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

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°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 significant. [14C]HCO3/- efflux was determined at pH 7.8, 38°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, J(eff) = J(max)/(eff) · C · (C + K(1/2)-1, used to describe the concentration dependence of J(eff,o) when only C((o)) varied, yields at C((i)) = 50 mM: K(1/2)/(o) = 3.8 mMJ, J(max)/(eff,o)= 20 nmol cm-2 s-1; at C((i)) = 165 mM; K(1/2)/(o) = 10 mM, J(max)/(eff,o) = 32 nmol cm-2 s-1. When C((i)) varied, noncompetitive self inhibition by HCO3/- binding (inhibitor constant K(I)) to an intracellular site was included (MS-fit). Under conditions of (a) symmetry; C((i=o)) = 9-600 mM, K(1/2)/(s) = 173 mM, K(I) = 172 mM, and J(max)/(eff,s) = 120 nmol cm-2 s-1, (b) asymmetry; C((o)) = 50 mM, K(1/2)/(i) = 116 mM, K(I) = 136 mM, and J(max)/(eff,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 K(1/2) and J(max)/(eff) 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°C, the system is asymmetric; 8-10 times more unloaded transport sites face inward than outward when C((i=o)), J(eff,s) was not monoexponentially dependent on temperature at 0-38°C, indicating that the transmembrane anion transport is controlled by several rate constants with different temperature dependencies, J(eff,s) was not significantly affected by increasingly pH((i=o)) from 7.4 to 7.8, but it decreased by 50% when pH was raised to 9.0.