RED-CELL ECHINOCYTOGENESIS IS CORRELATED TO THE RECRUITMENT OF EXTERNAL BAND-3 CONFORMATIONS

For human erythrocytes the bilayer-couple model predicts a reversible crenation for increasing ratios of external to internal membrane monolayer area. This can be proven experimentally by changing the ratio of lipids in the two monolayers. Under physiological conditions with no addition of membrane-active compounds, active lipid translocation and passive flip-flop are comparatively slow. Nevertheless, individual erythrocytes underwent dramatic shape changes within seconds when the suspension medium was changed. After cell suspension in low ionic strength solutions a rapid formation of echinocytes was observed. This suspension effect was also found at high ionic concentrations when Cl- was replaced by SO42-, an ion transported very slowly by band 3. The band-3 anion-exchange protein occupies about 10% of the total membrane area. Band-3 conformers are asymmetrically distributed with about 90% of the transport sites facing the inside. Specific band-3 inhibitors can induce echinocytes. These inhibitors are known to recruit the band-3 conformation with he transport site exposed to the external medium, e.g. 4,4'-diisothio-cyanatostilbene-2,2'-disulfonic acid, 4-acet-amido-4'-isothiocyana-tostilbene-2,2'-disulfonic acid, flufenamic acid, furosemide and salicylic acid. Other inhibitors, e.g. phenylglyoxal and niflumic acid, which do not recruit a certain conformation, are not echinocytogenic. Physiologically, band 3 ensures the equilibration of internal and external anions like Cl- and HCO3- which are distributed according to the transmembrane potential (TMP). In the literature a correlation of the TMP and the band-3 conformation, as well as a correlation of the TMP and the erythrocyte shape, is described. In the proposed model, conformational changes may significantly alter the monolayer area ratio. Low external Cl- concentrations, negative TMPs or inhibitors may recruit the band-3 transport site towards the outside solution. It is proposed that this increases the bulk of the protein in the external monolayer and subsequently induces an echinocytogenic shape transformation according to the bilayer couple model.