Treatment of hyphae of Neurospora crassa with antifungal plant defensins, i.e. Rs-AFP2 and Dm-AMP1 isolated from radish and dahlia seed, respectively, induced a rapid K+ efflux, Ca2+ uptake, and alkalinization of the incubation medium. The Rs-AFP2-induced alkalinization of the incubation medium could be inhibited with G-protein inhibitors. alpha-Hordothionin, an antifungal thionin from barley seed, caused a sustained increased Ca2+ uptake at subinhibitory concentrations but only a transient increased uptake at inhibitory concentrations. alpha-Hordothionin also caused increased K+ efflux and alkalinization of the medium, but these fluxes occurred more rapidly compared to those caused by plant defensins. Furthermore, alpha-hordothionin caused permeabilization of fungal hyphae to the non-metabolite alpha-aminoisobutyric acid and, in addition, altered the electrical properties of artificial lipid bilayers, consistently leading to rupture of the lipid bilayers. The plant defensins did not form ion-permeable pores in artificial membranes and did not exhibit substantial hyphal membrane permeabilization activity. Our results are consistent with the notion that thionins inhibit fungal growth as a result of direct protein-membrane interactions, whereas plant defensins might act via a different, possibly receptor-mediated, mechanism.

The outer membrane protein PulD (secretin) of Klebsiella oxytoca is required for transport of pullulanase across this membrane. We have purified a multimeric PulD complex from an Escherichia coli strain expressing all the proteins involved in pullulanase secretion. The outer membrane-anchored lipoprotein PulS was found to copurify with PulD. The molar ratio of the two proteins is close to 1:1, and the size of the complex is Ϸ1 MDa. Scanning transmission electron and cryo-electron microscopy analyses showed that the purified complex is a cylindrical structure having a central cavity of Ϸ7.6 nm and peripheral radial spokes. Fusion of proteoliposomes containing the purified complex with a planar lipid bilayer resulted in the appearance of small, voltage-activated, ion-conducting channels. We conclude that the central cavity seen in the electron microscope is part of a large gated channel and propose that the observed current f luctuations correspond to voltage-induced, relatively minor displacements of domains in the purified complex rather than to a complete opening of the secretin channel.

Mechanosensitive ion channels from Escherichia coli were studied in giant proteoliposomes reconstituted from an inner membrane fraction, or in giant round cells in which the outer membrane and the cell wall had been disrupted by a lysozyme-EDTA treatment and a mild osmotic shock. Patch-clamp experiments revealed the presence in these two preparations of an array of different conductances (100 to 2,300 pS in 0.1 M KCl) activated by stretch. The electrical activity induced by stretch in the native membrane was complex, due to the activation of several different conductances. In contrast, patches of proteoliposomes generally contained clusters of identical conductances, which differed from patch to patch. These experiments are consistent with the notion that these different conductances correspond to different proteins in the plasma membrane of E. coli, which segregate into clusters of identical channels on dilution involved in reconstitution in proteoliposomes. These conductances could be grouped into three subfamilies of poorly selective channels. In both preparations, the higher the conductance, the higher was the negative pressure needed for activation. We discuss the putative role of these channels as parts of a multicomponent osmoregulatory system.