Membrane Depolarization Prevents Cell Invasion by Bordetella pertussis Adenylate Cyclase Toxin

Abstract

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. 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. INTRODUCTIONAC1 1The abbreviation used is: ACadenylate cyclase. toxin is an important virulence factor for Bordetella pertussis, the causative agent of whooping cough (1Weiss A. Hewlett E.L. Myers G. Falkow S. J. Infect. Dis. 1984; 150: 219-222Crossref PubMed Scopus (147) Google Scholar, 2Weiss A.A. Hewlett E.L. Annu. Rev. Microbiol. 1986; 40: 661-686Crossref PubMed Scopus (232) Google Scholar, 3Cherry J.D. Brunell P.A. Golden G.S. Karzon D. Pediatrics. 1988; 81: 939-984Google Scholar, 4Khelef N. Sakamoto H. Guiso N. Microb. Pathog. 1992; 12: 227-235Crossref PubMed Scopus (93) Google Scholar, 5Hewlett E.L. Maloney N.J. Iglewski B. Moss J. Tu A.T. Vaughan M. Handbook of Natural Toxins, Volume 8: Microbial Toxins. Marcel Dekker, Inc., New York1994: 425-439Google Scholar). In vitro this potent toxin induces massive increases in the intracellular levels of cAMP in phagocytes and is postulated to use this mechanism to disarm the host's response to infection (6Confer D. Eaton J. Science. 1982; 217: 948-950Crossref PubMed Scopus (287) Google Scholar, 7Pearson R.D. Symes P. Conboy M. Weiss A.A. Hewlett E.L. J. Immunol. 1987; 139: 2749-2754PubMed Google Scholar). Productive interaction between AC toxin and target cells requires calcium ions and a post-translational modification that is dependent upon the product of another gene, cyaC (8Confer D. Slungaard A. Graf E. Panter S. Eaton J. Adv. Cyclic Nucleotide Protein Phosphorylation Res. 1984; 17: 183-187PubMed Google Scholar, 9Hanski E. Farfel Z. J. Biol. Chem. 1985; 260: 5526-5532Abstract Full Text PDF PubMed Google Scholar, 10Gentile F. Knipling L.G. Sackett L. Wolff J. J. Biol. Chem. 1990; 265: 10686-10692Abstract Full Text PDF PubMed Google Scholar, 11Hewlett E.L. Gray L. Allietta M. Ehrmann I. Gordon V.M. Gray M.C. J. Biol. Chem. 1991; 266: 17503-17508Abstract Full Text PDF PubMed Google Scholar, 12Barry E.M. Weiss A.A. Ehrmann I.E. Gray M.C. Hewlett E.L. Goodwin M.S. J. Bacteriol. 1991; 173: 720-726Crossref PubMed Google Scholar, 13Hewlett E.L. Gray M.C. Ehrmann I.E. Maloney N.J. Otero A.S. Gray L. Allietta M. Szabo G. Weiss A.A. Barry E.M. J. Biol. Chem. 1993; 268: 7842-7848Abstract Full Text PDF PubMed Google Scholar, 14Hackett M. Guo L. Shabanowitz J. Hunt D. Hewlett E. Science. 1994; 266: 433-435Crossref PubMed Scopus (193) Google Scholar). A specific cell surface receptor for the toxin has never been identified; rather, there is evidence that the first step in the intoxication process may involve an interaction with negatively charged lipids (15Gordon V.M. Leppla S.H. Hewlett E.L. Infect. Immun. 1988; 56: 1066-1069Crossref PubMed Google Scholar, 16Gordon V.M. Young W.W. Lechler S.M. Gray M.C. Leppla S.H. Hewlett E.L. J. Biol. Chem. 1989; 264: 14792-14796Abstract Full Text PDF PubMed Google Scholar, 17Szabo G. Gray M.C. Hewlett E.L. J. Biol. Chem. 1994; 269: 22496-22499Abstract Full Text PDF PubMed Google Scholar). As observed with several other bacterial toxins (18Hoch D.H. Romero-Mira M. Ehrlich B.E. Finkelstein A. DasGupta B.R. Simpson L.L. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 1692-1696Crossref PubMed Scopus (251) Google Scholar, 19Blaustein R.O. Koehler T.M. Collier R.J. Finkelstein A. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 2209-2213Crossref PubMed Scopus (239) Google Scholar, 20Menestrina G. Bashford C.L. Pasternak C.A. Toxicon. 