Hydrogen ion gradients across the mitochondrial, endosomal and plasma membranes in bloodstream forms of Trypanosoma brucei

Abstract

Conditions for the use of both [ 14 C]methylamine and 5,5-dimethyl[ 14 C]oxa-azolidine-2,4-dione (DMO) to measure the H 1 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 t 1/2 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 1 concentration outside the external pH range 7.0±7.5, and no evidence for the presence of an H 1 /Na 1 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 1 and the internal pH was unaffected by changes in Dc p , 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 Dc p . Oligomycin abolished the mitochondrial pH gradient (DpH m ) selectively, whereas chloroquine abolished only the endosomal pH gradient (DpH e ). The pH gradient across the plasma membrane (DpH p ) alone could be abolished by careful osmotic swelling of cells. The plasma membrane had an inwardly directed proton-motive force (DP p ) of 252 mV and an inwardly directed sodium-motive force (DN p ) of 2149 mV, whereas the mitochondrial inner membrane had only an inwardly directed DP m of 2195 mV. The pH gradient across the endosomal membranes was not accompanied by an electrical gradient. Consequently, endosomal membranes had an algebraically average outwardly directed DP l within the range 1 89 to 1 110 mV, depending on the measurement method.Keywords: membrane potential; pH gradients; proton-motive force; sodium-motive force; Trypanosoma bruceiThe study of H 1 -dependent events in cells was greatly stimulated by the pioneering work of Mitchell [1±4],who first focused attention on the cellular role of transmembrane H 1 gradients, and also by the development of simple and reliable techniques for the measurement of the concentration of H 1 in cells [5,6]. These advances have led to a general consensus that, in terms of the concentration of H 1 , most animal cells represent a three-compartment system consisting of the cyto...

Dissecting the Catalytic Mechanism of Trypanosoma brucei Trypanothione Synthetase by Kinetic Analysis and Computational Modeling

“…Based on published data (32–34), a phosphate buffer system was established which mimics the cytosol of the cell and is composed of 100 m m potassium (32), 15 m m sodium (32), 10 m m magnesium, 120 m m chloride (32), 10 m m total phosphate and has a pH of 7.0 (33, 34). From the total magnesium content of T. brucei (35), a concentration of 6 m m magnesium in the cytosol of bloodstream cells could be estimated.…”

The Glycosylphosphatidylinositol-PLC in Trypanosoma brucei Forms a Linear Array on the Exterior of the Flagellar Membrane Before and After Activation

“…It appears that the two negatively charged sulphonate groups and the positively charged quaternary nitrogen on the dye are sufficient to prevent entry of the dye into the cell, particularly in the presence of a negative plasma membrane potential, which excludes net negatively charged molecules lacking a specific energy-coupled transport system, e.g. the thiocyanate anion [34]. The cellular distribution of the GPI-PLC is shown in Figure 4, panel D, images 2, 4 and 5 in an actively dividing and, consequently, biflagellate cell.…”

The Mitochondrion Is a Site of Trypanocidal Action of the Aromatic Diamidine DB75 in Bloodstream Forms of Trypanosoma brucei

“…The trypanosome mitochondrion alters the biochemistry of the parasite to facilitate life within the (38,40,41). For example, a major functional difference between the mitochondrion in fly and mammalian infective forms is the mechanism through which these forms generate and maintain an energized ⌬⌿ m (7,36,37,45,49). The oligomycin-sensitive F 1 F 0 -ATPase of fly-infective forms (procyclics) functions similarly to the mammalian ATPase, whereas the ATPase of T. brucei BFs functions differently.…”