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Abstract

The delta pH-dependent quenching of Acridine orange was used to characterize Na⁺-H⁺ exchange and K⁺ and H⁺ conductances in brush-border membrane vesicles isolated by precipitation with either CaCl₂ or MgCl₂ from rat kidney cortex. A transmembrane pH difference of 2.5 units (inside acidic) was imposed and the initial rate of its dissipation was followed after injecting a puls of tetramethylammonium gluconate (control) or sodium or potassium gluconate. In membranes isolated by CaCl₂, the Na⁺-H⁺ exchange was partially electroneutral (45% to 77% of the total exchange) and the rest was due to electrically coupled Na⁺ and H⁺ movements through conductive pathways in the membranes. In membranes prepared by MgCl₂, the rate of total Na⁺-H⁺ exchange was about twice as high as that in membranes obtained by CaCl₂ precipitation. However, total and electroneutral exchanges were equal indicating negligible electrically coupled Na⁺ and H⁺ movements in these membranes. K0.5 for Na⁺ in all preparations was in the same range, being in average 30 mM. Amiloride was a competitive inhibitor of Na⁺-H⁺ exchange in membranes obtained with both preparations; K_i values ranged between 0.1 and 0.58 mM. The rates of delta pH-dissipation with K⁺ gradients (± valinomycin) were by 50% to 150% higher in membranes prepared with CaCl₂ than in membranes isolated with MgCl₂ indicating much higher H⁺ and K⁺ conductances in membranes obtained with CaCl₂. Therefore, the rate of Na⁺-H⁺ exchange as well as the conductances for various ions in the isolated brush-border membranes depend on membrane preparation.