharmacological characteristics of the receptor and underlie the physiological differences between both cell types. Further understanding of these differences could shed a light on the mechanisms through which glia integrates neuronal inputs and participates in the modulation of synaptic activity.
Keywords: Muller cells • receptors: pharmacology/physiology • ion channels