1990; 28: 477-491Crossref PubMed Scopus (58) Google Scholar), AC toxin was recently shown to form an ion-conductive pathway in artificial lipid bilayers in a voltage-dependent manner (17Szabo G. Gray M.C. Hewlett E.L. J. Biol. Chem. 1994; 269: 22496-22499Abstract Full Text PDF PubMed Google Scholar). This finding raises the possibility that in vivo the membrane potential might control the translocation of AC toxin across the plasma membrane. Although studies using ionophores or agents that collapse the mitochondrial electrochemical gradient have suggested that the movement of proteins into or across intracellular membranes might be affected by membrane potential, the complexity of the eukaryotic transport systems and the limitations inherent in the methods employed have precluded direct evaluation of this hypothesis (21Hudson T.H. Scharff J. Kimak M.A.G. Neville D.M. J. Biol. Chem. 1988; 263: 4773-4781Abstract Full Text PDF PubMed Google Scholar, 22Chaumont F. O'Riordan V. Boutry M. J. Biol. Chem. 1990; 265: 16856-16862Abstract Full Text PDF PubMed Google Scholar, 23Martin J. Mahlke K. Pfanner N. J. Biol. Chem. 1991; 266: 18051-18057Abstract Full Text PDF PubMed Google Scholar, 24Hannavy K. Rospert S. Schatz G. Curr. Opin. Cell Biol. 1993; 5: 694-700Crossref PubMed Scopus (69) Google Scholar). In contrast, the effects of membrane voltage on entry of AC toxin into cells can be studied directly in single cardiac myocytes by monitoring cAMP-stimulated calcium currents with the patch clamp technique (13Hewlett E.L. Gray M.C. Ehrmann I.E. Maloney N.J. Otero A.S. Gray L. Allietta M. Szabo G. Weiss A.A. Barry E.M. J. Biol. Chem. 1993; 268: 7842-7848Abstract Full Text PDF PubMed Google Scholar). This approach allows full control of the potential across the plasma membrane, while the development of intoxication is measured with high sensitivity and time resolution. We report here the results of experiments demonstrating that intoxication of atrial cells by AC toxin is highly voltage-dependent, being abolished by membrane depolarization.EXPERIMENTAL PROCEDURESPurification of Adenylate Cyclase ToxinWild type B. pertussis (strain BP338) and a mutant in which Lys-58 was replaced with a methionine residue (strain BPLM58IE) were grown as described previously (25Hewlett E. Gordon V. McCaffery J. Sutherland W. Gray M. J. Biol. Chem. 1989; 264: 19379-19384Abstract Full Text PDF PubMed Google Scholar, 26Ehrmann I. Weiss A. Goodwin M.S. Gray M.C. Barry E. Hewlett E.L. FEBS Lett. 1992; 304: 51-56Crossref PubMed Scopus (21) Google Scholar). AC toxin was purified by urea extraction, phenyl-Sepharose chromatography, sucrose gradient centrifugation, and calmodulin-Sepharose chromatography (13Hewlett E.L. Gray M.C. Ehrmann I.E. Maloney N.J. Otero A.S. Gray L. Allietta M. Szabo G. Weiss A.A. Barry E.M. J. Biol. Chem. 1993; 268: 7842-7848Abstract Full Text PDF PubMed Google Scholar). Each toxin batch was dialyzed against external solution immediately prior to experiments and then diluted to the desired concentration with external solution. Enzyme activity was measured by the conversion of [α-32P]ATP to [32P]cAMP in a cell-free assay (13Hewlett E.L. Gray M.C. Ehrmann I.E. Maloney N.J. Otero A.S. Gray L. Allietta M. Szabo G. Weiss A.A. Barry E.M. J. Biol. Chem. 1993; 268: 7842-7848Abstract Full Text PDF PubMed Google Scholar). The cAMP formed was isolated as described by Salomon et al. (27Salomon Y. Londos C. Rodbell M. Anal. Biochem. 