KB-R7943, an inhibitor of the reverse Na+ /Ca2+ exchanger, blocks N-methyl-D-aspartate receptor and inhibits mitochondrial complex I

Br J Pharmacol. 2011 Jan;162(1):255-70. doi: 10.1111/j.1476-5381.2010.01054.x.


Background and purpose: An isothiourea derivative (2-[2-[4-(4-nitrobenzyloxy)phenyl]ethyl]isothiourea methane sulfonate (KB-R7943), a widely used inhibitor of the reverse Na(+) /Ca(2+) exchanger (NCX(rev)), was instrumental in establishing the role of NCX(rev) in glutamate-induced Ca(2+) deregulation in neurons. Here, the effects of KB-R7943 on N-methyl-D-aspartate (NMDA) receptors and mitochondrial complex I were tested.

Experimental approach: Fluorescence microscopy, electrophysiological patch-clamp techniques and cellular respirometry with Seahorse XF24 analyzer were used with cultured hippocampal neurons; membrane potential imaging, respirometry and Ca(2+) flux measurements were made in isolated rat brain mitochondria. KEY RESULTS KB-R7943 inhibited NCX(rev) with IC(50) = 5.7 ± 2.1 µM, blocked NMDAR-mediated ion currents, and inhibited NMDA-induced increase in cytosolic Ca(2+) with IC(50) = 13.4 ± 3.6 µM but accelerated calcium deregulation and mitochondrial depolarization in glutamate-treated neurons. KB-R7943 depolarized mitochondria in a Ca(2+) -independent manner. Stimulation of NMDA receptors caused NAD(P)H oxidation that was coupled or uncoupled from ATP synthesis depending on the presence of Ca(2+) in the bath solution. KB-R7943, or rotenone, increased NAD(P)H autofluorescence under resting conditions and suppressed NAD(P)H oxidation following glutamate application. KB-R7943 inhibited 2,4-dinitrophenol-stimulated respiration of cultured neurons with IC(50) = 11.4 ± 2.4 µM. With isolated brain mitochondria, KB-R7943 inhibited respiration, depolarized organelles and suppressed Ca(2+) uptake when mitochondria oxidized complex I substrates but was ineffective when mitochondria were supplied with succinate, a complex II substrate.

Conclusions and implications: KB-R7943, in addition to NCX(rev) , blocked NMDA receptors in cultured hippocampal neurons and inhibited complex I in the mitochondrial respiratory chain. These findings are critical for the correct interpretation of experimental results obtained with KB-R7943 and a better understanding of its neuroprotective action.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Cells, Cultured
  • Electron Transport Complex I / antagonists & inhibitors*
  • Hippocampus / cytology
  • Hippocampus / drug effects
  • Hippocampus / metabolism
  • Microscopy, Fluorescence
  • Neurons / drug effects
  • Neurons / metabolism
  • Patch-Clamp Techniques
  • Rats
  • Receptors, N-Methyl-D-Aspartate / antagonists & inhibitors*
  • Sodium-Calcium Exchanger / antagonists & inhibitors*
  • Thiourea / analogs & derivatives*
  • Thiourea / pharmacology


  • 2-(2-(4-(4-nitrobenzyloxy)phenyl)ethyl)isothiourea methanesulfonate
  • Receptors, N-Methyl-D-Aspartate
  • Sodium-Calcium Exchanger
  • Electron Transport Complex I
  • Thiourea