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Glutamate Transport and Not Glutamate Receptor Binding Is Stimulated by Gangliosides in a Ca 2+ ‐Dependent Manner in Rat Brain Synaptic Plasma Membranes
Author(s) -
Hollmann Michael,
Seifert Wilfried
Publication year - 1989
Publication title -
journal of neurochemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.75
H-Index - 229
eISSN - 1471-4159
pISSN - 0022-3042
DOI - 10.1111/j.1471-4159.1989.tb11763.x
Subject(s) - glutamate receptor , membrane , synaptic vesicle , biophysics , ganglioside , chemistry , osmotic concentration , binding site , vesicle , stimulation , biochemistry , receptor , biology , endocrinology
Crude as well as purified synaptic plasma membrane (SPM) preparations were analyzed for the influence of the ganglioside galactosyl‐ N ‐acetylgalactosalminyl‐( N ‐acetyl‐neuraminyl)‐galactosylglucosyl ceramide (GMl) on high‐affinity binding of L‐[ 3 H]glutamate. Assayed in two different buffer systems, SPM consistently exhibited increased (40–50%) binding upon incubation with GM1 plus Ca 2+ , as compared to controls without GM1. Incorporation experiments with 3 H‐labeled GM1 proved trypsin‐stable insertion of GM1 into SPM, with a maximum incorporation pf four times the endogenous amount (35 nmol/mg of protein). The observed increase in glutamate binding was not due to a change in the affinity of the binding sites, but to a change in the number of binding sites, and it was absolutely dependent on the presence of Ca 2+ . A pharmacological profile of the GM1/Ca 2+ ‐stimulated glutamate binding is presented. The original classification of the stimulatory effect as an effect on glutamate receptor binding had to be revised to take into account the observed temperature sensitivity of the ganglioside effect, its sensitivity to high osmolarity and to ultrasonication, and the lack of binding stimulation after detergent treatment of membranes or after receptor solubilization. Vesicular space measured in both SPM preparations was found to be around 7 μl/mg of protein, in ganglioside‐treated as well as in control membranes. From the data, it is concluded that a special. Na+‐ and C1‐independent form of glutamate transport into resealed membrane vesicles is stimulated by gangliosides in the presence of Ca 2+ .