Ouabain Modulates the Functional Interaction Between Na,K-ATPase and NMDA Receptor

Mol Neurobiol. 2020 Oct;57(10):4018-4030. doi: 10.1007/s12035-020-01984-5. Epub 2020 Jul 10.


The N-methyl-D-aspartate (NMDA) receptor plays an essential role in glutamatergic transmission and synaptic plasticity and researchers are seeking for different modulators of NMDA receptor function. One possible mechanism for its regulation could be through adjacent membrane proteins. NMDA receptors coprecipitate with Na,K-ATPase, indicating a potential interaction of these two proteins. Ouabain, a mammalian cardiotonic steroid that specifically binds to Na,K-ATPase and affects its conformation, can protect from some toxic effects of NMDA receptor activation. Here we have examined whether NMDA receptor activity and downstream effects can be modulated by physiological ouabain concentrations. The spatial colocalization between NMDA receptors and the Na,K-ATPase catalytic subunits on dendrites of cultured rat hippocampal neurons was analyzed with super-resolution dSTORM microscopy. The functional interaction was analyzed with calcium imaging of single hippocampal neurons exposed to 10 μM NMDA in presence and absence of ouabain and by determination of the ouabain effect on NMDA receptor-dependent long-term potentiation. We show that NMDA receptors and the Na,K-ATPase catalytic subunits alpha1 and alpha3 exist in same protein complex and that ouabain in nanomolar concentration consistently reduces the calcium response to NMDA. Downregulation of the NMDA response is not associated with internalization of the receptor or with alterations in its state of Src phosphorylation. Ouabain in nanomolar concentration elicits a long-term potentiation response. Our findings suggest that ouabain binding to a fraction of Na,K-ATPase molecules that cluster with the NMDA receptors will, via a conformational effect on the NMDA receptors, cause moderate but consistent reduction of NMDA receptor response at synaptic activation.

Keywords: Calcium; LTP; NMDAR; Na,K-ATPase; Ouabain; Protein-protein interaction; Super-resolution microscopy.

MeSH terms

  • Animals
  • Calcium / metabolism
  • Down-Regulation / drug effects
  • Hippocampus / cytology
  • Models, Biological
  • N-Methylaspartate / metabolism
  • Neurons / cytology
  • Neurons / drug effects
  • Neurons / metabolism
  • Ouabain / pharmacology*
  • Phosphorylation / drug effects
  • Phosphotyrosine / metabolism
  • Protein Binding / drug effects
  • Rats, Sprague-Dawley
  • Receptors, N-Methyl-D-Aspartate / metabolism*
  • Sodium-Potassium-Exchanging ATPase / metabolism*
  • Synapses / drug effects
  • Synapses / metabolism
  • src-Family Kinases / metabolism


  • Receptors, N-Methyl-D-Aspartate
  • Phosphotyrosine
  • Ouabain
  • N-Methylaspartate
  • src-Family Kinases
  • Sodium-Potassium-Exchanging ATPase
  • Calcium