SUMMARY1. The kinetics of photoresponses to flashes and steps of light of rods, from the retina of the newt Triturus cristatus, were analysed by recording the membrane current with a suction electrode.2. In dark-adapted conditions the relation between the normalized amplitude of the photoresponse at a fixed time 1 s after the onset of light and the light intensity could be fitted by an exponential or a polynomial relation. In the presence of a steady bright light the same relation could be fitted by a Michaelis-Menten relation.3. The kinetics of photoresponses to light stimuli was reconstructed using a model in which: (i) three molecules of guanosine 3',5'-cyclic monophosphate (cyclic GMP) open a light-sensitive channel; (ii) light activates the enzyme phosphodiesterase, which hydrolyses cyclic GMP, thus closing light-sensitive channels; (iii) Ca2" ions permeate through light-sensitive channels; and (iv) intracellular Ca21 inhibits, in a co-operative way, the enzyme cyclase, which synthesizes cyclic GMP. 4. The model reproduces the shortening of the time to peak of brief flash photoresponses from about 1080 ms to about 690 ms with brighter lights. The model also explains the shortening of the time to peak to 350 ms observed in the presence of a steady light and the lack of a further acceleration with brighter flashes of lights.5. The presence in the model of an intracellular calcium buffer accounts for the partial reactivation of the photocurrent following a step of light, lasting several seconds. The time course of this reactivation is not accelerated by a steady bright light both experimentally and in the model. 6. After the extinction to a long step of light the photocurrent showed a rapid partial reactivation, which was followed by a slow component of the photoresponse which extinguished with a rate constant of about 0 05 s-'. The model explains the origin of this slow component by assuming that the inactivation of excited rhodopsin is partially reversible. 7. The model is also able to explain the particular changes of kinetics when t To whom correspondence and reprint requests should be sent.

a-Toxin, a lethal hemolytic toxin secreted by Staphylococcus aureus, forms ionic channels of large size in lipid membranes. To investigate the mechanism of channel assembly we have studied the kinetics of pore formation on small unilamellar vesicles. We have used two assays of vesicle permeabilization: one is the release of a fluorescent molecule trapped in their inner compartment; the other is the dissipation of an imposed potential.Both methods indicate that the kinetics are complex consisting of an initial delay followed by a non-linear relaxation. The dependence of the pore formation rate and the extent of permeabilization on the toxin/vesicle ratio indicates that aggregation of 4 -10 preinserted toxin monomers underlies channel assembly.The pH dependence of permeabilization suggests that protonation of an acidic group of the toxin is a prerequisite to channel formation. Inclusion of cholesterol in the target vesicles potentiates a-toxin effects, in a dose-dependent way, possibly by facilitating its protonation.The location of the proton-binding site on the two adjacent aspartic acid residues in positions 127 and 128 of the toxin monomer is proposed.Staphylococcus aureus a-toxin is a lethal, hemolytic and dermonecrotic toxin secreted as a single water-soluble 33-kDa polypeptide [l -31. This protein has strong membranedamaging properties and causes erythrocyte lysis via a complex reaction which is currently thought to involve: (a) binding of native a-toxin to the cell membrane in monomeric form; (b) oligomerization of the toxin to form an amphiphilic hexameric complex that generates a transmembrane channel responsible for ion leakage; (c) colloid-osmotic shock ensuing from the leakage of ions and leading to the lysis of the cell.The oligomerization step is now well established. It has been shown in fact that a-toxin forms hexameric aggregates on interaction with mammalian cells [4 -61, artificial membranes [7, 81 as well as other hydrophobic agents such as deoxycholate [9] and plasma low-density lipoproteins [lo]. Also the formation of pores has been proved both on natural cells [5, 111 and on lipid bilayers [12, 131. The colloid-osmotic hypothesis has been demonstrated, at least for red blood cells, by the use of an osmotic protectant [14].Up to now, however, a direct demonstration that the toxin channel is indeed formed by the hexameric aggregate has not been carried out. To clarify this particular aspect we have studied the kinetics of pore formation on unilamellar lipid vesicles as a model system. MATERIALS AND METHODS ToxinsSamples of lyophilized S . aureus a-toxin were kindly donated by K. Hungerer (Behring, Marburg, FRG) and S. Inhibition experiments were performed using purified polyclonal anti-(a-toxin) antibodies raised in rabbit (a kind gift of Dr Bhakdi). Briefly, a-toxin was incubated for 30 min at room temperature with either saline (control) or antibodies (final toxin concentration was 10 pg/ml a-toxin in both cases); aliquots were used on small unilamellar vesicles and tested or red blood cel...

