Kinetics of pore formation by the Bacillus thuringiensis toxin Cry1Ac

AbstractAfter binding to specific receptors, Cry toxins form pores in the midgut apical membrane of susceptible insects. The receptors could form part of the pore structure or simply catalyze pore formation and consequently be recycled. To discriminate between these possibilities, the kinetics of pore formation in brush border membrane vesicles isolated from Manduca sexta was studied with an osmotic swelling assay. Pore formation, as deduced from changes in membrane permeability induced by Cry1Ac during a 60-min incubation period, was strongly dose-dependent, but rapidly reached a maximum as toxin concentration was increased. Following exposure of the vesicles to the toxin, the osmotic swelling rate reached a maximum shortly after a delay period. Under these conditions, at relatively high toxin concentrations, the maximal osmotic swelling rate increased linearly with toxin concentration. When vesicles were incubated for a short time with the toxin and then rapidly cooled to prevent the formation of new pores before and during the osmotic swelling experiment, a plateau in the rate of pore formation was observed as toxin concentration was increased. Taken together, these results suggest that the receptors do not act as simple catalysts of pore formation, but remain associated with the pores once they are formed

Activation of an ATP-dependent K+ conductance in Xenopus oocytes by expression of adenylate kinase cloned from renal proximal tubules

AbstractIn rabbit proximal convoluted tubules, an ATP-sensitive K+ (KATP) channel has been shown to be involved in membrane cross-talk, i.e. the coupling (most likely mediated through intracellular ATP) between transepithelial Na+ transport and basolateral K+ conductance. This K+ conductance is inhibited by taurine. We sought to isolate this K+ channel by expression cloning in Xenopus oocytes. Injection of renal cortex mRNA into oocytes induced a K+ conductance, largely inhibited by extracellular Ba2+ and intracellular taurine. Using this functional test, we isolated from our proximal tubule cDNA library a unique clone, which induced a large K+ current which was Ba2+-, taurine- and glibenclamide-sensitive. Surprisingly, this clone is not a K+ channel but an adenylate kinase protein (AK3), known to convert NTP+AMP into NDP+ADP (N could be G, I or A). AK3 expression resulted in a large ATP decrease and activation of the whole-cell currents including a previously unknown, endogenous K+ current. To verify whether ATP decrease was responsible for the current activation, we demonstrated that inhibition of glycolysis greatly reduces oocyte ATP levels and increases an inwardly rectifying K+ current. The possible involvement of AK in the KATP channel’s regulation provides a means of explaining their observed activity in cytosolic environments characterized by high ATP concentrations

Les phénomènes de surface du verre dans la mise au point d'une ultramicroélectrode à pH

Ce document n'a pas de résumé

Les phénomènes de surface du verre dans la mise au point d'une ultramicroélectrode à pH

Ce document n'a pas de résumé

Effect of Insect Larval Midgut Proteases on the Activity of Bacillus thuringiensis Cry Toxins▿

To test the possibility that proteolytic cleavage by midgut juice enzymes could enhance or inhibit the activity of Bacillus thuringiensis insecticidal toxins, once activated, the effects of different toxins on the membrane potential of the epithelial cells of isolated Manduca sexta midguts in the presence and absence of midgut juice were measured. While midgut juice had little effect on the activity of Cry1Aa, Cry1Ac, Cry1Ca, Cry1Ea, and R233A, a mutant of Cry1Aa from which one of the four salt bridges linking domains I and II of the toxin was eliminated, it greatly increased the activity of Cry1Ab. In addition, when tested in the presence of a cocktail of protease inhibitors or when boiled, midgut juice retained almost completely its capacity to enhance Cry1Ab activity, suggesting that proteases were not responsible for the stimulation. On the other hand, in the absence of midgut juice, the cocktail of protease inhibitors also enhanced the activity of Cry1Ab, suggesting that proteolytic cleavage by membrane proteases could render the toxin less effective. The lower toxicity of R233A, despite a similar in vitro pore-forming ability, compared with Cry1Aa, cannot be accounted for by an increased susceptibility to midgut proteases. Although these assays were performed under conditions approaching those found in the larval midgut, the depolarizing activities of the toxins correlated only partially with their toxicities

Real-time fluorimetric analysis of gramicidin D- and alamethicin-induced K+ efflux from Sf9 and Cf1 insect cells

NRC publication: Ye

Real-time fluorimetric analysis of gramicidin D- and alamethicin-induced K+ efflux from Sf9 and Cf1 insect cells

NRC publication: Ye