Maintenance of internal pH and an electrochemical gradient in Trypanosoma brucei

Abstract

The internal pH value (pH i) of the long-slender bloodstream form of Trypanosoma brucei was estimated from the distribution of 14C-labeled 5,5-dimethyl-2,4-oxazolidinedione or 14C-labeled methyl amine between the intracellular space of the cells and the medium. The pH i of T. brucei remained relatively constant at 7.0–7.2 throughout an extracellular pH (pH o) range of 6.0–8.0. The maintenance of an internal pH more acidic than the environment appears to be a unique feature. Preincubation of T. brucei with carbonyl cyanide m-chlorophenyl hydrazone (CCCP) or CCCP + valinomycin had no appreciable effect on the ΔpH across the T. brucei membrane when the external pH was 8.O. However, when the external pH was 6.0, CCCP abolished the observed ΔpH. Nigericin significantly dissipated the ΔpH across the T. brucei membrane at all pH o values. These data suggest that under physiological conditions, the maintenance of a ΔpH across the bloodstream-form T. brucei membrane may be by a mechanism other than an energy-dependent gradient, whereas an energy-dependent pump may be needed for maintaining the pH i in an acidic environment. The electrical potential (Δψ) across the trypanosomal plasma membrane was also estimated using the lipophilic cation, [ 3H]tetraphenyl-phosphonium bromide. It appears dependent on both the external pH and the external salt conditions. Under ionic conditions similar to the host bloodstream, it ranges from −76 to −160 mV over an external pH range of 6.0 to 8.0, with an estimated value of −155.5 ± 0.7 at the physiological pH. It is partially inhibited by N-ethylmaleimide, whereas CCCP, NaN 3, NaF, EDTA, and Ca 2+ had no effect. The proton motive force (Δa \\ ̃ gmH) across the membrane of bloodstream form T. brucei is estimated to be about −143.2 mV under physiological conditions, to which Δψ is the main contributing force.