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 medi um, but the reason for this striking asymmetry has remained obscure. T o resolve this question, we have measured the apparent Cl- affinity of the transport site of human red blood cell band 3 protein under vario us conditions by analyzing the Cl-35 NMR free induction decay (FID). T he [Cl-] that half-saturates the transport sites with [Cl-i] = [Cl-0] (K-1/2) in RBC membranes (ghosts) is 46 +/- 5 mM at 0 degrees C, while the K-1/2(0) (for half-saturation with [Cl-0] at constant [Cl-i]) 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, K-1/2 and K-1/2(0) are 41 +/- 14 and 4 6 +/- 12 mM, respectively. The EM-induced increase in K-1/2(0) with li ttle change in K-1/2 can be most simply interpreted as meaning that EM abolishes the effects of the translocation rate constants on K-1/2(0) so that K-1/2(0) and K-1/2 of EM-treated cells now both reflect the t rue dissociation constant for binding of Cl- to the external transport site, K-0. The fact that K-0 for a slowly transported anion, iodide, is nearly the same in EM-treated as in control cells indicates that EM does not significantly affect K-0 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, b oth unloaded and Cl-loaded transport sites are asymmetrically oriented toward the inside, and K-1/2(0) (in untreated cells) is much lower th an K-0.