Mode of blockade by MK-801 of N-methyl-d-aspartate-induced increase in intracellular Ca²⁺ in cultured mouse hippocampal neurons

Microfluorometry with fura-2 was applied to study the action of the anticonvulsant (+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine maleate (MK-801) on N-methyl-d-aspartate (NMDA)-induced increase in intracellular Ca²⁺ concentration ([Ca²⁺]₁) in cultured mouse hippocampal neurons. MK-801 caused a potent and long-lasting blockade of the NMDA-activated [Ca²⁺]_i elevation in a selective manner, not affecting the [Ca²⁺]_i rise induced by quisqualate or kainate. Blockade and recovery from the blockade by MK-801 showed use dependency; the degree of blockade was dependent on the presence of NMDA. The use-dependent onset of antagonism was, however, highly sensitive to the bath temperature. MK-801 applied in the absence of NMDA had no effect on the response to subsequent application of NMDA at 22°C, whereas it reduced the subsequent response to NMDA significantly at 37°C. MK-801 interacted with the receptor-ion channel complex even when Mg²⁺, which is considered to block the open channel, had already blocked the NMDA-induced [Ca²⁺]_i. The recovery from blockade by MK-801 was not accelerated by the application of 10 mM Mg²⁺ for 5 min. These results suggest that MK-801 can gain access to its binding site in the absence of NMDA at physiological temperature, and that this binding site is distinct from that for Mg²⁺.