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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptHHS Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
Mol Biochem Parasitol. Author manuscript; available in PMC 2010 July 1.
Published in final edited form as:
PMCID: PMC2768547

P. falciparum Na+ / H+ Exchanger (PfNHE) Function and Quinine Resistance (QNR)

Sir: Two recent papers in this journal differ on connections between PfNHE function and QNR for P. falciparum [1, 2]. Authors of [2] challenge that PfNHE function is measureable for P. falciparum via Na+ induced recovery of cytosolic pH (pHcyt), and question our interpretation [1] that PfNHE is localized to plasma membrane (pm) and helps to regulate pHcyt. However, the closest known PfNHE homologue is T. gondii NHE1 which has clearly been localized to pm [3]. Structural criteria for predicting localization also points very strongly to pm for PfNHE [4].

Quantification of NHE function requires transient cytosolic acidosis. Various methods exist, including weak base loading [5] and nigericin - BSA treatment [6]. In [2], concerns regarding the latter are raised; however, we note that in [1] BSA is administered by perfusion, rather than added as a single dose into a cuvette as in [2]. Use of perfusion ensured that constant free BSA was always present since BSA - nigericin is washed away. Other variables (e.g. fatty acid free BSA) may also affect reliability. These different procedures likely lead to different efficiencies in quenching nigericin. Indeed, in [2] pHcyt “… reach[ed] a final pHi which was … equal to the extracellular pH …”. In [1] this is not the case, suggesting our approach clamps pHcyt, as shown (Fig. 4A, ref. [1]). Another key difference [1, 2] is the pHcyt at which NHE activity is initiated. The lower the pH prior to adding Na+, the faster Na+/H+ exchange will be. Calibration of pHcyt is therefore critical, as is how one distinguishes “faster” vs. “slower”. Calibration is not shown in [2], but was presumably obtained via bulk populations of detergent excised parasites injected into cuvettes (- CO2/HCO3). Calibration was shown in detail in [1], and was done under perfusion (+ CO2/HCO3) in real time for each live, intact cell whose pH or NHE activity is measured, eliminating the requirement that multiple bulk samples of detergent treated cells [2] behave similarly. Also, proper quantification of “faster” is multiplication of the measured change in pH vs. time by the cytosolic buffering capacity (βi). In [1] NHE is quantified this way and averaged for many replicate experiments and 13 strains. In [2] βi is not measured and “representative” recovery from acidosis is plotted as pH units vs. time for only 1 strain. Also, representative recovery from acidosis is shown only for samples with pHcyt ≥ 6.6 in [2]. In [1] it is quantified at many pHcyt including 6.2. NHE activity is much stimulated at lower pHcyt. Proper quantification of NHE [1] requires βi measurements and H+ flux data at many pHcyt values between (typically) 6.0 and 7.0.

We measure a Ki of ~ 0.9 μM for ethyl isopropyl amiloride (EIPA) for Na+ dependent recovery from acidosis. In [2] EIPA does not inhibit, however, only a single dose is examined. We have no simple explanation, but differences in assay precision, e.g. single iRBC SCP under physiologic perfusion [1] vs. a bulk detergent extracted parasite and cuvette– based approach [2] have likely led to distinct interpretations.

Notably [2] ignores that [1] correlates both PfNHE activity and pHcyt vs. genetically characterized QNR status [7]. Chr 13 × chr 9 epistatic loci defined for QNR correlate with elevated pHcyt [1] and the absence of any reference in [2] to extensive strain analysis in [1] indicates these authors do not disagree. Any interpretation of [1] should acknowledge that the activity we measure must reside in the chr 13 region and interact epistatically with chr 9. But a key experiment missing in [1] is measurement of steady state PfNHE activity. We do not yet know the relative importance of PfNHE in setting resting pHcyt. Clearly, the pm VATPase plays a key role [8], thus [2] is correct to further highlight VATPase. It may be that at certain pHcyt the more essential role of PfNHE is to balance Na+ transport, and that VATPase is more essential to pH equilibrium per se. We are grateful that Kirk and colleagues have raised these important points, nevertheless we are not convinced that [2] has ruled out our observation of a role for PfNHE in pHcyt regulation.


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1. Bennett TN, Patel J, Ferdig MT, Roepe PD. Plasmodium falciparum Na+/H+ exchanger activity and quinine resistance. Mol Biochem Parasitol. 2007;153(1):48–58. [PMC free article] [PubMed]
2. Spillman NJ, Allen RJ, Kirk K. Acid extrusion from the intraerythrocytic malaria parasite is not via a Na(+)/H(+) exchanger. Mol Biochem Parasitol. 2008;162(1):96–9. [PubMed]
3. Arrizabalaga G, Ruiz F, Moreno S, Boothroyd JC. Ionophore-resistant mutant of Toxoplasma gondii reveals involvement of a sodium/hydrogen exchanger in calcium regulation. J Cell Biol. 2004;165(5):653–62. [PMC free article] [PubMed]
4. Brett CL, Donowitz M, Rao R. Evolutionary origins of eukaryotic sodium/proton exchangers. Am J Physiol. 2005;288(2):C223–39. [PubMed]
5. Roos A, Boron WF. Intracellular pH. Physiol Rev. 1981;61(2):296–434. [PubMed]
6. Grinstein S, Cohen S, Rothstein A. Cytoplasmic pH regulation in thymic lymphocytes by an amiloride-sensitive Na+/H+ antiport. J Gen Physiol. 1984;83(3):341–69. [PMC free article] [PubMed]
7. Ferdig MT, Cooper RA, Mu J, Deng B, Joy DA, Su XZ, Wellems TE. Dissecting the loci of low-level quinine resistance in malaria parasites. Mol Microbiol. 2004;52(4):985–97. [PubMed]
8. Hayashi M, Yamada H, Mitamura T, Horii T, Yamamoto A, Moriyama Y. Vacuolar H(+)-ATPase localized in plasma membranes of malaria parasite cells, Plasmodium falciparum, is involved in regional acidification of parasitized erythrocytes. J Biol Chem. 2000;275(44):34353–8. [PubMed]