Chlorotetracycline induces calcium mediated shape changes in human erythrocytes. Is Ca asymmetrically distributed in the red cell membrane?

Calcium was localized in the red cell membrane by light microscopy using chlorotetracycline hydrochloride (CTC) as chelate probe. Treating human erythrocytes with CTC dissolved in saline free of divalent cations, leads to a 530 nm fluorescence emission in the cell border and to characteristic cell shape changes which were evaluated to assess intramembrane calcium distribution. CTC prevented and reverted erythrocyte crenation induced either by washing or superfusing the cells with saline. The ionophore A23187, EGTA and glucose depletion depressed the shape modifying effect of CTC. Thus, CTC appears to act on red cell shape by complex formation with membrane associated calcium. This is further confirmed by the failure of degraded CTC, devoid of metal binding capacity, to modify the crenated shape. The CTC effect can be reverted by superfusing the erythrocytes with CTC-free medium. Thus, calcium binds more tightly to the membrane than to CTC and is not displaced by the antibiotic. If the bilayer couple hypothesis [Sheetz, M.P., Singer, S.J., Proc. Natl. Acad. Sci. USA 71, 4457-4461 (1974)]applies, crenation is reverted by expansion of the inner membrane half relative to the outer membrane half. Expansion of the inner membrane half results from intercalation of CTC which binds to calcium. Thus, calcium in the red cell membrane preferentially occupies the inner leaflet of the bilayer.