A calmodulin-stimulated Ca2+ pump in plasma-membrane vesicles from Trypanosoma brucei; selective inhibition by pentamidine

Abstract

Despite previous reports [McLaughlin (1985) Mol. Biochem. Parasitol. 15, 189-201; Ghosh, Ray, Sarkar and Bhaduri (1990) J. Biol. Chem. 265, 11345-11351; Mazumder, Mukherjee, Ghosh, Ray and Bhaduri (1992) J. Biol. Chem. 267, 18440-18446] that the plasma membrane of different trypanosomatids only contains Ca(2+)-ATPase that does not show any demonstrable dependence on Mg2+, a high-affinity (Ca(2+)-Mg2+)-ATPase was demonstrated in the plasma membrane of Trypanosoma brucei. The enzyme became saturated with micromolar amounts of Ca2+, reaching a Vmax. of 3.45 +/- 0.66 nmol of ATP/min per mg of protein. The Km,app. for Ca2+ was 0.52 +/- 0.03 microM. This was decreased to 0.23 +/- 0.05 microM, and the Vmax. was increased to 6.36 +/- 0.22 nmol of ATP/min per mg of protein (about 85%), when calmodulin was present. T. brucei plasma-membrane vesicles accumulated Ca2+ on addition of ATP only when Mg2+ was present, and released it to addition of the Ca2+ ionophore A23187. In addition, this Ca2+ transport was stimulated by calmodulin. Addition of NaCl to Ca(2+)-loaded T. brucei plasma-membrane vesicles did not result in Ca2+ release, thus suggesting the absence of a Na+/Ca2+ exchanger in these parasites. Therefore the (Ca(2+)-Mg2+)-ATPase would be the only mechanism so far described that is responsible for the long-term fine tuning of the intracellular Ca2+ concentration of these parasites. The trypanocidal drug pentamidine inhibited the T. brucei plasma-membrane (Ca(2+)-Mg2+)-ATPase and Ca2+ transport at concentrations that had no effect on the Ca(2+)-ATPase activity of human or pig erythrocytes. In this latter case, pentamidine behaved as a weak calmodulin antagonist, since it inhibited the stimulation of the erythrocyte Ca(2+)-ATPase by calmodulin.

Adaptor Protein-3 (AP-3) Complex Mediates the Biogenesis of Acidocalcisomes and Is Essential for Growth and Virulence of Trypanosoma brucei

“…Although acidocalcisome numbers declined more markedly as compared with the changes in acidocalcisome PP i and polyP, this discordance can be explained by the differences in cell volume of the remaining acidocalcisomes. These phenotypic changes did not lead to significant changes in intracellular Ca 2ϩ levels, in agreement with the reported major role of the plasma membrane in the regulation of intracellular Ca 2ϩ concentration under steady state conditions (65,66). Loss of polyP is in agreement with the presence in acidocalcisomes of the vacuolar transporter chaperone complex (31), which is involved in its synthesis.…”

Trypanosoma brucei Plasma Membrane-Type Ca2+-ATPase 1 (TbPMC1) and 2 (TbPMC2) Genes Encode Functional Ca2+-ATPases Localized to the Acidocalcisomes and Plasma Membrane, and Essential for Ca2+ Homeostasis and Growth

“…This characteristic places the enzymes in a novel category together with the acidocalcisomal Ca 2ϩ -ATPase described in T. cruzi (5), D. discoideum (4,8), and T. gondii (6), and the vacuolar Ca 2ϩ -ATPases of S. cerevisiae (3), and E. histolytica (7). The previous detection of a calmodulin-stimulated Ca 2ϩ pump in plasma membrane vesicles from T. brucei bloodstream forms (16) suggests that calmodulin regulates TbPMC2 activity by interacting with as yet unidentified sequences on the enzyme, or that another Ca 2ϩ pump is present in the plasma membrane. Since calmodulininteracting proteins cannot be identified by simple bioinformatic analyses, as their calmodulin-binding domains do not share a common sequence motif (46), further biochemical work with the separate enzymes expressed in yeast could solve this discrepancy.…”

“…The relative importance of this protein, however, has not been determined nor has it been demonstrated that this protein functions as a Ca 2ϩ pump. A more recent report (16) showed the presence of a calmodulin-stimulated Ca 2ϩ pump in plasma membrane vesicles from T. brucei bloodstream forms. It is evident that a more thorough molecular characterization of the Ca 2ϩ -ATPases present in T. brucei will be essential for structure-function analysis and further development of potential chemotherapeutic agents.…”

Selective Transfer of Calcium from an Acidic Compartment to the Mitochondrion of Trypanosoma brucei

“…Conversely, prolonged exposure to elevated [Ca 2ϩ ] cyt can result in cell death (1,2). Trypanosome survival therefore depends upon redundant energy-dependent processes that carefully control cytosolic free Ca 2ϩ concentrations ([Ca 2ϩ ] cyt ) 1 (3)(4)(5)(6)(7)(8). The present study examines within intact trypanosomes the relative importance of several Ca 2ϩ -transporting organelles.…”