Evidence for a protective role of the Gardos channel against hemolysis in murine spherocytosis

Abstract

It has been shown that mice with complete deficiency of all 4.1R protein isoforms (4.1-/-) exhibit moderate hemolytic anemia, with abnormal erythrocyte morphology (spherocytosis) and decreased membrane stability. Here, we characterized the Gardos channel function in vitro and in vivo in erythrocytes of 4.1-/- mice. Compared with wild-type, the Gardos channel of 4.1-/- erythrocytes showed an increase in Vmax (9.75 +/- 1.06 vs 6.08 +/- 0.09 mM cell x minute; P < .04) and a decrease in Km (1.01 +/- 0.06 vs 1.47 +/- 1.02 microM; P < .03), indicating an increased sensitivity to activation by intracellular calcium. In vivo function of the Gardos channel was assessed by the oral administration of clotrimazole, a well-characterized Gardos channel blocker. Clotrimazole treatment resulted in worsening of anemia and hemolysis, with decreased red cell survival and increased numbers of circulating hyperchromic spherocytes and microspherocytes. Clotrimazole induced similar changes in 4.2-/- and band 3+/- mice, indicating that these effects of the Gardos channel are shared in different models of murine spherocytosis. Thus, potassium and water loss through the Gardos channel may play an important protective role in compensating for the reduced surface-membrane area of hereditary spherocytosis (HS) erythrocytes and reducing hemolysis in erythrocytes with cytoskeletal impairments.

Figure 1.

Osmotic fragility curves and phthalate density profiles for 4.1^-/-, 4.2^-/-, β-adducin^-/-, and control erythrocytes. (Left) Percentage lysis is plotted compared with osmolarity of incubation medium for 4.1^-/- (•), 4.2^-/- (○), β-addu-cin^-/- (□), and control erythrocytes (▪). (Right) Phthalate density profile for 4.1^-/-, 4.2^-/-, β-adducin^-/-, and control. Symbols are as in panel A. Error bars depict SD.

Figure 2.

Calcium-activated potassium channel (Gardos channel) in control, 4.2^+/-, 4.2^-/-, and 4.1^-/- mouse erythrocytes. Gardos channel, ChTX (50 nM)-sensitive potassium influx was measured at varying concentrations of extracellular calcium in the presence of A23187. Data are expressed as mean ± SD of 3 separate experiments.

Figure 3.

Histograms for cell volume and cell hemoglobin concentration at baseline and after treatment with Gardos channel blockers. Histograms for erythrocyte volume (RBC volume) and cell hemoglobin concentration (RBC HC) and plot of RBC HC (x-axis) versus RBC volume (y-axis) are presented at baseline (left side) and after the specified in vivo treatment (right side). Rows from top to bottom: control mouse before and after 11 days of treatment with clotrimazole (CLT, 80 mg/kg twice a day); 4.1^-/- mouse before and after 11 days of treatment with the vehicle use for drug administration; 4.1^-/- mouse before and after 11 days of treatment with CLT (80 mg/kg twice a day); 4.1^-/- mouse before and after 7 days of treatment with CDA (100 mg/kg twice a day); 4.2^-/- mouse before and after 6 days of treatment with CLT (160 mg/kg twice a day); and band 3^+/- mouse before and after 7 days of treatment with CLT (80 mg/kg twice a day). Data obtained with the Bayer ADVIA 120 hematology analyzer. One representative mouse shown for each condition; similar results were obtained for the other animals.

Figure 4.

Effects of CLT administration on erythrocyte lifespan for 4.1^-/- mice. Compared with healthy controls, 4.1^-/- erythrocytes have a markedly reduced half-life.20 Mice were treated with CLT at a dose of 80 mg/kg twice a day for 11 days. Biotinylation was carried out as described in “Materials and methods,” and animals were bled at specified times. The percentage of biotinylated erythrocytes was determined by flow cytometry and is shown here for 2 representative animals. Data were obtained from 3 untreated and 4 CLT-treated 4.1^-/- mice (7 days of treatment).

Figure 5.

Effects of CLT administration on 4.1^-/- mice renal tubular iron accumulation. Micrographs (original magnification, 40 ×) of iron-stained kidney sections of vehicle (left) and CLT-treated (right) 4.1^-/- animals demonstrating diminished iron staining in the drug-treated group.