Characterization of the mitochondrial Na +H + exchange. The effect of amiloride analogues

Abstract

The kinetic properties and inhibitor sensitivity of the Na +H + exchange activity present in the inner membrane of rat heart and liver mitochondria were studied. (1) Na +-induced H + efflux from mitochondria followed Michaelis-Menten kinetics. In heart mitochondria, the K m for Na + was 24 ± 4 mM and the V max was 4.5 ± 1.4 nmol H +/mg protein per s ( n = 6). Basically similar values were obtained in liver mitochondria ( K m = 31 ± 2 mM, V max = 5.3 ± 0.2 nmol H +/mg protein per s, n = 4). (2) Li + proved to be a substrate ( K m = 5.9 mM, V max = 2.3 nmol H +/mg protein per s) and a potent competitive inhibitor with respect to Na + ( K i ≈ 0.7 mM). (3) External H + inhibited the mitochondrial Na +H + exchange competitively. (4) Two benzamil derivatives of amiloride, 5-( N-4-chlorobenzyl)- N-(2′,4′-dimethyl)benzamil and 3′,5′-bis(trifluoromethyl)benzamil were efective inhibitors of the mitochondrial Na +H + exchange (50% inhibition was attained by approx. 60 μM in the presence of 15 mM Na +). (5) Three 5- amino analogues of amiloride, which are very strong Na +H + exchange blockers on the plasma membrane, exerted only weak inhibitory activity on the mitochondrial Na +H + exchange. (6) The results indicate that the mitochondrial and the plasma membrane antiporters represent distinct molecular entities.