Competitive Inhibition of NMDA Receptor–Mediated Currents by Extracellular Calcium Chelators

Calcium chelators have been widely used in electrophysiological recordings of N-methyl-d-aspartate (NMDA) receptor–mediated currents, as well as in studies of excitotoxicity. Intracellularly applied calcium chelators are known to inhibit, at least in part, such calcium-dependent processes as calmodulin-dependent inactivation, calcineurin-dependent desensitization, and rundown of NMDA receptors. On the other hand, the functional consequences and potential nonspecific effects of extracellularly applied chelators have not been extensively investigated. In whole-cell patch-clamp recordings from human embryonic kidney (HEK) 293 cells transiently transfected with recombinant NMDA receptors, we found that addition of calcium chelators such as EGTA shifted the glutamate dose-response curve to the right, from an EC50for NR1A/NR2A of 8 μM in 1.8 mM Ca2+to ∼24 μM in a solution containing nominal 0 Ca2+/5 mM EGTA and further to ∼80 μM in 20 mM EGTA. A similar shift in glutamate dose-response was observed for NR1A/NR2B currents. This dose-response shift was not due to a decrease in extracellular Ca2+concentration because there was no change in the glutamate EC50at Ca2+concentrations ranging from 10 mM to nominal 0/200 μM 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 chelators, containing four free carboxyl moieties, competitively inhibit glutamate binding to NMDA receptors.