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

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+/Ca2+ exchanger (NCXrev), was instrumental in establishing the role of NCXrev in glutamate‐induced Ca2+ 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 Ca2+ flux measurements were made in isolated rat brain mitochondria. KEY RESULTS KB‐R7943 inhibited NCXrev with IC50= 5.7 ± 2.1 µM, blocked NMDAR‐mediated ion currents, and inhibited NMDA‐induced increase in cytosolic Ca2+ with IC50= 13.4 ± 3.6 µM but accelerated calcium deregulation and mitochondrial depolarization in glutamate‐treated neurons. KB‐R7943 depolarized mitochondria in a Ca2+‐independent manner. Stimulation of NMDA receptors caused NAD(P)H oxidation that was coupled or uncoupled from ATP synthesis depending on the presence of Ca2+ 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 IC50= 11.4 ± 2.4 µM. With isolated brain mitochondria, KB‐R7943 inhibited respiration, depolarized organelles and suppressed Ca2+ 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 NCXrev, 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.