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 functions^1–4. Various observations indicate that band 3 proteins may exist as dimers in the membrane^5–9. 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 membrane^10,11, 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.