Kinetic properties of the sodium/hydrogen ion antiport of heart mitochondria

Abstract

The fluorescence of 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein (BCECF) has been used to follow the Na+/H+ antiport activity of isolated heart mitochondria as a Na+-dependent extrusion of matrix H+. The antiport activity measured in this way shows a hyperbolic dependence on external Na+ or Li+ concentration when the external pH (pHo) is 7.2 or higher. The apparent Km for Na+ decreases with increasing pHo to a limit of 4.6 mM. The Ki for external H+ as a competitive inhibitor of Na+/H+ antiport averages 3.0 nM (pHo 8.6). The Vmax at 24 degrees C is 160 ng ion of H+ min-1 (mg of protein)-1 and does not vary with pHo. Li+ reacts with the antiporter with higher affinity, but much lower Vmax, and is a competitive inhibitor of Na+/H+ antiport. The rate of Na+/H+ antiport is optimal when the pHi is near 7.2. When pHo is maintained constant, Na+-dependent extrusion of matrix H+ shows a hyperbolic dependence on [H+]i with an apparent Km corresponding to a pHi of 6.8. The Na+/H+ antiport is inhibited by benzamil and by 5-N-substituted amiloride analogues with I50 values in the range from 50 to 100 microM. The pH profile for this inhibition seems consistent with the availability of a matrix binding site for the amiloride analogues. The mitochondrial Na+/H+ antiport resembles the antiport found in the plasma membrane of mammalian cells in that Na+, Li+, and external H+ appear to compete for a common external binding site and both exchanges are inhibited by amiloride analogues.(ABSTRACT TRUNCATED AT 250 WORDS)

Molecular Cloning and Characterization of a Novel (Na+,K+)/H+ Exchanger Localized to the trans-Golgi Network

“…NHE7 HA was also insensitive to low concentrations (100 M) of benzamil, an amiloride analogue, although it was blocked significantly at high concentrations (1 mM). Since 100 M benzamil is known to block the Na ϩ -selective Na ϩ /H ϩ exchanger in isolated mitochondria (47), the fluxes measured under our conditions are unlikely to include uptake into mitochondria, more so given that the mitochondrial matrix is alkaline under steady-state conditions.…”

Identification of a Mitochondrial Na+/H+Exchanger

“…ϩ /H ϩ Exchangers 6956 preferentially inhibit the mammalian mitochondrial Na ϩ -selective NHE at the concentration (0.1 mM) used in this study (16), whereas it is a poor inhibitor of plasma membrane NHEs. This suggests that yeast Nha2 has pharmacological properties similar to those of its mammalian homologue.…”

The role of Na dysregulation in cardiac disease and how it impacts electrophysiology

“…The stoichiometry of the mNCE has been debated, but later evidence suggests that it may be electrogenic [4]. Na + entering the mitochondria is extruded by H + /Na + exchange (mNHE) [5][6][7]. Na + transport across other intracellular organelles (e.g.…”