Chloride conductive pathways which support electrogenic H+ pumping by Leishmania major promastigotes

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 (pHe 7.4, 28 degrees 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.