The plasma membrane Na+/Ca2+ exchange inhibitor KB‐R7943 is also a potent inhibitor of the mitochondrial Ca2+ uniporter

Abstract

Background and purpose: The thiourea derivative KB‐R7943, originally developed as inhibitor of the plasma membrane Na⁺/Ca²⁺ exchanger, has been shown to protect against myocardial ischemia‐reperfusion injury. We have studied here its effects on mitochondrial Ca²⁺ fluxes. Experimental approach. [Ca²⁺] in cytosol, mitochondria and endoplasmic reticulum (ER), and mitochondrial membrane potential were monitored using both luminescent (targeted aequorins) and fluorescent (fura‐2, tetramethylrhodamine ethyl ester) probes in HeLa cells. Key results: KB‐R7943 was also a potent inhibitor of the mitochondrial Ca²⁺ uniporter (MCU). In permeabilized HeLa cells, KB‐R7943 inhibited mitochondrial Ca²⁺ uptake with a Ki of 5.5±1.3 μM (mean±S.D.). In intact cells, 10μM KB‐R7943 reduced by 80% the mitochondrial [Ca²⁺] peak induced by histamine. KB‐R7943 did not modify the mitochondrial membrane potential and had no effect on the mitochondrial Na⁺/Ca²⁺ exchanger. KB‐R7943 inhibited histamine‐induced ER‐Ca²⁺ release in intact cells, but not in cells loaded with a Ca²⁺‐chelator to damp cytosolic [Ca²⁺] changes. Therefore, inhibition of ER‐Ca²⁺‐release by KB‐R7943 was probably due to the increased feedback Ca²⁺‐inhibition of inositol 1,4,5‐trisphosphate receptors after MCU block. This mechanism also explains why KB‐R7943 reversibly blocked histamine‐induced cytosolic [Ca²⁺] oscillations in the same range of concentrations required to inhibit MCU. Conclusions and Implications: Inhibition of MCU by KB‐R7943 may contribute to its cardioprotective activity by preventing mitochondrial Ca²⁺‐overload during ischemia‐reperfusion. In addition, the effects of KB‐R7943 on Ca²⁺ homeostasis provide new evidence for the role of mitochondria modulating Ca²⁺‐release and regenerative Ca²⁺‐oscillations. Search for permeable and selective MCU inhibitors may yield useful pharmacological tools in the future. British Journal of Pharmacology (2007) 151, 647–654; doi:10.1038/sj.bjp.0707260