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  • mitoxantrone Introduction Erythrocyte membrane proteins or t

    2021-11-30

    Introduction Erythrocyte membrane proteins or their orthologs are found in almost all mitoxantrone of the body [1,2]. Because of this compositional similarity and the erythrocyte membrane's accessibility, the red blood cell membrane (RBCM) has served as a crude model for mammalian plasma membranes for many years [2,3,4,5]. Current models of the red blood cell membrane (RBCM) depict a lipid bilayer embedded with a diversity of membrane-spanning proteins anchored to a cortical spectrin cytoskeleton via several bridging molecules such as ankyrin, adducin, protein 4.1 and protein 4.2 [3,4]. Most diagrams of the membrane also show a glucose transporter (GLUT1) anchored to adducin at the junctional spectrin-actin complex [3,4]. Indeed, an interaction between GLUT1, adducin and dematin seems assured based on surface labeling, immunoprecipitation, and proteomic studies [5]. However, GLUT1 also has been proposed to interact with other RBCM proteins, including the cytoplasmic domain of band 3 [6] and stomatin [7], suggesting its association with the membrane may not be so simplistic. Based on past studies demonstrating that different motile populations of membrane-spanning proteins can be characterized by “single particle tracking” experiments, where the diffusion paths of the different populations are imaged and their diffusions coefficients calculated from diffusion trajectories as a function of time [8], we undertook to determine the motile population(s) of GLUT1 in the RBCM. In the studies below, we covalently react a GLUT1-specific biotinylated ligand, ATB-BMPA [9] with glucose transporters in intact RBCs and then employ a streptavidin-linked fluorophore to track the movement of single GLUT1 molecules in intact RBCs. The data we obtain demonstrate that there are at least 3 subpopulations of GLUT1 in the membranes of whole RBCs and that only one subpopulation diffuses with a diffusion coefficient similar to that of adducin-associated band 3.
    Materials and methods
    Results and discussion
    Conclusion Current illustrations of the structure of the human erythrocyte membrane display two major complexes of membrane-spanning proteins [3,4], one comprised of band 3, band 4.1, glycophorins A and C, Duffy antigen, Kx, GLUT1, and stomatin, which are anchored directly or indirectly to the junctional complex, and a second containing band 3, glycophorin A, Rh proteins, CD47, LW, and glycophorin B tethered to the ankyrin complex. However, this standard depiction is almost certainly over-simplified. Thus, as demonstrated in this paper, GLUT1 exists in at least three different subpopulations, with other subpopulations obviously possible but not resolvable using our technique. While no information is available on the compositions of these subpopulations, they almost certainly correspond to interactions of GLUT1 with different membrane protein complexes, as the three populations are not likely different sized oligomers of GLUT1, since Saffman and Delbruck [29] have shown that changes in the cross-sectional area of a membrane-spanning protein will only change its diffusion coefficient by a factor proportional to the square root of the area increase. Thus, if a tetrameric GLUT1 were to associate into octamers, the diffusion coefficient would only decrease by 40%. Because the differences in Dμ are 3 orders of magnitude, these differences are almost certainly not solely due to different oligomeric forms of the transporter. By the same argument, small changes in the sizes of the GLUT1-containing membrane-spanning protein complexes cannot account for the changes in Dμ. Instead, the three subpopulations must derive from either major differences in the sizes of its membrane-spanning protein complexes or differences in their tethers to the spectrin-actin cytoskeleton (or both). Moreover, the fact that two of the motile subpopulations of GLUT1 do not coincide with the motile subpopulations of band 3 argues that at least two of the subpopulations of GLUT1 cannot be associated with band 3. Taken together, these data suggest that the standard diagrams of the human erythrocyte membrane remain oversimplified, even with the increasing complexity associated with each new iteration, suggesting that there is still a lot to learn about this simple model of human plasma membranes.