Effect of membrane potential on band 3 conformation in the human erythrocyte membrane detected by triplet state quenching experiments.
The triplet lifetime and absorption anisotropy decay of eosin-labeled band 3 was measured in resealed erythrocyte ghosts. Membrane potentials were generated by the addition of valinomycin in the presence of a K+ gradient. Neither negative nor positive membrane potentials had any detectable effect on the rotational diffusion of band 3 nor on the eosin triplet lifetime. The membrane potential did, however, affect quenching of the eosin triplet state by I- and TEMPO (2,2,6,6-tetramethylpiperidine-N-oxyl). Quenching was enhanced by a negative membrane potential (negative inside) and reduced by a positive membrane potential. In addition, it was found that a negative membrane potential enhanced the efficiency of eosin labeling of band 3 in intact erythrocytes. A positive membrane potential had the opposite effect. These results indicate that the eosin binding site on band 3 becomes more accessible to the extracellular aqueous phase in the presence of a negative membrane potential and less accessible in the presence of a positive membrane potential. Quenching by I- and TEMPO of the triplet state of eosin-labeled band 3 was further investigated as a function of pH. Quenching by TEMPO and its dependence on membrane potential were relatively insensitive to pH. In contrast, the rate of quenching by I- showed a marked decrease over the range pH 5.5-9.5. Moreover, the effect of a negative membrane potential on I- quenching also varied with pH. These results are discussed on the supposition that the eosin probe is located in the anion access channel of band 3.(ABSTRACT TRUNCATED AT 250 WORDS)
Wyatt
K
K
Department of Chemistry and Biological Chemistry, University of Essex, Colchester, U.K.
Cherry
R J
RJ
D001457:Anion Exchange Protein 1, Erythrocyte / Q000737:chemistry*; D004801:Eosine Yellowish-(YS) / Q000031:analogs & derivatives; D004910:Erythrocyte Membrane / Q000737:chemistry*; D005454:Fluorescence Polarization; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008564:Membrane Potentials / Q000502:physiology*; D011193:Potassium Iodide / Q000737:chemistry; D011487:Protein Conformation; D013050:Spectrometry, Fluorescence; D013113:Spin Labels; D013329:Structure-Activity Relationship
D001457:Anion Exchange Protein 1, Erythrocyte; D013113:Spin Labels; D011193:Potassium Iodide; C033531:eosin maleimide; D004801:Eosine Yellowish-(YS)