Cryohydrocytosis: increased activity of cation carriers in red cells from a patient with a band 3 mutation

Abstract

Background: Cryohydrocytosis is an inherited dominant hemolytic anemia characterized by mutations in a transmembrane segment of the anion exchanger (band 3 protein). Transfection experiments performed in Xenopus oocytes suggested that these mutations may convert the anion exchanger into a non-selective cation channel. The present study was performed to characterize so far unexplored ion transport pathways that may render erythrocytes of a single cryohydrocytosis patient cation-leaky.

Design and methods: Cold-induced changes in cell volume were monitored using ektacytometry and density gradient centrifugation. Kinetics, temperature and inhibitor-dependence of the cation and water movements in the cryohydrocytosis patient's erythrocytes were studied using radioactive tracers and flame photometry. Response of the membrane potential of the patient's erythrocyte membrane to the presence of ionophores and blockers of anion and cation channels was assessed.

Results: In the cold, the cryohydrocytosis patient's erythrocytes swelled in KCl-containing, but not in NaCl-containing or KNO(3)-containing media indicating that volume changes were mediated by an anion-coupled cation transporter. In NaCl-containing medium the net HOE-642-sensitive Na(+)/K(+) exchange prevailed, whereas in KCl-containing medium swelling was mediated by a chloride-dependent K(+) uptake. Unidirectional K(+) influx measurements showed that the patient's cells have abnormally high activities of the cation-proton exchanger and the K(+),Cl(-) co-transporter, which can account for the observed net movements of cations. Finally, neither chloride nor cation conductance in the patient's erythrocytes differed from that of healthy donors. Conclusions These results suggest that cross-talk between the mutated band 3 and other transporters might increase the cation permeability in cryohydrocytosis.

Figure 1.

Deformability profiles and density gradients of whole RBC populations kept in either sodium- or potassium-containing isotonic buffer on ice. (A) Deformability profiles at increasing osmolarity were run at room temperature in 18% dextran buffers containing exclusively sodium ions as cations. Osmoscans were recorded from RBC that were resuspended and stored at 0°C in buffers containing the indicated cations, from the patient A (AN: NaCl buffer, AK: KCl buffer), from the patient’s mother (BN: NaCl buffer, BK: KCl buffer), the patient’s father (CN: NaCl buffer, CK: KCl buffer), and from a random control sample from a healthy donor (con). The corresponding Percoll density profiles for the RBC of the CHC patient and his mother are shown next to the deformability profiles. (B). Density distribution of RBC stored for 1 h as indicated (RT for room temperature or at 0°C) and centrifuged at 0°C in isotonic buffer containing either Na⁺ or K⁺ as cations. (C) As in (B) except that the buffer and the Percoll solution were supplemented with 100 μM SITS where indicated.

Figure 2.

Plasma cation content, MCV, and hyperchromic RBC following incubation at different temperatures. (A) Changes in plasma K⁺, hyperchromic cell number and MCV during storage of blood from a healthy donor and the CHC patient at various temperatures with and without glucose. (B) Changes in plasma K⁺ concentration in patient’s whole blood (Hct 40%) stored at 0°C in the absence (□) and in the presence of 100 μM DIDS (▪) or 500 μM HOE-642 (▴). Curve fitting was done using a logistic sigmoid function. (C) Changes in plasma Na⁺ concentration in whole blood of the CHC patient during storage at 0°C in the absence (□) or the presence of 100 μM DIDS (▪) or 500 μM HOE-642 (▴). Linear regression was used for the curve fitting. Measurements were performed in triplicate and are shown as mean±SEM.

Figure 3.

Unidirectional potassium (⁸⁶Rb) influx into RBC. (A) Residual (ouabain-bumetanide-EGTA-insensitive) K⁺ influx in RBC from blood of a control, the patient’s mother, his brother and the CHC patient. Erythrocytes were resuspended in NaCl-containing medium and incubated at 37°C or 0°C (on ice) in the presence of ⁸⁶Rb. (B) Residual K⁺ influx in fresh and stored patient’s RBC in NaCl − and NaCH₃SO₄⁻containing medium at 37°C or at 0°C. Measurements were done in triplicate on each occasion and are presented as means ± SEM.