Dimeric association of band 3 in the erythrocyte membrane demonstrated by protein diffusion measurements.

Abstract

BAND 3, a major constituent of the human erythrocyte membrane, comprises a class of membrane-spanning proteins which are implicated in anion and possibly other transport functions1–4. Various observations indicate that band 3 proteins may exist as dimers in the membrane5–9. The most direct indication has been obtained from chemical cross-linking experiments. The band 3 dimer is an early product of various cross-linking reactions7, and the particular case of cross-linking by oxidative disulphide bridge formation results in almost quantitative dimerisation of band 3 (ref. 8). However, cross-linking experiments are in general inconclusive for they do not indicate whether a successful cross-linkage reflects a naturally occurring stable complex, or is the result of collisions between independent proteins which rapidly diffuse over small distances. In the recent elegant studies of Kiehm and Ji9, rapid photochemical cross-linking was achieved by photolysing with a millisecond flash. Whilst these experiments undoubtedly provide the best evidence so far obtained for the existence of the band 3 dimer, they still leave some doubts concerning the possibility of collision-induced crosslinks. These can only be ruled out if the mean time between collisions is shown to be much longer than the lifetime of the flash-induced reactive species. The latter time was assumed to be short (of the order of 1 ms) on the basis of solution studies but was not measured in the experimental conditions. The collision frequency is a function of the local lateral diffusion coefficient. Band 3 diffusion over distances >1 µm seems to be slow in the erythrocyte membrane10,11, but as discussed elsewhere12, this may reflect restrictions to long range diffusion and does not rule out the possibility of more rapid local diffusion. If the local diffusion coefficient is in the range 10−9 to 10−10 cm2 s−1, the mean time between collisions is also of the order of 1 ms (ref. 9). A lateral diffusion coefficient in this range is consistent with the rotational diffusion of band 3 (ref. 12). Here we present a new physical approach to determining the self-association of band 3, based on the measurement of rotational diffusion. The rotational diffusion coefficient of an intrinsic membrane protein is strongly dependent on the diameter of its cross-section in the plane of the membrane. We show that cross-linking band 3 into dimers produces no observable decrease in the rotational diffusion coefficient of band 3 in the membrane. We therefore conclude that the dimer pre-exists in the membrane and is not a product of the cross-linking reaction following random collisions.