TY - JOUR

T1 - Chloride Gradient Is Involved in Ammonium Influx in Human Erythrocytes

AU - Sudnitsyna, Julia

AU - Ruzhnikova, Tamara O.

AU - Panteleev, Mikhail A.

AU - Kharazova, Alexandra

AU - Gambaryan, Stepan

AU - Mindukshev, Igor V.

PY - 2024/7/5

Y1 - 2024/7/5

N2 - The ammonia/ammonium (NH3/NH4+, AM) concentration in human erythrocytes (RBCs)is significantly higher than in plasma. Two main possible mechanisms for AM transport, includingsimple and facilitated diffusion, are described; however, the driving force for AM transport is notyet fully characterized. Since the erythroid ammonium channel RhAG forms a structural unit withanion exchanger 1 (eAE1) within the ankyrin core complex, we hypothesized the involvement ofeAE1 in AM transport. To evaluate the functional interaction between eAE1 and RhAG, we used aunique feature of RBCs to swell and lyse in isotonic NH4+ buffer. The kinetics of cell swelling andlysis were analyzed by flow cytometry and an original laser diffraction method, adapted for accuratevolume sensing. The eAE1 role was revealed according to (i) the changes in cell swelling and lysiskinetics, and (ii) changes in intracellular pH, triggered by eAE1 inhibition or the modulation of eAE1main ligand concentrations (Cl− and HCO3−). Additionally, the AM import kinetics was analyzedenzymatically and colorimetrically. In NH4+ buffer, RBCs concentration-dependently swelled andlysed when [NH4+] exceeded 100 mM. Cell swelling and hemolysis were tightly regulated by chlorideconcentration. The complete substitution of chloride with glutamate prevented NH4+-induced cellswelling and hemolysis, and the restoration of [Cl−] dose-dependently amplified the rates of RBCswelling and lysis and the percentage of hemolyzed cells. Similarly, eAE1 inhibition impeded cellswelling and completely prevented hemolysis. Accordingly, eAE1 inhibition, or a lack of chlorideanions in the buffer, significantly decreased NH4+ import. Our data indicate that the eAE1-mediatedchloride gradient is required for AM transport. Taken together, our data reveal a new player in AMtransport in RBCs.

AB - The ammonia/ammonium (NH3/NH4+, AM) concentration in human erythrocytes (RBCs)is significantly higher than in plasma. Two main possible mechanisms for AM transport, includingsimple and facilitated diffusion, are described; however, the driving force for AM transport is notyet fully characterized. Since the erythroid ammonium channel RhAG forms a structural unit withanion exchanger 1 (eAE1) within the ankyrin core complex, we hypothesized the involvement ofeAE1 in AM transport. To evaluate the functional interaction between eAE1 and RhAG, we used aunique feature of RBCs to swell and lyse in isotonic NH4+ buffer. The kinetics of cell swelling andlysis were analyzed by flow cytometry and an original laser diffraction method, adapted for accuratevolume sensing. The eAE1 role was revealed according to (i) the changes in cell swelling and lysiskinetics, and (ii) changes in intracellular pH, triggered by eAE1 inhibition or the modulation of eAE1main ligand concentrations (Cl− and HCO3−). Additionally, the AM import kinetics was analyzedenzymatically and colorimetrically. In NH4+ buffer, RBCs concentration-dependently swelled andlysed when [NH4+] exceeded 100 mM. Cell swelling and hemolysis were tightly regulated by chlorideconcentration. The complete substitution of chloride with glutamate prevented NH4+-induced cellswelling and hemolysis, and the restoration of [Cl−] dose-dependently amplified the rates of RBCswelling and lysis and the percentage of hemolyzed cells. Similarly, eAE1 inhibition impeded cellswelling and completely prevented hemolysis. Accordingly, eAE1 inhibition, or a lack of chlorideanions in the buffer, significantly decreased NH4+ import. Our data indicate that the eAE1-mediatedchloride gradient is required for AM transport. Taken together, our data reveal a new player in AMtransport in RBCs.

KW - erythrocytes; anion exchanger 1; band 3; DIDS; DIBAC4(3)

KW - RhAG; ammonia; ammonium; chloride gradient 1. Introduction

KW - DIBAC4(3)

KW - DIDS

KW - RhAG

KW - ammonia

KW - ammonium

KW - anion exchanger 1

KW - band 3

KW - chloride gradient

KW - erythrocytes

UR - https://www.mendeley.com/catalogue/c75b8857-51b6-3277-a462-25467305c505/

U2 - 10.3390/ijms25137390

DO - 10.3390/ijms25137390

M3 - Article

VL - 25

JO - International Journal of Molecular Sciences

JF - International Journal of Molecular Sciences

SN - 1422-0067

IS - 13

M1 - 7390

ER -