Role of Polyamine Structure in Inhibition of K+-Cl- Cotransport in Human Red Cell Ghosts

1. K+-Cl- cotransport in human red cell ghosts is inhibited by divalent inorganic cations, soluble polycations and amphipathic organic cations. These findings suggest a common mechanism of inhibition, namely, binding of the cations to negative charges at the surface of a hydrophobic structure. 2. We have characterized the inhibitory capacity of a number of polyamines in order to obtain information about the nature of the charges with which they interact. Neomycin inhibited swelling-stimulated cotransport. The diquaternary amines dimethonium and decamethonium were relatively ineffective inhibitors. These compounds are thought to shield negative charges, but not bind to them. 3. Comparison of a homologous series of polyamines indicated that primary amines were better inhibitors than secondary amines, that inhibition increased with the charge of the polyamine, and that inhibition increased as the distance separating the amines increased. 4. The results indicate that the negative charges to which polycations bind are multiple and mobile. Since they must be associated with a hydrophobic environment, it is likely that they are negatively charged phospholipids located in the inner leaflet of the bilayer membrane. 5. Heating red cells or ghosts to 49 C denatures spectrin. Heating markedly increased K+ uptake in swollen ghosts but not in shrunken ghosts. The increase in uptake was reversed when swollen ghosts were shrunk even though denaturation of spectrin was not reversed. Polyamines, which inhibited swelling-activated K+ uptake in control ghosts, similarly inhibited the increased uptake in heated ghosts. 6. We speculate that spectrin, which is closely associated with the inner bilayer leaflet, shields negative charges in a volume-dependent manner and so regulates volume-sensitive K+ transport.