Adenylate cyclase (AC) toxin from Bordetella pertussis delivers its catalytic domain to the interior of target cells where it converts host ATP to cAMP in a process referred to as intoxication. This toxin also hemolyzes sheep erythrocytes by a mechanism presumed to include pore formation and osmotic lysis. Intoxication and hemolysis appear at strikingly different toxin concentrations and evolve over different time scales, suggesting that different molecular processes may be involved. The present study was designed to test the hypothesis that intoxication and hemolysis occur by distinct mechanisms.Although the hemolytic activity of AC toxin has a lag of >1 h, intoxication starts immediately. Because of this difference, we sought a surrogate or precursor lesion that leads to hemolysis, and potassium efflux has been observed from erythrocytes treated with other poreforming toxins. AC toxin elicits an increase in K ؉ efflux from sheep erythrocytes and Jurkat cells, a human Tcell leukemia line, that begins within minutes of toxin addition. The toxin concentration dependence along with the analysis of the time course suggest that toxin monomers are sufficient to elicit release of K ؉ and to deliver the catalytic domain to the cell interior. Hemolysis, on the other hand, is a highly cooperative event that likely requires a subsequent oligomerization of these individual units. Although induction of K ؉ efflux shares some structural and environmental requirements with both intoxication and hemolysis, it can occur under conditions in which intoxication is reduced or prevented. The data presented here suggest that the transmembrane pathway by which K ؉ is released is separate and distinct from the structure required for intoxication but may be related to, or a precursor of, that which is ultimately responsible for hemolysis.

Bordetella pertussis adenylate cyclase (AC) toxin belongs to the RTX family of toxins but is the only member with a known catalytic domain. The principal pathophysiologic function of AC toxin appears to be rapid production of intracellular cyclic AMP (cAMP) by insertion of its catalytic domain into target cells (referred to as intoxication). Relative to other RTX toxins, AC toxin is weakly hemolytic via a process thought to involve oligomerization of toxin molecules. Monoclonal antibody (MAb) 3D1, which binds to an epitope (amino acids 373 to 399) at the distal end of the catalytic domain of AC toxin, does not affect the enzymatic activity of the toxin (conversion of ATP into cAMP in a cell-free system) but does prevent delivery of the catalytic domain to the cytosol of target erythrocytes. Under these conditions, however, the ability of AC toxin to cause hemolysis is increased three-to fourfold. To determine the mechanism by which the hemolytic potency of AC toxin is altered, we used a series of deletion mutants. A mutant toxin, ⌬AC, missing amino acids 1 to 373 of the catalytic domain, has hemolytic activity comparable to that of wild-type toxin. However, binding of MAb 3D1 to ⌬AC enhances its hemolytic activity three-to fourfold similar to the enhancement of hemolysis observed with 3D1 addition to wild-type toxin. Two additional mutants, ⌬N489 (missing amino acids 6 to 489) and ⌬N518 (missing amino acids 6 to 518), exhibit more rapid hemolysis with quicker onset than wild-type toxin does, while ⌬N549 (missing amino acids 6 to 549) has reduced hemolytic activity compared to wild-type AC toxin. These data suggest that prevention of delivery of the catalytic domain or deletion of the catalytic domain, along with additional amino acids distal to it, elicits a conformation of the toxin molecule that is more favorable for hemolysis.

Adenylate cyclase toxin from Bordetella pertussis is a 177-kDa calmodulin-activated enzyme that has the ability to enter eukaryotic cells and convert endogenous ATP into cAMP. Little is known, however, about the mechanism of cell entry. We now demonstrate that intoxication of cardiac myocytes by adenylate cyclase toxin is driven and controlled by the electrical potential across the plasma membrane. The steepness of the voltage dependence of intoxication is comparable with that previously observed for the activation of K+ and Na+ channels of excitable membranes. The voltage-sensitive process is downstream from toxin binding to the cell surface and appears to correspond to the translocation of the catalytic domain across the membrane.

Intracellular perfusion of atrial myocytes with adenosine 5'-(gamma-thio) triphosphate (ATP gamma S), an ATP analog, elicits a progressive increase of the muscarinic potassium channel current, IK(M), in the absence of agonists. In this respect, ATP gamma S mimics the actions of guanosine triphosphate (GTP) analogs, which produce direct, persistent activation of the guanyl nucleotide-binding (G) protein controlling the K+(M) channel. The effect of ATP gamma S on IK(M), however, differs from that produced by GTP analogs in two aspects: it requires relatively large ATP gamma S concentrations, and it appears after a considerable delay, suggesting a rate-limiting step not present in similar experiments performed with guanosine 5'-(gamma-thio) triphosphate (GTP gamma S). Incubation of atrial homogenates with [35S]ATP gamma S leads to formation of significant amounts of [35S]GTP gamma S, suggesting that activation of IK(M) by ATP gamma S arises indirectly through its conversion into GTP gamma S by cellular enzymes. ATP gamma S is often used to demonstrate the involvement of protein phosphorylation in the control of various cellular processes. The finding that cytosolic application of ATP gamma S can also lead to G-protein activation implies that experiments with ATP gamma S must be interpreted with caution.

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