Kim Bongju, Matsuoka Satoshi
Department of Physiology and Biophysics, Graduate School of Medicine, Kyoto University, Yoshida-Konoe-cho, Sakyo-ku, Kyoto 606-8501, Japan.
J Physiol. 2008 Mar 15;586(6):1683-97. doi: 10.1113/jphysiol.2007.148726. Epub 2008 Jan 24.
To clarify the role of mitochondrial Na(+)-Ca(2+) exchange (NCX(mito)) in regulating mitochondrial Ca(2+) (Ca(2+)(mito)) concentration at intact and depolarized mitochondrial membrane potential (DeltaPsi(mito)), we measured Ca(2+)(mito) and DeltaPsi(mito) using fluorescence probes Rhod-2 and TMRE, respectively, in the permeabilized rat ventricular cells. Applying 300 nm cytoplasmic Ca(2+) (Ca(2+)(c)) increased Ca(2+)(mito) and this increase was attenuated by cytoplasmic Na(+) (Na(+)(c)) with an IC(50) of 2.4 mm. To the contrary, when DeltaPsi(mito) was depolarized by FCCP, a mitochondrial uncoupler, Na(+)(c) enhanced the Ca(2+)(c)-induced increase in Ca(2+)(mito) with an EC(50) of about 4 mm. This increase was not significantly affected by ruthenium red or cyclosporin A. The inhibition of NCX(mito) by CGP-37157 further increased Ca(2+)(mito) when DeltaPsi(mito) was intact, while it suppressed the Ca(2+)(mito) increase when DeltaPsi(mito) was depolarized, suggesting that DeltaPsi(mito) depolarization changed the exchange mode from forward to reverse. Furthermore, DeltaPsi(mito) depolarization significantly reduced the Ca(2+)(mito) decrease via forward mode, and augmented the Ca(2+)(mito) increase via reverse mode. When the respiratory chain was attenuated, the induction of the reverse mode of NCX(mito) hyperpolarized DeltaPsi(mito), while DeltaPsi(mito) depolarized upon inducing the forward mode of NCX(mito). Both changes in DeltaPsi(mito) were remarkably inhibited by CGP-37157. The above experimental data indicated that NCX(mito) is voltage dependent and electrogenic. This notion was supported theoretically by computer simulation studies with an NCX(mito) model constructed based on present and previous studies, presuming a consecutive and electrogenic Na(+)-Ca(2+) exchange and a depolarization-induced increase in Na(+) flux. It is concluded that Ca(2+)(mito) concentration is dynamically modulated by Na(+)(c) and DeltaPsi(mito) via electrogenic NCX(mito).
为了阐明线粒体钠钙交换体（NCX(mito)）在完整和去极化线粒体膜电位（ΔΨ(mito)）条件下调节线粒体钙（Ca(2+)(mito)）浓度中的作用，我们分别使用荧光探针罗丹明-2和四甲基罗丹明乙酯（TMRE）在透化的大鼠心室细胞中测量了Ca(2+)(mito)和ΔΨ(mito)。施加300 nM的胞质钙（Ca(2+)(c)）可增加Ca(2+)(mito)，而这种增加会被胞质钠（Na(+)(c)）减弱，半数抑制浓度（IC(50)）为2.4 mM。相反，当线粒体解偶联剂羰基氰化物间氯苯腙（FCCP）使ΔΨ(mito)去极化时，Na(+)(c)增强了Ca(2+)(c)诱导的Ca(2+)(mito)增加，半数有效浓度（EC(50)）约为4 mM。这种增加不受钌红或环孢素A的显著影响。当ΔΨ(mito)完整时，CGP-37157对NCX(mito)的抑制进一步增加了Ca(2+)(mito)，而当ΔΨ(mito)去极化时，它抑制了Ca(2+)(mito)的增加，这表明ΔΨ(mito)去极化使交换模式从正向转变为反向。此外，ΔΨ(mito)去极化显著减少了通过正向模式的Ca(2+)(mito)降低，并增强了通过反向模式的Ca(2+)(mito)增加。当呼吸链减弱时，诱导NCX(mito)的反向模式会使ΔΨ(mito)超极化，而诱导NCX(mito)的正向模式时ΔΨ(mito)会去极化。ΔΨ(mito)的这两种变化均被CGP-37157显著抑制。上述实验数据表明NCX(mito)是电压依赖性且产电的。基于当前和先前研究构建的NCX(mito)模型进行的计算机模拟研究从理论上支持了这一观点，该模型假定存在连续且产电的钠钙交换以及去极化诱导的钠通量增加。得出的结论是，Ca(2+)(mito)浓度通过产电的NCX(mito)由Na(+)(c)和ΔΨ(mito)动态调节。
