The Journal of Membrane Biology

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.

The question of whether a single molecule can account for every observed swelling-activated Cl− current deserves to be addressed and biophysical description seems to be an adequate criterion to classify these channels. We studied the biophysical properties of swelling-activated Cl− currents in 9HTEo-cells using whole-cell and outside-out patch clamp recordings. Hypotonic shock activated outwardly rectifying currents that inactivated at potentials higher than 20 mV. The decay phase of the current was well fitted by two exponential functions and both time constants were voltage-dependent. Two voltage-dependent time constants were also necessary to describe reactivation. The midpoint of current inactivation was 54 mV. The voltage dependence of kinetics did not significantly change by modifying the extracellular NaCl concentration while the inactivation midpoint slightly shifted. In conclusion, our results indicate that the voltage-dependent properties of the swelling-activated Cl− currents in 9HTEo- cells are largely independent from the extracellular ionic strength and the extracellular Cl− concentration. Excised patches from cells exposed to hypotonic shock showed single channel currents that inactivated at positive membrane potentials and displayed chord conductance of ∼60 pS at 100 mV and of ∼20 pS at −80 mV. The permeability sequence for the single channel was I− > Br− > Cl− > gluconate and currents were blocked by Reactive blue 2. These properties indicate that intermediate conductance outwardly rectifying channels are responsible for the macroscopic swelling-activated current.

We investigated the effect of several parameters, such as temperature, pH and proteins, on the fusion between synaptosomes, freshly isolated from rat brain cortex, and large unilamellar phosphatidylserine liposomes. These studies were carried out in both peroxidized and nonperoxidized synaptosomes. Mixing of membrane lipids was monitored using a fluorescence resonance energy transfer assay. Ascorbate (0.8 mm)/ Fe2+ (2.5 μm)-induced peroxidation of synaptosomes enhanced the fusion process (twofold) which may reflect an increase in synaptosomal protein hydrophobicity and hence a facilitation of intermembrane aggregation. The fusion process was shown to be temperature sensitive, a reduction in the extent being observed (twofold) as the temperature was lowered from 37 to 25°C. This effect may be due to changes in membrane fluidity. The fusion process is pH dependent, an increase in both kinetics and extent being observed when the pH was lowered from 7.4 to 5.5. A significant inhibition (92% at pH 7.4; 35% at pH 5.5) of the interaction between synaptosomes and liposomes by trypsin pretreatment of synaptosomes was found, thus indicating that the fusion reaction is a protein-mediated process. The inhibitory effect of trypsin at pH 5.5 is not so strong as that at physiological pH. These results suggest that, in addition to the involvement of proteins, nonspecific interactions between the synaptosomal and liposomal membranes under acidic conditions may also play a role in the fusion process. The investigation of binding of synaptosomes to liposomes under several experimental conditions provided evidence for the participation of proteins in membrane aggregation, as well as for the role of electrostatic forces in this process, at mild acidic pH.