심근 Mitochondria의 Na⁺-Ca⁺⁺교환에 관한 연구

The Study on Na⁺-Ca⁺⁺ Exchange in Heart Mitochondria

The Na⁺-and K⁺-induced Ca⁺⁺ release was measured isotopically by millipore filter technique in pig heart mitochondria. With EGTA-quenching technique, the characteristics of mitochondrial Ca⁺⁺-pool and the sources of Ca⁺⁺ released from mitochondria by Na⁺ or K⁺ were analyzed. The mitochondrial Ca⁺⁺-pool could be distinctly divided into two components: internal and external ones which were represented either by uptake through inner membrane, or by energy independent passive binding to external surface of mitochondria, respectively. In energized mitochondria, a large portion of Ca⁺⁺was transported into internal pool with little external binding, while in de-enerigzed state, a large portion of transported Ca⁺⁺ existed in the external pool with limited amount of Ca⁺⁺ in the internal pool which was possibly transported through the Ca⁺⁺-carrier present in the inner membrane. Na⁺ induced the Ca⁺⁺ release from both internal pool and external pool and external binding pool of mitochondria. In contrast, K⁺ did not affect Ca⁺⁺ of the internal pool, but, displaced Ca⁺⁺ bound to external surface of the mitochondria. When the Ca⁺⁺-reuptake was blocked by EGTA, the Ca⁺⁺ release from the internal pool by Na⁺ was rapid; the rate of Ca⁺⁺-efflux appeared to be a function of [Na⁺]² and about 8mM Na⁺ was required to elicit half-maximal velocity of Ca⁺⁺-efflux. So it was revealed that Ca⁺⁺-efflux velocity was particulary sensitive to small changes of the Na⁺ concentration in physiological range. Energy independent Ca⁺⁺-binding sites of mitochondrial external surface showed unique characteristics. The total number of external Ca⁺⁺-binding sites of pig heart mitochondria was 29 nmoles per mg protein and the dissociation constant(Kd) was 34μM. The Ca⁺⁺-binding to the external sites seemed to be competitively inhibited by Na⁺ and K⁺; the inhibition constant(Ki) were 9.7 mM and 7.1 mM respectively. Considering the intracellular ion concentrations and large proportion of Ca⁺⁺ uptake in energized mitochondria, the external Ca⁺⁺-binding pool of the mitochondria did not seem to play a significant role on the regulation of intracellular free Ca⁺⁺ concentration. From this experiment, it was suggested that a small change of intracellular free Na⁺ concentration might play a role on regulation of free Ca⁺⁺ concentration in cardiac cell by influencing Ca⁺⁺-efflux from the internal pool of mitochondria.