The ammonia/ammonium (NH3/NH4
+, AM) concentration in human erythrocytes (RBCs)
is significantly higher than in plasma. Two main possible mechanisms for AM transport, including
simple and facilitated diffusion, are described; however, the driving force for AM transport is not
yet fully characterized. Since the erythroid ammonium channel RhAG forms a structural unit with
anion exchanger 1 (eAE1) within the ankyrin core complex, we hypothesized the involvement of
eAE1 in AM transport. To evaluate the functional interaction between eAE1 and RhAG, we used a
unique feature of RBCs to swell and lyse in isotonic NH4
+ buffer. The kinetics of cell swelling and
lysis were analyzed by flow cytometry and an original laser diffraction method, adapted for accurate
volume sensing. The eAE1 role was revealed according to (i) the changes in cell swelling and lysis
kinetics, and (ii) changes in intracellular pH, triggered by eAE1 inhibition or the modulation of eAE1
main ligand concentrations (Cl− and HCO3
−). Additionally, the AM import kinetics was analyzed
enzymatically and colorimetrically. In NH4
+ buffer, RBCs concentration-dependently swelled and
lysed when [NH4
+] exceeded 100 mM. Cell swelling and hemolysis were tightly regulated by chloride
concentration. The complete substitution of chloride with glutamate prevented NH4
+-induced cell
swelling and hemolysis, and the restoration of [Cl−] dose-dependently amplified the rates of RBC
swelling and lysis and the percentage of hemolyzed cells. Similarly, eAE1 inhibition impeded cell
swelling and completely prevented hemolysis. Accordingly, eAE1 inhibition, or a lack of chloride
anions in the buffer, significantly decreased NH4
+ import. Our data indicate that the eAE1-mediated
chloride gradient is required for AM transport. Taken together, our data reveal a new player in AM
transport in RBCs.