Inhibition of Plasma Membrane Na/Ca-Exchanger by KB-R7943 or Lithium Reveals Its Role in Ca-Dependent N-methyl-d-aspartate Receptor Inactivation

J Pharmacol Exp Ther. 2015 Dec;355(3):484-95. doi: 10.1124/jpet.115.227173. Epub 2015 Sep 21.

Abstract

To evaluate the possible role of the plasma membrane Na(+)/Ca(2+)-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 Ca(2+)-free solution and was not observed in the presence of extracellular Ca(2+) on 1,2-Bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-loaded neurons, suggesting that Ca(2+) 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 Ca(2+). Most likely, the accumulation of intracellular Ca(2+) is caused by the inhibition of Ca(2+) extrusion via NCX. Thus, KBR and Li(+) provoke Ca(2+)-dependent receptor inactivation due to the disruption of Ca(2+) extrusion by the NCX. The data reveal the role of NCX in regulation of Ca(2+)-dependent inactivation of NMDARs.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Calcium / metabolism*
  • Cell Membrane / drug effects*
  • Cerebral Cortex / cytology
  • Cerebral Cortex / drug effects
  • Chelating Agents / pharmacology
  • Egtazic Acid / analogs & derivatives
  • Egtazic Acid / pharmacology
  • Excitatory Amino Acid Antagonists / pharmacology
  • Female
  • Glycine / pharmacology
  • HEK293 Cells
  • Humans
  • Lithium Compounds / pharmacology*
  • Neurons / drug effects
  • Patch-Clamp Techniques
  • Rats
  • Rats, Wistar
  • Receptors, N-Methyl-D-Aspartate / antagonists & inhibitors*
  • Sodium-Calcium Exchanger / antagonists & inhibitors*
  • Thiourea / analogs & derivatives*
  • Thiourea / pharmacology

Substances

  • 2-(2-(4-(4-nitrobenzyloxy)phenyl)ethyl)isothiourea methanesulfonate
  • Chelating Agents
  • Excitatory Amino Acid Antagonists
  • Lithium Compounds
  • Receptors, N-Methyl-D-Aspartate
  • Sodium-Calcium Exchanger
  • 1,2-bis(2-aminophenoxy)ethane N,N,N',N'-tetraacetic acid acetoxymethyl ester
  • Egtazic Acid
  • Thiourea
  • Calcium
  • Glycine