Inorganic phosphate uptake in Trypanosoma cruzi is coupled to K cycling and to active Na extrusion |
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Authors: | CF Dick ALA Dos-Santos D Majerowicz LS Paes NL Giarola KC Gondim A Vieyra JR Meyer-Fernandes |
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Institution: | 1. Instituto de Microbiologia Professor Paulo de Góes, Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ, Brazil;2. Laboratório de Bioquímica Celular, Instituto de Bioquímica Médica, Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ, Brazil;3. Instituto Nacional de Ciência e Tecnologia em Biologia Estrutural e Bioimagem, Rio de Janeiro, RJ, Brazil;4. Laboratório de Bioquímica e Fisiologia de Insetos, Instituto de Bioquímica Medica, Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ, Brazil;5. Instituto Nacional de Ciência e Tecnologia em Entomologia Molecular, Rio de Janeiro, RJ, Brazil;6. Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ, Brazil |
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Abstract: | BackgroundOrthophosphate (Pi) is a central compound in the metabolism of all organisms, including parasites. There are no reports regarding the mechanisms of Pi acquisition by Trypanosoma cruzi.Methods32Pi influx was measured in T. cruzi epimastigotes. The expression of Pi transporter genes and the coupling of the uptake to Na+, H+ and K+ fluxes were also investigated. The transport capacities of different evolutive forms were compared.ResultsEpimastigotes grew significantly more slowly in 2 mM than in 50 mM Pi. Influx of Pi into parasites grown under low Pi conditions took place in the absence and presence of Na+. We found that the parasites express TcPho84, a H+:Pi-symporter, and TcPho89, a Na+:Pi-symporter. Both Pi influx mechanisms showed Michaelis–Menten kinetics, with a one-order of magnitude higher affinity for the Na+-dependent system. Collapsing the membrane potential with carbonylcyanide-p-trifluoromethoxyphenylhydrazone strongly impaired the influx of Pi. Valinomycin (K+ ionophore) or SCH28028 (inhibitor of (H+ + K+)ATPase) significantly inhibited Pi uptake, indicating that an inwardly-directed H+ gradient energizes uphill Pi entry and that K+ recycling plays a key role in Pi influx. Furosemide, an inhibitor of the ouabain-insensitive Na+-ATPase, decreased only the Na+-dependent Pi uptake, indicating that this Na+ pump generates the Na+ gradient utilized by the symporter. Trypomastigote forms take up Pi inefficiently.ConclusionsPi starvation stimulates membrane potential-sensitive Pi uptake through different pathways coupled to Na+ or H+/K+ fluxes.General significanceThis study unravels the mechanisms of Pi acquisition by T. cruzi, a key process in epimastigote development and differentiation to trypomastigote forms. |
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Keywords: | FCCP carbonylcyanide-p-trifluoromethoxyphenylhydrazone HEPES 4-(2-hydroxyethyl)-1-piperazine-ethanesulfonic acid LIT liver infusion tryptose medium MES 2-(N-morpholino)ethanesulfonic acid PSG phosphate buffered saline containing glucose |
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