Source of transport site asymmetry in the band 3 anion exchange protein determined by NMR measurements of external Cl- affinity.
Flux measurements indicate that a far greater number of unloaded band 3 anion transport sites face the cytoplasm than face the external medium, but the reason for this striking asymmetry has remained obscure. To resolve this question, we have measured the apparent Cl- affinity of the transport site of human red blood cell band 3 protein under various conditions by analyzing the 35Cl NMR free induction decay (FID). The [Cl-] that half-saturates the transport sites with [Cli] = [Clo] (K1/2) in RBC membranes (ghosts) is 46 +/- 5 mM at 0 degree C, while the Ko1/2 (for half-saturation with [Clo] at constant [Cli]) of intact cells is 3.2 +/- 2.1 mM. When cells were pretreated with EM, an inhibitor of band 3 anion exchange that does not prevent Cl- binding to the external transport site, K1/2 and Ko1/2 are 41 +/- 14 and 46 +/- 12 mM, respectively. The EM-induced increase in Ko1/2 with little change in K1/2 can be most simply interpreted as meaning that EM abolishes the effects of the translocation rate constants on Ko1/2 so that Ko1/2 and K1/2 of EM-treated cells now both reflect the true dissociation constant for binding of Cl- to the external transport site, Ko. The fact that Ko for a slowly transported anion, iodide, is nearly the same in EM-treated as in control cells indicates that EM does not significantly affect Ko for chloride. Our results indicate that the true dissociation constants for Cl- at the inside and outside are very similar but that the rate constant for inward translocation is much larger than that for outward translocation. For this reason, both unloaded and Cl-loaded transport sites are asymmetrically oriented toward the inside, and Ko1/2 (in untreated cells) is much lower than Ko.
Liu
D
D
Department of Biophysics, University of Rochester Medical Center, New York 14642, USA.
Kennedy
S D
SD
Knauf
P A
PA
D001457:Anion Exchange Protein 1, Erythrocyte / Q000378:metabolism*; D001692:Biological Transport; D002712:Chlorides / Q000378:metabolism*; D004801:Eosine Yellowish-(YS) / Q000031:analogs & derivatives / Q000494:pharmacology; D004910:Erythrocyte Membrane / Q000187:drug effects / Q000378:metabolism; D004912:Erythrocytes / Q000187:drug effects / Q000378:metabolism*; D006801:Humans; D007455:Iodine / Q000378:metabolism; D009682:Magnetic Resonance Spectroscopy; D011487:Protein Conformation
D001457:Anion Exchange Protein 1, Erythrocyte; D002712:Chlorides; C033531:eosin maleimide; D007455:Iodine; D004801:Eosine Yellowish-(YS)