We recently reported that human SLC4A11 mediates electrogenic transmembrane NH
3:H
+ fluxes,
27 and this result has been verified by two other independent groups.
25,26 Furthermore, a recent report from a third group shows murine Slc4a11 mediates similar electrogenic transmembrane NH
3:H
+ fluxes.
24 So we next set out to determine if the Slc4a11-mediated NH
3:H
+ flux is impaired in
Slc4a11−/− MCECs (
Fig. 4A). Cells were initially incubated with BF Ringer's, pulsed with 10 mM NH
4Cl in BF Ringer's, and then switched back to BF Ringer's. Once dissolved in solution, a small fraction of NH
4Cl forms NH
3, a small noncharged molecule that is readily membrane diffusible. On entry, NH
3 instantaneously reacts with intracellular H
2O to form NH
4+ and releases OH
−. The latter rapidly alkalinizes the cell, causing the initial rapid alkalinizing phase (100–120 seconds) on NH
4Cl application. Eventually the cell will reach an equilibrium where intracellular [NH
3] equals extracellular [NH
3]. Then a slow acidification occurs in the midphase (120–400 seconds) of the NH
4Cl pulse, indicating there is an additional weak acid NH
4+ (or NH
3:H
+ equivalently) flux entering the cell.
38 In this case, Slc4a11 activity brings more NH
3:H
+ into the cell.
Figure 4B shows that the rate of this slow acidification is significantly faster in
Slc4a11+/+ MCECs compared with
Slc4a11−/− MCECs, consistent with the additional NH
3:H
+ influx provided by Slc4a11. Then, when the NH
4Cl was washed away by BF Ringer's, there is a pronounced and rapid acidification on NH
4Cl removal (400–440 seconds) (
Fig. 4B). This is due to the rapid exit of NH
3 gas and the conversion of accumulated NH
4+ to NH
3 + H
+. The rapid acid loading immediately after NH
4Cl removal is a reflection of the amount of weak acid (NH
4+ or NH
3:H
+) that has entered the cell during the NH
4Cl pulse.
38 And even though NH
4Cl was removed in this phase on the outside, there is NH
4+ temporarily trapped intracellularly.
38 The amount of NH
4+ trapped is directly correlated with the extent of the acidification according to the Henderson-Hasselbalch equation.
38 Figure 4A shows that the depth of this acid load is much greater in
Slc4a11+/+ MCECs. The pH
i recovery (440–520 seconds) from this acid load is a phenomenon of the collective effect from Slc4a11-mediated NH
3:H
+ efflux and Na
+/H
+ exchanger-mediated H
+ extrusion. We measured the initial rate of pH
i recovery as a measure of apparent NH
3:H
+ efflux primarily attributed to Slc4a11 activity. We observed a rapid recovery in
Slc4a11+/+ MCECs, but significantly slower recovery in
Slc4a11−/− MCECs (
Fig. 4B). To more accurately represent the Slc4a11-mediated NH
3:H
+ efflux, we performed further analysis by subtracting the Na
+/H
+ exchanger-mediated apparent pH
i recovery (
Fig. 3C), to obtain the adjusted NH
3:H
+ efflux. The average of apparent Na
+/H
+ exchanger activity (0.0014/s) between
Slc4a11+/+ and
Slc4a11−/− MCECs was used given there was no statistical significance between the two cell lines.
Figure 4C shows that the adjusted NH
3:H
+ efflux is 0.0044 ± 0.0015/s (
n = 5) in
Slc4a11+/+ MCECs, and significantly smaller 0.0005 ± 0.0003/s (
n = 5,
P = 0.0347) in
Slc4a11−/− MCECs.