Abstract
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. To accomplish 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 +/- 14 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.
Highlights
In its simplest representation, the human erythrocyte membrane is thought to be comprised of a two dimensional cortical membrane skeleton connected to a lipid bilayer by multiple proteinprotein and protein-lipid interactions [1]
Concentration dependence and kinetics of cdb3(His)6 displacement of ankyrin from inside-out erythrocyte membrane vesicles (IOVs) – Before determining the kinetics of dissociation of the band 3-ankyrin bridge in IOVs, it was necessary to estimate the concentration of cdb3-(His)6 that would lead to maximal displacement of endogenous ankyrin under equilibrium conditions
We have provided evidence that the band 3-ankyrin dissociation constant in erythrocytes is 0.060 ± 0.019 min-1 (t1/2 = 12 ± 3.6 min), regardless of the presence or absence of shear stress
Summary
The human erythrocyte membrane is thought to be comprised of a two dimensional cortical membrane skeleton connected to a lipid bilayer by multiple proteinprotein and protein-lipid interactions [1]. While the equilibrium binding constants for most protein-protein interactions in the human red cell membrane have been reported, little information is available on the dynamics of the same protein-protein associations. Such dynamics are, very important because the “on” and “off” rates of major protein interactions determine the elasticity versus plasticity of a cell [11]. Any membrane that is stabilized by slow “off” rate constants in its protein scaffold will be unable to rearrange during short periods of deformation This resistance to rearrangement will result in the membrane’s elastic return to its original morphology upon removal of the external deforming force. Because the red cell must frequently pass through blood vessels that are less than half its ~ 8 μm diameter, it seemed important to characterize the “off” rate constants of the most structurally critical interactions in order to determine their contributions to the elastic and plastic properties of the cell
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