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 [H-3]5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine (MK-801) binding before equilibrium to an ion channel associated with the N-methyl-D-aspartate (NMDA) receptor complex in brain synaptic membranes extensively washed and treated with Triton X-100. The binding dependent on Glu almost linearly increased in proportion to decreasing proton concentrations at a pH range of 6.0 to 9.0 in external incubation medium, while a Gly-dependent portion of the binding increased with decreasing proton concentrations up to a pH of 7.5 with a plateau thereafter. In contrast, the SPD-dependent binding increased in proportion to decreasing proton concentrations up to a pH of 7.0 with a gradual decline thereafter. Similar profiles were also obtained with [3H]MK-801 binding at equilibrium, with an exception that significant binding of [H-3]MK-801 was detected in the absence of any added agonists. The potency of SPD to potentiate [H-3]MK-801 binding before equilibrium increased in proportion to decreasing proton concentrations, with those of both Glu and Gly being unchanged. In contrast, the ability of (+)MK-801 to displace [H-3]MK-801 binding at equilibrium was not significantly affected by a decrement of 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 external proton concentrations did not similarly affect binding of several radioligands for the NMDA and Gly domains on the receptor complex. Decreasing proton concentrations were effective in exponentially potentiating binding of [H-3]SPD at a pH range of 6.0 to 9.0 without virtually altering [H-3]D,L-alpha-amino-3-hydroxy-5-methyl-isoxazole-4-propionic acid binding. In addition, [3H]kainic acid binding markedly decreased with decreasing proton concentrations only in the presence of Ca2+ ions. These results suggest that protons negatively modulate neuronal responses mediated by the NMDA receptor ionophore complex through interference with opening mechanisms of the channel domain without disturbing association processes of the endogenous agonists with the respective recognition domains in rat brain. Moreover, possible modulation by protons of responses mediated by the kainate receptor in the presence of Ca2+ ions at concentrations that occur in vivo is also suggested.