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

Abstract

Band 3-mediated C1-exchange in human red blood cells and resealed ghosts was measured at 38°C by the continuous flow tube method. When external C1-concentration, C (°), is varied with constant internal C1-o concentration, C (i), the flux fits a simple Michaelis-Menten saturation curve (MM fit), with KI/2 = 3.8 2 0.4 mM. When the CI-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 C1-, and then decreases at very high C li=°). An MM fit to the data with C {i = o/ < 500 mM gives KI~ 2 of 106 + 13 mM; fits including modifier site inhibition (MS fit) give an over threefold higher K~) 2. Despite this uncertainty, the intrinsic asymmetry of unloaded transport sites, A (defined as E{°)/E I~) with C ~) = C °~, where E (i) is the fraction of unloaded inward-facing sites and E ~°) is the fraction of unloaded outward-facing sites), calculated from KIll2 and KI/°2, ranges only from 0.046 to 0.107. A new method, which uses the initial slope of a plot of C1 flux versus C ~i -°), gives A values of 0.023 to 0.038. Flufenamic acid (FA) inhibits C1-exchange by binding to an external site different from the transport site. At 38°C, FA binds 24-36 times more tightly to E ~°) 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 0/ than in the E ~°) 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.

The permeability of red blood cells to chloride, urea, and water

“…Both k and P depend in a complex manner on the intracellular and extracellular chloride concentrations and the asymmetric affinities of chloride to the transporter (see Knauf and Brahm, 1989;Gasbjerg et al, 1996;Knauf et al, 1996). The concentration-dependent ('apparent') P Cl was determined under equilibrium conditions with fixed extracellular chloride concentrations of 127 (Amphiuma) or 150mmoll -1 (chick, duck, dog and human), where the transport system is almost completely saturated in human RBC.…”

Mouse Ae1 E699Q mediates SO42−i/aniono exchange with [SO42−]i-dependent reversal of wild-type pHo sensitivity

“…The modifier site likely has no physiological significance in the red blood cell, since Cl Ϫ and HCO 3 Ϫ at their physiological systemic plasma concentrations interact with it only weakly. Model-dependent estimates of modifier site K 1/2 for Cl Ϫ o range from 270 to 375 mM (20). However, the modifier site on kidney AE1 of the type A intercalated cell might gain importance under antidiuretic conditions, in which renal medullary interstitial [Cl Ϫ ] can be sustained at 300 mM in the human and at 600 -1,200 mM or more in rodents.…”

Cysteine-directed cross-linking localizes regions of the human erythrocyte anion-exchange protein (AE1) relative to the dimeric interface

“…Human erythrocyte AE isoform 1 (AE1) is the bestcharacterized member [1,2]. Although the kinetics of anion exchange have been studied extensively for AE1 [3,4], relating these kinetic data to a mechanistic model for protein function requires structural information describing the anion-translocation channel. Considerable data are available concerning the topology of the AE1 membrane domain.…”

“…TwoAbbreviations used : AE, anion exchanger ; AE1, AE isoform 1 ; AE1C − , cysteineless form of AE1 ; BCECF-AM, 2h,7h-bis-(2-carboxyethyl)-(5 and 6)-carboxyfluorescein acetoxymethyl ester ; biotin maleimide, 3-(N-maleimidylpropionyl)biocytin ; BMH, bismaleimidohexane ; C 12 E 8 , octaethylene glycol monodecyl ether ; CuP, Cu(II)-(1,10-phenanthroline) 3 ; DTT, dithiothreitol ; EC, extracellular loop ; IC, intracellular loop ; o-PDM, N,Nh-ophenylenedimaleimide ; p-PDM, N,Nh-p-phenylenedimaleimide ; TM, transmembrane segment. 1 Present address: Department of Clinical Laboratory Sciences, John Radcliffe Hospital, University of Oxford, Oxford OX3 9DU, U.K. 2 To whom correspondence should be addressed (e-mail joe.casey!ualberta.ca).…”