Eosin-5-maleimide inhibits red cell Cl- exchange at a noncompetitive site that senses band 3 conformation

Abstract

Eosin-5-maleimide (EM) has been used as a fluorescent probe for the external-facing transport site of the human erythrocyte band 3 protein. Changes in chloride concentration at both sides of the membrane have no significant effect on the inhibitory potency of EM as a reversible inhibitor of Cl- exchange at 0 degrees C, however, demonstrating that it is not a competitive inhibitor. The affinity of EM for the form of band 3 with the transport site facing outward is approximately five times greater than for the form with the transport site facing the cytoplasm; binding of iodide to the external transport site causes no statistically significant decrease in affinity for EM. Eosin, without the maleimide moiety, is a slightly more potent inhibitor than is EM. Erythrosin, an analogue with four iodide atoms replacing the four bromide atoms in eosin, is a much more potent inhibitor, with a half-inhibitory concentration of only 3.1 microM, > 30 times lower than that of EM. Neither eosin nor erythrosin inhibition is affected by changes in chloride concentration as would be expected for a competitive inhibitor. Thus EM and the other eosin derivatives bind to a site separate from the external transport site, but one that is affected by the changes of transport site conformation from the inward-facing to the outward-facing state.

Selective inhibition of Cl− conductance in toad skin by blockers of Cl− channels and transporters

“…This kind of interference is surprising for agents that are supposed to be inhibitors of cotransporters or antiporters for Cl Ϫ movement across membranes. Erythrosine and eosin inhibit the anion exchanger (band 3) in red blood cell ghosts (13), and furosemide is well known as a selective blocker of the Na-K-2Cl symporter (6). Carrier-mediated transport affected by erythrosine, eosin, and furosemide is electroneutral, whereas G Cl represents the conductive translocation of Cl Ϫ across channels or channel-like structures.…”

“…Particularly effective is the substitution by I Ϫ on the anthracene ring. For the interference with the band 3 anion exchanger, it has been suggested that the larger lipophilicity of the I Ϫ moiety in erythrosine compared with that of Br Ϫ in eosin could facilitate the access to a binding site within hydrophobic environments (13). It should be noted that a band 3-like protein has been localized immunologically in toad skin MR cells (10).…”

“…context that the inhibition of the band 3 anion exchanger by eosin or erythrosine occurs by interference with a site separate from the transport site for anions (13). The resulting shift of the G/V relationship is the first example of such an inactivation and would be opposite to the effect exerted by theophylline (12), IBMX, and other xanthine derivatives lacking inhibition of the phosphodiesterase (Nagel, unpublished observations).…”

“…In the present study, we analyze the response of G Cl to application of different Cl Ϫ channel blockers from the group of arylaminobenzoates and particular inhibitors of cotransporters or antiporters involved in transmembranal Cl Ϫ movement. As inhibitors of Cl Ϫ -dependent cotransport, the loop diuretics furosemide and bumetanide (31) were selected, along with two derivatives of fluorescein, eosin and erythrosine, which inhibit the band 3 anion exchanger in red blood cells (13). The inclusion of the latter compounds was prompted by our observation that eosin-5-maleimide reversibly blocks the voltageactivated G Cl in toad skin (23).…”

Detection of Cl- binding to band 3 by double-quantum-filtered 35Cl nuclear magnetic resonance

“…To achieve nearly complete binding of band 3 with DNDS, enough DNDS was added to the samples to achieve more than 98% inhibition of Clexchange. Based on the measurement that ID50 for DNDS is 4.11 ,uM in 150 mM Clmedium (Knauf et al, 1993b), the final concentration of DNDS in the sample was at least 400 p.M (based on total sample volume).…”