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A plasma membrane P-type H(+)-ATPase regulates intracellular pH in Leishmania mexicana amazonensis
Norma Marchesini1, Roberto Docampo
1Laboratory of Molecular Parasitology, Department of Pathobiology, College of Verterinary Medicine, University of Illinois at Urbana Champaign, 2001 South, Lincoln Avenue, Urbana, IL 61802, USA.
Abstract:
A recent report (Mukherjee et al., J. Biol. Chem. 276 (2001) 5563) has proposed that the plasma membrane Mg(+)-ATPase of promastigotes of Leishmania donovani, that is involved in its intracellular pH regulation, is an electroneutral H(+)/K(+) antiporter rather than an electrogenic H(+) pump. Since this proposition has important implications for the use of the pump as a target for chemotherapy, we investigated its nature in the mammalian stage (amastigote) of L. mexicana amazonensis and compared it with that present in promastigotes. Intracellular pH and H(+) efflux were measured using the acetotoxymethyl ester and the free form of 2',7'-bis-(carboxyethyl)-5(and-6)-carboxyfluorescein, respectively. Intracellular pH in amastigotes (at an external pH of 5.5) and promastigotes (at an external pH of 7.4) was 6.36+/-0.02 and 6.83+/-0.07, respectively. Differences in the mechanisms for regulation of intracellular pH were noted between amastigote and promastigote forms. Amastigotes maintained their intracellular pH neutral over a wide range of external pHs in the absence of K(+) or Na(+). The H(+)-ATPase inhibitors N,N'-dicyclohexylcarbodi-imide, diethylstilbestrol and N-ethylmaleimide, substantially decreased their steady-state intracellular pH, inhibited proton efflux, and their recovery from acidification. The data support the presence of an H(+)-ATPase as the major regulator of intracellular pH in amastigotes. In contrast, promastigotes were unable to maintain a neutral pH under acidic conditions and although their steady-state intracellular pH and recovery from acidification were affected by H(+)-ATPase inhibitors, bicarbonate was able to overcome intracellular acidification. Bicarbonate was also able to raise the steady-state intracellular pH from 6.80+/-0.03 to 7.25+/-0.09 and induce membrane hyperpolarization. No evidence was found of the possible involvement of a K(+)/H(+)-ATPase in intracellular pH regulation in both developmental stages of L. m. amazonensis.
Insights
Investigating Leishmania pH regulation, this study found amastigotes use H(+)-ATPase, while promastigotes utilize bicarbonate. This clarifies potential chemotherapy targets by differentiating proton pump mechanisms in parasite stages.
Area of Science:
- Parasitology
- Molecular Biology
- Biochemistry
Background:
- Previous research suggested Leishmania donovani promastigote plasma membrane Mg(2+)-ATPase functions as an electroneutral H(+)/K(+) antiporter.
- This proposed mechanism has significant implications for developing chemotherapy targeting parasite pH regulation.
- Understanding the precise mechanism is crucial for identifying effective drug targets.
Purpose of the Study:
- To investigate the nature of the proton pump in the mammalian stage (amastigote) of Leishmania mexicana amazonensis.
- To compare the pH regulatory mechanisms between amastigote and promastigote forms of Leishmania.
- To evaluate the role of H(+)-ATPase versus H(+)/K(+) antiporter in intracellular pH homeostasis.
Main Methods:
- Measurement of intracellular pH using 2',7'-bis-(carboxyethyl)-5(and-6)-carboxyfluorescein acetotoxymethyl ester.
- Quantification of proton (H(+)) efflux using the free form of the same fluorescent dye.
- Assessment of intracellular pH regulation under varying external pH conditions and in the presence of specific inhibitors (N,N'-dicyclohexylcarbodi-imide, diethylstilbestrol, N-ethylmaleimide) and ions (K(+), Na(+), bicarbonate).
Main Results:
- Amastigotes maintained a neutral intracellular pH across a wide range of external pHs, independent of K(+) or Na(+), and their pH regulation was significantly inhibited by H(+)-ATPase inhibitors.
- Promastigotes exhibited impaired pH regulation under acidic conditions and, while affected by H(+)-ATPase inhibitors, their intracellular acidification was overcome by bicarbonate, which also induced membrane hyperpolarization.
- No evidence supported the involvement of a K(+)/H(+)-ATPase in the pH regulation of either developmental stage.
Conclusions:
- The study strongly supports the presence of a proton-transporting ATPase (H(+)-ATPase) as the primary regulator of intracellular pH in Leishmania amastigotes.
- Promastigotes rely on bicarbonate-mediated mechanisms for pH homeostasis, differing significantly from amastigotes.
- These distinct pH regulatory pathways in different Leishmania developmental stages offer specific targets for anti-parasitic chemotherapy.