Competitive inhibition of NMDA receptor-mediated currents by extracellularcalcium chelators

Calcium chelators have been widely used in electrophysiological recordings of N-methyl-D-aspartate (NMDA) receptor-mediated currents, as well as in st udies of excitotoxicity. Intracellularly applied calcium chelators are know n to inhibit, at least in part, such calcium-dependent processes as calmodu lin-dependent inactivation, calcineurin-dependent desensitization, and rund own of NMDA receptors. On the other hand, the functional consequences and p otential nonspecific effects of extracellularly applied chelators have not been extensively investigated. In whole-cell patch-clamp recordings from hu man embryonic kidney (HEK) 293 cells transiently transfected with recombina nt NMDA receptors, we found that addition of calcium chelators such as EGTA shifted the glutamate dose-response curve to the right, from an EC50 for N R1A/NR2A of 8 mu M in 1.8 mM Ca2+ to similar to 24 mu M in a solution conta ining nominal 0 Ca2+/5 mM EGTA and further to similar to 80 mu M in 20 mM E GTA. A similar shift in glutamate dose-response was observed for NR1A/NR2B currents. This dose-response shift was not due to a decrease in extracellul ar Ca2+ concentration because there was no change in the glutamate EC50 at Ca2+ concentrations ranging from 10 mM to nominal 0/200 mu M EGTA. Moreover , addition of 5 mM EGTA fully chelated with 6.8 mM Ca2+ did not produce any shift in the glutamate dose-response curve. We propose that calcium chelat ors, containing four free carboxyl moieties, competitively inhibit glutamat e binding to NMDA receptors.