1974; 58: 541-548Crossref PubMed Scopus (3368) Google Scholar). Enzyme specific activities for wild type toxin, BP338, ranged from 0.8 to 1.2 mmol of cAMP/min/mg of toxin. Toxin activity of wild type toxin, as determined by quantitation of intracellular cAMP in Jurkat cells exposed to AC toxin for 30 min at 37°C (13Hewlett E.L. Gray M.C. Ehrmann I.E. Maloney N.J. Otero A.S. Gray L. Allietta M. Szabo G. Weiss A.A. Barry E.M. J. Biol. Chem. 1993; 268: 7842-7848Abstract Full Text PDF PubMed Google Scholar), was 12-15 μmol of cAMP/mg of Jurkat cell protein/mg of toxin. The mutant toxin, strain BPLM58IE, exhibited a 1000-fold reduction in enzymatic activity relative to wild type toxin and was unable to increase cAMP in target cells.Electrophysiologic Studies-Myocytes were enzymatically dissociated from bullfrog atria as described previously (28Otero A.S. Sweitzer N.M. Mol. Pharmacol. 1993; 44: 595-604PubMed Google Scholar). Membrane currents were recorded at 22−24°C in the whole-cell configuration of the gigaseal patch clamp technique (29Hamill O.P. Marty A. Neher E. Sakmann B. Sigworth F.J. Pflügers Arch. Eur. J. Physiol. 1981; 391: 85-100Crossref PubMed Scopus (15094) Google Scholar). The internal solution contained 120 mM CsCl (to block K+selective channels), 1 mM EGTA, 2.5 mM Mg+ATP, 0.2 mM Li4+GTP, 5 mM HEPES, adjusted to pH 7.4 with KOH, while the external solution contained 85 mM NaCl, 20 mM CsCl, 2 mM MgCl2, 2 mM CaCl2, 20 mM HEPES (pH 7.4 with NaOH), and 5 μM tetrodotoxin, a Na+channel blocker. The standard pulse protocol consisted of 0.5-s steps from a holding potential of −85 mV to −135, −75, −45, −15, 15, and 45 mV. The cell was kept at −85 mV for 2 s between pulses. The amplitude of ICawas measured as the difference between the peak inward current and the steady state current averaged during the last 10 ms of the pulse.RESULTS AND DISCUSSIONAC toxin, at concentrations of 3.5-4.5 μg/ml, elicits a large increase (11.7-fold ± 4.4; n = 16) in the amplitude of Ca2+currents (ICa) of frog atrial myocytes (13Hewlett E.L. Gray M.C. Ehrmann I.E. Maloney N.J. Otero A.S. Gray L. Allietta M. Szabo G. Weiss A.A. Barry E.M. J. Biol. Chem. 1993; 268: 7842-7848Abstract Full Text PDF PubMed Google Scholar) (Fig. 1), comparable in magnitude to the stimulation of ICainduced by β-adrenergic agonists (30Li Y. Hanf R. Otero A.S. Fischmeister R. Szabo G. J. Gen. Physiol. 1994; 104: 941-959Crossref PubMed Scopus (13) Google Scholar). This effect of AC toxin is characterized by a short latency period (0.59 ± 0.17 min; n = 6) followed by a slow increase of Ca2+currents, with a maximum being reached within 3.5-9 min. An enzyme-deficient AC toxin, in which lysine 58, a residue involved in catalysis, is replaced by a methionine (26Ehrmann I. Weiss A. Goodwin M.S. Gray M.C. Barry E. Hewlett E.L. FEBS Lett. 1992; 304: 51-56Crossref PubMed Scopus (21) Google Scholar) has no effect on ICa(Fig. 1). Thus, the increase in the amplitude of the Ca2+current is directly related to intracellular cAMP production, which is an intrinsic property of the wild type toxin and leads to activation of protein kinase A and phosphorylation of L-type Ca2+channels (31Trautwein W. Hescheler J. Annu. Rev. Physiol. 1990; 52: 257-274Crossref PubMed Scopus (317) Google Scholar).To determine the voltage dependence of intoxication, we applied AC toxin to cells for a fixed period while the membrane potential was clamped at the values specified in the figure legends. Calcium currents were measured at the beginning of the experiment, for determination of basal levels, and following the holding period. We found that the increase of ICais virtually complete following 5 min of toxin application to myocytes held at −85 mV (Fig. 2 A). In contrast, there is no increase in Ca2+currents when the membrane potential is kept at +45 mV during the first 5 min of AC toxin application (Fig. 2 B); upon return to the standard pulse protocol, ICais still at the basal level and only then starts to