A ligand binding study of the interactions of guanine nucleotides with non-NMDA receptors

The interactions of guanine nucleotides, and particularly GTP, with the [ 3H]-α-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA) and [ 3H]-kainate (KA) binding sites present on brain membranes was studied, using the ligand binding methodology and Scatchard analysis, in order to establish the competitive/non competitive nature of the interaction and determine whether guanine nucleotides, KA and AMPA share common binding sites. GTP was found to block [ 3H]-AMPA and [ 3H]-KA binding to rat cortical membranes with IC 50 values of 0.4 mM and 1 mM respectively and the [ 3H] KA-binding to chick cerebellar membranes with a IC 50 value of 20 μM. Scatchard analysis of [ 3H]-KA binding performed in the absence or presence of 1 mM GTP or 0.25 mM AMPA reveals that the high affinity [ 3H]-KA binding component is not affected by GTP but blocked in a non competitive fashion by AMPA while the low affinity [ 3H]-KA binding component is not affected by AMPA but blocked by GTP. Scatchard analysis of [ 3H]-KA binding to chick cerebellar membranes performed in the absence or presence of 33 μM GTP reveals a single binding site blocked in a competitive fashion by GTP. Scatchard analysis of [ 3H]-AMPA binding performed in the absence or presence of 0.5 mM GTP or 30 μM KA reveals that the high affinity [ 3H]-AMPA binding component is affected in a non competitive fashion by both GTP and KA while the low affinity [ 3H] AMPA binding component is affected in a competitive fashion by both GTP and KA. These results suggest that the proteins carrying the low affinity [ 3H]-AMPA binding site in rat cortical membranes and the [ 3H]-KA binding sites in chick cerebellar membranes harbour in their extracellular domain a single binding site for the radioactive ligand and GTP. The role of GTP as a brain endogenous glutamatergic ligand is discussed.