From the
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
AC
AC 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 Ca
On-line formulae not verified for accuracy
Experimental evidence indicates that B.
pertussis AC toxin invades target cells directly, rather than
depending on an endocytic pathway such as the one utilized by
Bacillus anthracis toxin, the only other invasive bacterial
toxin known to have adenylate cyclase activity
(15, 16, 33) . Therefore, a basic model of AC
toxin entry into cells involves a sequence of at least two steps: 1)
binding to the outer surface of the membrane and 2) insertion into the
membrane with translocation of the catalytic portion into the cell
interior
(5) . In principle, the membrane potential could affect
either or both these processes. To better define the step affected by
the voltage, we performed experiments similar to the one in
Fig. 2B, applying toxin for 5 min while holding the cell
at +45 mV. Before the pulse protocol was resumed, however, the
toxin-containing solution was replaced with standard external solution
and the cell was washed for 2-3 min. Were the toxin not able to
bind to the membrane at positive potentials, this washout period should
completely remove it from the cell surface as well as the bathing
solution, yielding no increase in I
We thank Sheila Van Cuyk for assistance with toxin
purification.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
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.
(
)
toxin is an important virulence
factor for Bordetella pertussis, the causative agent of
whooping cough
(1, 2, 3, 4, 5) .
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
(6, 7) . 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 (8, 9, 10, 11, 12, 13, 14) .
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
(15, 16, 17) . As observed with several
other bacterial toxins
(18, 19, 20) , AC toxin
was recently shown to form an ion-conductive pathway in artificial
lipid bilayers in a voltage-dependent manner
(17) . 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
(21, 22, 23, 24) . 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
(13) . 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.
Purification of Adenylate Cyclase Toxin
Wild
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
(25, 26) . AC toxin was purified by
urea extraction, phenyl-Sepharose chromatography, sucrose gradient
centrifugation, and calmodulin-Sepharose chromatography
(13) .
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
[-
P]ATP to [
P]cAMP
in a cell-free assay
(13) . The cAMP formed was isolated as
described by Salomon et al. (27) . 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
(13) , 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
(28) .
Membrane currents were recorded at 22-24 °C in the whole-cell
configuration of the gigaseal patch clamp technique
(29) . The
internal solution contained 120 mM CsCl (to block K
selective channels), 1 mM EGTA, 2.5 mM
Mg
ATP, 0.2 mM Li
GTP, 5 mM
HEPES, adjusted to pH 7.4 with KOH, while the external solution
contained 85 mM NaCl, 20 mM CsCl, 2 mM
MgCl
, 2 mM CaCl
, 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 I
was measured as the difference between the peak inward current
and the steady state current averaged during the last 10 ms of the
pulse.
currents
( I
) of frog atrial myocytes
(13) (Fig. 1), comparable in magnitude to the stimulation
of I
induced by
-adrenergic agonists
(30) . 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 Ca
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
(26) has no effect on I
(Fig. 1). Thus, the increase in the amplitude of the
Ca
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
Ca
channels
(31) .
Figure 1:
Time
course of stimulation of calcium currents by AC toxin. Peak inward
currents elicited by 0.5-s steps from a holding potential of 85
to 15 mV are plotted as a function of time; cells were superfused with
wild type AC toxin (4 µg/ml,
) or with mutant toxin, BPLM58IE
(5.2 µg/ml,
) for the interval indicated by the
horizontal bar.
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 Iis virtually complete following 5 min of toxin application to
myocytes held at
85 mV (Fig. 2 A). In contrast,
there is no increase in Ca
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, I
is 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, I(
) 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 I
was measured again.
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 Iwhile cells are held in the
range of 0 to +45 mV; as the holding potential becomes negative,
the toxin-induced increases in I
rise steeply,
reaching a maximum around
45 mV. The solid line is a fit of the data to the Boltzmann equation
(1) ,
Figure 3:
Voltage
dependence of Istimulation. 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 I
amplitude 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 I
.
To verify that the observed voltage
dependence is a property of AC toxin, and not a characteristic of one
or more components of the phosphorylation cascade triggered by cAMP, we
performed experiments in which Iwas stimulated
not by AC toxin but by isoproterenol, a
-adrenergic receptor
agonist, which increases cAMP production through stimulation of the
endogenous adenylate cyclase. As seen in Fig. 3, enhancement of
cardiac Ca
currents by isoproterenol shows little
influence of the membrane potential, confirming that the striking
voltage dependence observed with AC toxin is a characteristic of the
intoxication process.
after pulsing
is resumed. In contrast, if binding occurs normally at positive
potentials and only the translocation step is prevented,
I
should increase at the end of the holding
period even if the toxin is no longer present in the bath. Indeed,
Fig. 4
shows that even when the toxin is removed from the bath,
I
increases upon return to the standard pulse
protocol, indicating that during the period at +45 mV toxin
molecules attached themselves to the plasma membrane in a manner that
was resistant to subsequent washout. The results obtained under these
conditions ( n = 3) are qualitatively identical to those
in Fig. 2 B, establishing that the voltage-sensitive step
in toxin delivery is downstream from binding and is likely to represent
translocation of the catalytic site to the cytoplasm.
Figure 4:
Effect of AC toxin application and washout
at 45 mV. The experimental protocol and symbols are the same
as in Fig. 2, except for the duration of toxin application and the
inclusion of a washout period as indicated by the horizontal bar.
Our results
demonstrate that the delivery of AC toxin to its site of action is
driven and controlled by the electric field across the plasma membrane.
Intoxication of atrial cells is readily observed within minutes of
exposure to adenylate cyclase toxin when myocytes are held at negative
potentials but hindered at membrane potentials positive to 45 mV
and is totally prevented when the membrane is kept at positive
potentials. This steep voltage dependence of intoxication (5.5 mV per
e-fold increase in intoxication) is comparable with that of
the voltage-gated K
and Na
channels
of squid axon (4.8 and 3.9 mV per e-fold increase in
conductance, respectively (Ref.32 and references therein)) and places
AC toxin delivery among the most voltage-sensitive biological processes
known. In our studies, the ability to follow the time course of
intoxication of single cells by AC toxin has allowed direct
demonstration of a role for membrane potential in toxin delivery. This
novel experimental system should help expand our present understanding
of the molecular mechanisms underlying membrane translocation of
proteins in general and of bacterial toxins in particular.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.