Rate of rupture and reattachment of the band 3-ankyrin bridge on the human erythrocyte membrane

The principal bridge connecting the erythrocyte membrane to the spectrin-based skeleton is established by band 3 and ankyrin; mutations leading to reduced bridge formation or increased bridge rupture result in morphological and mechanical abnormalities. Because membrane mechanical properties are determined in part by the protein interactions that stabilize the membrane, we have evaluated the rates of rupture and reattachment of band 3-ankyrin bridges under both resting and mechanically stressed conditions. Toaccomplish this, we have examined the rate of ankyrin displacement from inside-out vesicles by the hexahistidine-tagged cytoplasmic domain of band 3, cdb3-(His)₆ and the rate of substitution of cdb3-(His)₆ into endogenous band 3-ankyrin bridges in resealed erythrocytes in the presence and absence of shear stress. We demonstrate that 1) exogenous cdb3-(His)₆ displaces endogenous ankyrin from IOVs with a half-time and first order rate constant of 42 ± 4 min and 0.017 ± 0.0058 min^-1, respectively; 2) exogenous cdb3-(His)₆ substitutes endogenous band 3 in its linkage to ankyrin in resealed cells with a half-time and first order rate constant of 12 ± 3.6 min and 0.060 ± 0.019 min^-1, respectively; 3) cdb3-(His)₆-mediated rupture of the band 3-ankyrin bridge in resealed cells results in decreased membrane mechanical stability, decreased deformability, abnormal morphology, and spontaneous vesiculation of the cells; and 4) the above on/off rates are not significantly accelerated by mechanical shear stress. We conclude that the off rates of the band 3-ankyrin interaction are sufficiently slow to allow sustained erythrocyte deformation without loss of elasticity.