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- W2025838739 abstract "The proton extrusion mechanisms of Leishmania promastigotes were studied in terms of electrogenic movements of protons and anions (Cl− and HCO3−). Changes in membrane potential (Vm) and intracellular pH (pHi) were monitored fluorimetrically with the potential sensitive dye bis-oxonol and the pH-sensitive dye tetraacethoxymethyl 2′,7′-bis-(carboxyethyl)-5,6-carboxyfluorescein, respectively. In nominal bicarbonate-free medium (pHe7.4, 28°C), Vm and pHi of Leishmania promastigotes were maintained at −113 ± 4 mV and 6.75 ± 0.02, respectively. In Cl− free (gluconate-based) medium, cells underwent a time-dependent acidification (0.3 pH units) and a long term membrane hyperpolarization (7-10 mV), both of which were greatly enhanced in the presence of the anion blocker, 4,4′-diisothiocyanodihydrostilbene-2,2′-disulfonic acid (H2DIDS). Cells in Cl−-free medium underwent a marked depolarization upon treatment with the H+-ATPase inhibitor dicyclohexylcarbodiimide (DCCD), but hyperpolarized after repletion with Cl−. In Cl−-depleted cells, replenishment of Cl− led to a H2DIDS-sensitive cytoplasmic alkalinization and a small initial hyperpolarization. Cells exposed either to DCCD or to the H+ uncoupler carbonylcyanide chlorophenylhydrazone caused a marked cytoplasmic acidification and membrane depolarization. In the presence of 25 mM HCO3−, promastigotes maintained an almost neutral cytosol, irrespective of H+ pump action or ionic composition of the medium. The present observations provide evidence for the operation of a DCCD-sensitive electrogenic H+-ATPase which contributes to the maintenance of a highly hyperpolarized plasma membrane in Leishmania promastigotes. H+ pump activity required a parallel pathway of Cl− ions in order to dissipate the pump generated electrical potential. In nominally CO2-free media, the two electrogenic systems are implicated in the maintenance of cell pH and indirectly in electrochemically driven nutrient uptake. In physiological CO2/HCO3−-containing media, the H+ pump and Cl− channel play a role only secondary to that of HCO3− in pHi homeostasis. The proton extrusion mechanisms of Leishmania promastigotes were studied in terms of electrogenic movements of protons and anions (Cl− and HCO3−). Changes in membrane potential (Vm) and intracellular pH (pHi) were monitored fluorimetrically with the potential sensitive dye bis-oxonol and the pH-sensitive dye tetraacethoxymethyl 2′,7′-bis-(carboxyethyl)-5,6-carboxyfluorescein, respectively. In nominal bicarbonate-free medium (pHe7.4, 28°C), Vm and pHi of Leishmania promastigotes were maintained at −113 ± 4 mV and 6.75 ± 0.02, respectively. In Cl− free (gluconate-based) medium, cells underwent a time-dependent acidification (0.3 pH units) and a long term membrane hyperpolarization (7-10 mV), both of which were greatly enhanced in the presence of the anion blocker, 4,4′-diisothiocyanodihydrostilbene-2,2′-disulfonic acid (H2DIDS). Cells in Cl−-free medium underwent a marked depolarization upon treatment with the H+-ATPase inhibitor dicyclohexylcarbodiimide (DCCD), but hyperpolarized after repletion with Cl−. In Cl−-depleted cells, replenishment of Cl− led to a H2DIDS-sensitive cytoplasmic alkalinization and a small initial hyperpolarization. Cells exposed either to DCCD or to the H+ uncoupler carbonylcyanide chlorophenylhydrazone caused a marked cytoplasmic acidification and membrane depolarization. In the presence of 25 mM HCO3−, promastigotes maintained an almost neutral cytosol, irrespective of H+ pump action or ionic composition of the medium. The present observations provide evidence for the operation of a DCCD-sensitive electrogenic H+-ATPase which contributes to the maintenance of a highly hyperpolarized plasma membrane in Leishmania promastigotes. H+ pump activity required a parallel pathway of Cl− ions in order to dissipate the pump generated electrical potential. In nominally CO2-free media, the two electrogenic systems are implicated in the maintenance of cell pH and indirectly in electrochemically driven nutrient uptake. In physiological CO2/HCO3−-containing media, the H+ pump and Cl− channel play a role only secondary to that of HCO3− in pHi homeostasis." @default.
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- W2025838739 date "1995-03-01" @default.
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- W2025838739 title "Chloride Conductive Pathways Which Support Electrogenic H+ Pumping by Leishmania major Promastigotes" @default.
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- W2025838739 doi "https://doi.org/10.1074/jbc.270.10.5299" @default.
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