Abstract

To evaluate the possible role of the plasma membrane Na⁺/Ca²⁺-exchanger (NCX) in regulation of N-methyl-d-aspartate (NMDA) receptors (NMDARs), we studied effects of 2-[2-[4-(4-nitrobenzyloxy) phenyl]ethyl]isothiourea methanesulfonate (KB-R7943; KBR) and lithium (inhibitors of NCX) on NMDA-elicited whole-cell currents using the patch-clamp technique on rat cortical neurons and human embryonic kidney 293T cells expressing recombinant NMDARs. KBR inhibited NMDAR currents in a voltage-independent manner with similar potency for receptors of GluN1/2A and GluN1/2B subunit compositions that excludes open-channel block and GluN2B-selective inhibition. The inhibition by KBR depended on glycine (Gly) concentration. At 30 μM NMDA, the KBR IC₅₀ values were 5.3 ± 0.1 and 41.2 ± 8.8 μM for 1 and 300 μM Gly, respectively. Simultaneous application of NMDA + KBR in the absence of Gly induced robust inward NMDAR currents that peaked and then rapidly decreased. KBR, therefore, is an agonist (EC₅₀ is 1.18 ± 0.16 µM) of the GluN1 subunit coagonist binding sites. The decrease of NMDA-elicited currents in the presence of KBR was abolished in Ca²⁺-free solution and was not observed in the presence of extracellular Ca²⁺ on 1,2-Bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid-loaded neurons, suggesting that Ca²⁺ affects NMDARs from the cytosol. In agreement, the substitution of Li⁺ for extracellular Na⁺ caused a considerable decrease of NMDAR currents, which was not observed in the absence of extracellular Ca²⁺. Most likely, the accumulation of intracellular Ca²⁺ is caused by the inhibition of Ca²⁺ extrusion via NCX. Thus, KBR and Li⁺ provoke Ca²⁺-dependent receptor inactivation due to the disruption of Ca²⁺ extrusion by the NCX. The data reveal the role of NCX in regulation of Ca²⁺-dependent inactivation of NMDARs.