increase, following a time course similar to that shown in Fig. 1.Figure 2:Effect of AC toxin application on cells held at constant voltages. After the establishment of the whole-cell recording mode, ICa(•) was elicited by 0.5-s voltage steps to 15 mV from a holding potential of −85 mV, and basal currents were measured. The cell was then clamped at −85 mV (A) or +45 mV (B) for the period indicated by dotted lines. AC toxin (4 μg/ml) was applied for the period indicated by the horizontal bar. After 5 min of toxin exposure at a constant voltage, the pulse protocol was resumed directly and ICawas measured again.View Large Image Figure ViewerDownload (PPT)Fig. 3 shows the percent increase in calcium currents observed for cells held at various potentials as a function of the membrane potential. The results indicate that there is essentially no stimulation of ICawhile cells are held in the range of 0 to +45 mV; as the holding potential becomes negative, the toxin-induced increases in ICarise steeply, reaching a maximum around −45 mV. The solid line is a fit of the data to the Boltzmann equation (1Weiss A. Hewlett E.L. Myers G. Falkow S. J. Infect. Dis. 1984; 150: 219-222Crossref PubMed Scopus (147) Google Scholar), I=I\textmax/1+eβ(V-V0.5)(Eq. 1) Figure 3:Voltage dependence of ICastimulation. Experiments followed the protocol illustrated in Fig. 2. During the first 5 min of exposure to AC toxin (4 μg/ml) the voltage was kept at the values shown. Calcium currents recorded immediately after the pulse protocol was resumed were normalized to the maximal ICaamplitude in each cell (♦). Plotted are results obtained with individual cells; symbols with error bars are means ± S.E. from three separate experiments. Also shown are the results of experiments following the same protocol, but using 0.2 μM isoproterenol (□) instead of AC toxin to stimulate ICa.View Large Image Figure ViewerDownload (PPT)Yielding Imax = 85.56%, β = 0.181 mV1, and V0.5 (voltage for half-maximal stimulation of Ca) = 16.6 mV. The slope factor β is equal to ze/kT, where k is the Boltzmann constant, e is elementary charge, T the absolute temperature, and z the number of elementary charges involved in toxin delivery. From the inverse of β we obtain a voltage dependence (32Hille B. Ionic Channels of Excitable Membranes. 2nd Ed. Sinauer Associates, Inc., Sunderland, MA1992: 23-58Google Scholar) of mV increase in = mV, can be that elementary charges across the membrane during toxin delivery. This is with the movement of toxin with charges or a with charges protein The possibility with a dependence of 3 for by AC toxin in lipid bilayers (17Szabo G. Gray M.C. Hewlett E.L. J. Biol. Chem. 1994; 269: 22496-22499Abstract Full Text PDF PubMed Google Scholar).To that the observed voltage dependence is a property of AC toxin, and a of or of the phosphorylation by we experiments in which ICawas by AC toxin but by a β-adrenergic receptor which increases cAMP stimulation of the endogenous adenylate cyclase. As in Fig. of cardiac Ca2+currents by isoproterenol shows of the membrane potential, that the voltage dependence observed with AC toxin is a of the intoxication evidence that B. pertussis AC toxin target cells on an pathway as the by toxin, the only other bacterial toxin to have adenylate cyclase activity (15Gordon V.M. Leppla S.H. Hewlett E.L. Infect. Immun. 