SUPPORTING EVIDENCE FOR NEGATIVE MODULATION BY PROTONS OF AN ION-CHANNEL ASSOCIATED WITH THE N-METHYL-D-ASPARTATE RECEPTOR COMPLEX IN RAT-BRAIN USING LIGAND-BINDING TECHNIQUES

The addition of L-glutamic acid (Glu) alone, both Glu and glycine (Gly ) or Glu/Gly/spermidine (SPD) was effective in potentiating 0,11-dihyd ro-5H-dibenzo[a,d]cyclohepten-5,10-imine (MK-801) binding before equil ibrium to an ion channel associated with the N-methyl-D-aspartate (NMD A) receptor complex in brain synaptic membranes extensively washed and treated with Triton X-100. The binding dependent on Glu almost linear ly increased in proportion to decreasing proton concentrations at a pH range of 6.0 to 9.0 in external incubation medium, while a Gly-depend ent portion of the binding increased with decreasing proton concentrat ions up to a pH of 7.5 with a plateau thereafter. In contrast, the SPD -dependent binding increased in proportion to decreasing proton concen trations up to a pH of 7.0 with a gradual decline thereafter. Similar profiles were also obtained with [3H]MK-801 binding at equilibrium, wi th an exception that significant binding of [H-3]MK-801 was detected i n the absence of any added agonists. The potency of SPD to potentiate [H-3]MK-801 binding before equilibrium increased in proportion to decr easing proton concentrations, with those of both Glu and Gly being unc hanged. In contrast, the ability of (+)MK-801 to displace [H-3]MK-801 binding at equilibrium was not significantly affected by a decrement o f external proton concentrations from pH 7.5 to pH 8.5 in the presence of Glu/Gly and Glu/Gly/SPD added. However, similar changes in externa l proton concentrations did not similarly affect binding of several ra dioligands for the NMDA and Gly domains on the receptor complex. Decre asing proton concentrations were effective in exponentially potentiati ng binding of [H-3]SPD at a pH range of 6.0 to 9.0 without virtually a ltering pha-amino-3-hydroxy-5-methyl-isoxazole-4-propionic acid bindin g. In addition, [3H]kainic acid binding markedly decreased with decrea sing proton concentrations only in the presence of Ca2+ ions. These re sults suggest that protons negatively modulate neuronal responses medi ated by the NMDA receptor ionophore complex through interference with opening mechanisms of the channel domain without disturbing associatio n processes of the endogenous agonists with the respective recognition domains in rat brain. Moreover, possible modulation by protons of res ponses mediated by the kainate receptor in the presence of Ca2+ ions a t concentrations that occur in vivo is also suggested.