Dynamics and interactions of the anion channel in intact human erythrocytes: an electron paramagnetic resonance spectroscopic study employing a new membrane-impermeant bifunctional spin-label.
We have developed a new membrane-impermeant, bifunctional spin-labeling reagent, bis-(sulfo-N-succinimidyl) doxyl-2-spiro-4'-pimelate (BSSDP), and employed it in an electron paramagnetic resonance (EPR) study of the rotational diffusion of the anion-exchange channel (band 3) in intact human erythrocytes. BSSDP reacts in a covalent manner and with high specificity with the extracytoplasmic domain of band 3, forming a complex in which the spin-label is immobilized on the protein. The linear EPR spectrum of BSSDP-labeled intact erythrocytes is characteristic of a highly immobilized, spatially isolated nitroxide probe. The saturation-transfer EPR spectrum of the same sample indicates that the anion channel in intact erythrocytes exhibits rotational dynamics in the 0.1-1 ms correlation time range at 20 degrees C. Rotational dynamics in this motional domain are consistent with a strong interaction of the anion-exchange channel with the erythrocyte cytoskeleton. The saturation-transfer EPR spectrum of ghosts prepared from BSSDP-labeled erythrocytes indicates a significant increase in rotational mobility of the anion channel, suggesting a significant disruption on lysis of interactions between the anion channel and the cytoskeleton.
D001457:Anion Exchange Protein 1, Erythrocyte / Q000378:metabolism; D000838:Anions; D004578:Electron Spin Resonance Spectroscopy / Q000379:methods; D004912:Erythrocytes / Q000378:metabolism*; D006801:Humans; D007202:Indicators and Reagents; D007473:Ion Channels / Q000378:metabolism*; D007700:Kinetics; D013058:Mass Spectrometry; D010446:Peptide Fragments / Q000032:analysis; D013113:Spin Labels / Q000138:chemical synthesis*; D013388:Succinimides / Q000138:chemical synthesis* / Q000378:metabolism
D001457:Anion Exchange Protein 1, Erythrocyte; D000838:Anions; D007202:Indicators and Reagents; D007473:Ion Channels; D010446:Peptide Fragments; D013113:Spin Labels; D013388:Succinimides; C049668:bis(sulfo-N-succinimidyl)doxyl-2-spiro-4'-pimelate