CHLORIDE CONDUCTIVE PATHWAYS WHICH SUPPORT ELECTROGENIC H-MAJOR PROMASTIGOTES( PUMPING BY LEISHMANIA)

The proton extrusion mechanisms of Leishmania promastigotes were studi ed in terms of electrogenic movements of protons and anions (Cl- and H CO3-). Changes in membrane potential (V-m) and intracellular pH (pH(i) ) were monitored fluorimetrically with the potential sensitive dye bis oxonol and the pH-sensitive dye tetraacethoxymethyl 2',7'-bis-(carbox yethyl)-5,6 carboxyfluorescein, respectively. In nominal bicarbonate-f ree medium (pH(e) 7.4, 28 degrees C), V-m and pH(i) of Leishmania prom astigotes were maintained at -113 +/- 4 mV and 6.75 +/- 0.02, respecti vely. In Cl- free (gluconate based) medium, cells underwent a time dep endent acidification (0.3 pH units) and a long term membrane hyperpola rization (7-10 mV), both of which were greatly enhanced in the presenc e of. the anion blocker, 4,4'-diisothiocyanodihydrostilbene-2,2'-disul fonic acid (H2DIDS). Cells in Cl--free medium underwent a marked depol arization upon treatment with the H+-ATPase inhibitor dicyclohexylcarb odiimide (DCCD), but hyperpolarized after repletion with Cl-. In Cl--d epleted cells, replenishment of Cl- led to a H2DIDS-sensitive cytoplas mic alkalinization and a small initial hyperpolarization. Cells expose d either to DCCD or to the H+ uncoupler carbonylcyanide chloraphenylhy drazone caused a marked cytoplasmic acidification and membrane depolar ization. In the presence of 25 mM HCO3-, promastigotes maintained an a lmost neutral cytosol, irrespective of H+ pump action or ionic composi tion 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 Leis hmania 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 implic ated 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 HCO 3-, in pH(i) homeostasis.