Band 3, the anion exchanger of the erythrocyte membrane, is also a flippase

Abstract

The transbilayer reorientation (flip-flop) of the long-chain amphiphilic anion DENSA ( 5-(N- decyl)aminonaphthalene-2- sulfonic acid ) in the erythrocyte membrane was studied by fluorescence spectroscopy. DENSA intercalates into the membrane at a high membrane/water partition coefficient (3.2 · 10 5) and rapidly reorients from the outer to the inner layer in a first order process ( k = 0.11 min −1 , 37°C, pH 7.4) leading to a steady-state distribution inner:outer layer of about 30:70. The activation energy of the fully reversible and symmetric flip process is about 110 kJ/mol. DIDS and various other established covalent and non-covalent inhibitors of anion transport via the erythrocyte anion exchanger, band 3 (AE 1), suppress the flip to a minimum of about 30–35% of the control. The flip is also inhibited by Cl − with a half maximal inhibitory concentration equal to that required for the inhibition of the exchange flux of ordinary anions via band 3. These findings indicate the involvement of a band 3 mediated (DIDS-sensitive) component of the flip and a DIDS-insensitive one, possibly involving, at least to some extent, simple transbilayer ‘diffusion’. This latter component is stimulated by diamide, an SH oxidant known to increase the permeability of the membrane lipid domain of the erythrocyte. Alcohols (butanol, hexanol) accelerate both flip components. Papain treatment, known to inhibit ‘ordinary’ anion exchange, accelerates both flip and flop. The results suggest that band 3 protein, besides being a conventional transporter of anions, can act as a flippase translocating anionic, membrane-intercalated amphiphiles approaching the transporter from the lipid domain. The flippase mode of operation of band 3 must, however, differ in its mechanism from the conventional exchange mode.