The distribution of permeant ions demonstrates the presence of at least two distinct electrical gradients in bloodstream forms of Trypanosoma brucei

Abstract

The distribution of 86Rb+ and the radiolabelled lipophilic cation [3H]methyltriphenylphosphonium (MePh3P+) was used to investigate the membrane potentials that exist in bloodstream forms of Trypanosoma brucei. Even after correction for binding to cellular constituents, the accumulation of MePh3P+ was approximately tenfold greater than the accumulation of Rb+ under resting conditions. The addition of low concentrations of carbonylcyanide p‐trifluoromethoxyphenylhydrazone or valinomycin reduced the accumulation of MePh3P+ tenfold without perturbing the accumulation of Rb+. Although selective permeabilization of the plasma membrane abolished the accumulation of Rb+ and caused a substantial decrease in the accumulation of MePh3P+, a significant carbonylcyanide‐p‐trifluoromethoxyphenylhydrazone‐sensitive accumulation of MePh3P+ persisted under these conditions. These data were consistent with the presence of at least two distinct membrane potentials (Ψ) in bloodstream forms of T. brucei; a potential across the plasma membrane (Ψp) and an additional Ψ, generated by the electrogenic movement of H+, across the membrane of an intracellular organelle that possesses no electrical permeability to Rb+ or K+.

The mitochondrion in bloodstream forms of Trypanosoma brucei is energized by the electrogenic pumping of protons catalysed by the F1F0‐ATPase

“…and either ['HH]MePh3P+ (10 nM, 30 pCi/ nmol) or [14C]DM0 (20 pM, 54 Ci/mol). After 10 min, the suspension was centrifuged at 8000 g for 20 s through a layer of silicon oil and the accumulation of [3H]MePh3P+ or ['4C]DM0 was measured in an analogous fashion to that described previously for intact trypanosomes (Nolan and Voorheis, 1991). The intramitochondrial and cxtramitochondrial volumes of pellets of isolated mitochondrial vesicles were measured using 3 H 2 0 (0.2 pCi/ml) and [U-'4C]sucrose (0.45 pM, 555 Ci/M).…”

“…In every case parallel experiments were performed to measure the intracellular and extracellular spaces in the pellets of cells using 3 H 2 0 and [U-'4C]sucrose as describcd previously (Nolan and Voorheis, 1991). Radiolabelled-ion uptake by mitochondrial vesicles was measured following resuspension of the mitochondrial fraction, isolated from bloodstream forms of T. hrucei (1 mg protein/ ml), in a medium containing 20 mM Mops, pH 7.0, 300 mM sucrose, 2 mM MgC12, fatty-acid-free bovine serum albumin (0.2%, mass/vol.)…”

“…The accumulation of [3H]MePh3P+ and 86Rb+ by bloodstream forms of T. hrucei, incubated in 1 I8 mM NaCl, 5 m M KC1, 1.2mM MgS04, 16mM Na2HP04, 1.2mM KHJ' O,, 5 mM NaHCO,, 10 mM glucose and 30 mM Tes, adjusted to pH 7.5 with 1 M NaOH (solution A), was measured using a centrifugation method as described by Nolan and Voorheis (1991). In every case parallel experiments were performed to measure the intracellular and extracellular spaces in the pellets of cells using 3 H 2 0 and [U-'4C]sucrose as describcd previously (Nolan and Voorheis, 1991).…”

“…Recently, the presence of an energized intracellular compartment in bloodstream forms of Trypanosoma brucei was demonstrated but its identity was not determined (Nolan and Voorheis, 1991). The single mitochondrion in these parasitic protozoons could be this energized compartment (Vickerman, 1962(Vickerman, ,1965.…”

“…The mitochondrial membrane potential generated by the mitochondrial F,F,-ATPase in intact trypanosomes was calculated after solving the two-compartment problem for the uptake of the lipophilic cation, methyltriphenylphosphonium (MePh,P+) and was shown to have a value of approximately 150mV. When the value for the dym is combined with that for the mitochondrial pH gradient (Nolan and Voorheis, 1990), the mitochondrial proton-motive force was calculated to be greater than 190 mV. It seems likely that this mitochondria1 proton-motive force serves a role in the directional transport of ions and rnctabolites across thc promitochondrial inner membrane during the bloodstream stage of the life cycle, as well as promoting the import of nuclearencoded protein into the promjtochondrion during the transformation of bloodstream forms into the next stage of the life cycle of T. hrucei.…”