The erythrocyte anion exchange channel (band 3) is responsible for rapid transmembrane exchange of chloride and bicarbonate and is therefore a crucial component in the transport of carbon dioxide (as bicarbonate) from the distal tissues to the lungs. It is also a structurally important component of the erythrocyte membrane, comprising an estimated 25-30% of the total membrane-associated proteins. Further, it appears to be an important organizing center for the cell, having binding sites for a number of glycolytic enzymes and for cytoskeletal proteins. The long term goals are to elucidate the structural basis of the functions of band 3 as an organizing center for membrane and cytoskeletal proteins and as an anion exchange channel. Our modus operandi is to develop new structural and spectroscopic reagents which allow us to probe previously inaccessible aspects of these functions. One of these structural probes, bis(sulfosuccinimidyl suberate) (BS3), has been shown to form intersubunit and intrasubunit cross-links in the extracytoplasmic domain of the band 3 dimer. Further, BS3 reacts with residues which have been shown to play a functional role in anion transport. Both the functionally important intrasubunit site and the structurally important intersubunit site will be identified by labeling with [14C]BS3, digesting the labeled band 3, sequencing the labeled peptides, and aligning the peptide sequences with the sequence of band 3. A new heterobifunctional cross-linker will also be synthesized to probe the interaction of the anion channel with ankyrin, a cytoskeletal attachment protein, in intact human erythrocytes. The new membrane-impermeant, bifunctional spin label bis(sulfosuccinimidyl)-4-doxylpimelate (BSSDP) has been shown to covalently bind to the extracytoplasmic domain of band 3 in intact human erythrocytes. Electron paramagnetic resonance (EPR) spectroscopic techniques can then be used to monitor the rotational mobility of band 3 in intact erythrocytes. Hindered mobility of band 3 reflects anion channel-cytoskeleton interactions. Using this technique, the effects on these dynamic interactions of changes in the energy and ion balance of the intact cell and of selective disruption of cytoskeletal elements in ghosts will be studied. For this study, structural and isotopic homologs of BSSDP will be synthesized, and immunochemical methods for identifying BSSDP-labeled proteins will be developed.
