Anion Transport in Red Blood Cells. III. Sites and Sidedness of Inhibition by High-Affinity Reversible Binding Probes

Abstract

Studies of binding of the reversible inhibitor DNDS (for abbreviations, see Nomenclature) and red blood cell membranes revealed 8.6 ± 0.7 × 10⁵ high-affinity binding sites per cell (K_D = 0.8 ± 0.4 μM). Under conditions of “mutual depletion,” inhibition studies of anion exchange revealed 8.0 ± 0.7 × 10⁵ DNDS inhibitory sites per cell (K_D = 0.87 ± 0.04 μM). Binding and kinetic studies with DNDS indicate that there are 0.8 - 0.9 × 10⁶ functional anion transport sites per red blood cell. The transport of DNDS displayed high temperature and concentration dependencies, chemical specificity, susceptibility to inhibition by DIDS, and differences between egress and ingress properties. Under conditions of no DNDS penetration (e.g., 0°C), inhibition of anion exchange by DNDS showed marked sidedness from the outside inhibitions and were demonstrable at micromolar concentrations, whereas from the inside no inhibition occurred even at millimolar concentrations.