The influence of electrochemical gradients of Na+ and K+ upon the membrane binding and pore forming activity of the terminal complement proteins

The hemolytic activity of the terminal complement proteins (C5b-9) towards erythrocytes containing high potassium concentration has been reported to be dramatically increased when extracellular Na+ is substituted isotonically by K+ (Dalmasso, A.P., et al., 1975,J. Immunol. 115:63–68). This phenomenon was now further investigated using resealed human erythrocyte ghosts (ghosts), which can be maintained at a nonlytic osmotic steady state subsequent to C5b-9 binding: (1) The functional state of C5b-9-treated ghosts was studied from their ability to retain trapped [14C]-sucrose or [3H]-inulin when suspended either in the presence of Na+ or K+. A dramatic increase in the permeability of the ghost membrane to both nonelectrolytes-in the absence of significant hemoglobin release-was observed for C5b-9 assembly in the presence of external K+. (2) The physical binding of the individual125I-labeled terminal complement proteins to ghost membranes was directly measured as a function of intra- and extracellular K+ and Na+. The uptake of125I-C7,125I-C8, and125I-C9 into membrane C5b-9 was unaltered by substitution of Na+ by K+. (3) The binding of the terminal complement proteins to ghosts subjected to a transient membrane potential generated by the K+-ionophore valinomycin (in the presence of K+ concentration gradients) was measured. No significant change in membrane binding of any of the C5b-9 proteins was detected under the influence of both depolarizing and hyperpolarizing membrane potentials. It can be concluded that the differential effect of Na+ versus K+ upon the erythrocyte membrane isnot due to an effect upon the binding of the complement proteins to the membraneper se, but upon the functional properties of the assembled C5b-9 pore site.