Kinetic properties of the K+/H+ antiport of heart mitochondria.
The fluorescence of 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein (BCECF) has been used to follow a K(+)-dependent alkaline shift in the matrix pH (pHi) of isolated heart mitochondria. The K(+)-dependent pHi change has properties consistent with an inward exchange of K+ for matrix H+ on the K+/H+ antiport of the mitochondrion. The reaction is activated by depletion of matrix Mg2+ with A23187 and by an alkaline external pH (pHo) and hypotonic conditions. The exchange is inhibited by quinine, dicyclohexylcarbodiimide, and exogenous Mg2+, but not by Li+. The rate of K+/H+ antiport measured in this way increases with increasing pHo to a maximum near pHo 9. The rate is a hyperbolic function of [K+] at pHo values above 8.3 with an apparent Km of 30 mM at pHo 8.4 and 14 mM at pHo 8.8. External H+ acts as a mixed-type inhibitor of the K+/H+ antiport under these conditions with a Ki equivalent to pHo 8.6-8.8. When pHo is kept constant, the reaction is relatively insensitive to matrix pH (pHi) in the range from 7.0 to 7.5. Above this pHi, the K(+)-dependent H+ extrusion shows a hyperbolic dependence on [H+]i with an apparent Km equivalent to pHi 8.1. The activated antiport shows an affinity sequence of Li+ greater than K+ = Rb+ greater than Cs+. The inward antiport of K+ is inhibited noncompetitively by NH4+ and is also sensitive to benzamil and to 5-N-substituted amiloride analogues with I50 values near 20 microM. Both NH4+ and the amiloride analogues increase pHi at constant pHo and appear to be concentrated in the matrix under these conditions.
Brierley
G P
GP
Department of Physiological Chemistry, Ohio State University Medical Center, Columbus 43221.
Jung
D W
DW
D000584:Amiloride / Q000031:analogs & derivatives / Q000494:pharmacology; D000818:Animals; D001692:Biological Transport / Q000187:drug effects; D000001:Calcimycin / Q000494:pharmacology; D002352:Carrier Proteins / Q000378:metabolism*; D002414:Cations, Monovalent; D002417:Cattle; D002794:Choline; D004024:Dicyclohexylcarbodiimide / Q000494:pharmacology; D005452:Fluoresceins; D005456:Fluorescent Dyes; D006863:Hydrogen-Ion Concentration; D007038:Hypotonic Solutions; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology; D008929:Mitochondria, Heart / Q000378:metabolism*; D011188:Potassium / Q000494:pharmacology; D027961:Potassium-Hydrogen Antiporters; D011803:Quinine / Q000494:pharmacology
D002352:Carrier Proteins; D002414:Cations, Monovalent; D005452:Fluoresceins; D005456:Fluorescent Dyes; D007038:Hypotonic Solutions; D027961:Potassium-Hydrogen Antiporters; C013407:benzamil; D000001:Calcimycin; D004024:Dicyclohexylcarbodiimide; D000584:Amiloride; C043829:2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein; D011803:Quinine; D008274:Magnesium; D002794:Choline; D011188:Potassium