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Sir: Two recent papers in this journal differ on connections between PfNHE function and QNR for P. falciparum [1, 2]. Authors of  challenge that PfNHE function is measureable for P. falciparum via Na+ induced recovery of cytosolic pH (pHcyt), and question our interpretation  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 . Structural criteria for predicting localization also points very strongly to pm for PfNHE .
Quantification of NHE function requires transient cytosolic acidosis. Various methods exist, including weak base loading  and nigericin - BSA treatment . In , concerns regarding the latter are raised; however, we note that in  BSA is administered by perfusion, rather than added as a single dose into a cuvette as in . 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  pHcyt “… reach[ed] a final pHi which was … equal to the extracellular pH …”. In  this is not the case, suggesting our approach clamps pHcyt, as shown (Fig. 4A, ref. ). 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 , but was presumably obtained via bulk populations of detergent excised parasites injected into cuvettes (- CO2/HCO3). Calibration was shown in detail in , 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  behave similarly. Also, proper quantification of “faster” is multiplication of the measured change in pH vs. time by the cytosolic buffering capacity (βi). In  NHE is quantified this way and averaged for many replicate experiments and 13 strains. In  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 . In  it is quantified at many pHcyt including 6.2. NHE activity is much stimulated at lower pHcyt. Proper quantification of NHE  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  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  vs. a bulk detergent extracted parasite and cuvette– based approach  have likely led to distinct interpretations.
Notably  ignores that  correlates both PfNHE activity and pHcyt vs. genetically characterized QNR status . Chr 13 × chr 9 epistatic loci defined for QNR correlate with elevated pHcyt  and the absence of any reference in  to extensive strain analysis in  indicates these authors do not disagree. Any interpretation of  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  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 , thus  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  has ruled out our observation of a role for PfNHE in pHcyt regulation.
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