We investigated the interaction of tetanus toxin with small unilamellar vesicles composed of different phospholipids as a function of pH, toxin concentration, temperature, and ionic strength of the solution. Tetanus toxin increased the permeability of the vesicles to fluorescent markers of molecular weight up to 700. The time course of the permeabilization was described as the sum of two exponential components of which the faster accounts for more than 70% of the total effect. Both time constants decreased when the pH of the solution was lowered and when vesicles contained negative lipids. These results can be explained in terms of a phenomenological model based on reaction rate theory. The model assumes that tetanus toxin, after equilibrating with the local pH existing at the surface of the vesicles, inserts into the lipid bilayer forming an ionic channel through which solutes can diffuse. Trigger event for the insertion of the toxin is the protonation, and consequent neutralization of one charged group which makes the molecule more hydrophobic. The intrinsic pK of this group was found to be 3.4 +/- 0.2, suggesting that it may be a carboxyl group. Since the toxin equilibrates with the local pH, the enhancing effect of acidic phospholipids is merely explained by the creation of a negative surface potential which increases the local proton concentration. This was confirmed by the inhibitory effect of high Na+ concentration which reduced the surface charge by screening and specific binding. We found still small differences between the lipids tested and the following order of sensitivity to the action of the toxin: phosphatidylinositol greater than phosphatidylserine greater than phosphatidylcholine approximately cholesterol. The activation energy for the two time constants was found to be 19.8 and 14.8 kcal/mol, fast and slow component, respectively, i.e., slightly larger than that for pure diffusion through the bilayer. The permeabilization induced by tetanus toxin is a voltage-dependent process because vesicles bearing an inner negative potential were depolarized very quickly whereas those bearing an inner positive voltage were barely depolarized at all.

We have investigated the interaction of Pseudomonas exotoxin A with small unilamellar vesicles comprised of different phospholipids as a function of pH, toxin, and lipid concentration. We have found that this toxin induces vesicle permeabilization, as measured by the release of a fluorescent dye. Permeabilization is due to the formation of ion-conductive channels which we have directly observed in planar lipid bilayers. The toxin also produces vesicle aggregation, as indicated by an increase of the turbidity. Aggregation and permeabilization have completely different time course and extent upon toxin dose and lipid composition, thus suggesting that they are two independent events. Both time constants decrease by lowering the pH of the bulk phase or by introducing a negative lipid into the vesicles. Our results indicate that at least three steps are involved in the interaction of Pseudomonas exotoxin A with lipid vesicles. After protonation of one charged group the toxin becomes competent to bind to the surface of the vesicles. Binding is probably initiated by an electrostatic interaction because it is absolutely dependent on the presence of acidic phospholipids. Binding is a prerequisite for the subsequent insertion of the toxin into the lipid bilayer, with a special preference for phosphatidylglycerol-containing membranes, to form ionic channels. At high toxin and vesicle concentrations, bound toxin may also induce aggregation of the vesicles, particularly when phosphatidic acid is present in the lipid mixture. A quenching of the intrinsic tryptophan fluorescence of the protein, which is induced by lowering the pH of the solution, becomes more drastic in the presence of lipid vesicles. However, this further quenching takes so long that it cannot be a prerequisite to either vesicle permeabilization or aggregation. Pseudomonas exotoxin A shares many of these properties with other bacterial toxins like diphtheria and tetanus toxin.

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