Complement pore genesis observed in erythrocyte membranes by fluorescence microscopic single-channel recording

Abstract

The formation and opening of single complement pores could be directly observed in erythrocyte ghosts by confocal laser-scanning microscopy employing the recently introduced method of fluorescence microscopic single-channel recording. Resealed sheep erythrocyte ghosts were incubated with human complement. By limiting the concentration of C8, the eighth component of complement, the fraction of cells rendered permeable for the small polar fluorescent probe Lucifer Yellow was varied between 0.50 and 0.90. Under each condition the flux rate, k, of Lucifer Yellow was determined for a substantial number of ghosts. By analysing the sample population distribution of k the flux rate k1 of ghosts with a single pore was found to be (4.8 +/- 1.1) x 10(-3) s-1 consistent with a pore radius of about 3.5 nm (35 A). The genesis of single complement pores was studied by continuous influx measurements while triggering pore formation by a temperature shift. Pore genesis was found to be a very slow process, proceeding on a time scale of several minutes. During pore genesis the influx curves had a sigmoid shape, which excluded the possibility that the pore was preformed on the membrane surface and subsequently inserted. However, the influx curves could be well simulated by a model which assumed that pores grow stepwise by sequential incorporation of C9 monomers. The model predicts conditions under which the incorporation of single monomers can be directly revealed.