1988; 56: 1066-1069Crossref PubMed Google Scholar, 16Gordon V.M. Young W.W. Lechler S.M. Gray M.C. Leppla S.H. Hewlett E.L. J. Biol. Chem. 1989; 264: 14792-14796Abstract Full Text PDF PubMed Google Scholar, J. Biol. Chem. 1986; Full Text PDF PubMed Google Scholar). a of AC toxin entry into cells a of at 1) binding to the surface of the membrane and into the membrane with translocation of the catalytic into the cell E.L. Maloney N.J. Iglewski B. Moss J. Tu A.T. Vaughan M. Handbook of Natural Toxins, Volume 8: Microbial Toxins. Marcel Dekker, Inc., New York1994: 425-439Google Scholar). In the membrane potential or the step affected by the voltage, we experiments similar to the in Fig. toxin for 5 min while holding the cell at +45 mV. the pulse protocol was however, the solution was replaced with standard external solution and the cell was for min. the toxin to to the membrane at this period from the cell surface as as the no increase in is In contrast, binding at potentials and only the translocation step is increase at the of the holding period the toxin is no in the Fig. shows that when the toxin is from the upon return to the standard pulse protocol, that during the period at +45 mV toxin to the plasma membrane in a manner that was to The results obtained = are to in Fig. 2 that the voltage-sensitive step in toxin is downstream from binding and is to translocation of the catalytic to the of AC toxin application and at 45 mV. The protocol and symbols are the same as in Fig. for the of toxin application and the of a period as indicated by the horizontal Large Image Figure ViewerDownload results demonstrate that the of AC toxin to of is driven and controlled by the across the plasma membrane. of atrial cells is observed within of exposure to adenylate cyclase toxin when myocytes are held at potentials but at membrane potentials to −45 mV and is when the membrane is kept at This voltage dependence of intoxication mV increase in is comparable with that of the K+and Na+channels of and mV increase in and and AC toxin the voltage-sensitive In the ability to the time course of intoxication of single cells by AC toxin has direct of a for membrane potential in toxin delivery. This of the membrane translocation of proteins in and of bacterial toxins in INTRODUCTIONAC1 1The abbreviation used is: ACadenylate cyclase. toxin is an important virulence factor for Bordetella pertussis, the causative agent of whooping cough (1Weiss A. Hewlett E.L. Myers G. Falkow S. J. Infect. Dis. 1984; 150: 219-222Crossref PubMed Scopus (147) Google Scholar, 2Weiss A.A. Hewlett E.L. Annu. Rev. Microbiol. 1986; 40: 661-686Crossref PubMed Scopus (232) Google Scholar, 3Cherry J.D. Brunell P.A. Golden G.S. Karzon D. Pediatrics. 1988; 81: 939-984Google Scholar, 4Khelef N. Sakamoto H. Guiso N. Microb. Pathog. 1992; 12: 227-235Crossref PubMed Scopus (93) Google Scholar, 5Hewlett E.L. Maloney N.J. Iglewski B. Moss J. Tu A.T. Vaughan M. Handbook of Natural Toxins, Volume 8: Microbial Toxins. Marcel Dekker, Inc., New York1994: 425-439Google Scholar). In vitro this potent toxin induces massive increases in the intracellular levels of cAMP in phagocytes and is postulated to use this mechanism to disarm the host's response to infection (6Confer D. Eaton J. Science. 1982; 217: 948-950Crossref PubMed Scopus (287) Google Scholar, 7Pearson R.D. Symes P. Conboy M. Weiss A.A. Hewlett E.L. J. Immunol. 1987; 139: 2749-2754PubMed Google Scholar). Productive interaction between AC toxin and target cells requires calcium ions and a post-translational modification that is dependent upon the product of another gene, cyaC (8Confer D. Slungaard A. Graf E. Panter S. Eaton J. Adv. Cyclic Nucleotide Protein Phosphorylation Res. 1984; 17: 183-187PubMed Google Scholar, 9Hanski E. Farfel Z. J. Biol. Chem. 1985; 260: 5526-5532Abstract Full Text PDF PubMed Google Scholar, 10Gentile F. Knipling L.G. Sackett L. Wolff J. J. Biol. Chem. 1990; 265: 10686-10692Abstract Full Text PDF PubMed Google Scholar, 11Hewlett E.L. Gray L. Allietta M. Ehrmann I. Gordon V.M. Gray M.C. J. Biol. Chem. 1991; 266: 17503-17508Abstract Full Text PDF PubMed Google Scholar, 12Barry E.M. Weiss A.A. Ehrmann I.E. Gray M.C. Hewlett E.L. Goodwin M.S. J. Bacteriol. 