Inhibition of inorganic anion transport across the human red blood cell membrane by chloride-dependent association of dipyridamole with a stilbene disulfonate binding site on the band 3 protein

Abstract

The inhibition of inorganic anion transport by dipyridamole (2,6-bis(diethanolamino)-4,8-dipiperidinopyrimido[5,4- d]pyrimidine) takes place only in the presence of Cl −, other halides, nitrate or bicarbonate. At any given dipyridamole concentration, the anion flux relative to the flux in the absence of dipyridamole follows the equation: J rel = (1 + α 2[Cl −])/(1 + α 4[Cl −]) where α 2 and α 4 are independent of [Cl −] but dependent on dipyridamole concentration. At high [Cl −] the flux approaches α 2/α 4, which decreases with increasing dipyridamole concentration. Even when both [Cl −] and dipyridamole concentration assume large values, a small residual flux remains. The equation can be deduced on the assumption that Cl − binding allosterically increases the affinity for dipyridamole binding to band 3 and that the bound dipyridamole produces a non-competitive inhibition of sulfate transport. The mass-law constants for the binding of Cl − and dipyridamole to their respective-binding sites are about 24 mM and 1.5 μM, respectively (pH 6.9, 26 °C). Dipyridamole binding leads to a displacement of 4,4′-dibenzoylstilbene-2,2′-disulfonate (DBDS) from the stilbenedisulfonate binding site of band 3. The effect can be predicted quantitatively on the assumption that the Cl −-promoted dipyridamole binding leads to a competitive replacement of the stilbenedisulfonates. For the calculations, the same mass-law constants for binding of Cl − and dipyridamole can be used that were derived from the kinetic studies on Cl −-promoted anion transport inhibition. The newly described Cl − binding site is highly selective with respect to Cl − and other monovalent anion species. There is little competition with SO 4 2−, indicating that Cl − binding involves other than purely electrostative forces. The affinity of the binding site to Cl − does not change over the pH range 6.0–7.5. Dipyridamole binds only in its deprotonated state. Binding of the deprotonated dipyridamole is pH-independent over the same range as Cl − binding.