P. falciparum Na +/H + exchanger (PfNHE) function and quinine resistance (QNR) : [Reply to: Spillman et al. “Acid extrusion from the intraerythrocytic malaria parasite is not via a Na +/H + exchanger” Mol. Biochem. Parasitol. 2008 162 (1) 96–99

Abstract

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.