1991; 173: 720-726Crossref PubMed Google Scholar, 13Hewlett E.L. Gray M.C. Ehrmann I.E. Maloney N.J. Otero A.S. Gray L. Allietta M. Szabo G. Weiss A.A. Barry E.M. J. Biol. Chem. 1993; 268: 7842-7848Abstract Full Text PDF PubMed Google Scholar, 14Hackett M. Guo L. Shabanowitz J. Hunt D. Hewlett E. Science. 1994; 266: 433-435Crossref PubMed Scopus (193) Google Scholar). A specific cell surface receptor for the toxin has never been identified; rather, there is evidence that the first step in the intoxication process may involve an interaction with negatively charged lipids (15Gordon V.M. Leppla S.H. Hewlett E.L. Infect. Immun. 1988; 56: 1066-1069Crossref PubMed Google Scholar, 16Gordon V.M. Young W.W. Lechler S.M. Gray M.C. Leppla S.H. Hewlett E.L. J. Biol. Chem. 1989; 264: 14792-14796Abstract Full Text PDF PubMed Google Scholar, 17Szabo G. Gray M.C. Hewlett E.L. J. Biol. Chem. 1994; 269: 22496-22499Abstract Full Text PDF PubMed Google Scholar). As observed with several other bacterial toxins (18Hoch D.H. Romero-Mira M. Ehrlich B.E. Finkelstein A. DasGupta B.R. Simpson L.L. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 1692-1696Crossref PubMed Scopus (251) Google Scholar, 19Blaustein R.O. Koehler T.M. Collier R.J. Finkelstein A. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 2209-2213Crossref PubMed Scopus (239) Google Scholar, 20Menestrina G. Bashford C.L. Pasternak C.A. Toxicon. 1990; 28: 477-491Crossref PubMed Scopus (58) Google Scholar), AC toxin was recently shown to form an ion-conductive pathway in artificial lipid bilayers in a voltage-dependent manner (17Szabo G. Gray M.C. Hewlett E.L. J. Biol. Chem. 1994; 269: 22496-22499Abstract Full Text PDF PubMed Google Scholar). This finding raises the possibility that in vivo the membrane potential might control the translocation of AC toxin across the plasma membrane. Although studies using ionophores or agents that collapse the mitochondrial electrochemical gradient have suggested that the movement of proteins into or across intracellular membranes might be affected by membrane potential, the complexity of the eukaryotic transport systems and the limitations inherent in the methods employed have precluded direct evaluation of this hypothesis (21Hudson T.H. Scharff J. Kimak M.A.G. Neville D.M. J. Biol. Chem. 1988; 263: 4773-4781Abstract Full Text PDF PubMed Google Scholar, 22Chaumont F. O'Riordan V. Boutry M. J. Biol. Chem. 1990; 265: 16856-16862Abstract Full Text PDF PubMed Google Scholar, 23Martin J. Mahlke K. Pfanner N. J. Biol. Chem. 1991; 266: 18051-18057Abstract Full Text PDF PubMed Google Scholar, 24Hannavy K. Rospert S. Schatz G. Curr. Opin. Cell Biol. 1993; 5: 694-700Crossref PubMed Scopus (69) Google Scholar). In contrast, the effects of membrane voltage on entry of AC toxin into cells can be studied directly in single cardiac myocytes by monitoring cAMP-stimulated calcium currents with the patch clamp technique (13Hewlett E.L. Gray M.C. Ehrmann I.E. Maloney N.J. Otero A.S. Gray L. Allietta M. Szabo G. Weiss A.A. Barry E.M. J. Biol. Chem. 1993; 268: 7842-7848Abstract Full Text PDF PubMed Google Scholar). This approach allows full control of the potential across the plasma membrane, while the development of intoxication is measured with high sensitivity and time resolution. We report here the results of experiments demonstrating that intoxication of atrial cells by AC toxin is highly voltage-dependent, being abolished by membrane