The asymmetry of chloride transport at 38°C in human red blood cell membranes

Abstract

Band 3-mediated Cl- exchange in human red blood cells and resealed ghosts was measured at 38°C by the continuous flow tube method. When external Cl- concentration, C((o)), is varied with constant internal Cl- concentration, C((i)), the flux fits a simple Michaelis-Menten saturation curve (MM-fit), with K(1/2)/(o) = 3.8 ± 0.4 mM. When the Cl- concentration is varied simultaneously at both sides of the membrane in resealed ghosts (C((i)) = C((o)) = C((i=o)), the flux rises toward a flat maximum between 200 and 450 mM Cl-, and then decreases at very high C((i=o))). An MM-fit to the data with C((i=o)) < 500 mM gives K(1/2)/(s) of 106 ± 13 mM; fits including modifier site inhibition (MS fit) give an over threefold higher K(1/2)/(s). Despite this uncertainty, the intrinsic asymmetry of unloaded transport sites, A (defined as E((o))/E((i)) with C((i)) = C((o)), where E((i)) is the fraction of unloaded inward-facing sites and E((o)) is the fraction of unloaded outward-facing sites), calculated from K(1/2)/(s) and K(1/2)/(o), ranges only from 0.046 to 0.107. A new method, which uses the initial slope of a plot of Cl- flux versus C((i=o)), gives A values of 0.023 to 0.038. Flufenamic acid (FA) inhibits Cl- exchange by binding to an external site different from the transport site. At 38°C, FA binds 24-36 times more tightly to E((o)) than to E((i)). Estimates of A from FA inhibitory potency range from 0.01 to 0.05. All methods, including bicarbonate data from the preceding paper, indicate that at 38°C, like 0°C, far more band 3 molecules are in the E((i)) than in the E((o)) form. The agreement of various methods supports the ping-pong model for anion exchange, and demonstrates that the intrinsic asymmetry is very slightly, if at all, affected by temperature.