Hydrogen ion gradients across the mitochondrial, endosomal and plasma membranes in bloodstream forms of trypanosoma brucei solving the three-compartment problem

Abstract

Conditions for the use of both [14C]methylamine and 5, 5-dimethyl[14C]oxa-azolidine-2,4-dione (DMO) to measure the H+ concentration of intracellular compartments of monomorphic long thin bloodstream forms of Trypanosoma brucei were established. Neither probe was actively transported or bound to internal components of the cell and both probes equilibrated passively with a t1/2 close to 8 min. DMO was excluded from cells, while methylamine was accumulated but not metabolized. Solution of the three-compartment problem revealed that, when cells were respiring aerobically on glucose at an external pH of 7.5, the cytoplasmic pH was in the range 6.99-7.03, the pH of the mitochondrial matrix was 7.71-7.73, and the algebraic average pH of the various endosomal compartments was 5.19-5.50. Similar values were found when cells were respiring aerobically on glycerol. However, bloodstream forms of T. brucei could not maintain a constant internal H+ concentration outside the external pH range 7.0-7.5, and no evidence for the presence of an H+/Na+ exchanger was found. Full motility and levels of pyruvate production were maintained as the external pH was raised as high as 9.5, suggesting that these cells tolerate significant internal alkalinisation. However, both motility and pyruvate production were severely inhibited under acidic conditions, and the cells deteriorated rapidly below an external pH of 6.5. Physiologically, the plasma membrane of T. brucei had low permeability to H+ and the internal pH was unaffected by changes in Deltapsip, which is dominated by the potassium diffusion potential. However, in the presence of FCCP, the internal pH fell rapidly about 0.5 pH unit and came into equilibrium with Deltapsip. Oligomycin abolished the mitochondrial pH gradient (DeltapHm) selectively, whereas chloroquine abolished only the endosomal pH gradient (DeltapHe). The pH gradient across the plasma membrane (DeltapHp) alone could be abolished by careful osmotic swelling of cells. The plasma membrane had an inwardly directed proton-motive force (DeltaPp) of -52 mV and an inwardly directed sodium-motive force (DeltaNp) of -149 mV, whereas the mitochondrial inner membrane had only an inwardly directed DeltaPm of -195 mV. The pH gradient across the endosomal membranes was not accompanied by an electrical gradient. Consequently, endosomal membranes had an algebraically average outwardly directed DeltaPl within the range + 89 to +110 mV, depending on the measurement method.