Kinetics of bicarbonate and chloride transport in human red cell membranes

Unidirectional [14C]HCO3- and 36Cl- efflux from human red cells and ghosts was studied under self-exchange conditions at pH 7.8 and 0 degrees C by means of the Millipore-Swinnex filtering technique. Control bicarbonate experiments showed that 14CO2 loss from the cells to the efflux medium was insignificant. The anion flux was determined under (a) symmetric variations of the anion concentration (C(i) = C(o) = 5-700 mM), and (b) asymmetric conditions with CAn constant on one side and varied on the other side of the membrane. Simple Michaelis-Menten-like kinetics (MM fit: J(eff) = J(eff)max.C/(K1/2 + C)) was used to describe anion flux dependence on C for (a) C(i) = C(o) = 5-100 mM, (b) C(i) = 6-100 mM, C(o) = constant, and (c) C(i) = constant, C(o) = 1-25 mM. At higher cellular concentrations noncompetitive self-inhibition by anion binding (inhibition constant Ki mM) to an intracellular site was included in the model (MS fit): J(eff) = J(eff)max.C(i)/[(K1/2 + C(i)).(1 + C(i)/Ki)]. The MM fits show that the external half-saturation constant, Ko1/2 ( = C(o)An for J(eff,o) = 1/2.j(eff,o)max) at C(o) = 1-25 mM is 1.5-2.4 mM (HCO3-) and 1.8-2.6 mM (Cl-). At C(o) = 1-260 mM Ko1/2 is 1.2-1.5 mM (HCO3-) and 1.4-1.8 mM (Cl-). The respective maximum flux, J(eff,o)max (nmol/[cm2.s]), for C(o) = 1-25 mM is 0.41-0.51 (HCO3-) and 0.28-0.38 (Cl-), and for C(o) = 1-260 mM 0.39-0.44 (HCO3-) and 0.27-0.31 (Cl-). The internal half-saturation constant, Ki1/2 mM is: MM fit (C(i) = 6-100 mM, C(o) = 50 mM), 18.0 mM (HCO3-) and 23.8 mM (Cl-); MS fit (C(i) = 6-920 mM, C(o) = 50 mM), 32.0 mM (HCO3-) and 45.1 mM (Cl-). The maximum flux, J(eff,i)max (nmol/[cm2.s]) is: MM fit; 0.50 (HCO3-) and 0.34 (Cl-); MS fit, 0.70 (HCO-3) and 0.50 (Cl-). The half-inhibition constants of the MS fit, Ki, are 393 mM (HCO3-) and 544 mM (Cl-). The MM fit shows that the symmetric half-saturation constant, Ks1/2, is 20.2 (HCO-3) and 23.9 (Cl-) mM, and J(eff,s)max is 0.51 (HCO3-) and 0.32 (Cl-) nmol/(cm2.s). The MS fit shows that for C = 5-700 mM Ks1/2 is 30.4 nM (HCO3-) and 50.1 mM (Cl-), and Ki is 541 mM (HCO3-) and 392 mM (Cl-).(ABSTRACT TRUNCATED AT 400 WORDS)