Kinetics of bicarbonate transport in human red blood cell membranes at body temperature

J Gen Physiol. 1996 Dec;108(6):565-75. doi: 10.1085/jgp.108.6.565.

Abstract

We studied unidirectional [14C]HCO3- efflux from human resealed red cell ghosts with 1 mM acetazolamide under self-exchange conditions at pH = pH(i = o) 7.4-9.0 and 0-38 degrees C by means of the Millipore-Swinnex and continuous flow tube filtering techniques. 14CO2 loss from cells to efflux medium and further to the atmosphere was insignificant. [14C]HCO3- efflux was determined at pH 7.8, 38 degrees C under symmetric variation of the HCO3- concentrations (C(i = o)), and asymmetric conditions: C(i) varied, C(o) constant, or C(o) varied, C(i) constant. MM-fit, Jeff = Jmaxeff x C x (C + K1/2)-1, used to describe the concentration dependence of Jeff,o when only C(o) varied, yields at C(i) = 50 mM: K1/2o = 3.8 mMJ, Jmaxeff.o = 20 nmol cm-2 s-1; at C(i) = 165 mM: K1/2o = 10 mM, Jmaxeff.o = 32 nmol cm-2 s-1. When C(i) varied, noncompetitive self inhibition by HCO3- binding (inhibitor constant K1) to an intracellular site was included (MS-fit). Under conditions of (a) symmetry: C(i = o) = 9-600 mM, K1/2s = 173 mM, K1 = 172 mM, and Jmaxeff,s = 120 nmol cm-2 s-1, (b) asymmetry: C(o) = 50 mM, K1/2i = 116 mM, K1 = 136 mM, and Jmaxeff,i = 92 nmol cm-2 s-1. All flux parameters accord with the ping-pong model for anion exchange. The data for C(i) < 200 mM also fit well to the MM equation, but K1/2 and Jmaxeff are different from the MS-fit and are inconsistent with the ping-pong model. Thus, self-inhibition (MS-fit) must be included even at low concentrations. As at 0 degree C, the system is asymmetric: 8-10 times more unloaded transport sites face inward than outward when C(i = o). Jeff,s was not mono-exponentially dependent on temperature at 0-38 degrees C, indicating that the transmembrane anion transport is controlled by several rate constants with different temperature dependencies. Jeff,s was not significantly affected by increasing pH(i = o) from 7.4 to 7.8, but it decreased by 50% when pH was raised to 9.0.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • 4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid / pharmacology
  • Bicarbonates / antagonists & inhibitors
  • Bicarbonates / pharmacokinetics*
  • Biological Transport / drug effects
  • Body Temperature*
  • Erythrocyte Membrane / metabolism*
  • Humans
  • Hydrogen-Ion Concentration
  • Kinetics
  • Models, Biological
  • Temperature

Substances

  • Bicarbonates
  • 4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid