(Received for publication, May 19, 1994; and in revised form, October 26, 1994)
From the
Lophotoxin and the bipinnatins are members of the lophotoxin
family of marine neurotoxins, which covalently react with Tyr in the
-subunits of the nicotinic acetylcholine receptor.
Bipinnatin-A, -B, and -C are protoxins that have been shown to
spontaneously convert from inactive to active toxins during
preincubation in buffer. However, in this report, we show that
preincubation of lophotoxin did not result in an increase in the
subsequent rate of irreversible inhibition of nicotinic receptors.
Thus, unlike the bipinnatins, lophotoxin does not appear to be an
inactive protoxin. Lophotoxin preferentially inhibited one of the two
acetylcholine-binding sites on the receptor, and this preference
resulted from both a higher reversible affinity and a faster rate of
irreversible inhibition at this site. Association of
I-
-bungarotoxin in the presence of lophotoxin was
analyzed to obtain the apparent reversible association and dissociation
rate constants for lophotoxin. The apparent association rate constant
of lophotoxin was approximately 10
-fold slower than
expected for a diffusion-limited interaction, indicating that
lophotoxin is a slow binding irreversible inhibitor. The kinetic
constants that describe the interaction of lophotoxin with the receptor
did not change in the presence of dibucaine, suggesting that, unlike
agonists, the slow apparent association of lophotoxin does not result
from a slow transition of the receptor to a desensitized conformation.
Nicotinic acetylcholine receptors are ligand-gated ion channels
found on nerve and muscle cells. Two molecules of acetylcholine must
bind to the receptor to open the channel and allow ion flux across the
cell membrane. The muscle subtype of the nicotinic receptor is a
pentamer of four homologous protein subunits
() in a circular arrangement spanning
the membrane bilayer (Unwin, 1993). The two acetylcholine-binding sites
of the receptor are located near the
/
- and
/
-subunit interfaces and have distinct pharmacological
properties due to unique contributions of the
- and
-subunits
(Blount and Merlie, 1989; Sine and Claudio, 1991a).
The lophotoxins
are marine neurotoxins that share a similar 14-carbon cyclic structure
and demonstrate similar biological activity. A total of 12 active
lophotoxin analogs have been isolated from various species of the soft
corals Lophogorgia and Pseudopterogorgia (Fenical et al., 1981; Culver et al., 1985; Abramson et
al., 1989; Wright et al., 1989). A radiolabeled analog
was used to show that the lophotoxins irreversibly inhibit nicotinic
receptors by forming a covalent bond with Tyr in the
-subunits (
Tyr
) of the receptor (Abramson et al., 1988, 1989). The lophotoxins are unique among
naturally occurring neurotoxins in their selective irreversible
inhibition of a neurotransmitter receptor by covalent modification of a
specific amino acid. Understanding the mechanism of the irreversible
inhibition and the chemical nature of the covalent bond with
Tyr
may provide new insights for the design of novel
site-directed irreversible receptor antagonists.
The naturally
occurring lophotoxin analogs bipinnatin-A, -B, and -C are initially
inactive inhibitors (protoxins) that become active upon preincubation
in aqueous buffer (Groebe et al., 1994). Although the
bipinnatins presumably form an initial reversible complex with the
nicotinic receptor prior to formation of a covalent bond with
Tyr
, their initial reversible affinity is relatively
low (Groebe et al., 1994). However, the bipinnatins are quite
selective in that they 1) do not inhibit related neurotransmitter
receptors, 2) are covalently incorporated only into the
-subunits
of the nicotinic receptor, and 3) react with
Tyr
but
not with an adjacent
Tyr
(Abramson et al.,
1988, 1989). Due to the influence of the
- and
-subunits, the
two acetylcholine-binding sites are inhibited by the bipinnatins at
different rates. The bipinnatins preferentially inhibit the
acetylcholine-binding site located near the
/
-subunit
interface (Sine and Claudio, 1991a; Groebe et al., 1994). In
addition, structural differences among bipinnatin-A, -B, and -C have
profound effects on their apparent rates of irreversible inhibition and
on the extent to which they discriminate between the two
acetylcholine-binding sites (Culver et al., 1985;
Abramson et al., 1991; Groebe et al., 1994).
Lophotoxin has a chemical structure similar to the bipinnatins, and
it has been suggested that lophotoxin is also unstable in aqueous
buffer (Culver et al., 1984). However, in contrast to
bipinnatin-B, the IC of lophotoxin decreases with
increasing times of incubation with nicotinic receptors (Abramson, et al., 1991). This would be expected of a relatively stable
irreversible inhibitor. An investigation of the stability of lophotoxin
and the kinetics of its interaction with the nicotinic receptor is a
necessary prerequisite for understanding the structural requirements
for binding and, ultimately, the mechanism of irreversible inhibition.
In this study, we investigate the stability of lophotoxin and report
the kinetic constants for inhibition of nicotinic receptors on intact
BC
H-1 cells by lophotoxin.
The percentages of available acetylcholine-binding sites are
represented by A and B. The rate constants (k and k
)
represent the apparent pseudo first-order rates for irreversible
inhibition of the available acetylcholine-binding sites (Kitz and
Wilson, 1962; Culver et al., 1984). Since it is known that
lophotoxin preferentially inhibits one of the two acetylcholine-binding
sites, A and B in were each constrained
to equal 50 (Culver et al., 1984; Sine and Claudio, 1991a).
The interaction of lophotoxin with either acetylcholine-binding site can be described by ,
where L represents lophotoxin, R represents
the receptor, LR represents an initial reversible
complex, L-R represents an irreversible complex, K
represents the dissociation constant for
formation of the L
R complex, and k
represents the rate constant for formation of the L-R complex
(Kitz and Wilson, 1962; Culver et al., 1984; Rakitzis, 1984).
A plot of k
versus lophotoxin
concentration describes a hyperbolic curve in which the maximum k
is equal to k
, and the
concentration of toxin ([L]) at half-maximal k
is equal to K
().
The covalent reaction between lophotoxin and Tyr
prevents the binding of
I-
-BTX. If
I-
-BTX does not bind to the receptor when lophotoxin reversibly occupies the acetylcholine-binding site, then the
simultaneous interaction of both
I-
-BTX and
lophotoxin with the nicotinic receptor can be described by ,
where L, R, LR, L-R,
and k
are as defined for , B represents
I-
-BTX, B
R represents
a reversible complex between
I-
-BTX and the
receptor, k
and k
represent
the association and dissociation rate constants, respectively, for
lophotoxin, and k
and k
represent the association and dissociation rate constants,
respectively, for
I-
-BTX. Changes in ligand-bound
receptor complexes over time are described by the differential .
The variables [B], [L], and [R] in can be replaced with the conservation of mass ,
where [B],
[L
], and
[R
] represent total concentrations of
I-
-BTX, lophotoxin, and receptor, respectively. The
value for k
was experimentally determined from the
association of
I-
-BTX in the absence of competing
drugs (5.49 ± 0.430
10
M
s
, n = 4). The value for k
was previously
determined to be 3.3
10
s
(Sine and Claudio, 1991b). The value for k
was experimentally determined from an independent analysis of the
irreversible inhibition of nicotinic receptors by lophotoxin (Eq. 2).
Because of the limited ability of lophotoxin to distinguish between the
two acetylcholine-binding sites, an average of the values for k
was used in the analysis. Thus, the only unknown
parameters in are k
and k
. were numerically integrated, and the resultant curves were
fit to the experimental data using a Marquardt-Levenberg iterative
curve-fitting algorithm to determine the best-fit values of k
and k
. The analysis was
performed on a SparcStation 2 computer (Sun Microsystems) using the
data analysis software package PROPHET 4.0 (Bolt, Beranek, and Newman,
Inc.).
Bipinnatin-A, -B, and -C are protoxins that are spontaneously
activated during incubation in aqueous buffer (Groebe et al.,
1994). Since the structure of lophotoxin is similar to the bipinnatins (Fig. 1), it might be expected that lophotoxin would also
undergo spontaneous activation upon incubation in buffer. Indeed, the
instability of lophotoxin has already been proposed (Culver et
al., 1984). To investigate the stability and possible
activation of lophotoxin, the apparent rate of irreversible inhibition
of nicotinic receptors (k) by lophotoxin was
determined after preincubation of the toxin in buffer for 0, 2, and 8
h. Surprisingly, preincubation of lophotoxin in buffer for up to 8 h
did not result in a significant change in the apparent rate of
irreversible inhibition of receptors (Fig. 2). In addition, no
difference was detected by thin-layer chromatography between 100
µM lophotoxin in ethyl acetate and 100 µM lophotoxin incubated at room temperature for 1 h in water and 1%
dimethyl sulfoxide (data not shown). These results suggest that
lophotoxin does not undergo activation in aqueous buffer and that it is
more stable than the bipinnatins.
Figure 1: The chemical structures of the lophotoxins.
Figure 2:
Effect of preincubation of lophotoxin on
the rate of irreversible inhibition of nicotinic receptors. Receptors
were incubated with 20 µM fresh lophotoxin
(--) or with 20 µM lophotoxin that had
been allowed to pre-incubate in assay buffer for 2 h (-
-
- -) or 8 h (- -
- -). The
value of log
(100*R
/R
)
is the log of the percentage of specifically bound
I-
-BTX at each time point (R
) to the specific binding of
I-
-BTX to cells not exposed to lophotoxin (R
). Lines through the data were
obtained by nonlinear regression of a double first-order exponential
decay function ().
The apparent rate of irreversible
inhibition of nicotinic receptors by lophotoxin was determined over a
toxin concentration range of 0.5-50 µM. At low
concentrations of lophotoxin (<5 µM), two apparent
rates of irreversible inhibition were clearly observable on a semi-log
plot of the data (Fig. 3). At higher concentrations, the
difference between the two rates became less pronounced. The apparent
rates of irreversible inhibition for the two acetylcholine-binding
sites (k and k
) did not increase linearly with
increasing toxin concentration but approached upper limits at
approximately 50 µM toxin (Fig. 4). Thus, for each
binding site, the equilibrium dissociation constant (K
) and the rate of irreversible inhibition (k
) could be determined by nonlinear regression of to values of k
and k
as a function of lophotoxin
concentration (Table 1). It has been previously shown that
lophotoxin preferentially inhibits the site with lower affinity for
metocurine and that this site is located near the
/
-subunit
interface in nicotinic receptors from BC
H-1 cells (Culver et al., 1984; Sine and Claudio, 1991a). Analysis of k
as a function of lophotoxin concentration
suggests that this site preference resulted from both a higher
reversible affinity and a faster rate of irreversible inhibition at the
/
-site. Comparison of the bimolecular reaction constants (k
) for each acetylcholine-binding site reveals
that lophotoxin discriminated between the two sites by approximately
4-fold (Table 1).
Figure 3:
Irreversible inhibition of nicotinic
receptors by lophotoxin. Receptors were incubated with 0.5 µM (), 1.0 µM (
), 2.0 µM (
), 5.0 µM (
), 10.0 µM (
), 20.0 µM (
), and 50 µM (
) lophotoxin. The data shown are from representative
experiments at each concentration of toxin. The value of log
(100*R
/R
)
is the log of the percentage of specifically bound
I-
-BTX at each time point (R
) to the specific binding of
I-
-BTX to cells not exposed to lophotoxin (R
). Lines through the data were
obtained by nonlinear regression of a double first-order exponential
decay function ().
Figure 4:
Apparent rate of receptor inhibition by
lophotoxin versus toxin concentration. The values of k (panelA) or k
(panelB) are plotted versus toxin concentration. Each data point represents the
mean ± S.E. of 3-5 experiments. Lines through the
data were obtained by nonlinear regression of a saturation binding
function to a single class of binding sites ().
An alternative method that can be used to
determine the initial reversible affinity of lophotoxin is to examine
the ability of lophotoxin to inhibit the initial rate of I-
-BTX association (Weiland et al., 1977).
For example, if it is assumed that the association of lophotoxin is
diffusion limited (e.g.k
1
10
M
s
),
then 100 µM lophotoxin (K
=
4-11 µM) (Table 1) would be expected to
reversibly occupy greater than 90% of the total population of
acetylcholine-binding sites within 10
s (Motulsky
and Mahan, 1984). In comparison, 20 nM
I-
-BTX (k
= 5.49
10
M
s
) requires at least 50 min to occupy the same
number of sites. Thus, if 100 µM lophotoxin and 20 nM
I-
-BTX are simultaneously added to receptors,
lophotoxin would be expected to reversibly occupy almost all of the
acetylcholine-binding sites before
I-
-BTX was bound
to any significant extent. The result would be a marked reduction in
the apparent initial association rate of
I-
-BTX.
Surprisingly, however, 100 µM lophotoxin had little, if
any, effect on the apparent initial association rate of
I-
-BTX over the first 5 min (Fig. 5). These
results are inconsistent with a diffusion-limited association rate for
lophotoxin.
Figure 5:
Association of I-
-BTX
to nicotinic receptors in the presence and absence of lophotoxin.
Association of
I-
-BTX (20 nM) to nicotinic
receptors was determined in the absence of lophotoxin (
) or after
simultaneous addition of
I-
-BTX and 20 µM (
) or 100 µM (
) lophotoxin. Lines through the data were obtained by nonlinear regression of
numerically integrated differential equations as described under
``Experimental Procedures.''
The association (k) and
dissociation (k
) rate constants for unlabeled
reversible competitors can be obtained indirectly by using the
integrated rate equation that defines the association of a radiolabeled
ligand in the presence of an unlabeled reversible competitor (Motulsky
and Mahan, 1984; Contreras et al., 1986; Hawkinson and Casida,
1992). This method can be applied to any radiolabeled ligand and
unlabeled reversible competitor provided that measurable accumulation
of bound radioligand occurs before the unlabeled competitor reaches
equilibrium (Contreras et al., 1986). However, the
differential equations that describe the interaction of a radioligand
and an unlabeled irreversible competitor with a receptor () are not readily integrated. This limitation can be
overcome by numerical integration of the series of differential
equations that describe these reactions, coupled with nonlinear
regression analysis (Chandler et al., 1972; Williams et
al., 1979; Zimmerle and Frieden, 1989; Frieden, 1993). Nonlinear
regression of the numerical solutions of was applied to the complete
association of
I-
-BTX in the presence of lophotoxin
to determine k
and k
for
lophotoxin (Fig. 5). As expected, the values obtained for k
(1580 ± 255 M
min
, n = 6) and k
(0.027 ± 0.009 min
, n = 6) were independent of lophotoxin concentrations
between 20 and 100 µM. Due to the limited ability of
lophotoxin to distinguish between the two acetylcholine-binding sites (Table 1), the values for k
and k
at both sites could not be determined. However,
the average K
calculated from the ratio of k
/k
(16.6 ± 4.6
µM) was similar to the average K
(7.6
µM) of the two individual binding sites determined
independently (Table 1).
The observations that 1) the apparent
initial association rate of I-
-BTX was unaffected
for the first 5 min by saturating concentrations of lophotoxin and 2)
that lophotoxin has an apparent association rate constant that is
approximately 10
-fold slower than a diffusion-limited rate
indicate that lophotoxin is a slow binding inhibitor (Morrison and
Walsh, 1988). A slow conformational isomerization of the receptor from
a low affinity state to a higher affinity state is one possible
mechanism that could result in a slow apparent association rate
(Vauquelin et al., 1980; Contreras et al., 1986;
Morrison and Walsh, 1988; Lejczak et al., 1989). Indeed, a
slow transition of the nicotinic receptor from a low affinity state to
a higher affinity state is thought to occur upon the binding of
agonists, and this transition is facilitated by the noncompetitive
allosteric inhibitor dibucaine (Weiland et al., 1976, 1977;
Boyd and Cohen, 1980; Sine and Claudio, 1991a). For example, a low
concentration of carbamylcholine (20 µM) had relatively
little effect on the apparent initial association rate of
I-
-BTX (Fig. 6, upper panel).
However, in the presence of dibucaine, the same concentration of
carbamylcholine resulted in an immediate and profound decrease in the
apparent initial association rate of
I-
-BTX (Fig. 6, upper panel). In the absence of
carbamylcholine, dibucaine did not affect the association rate constant
of
I-
-BTX with the receptor (k
= 5.48
10
± 760 M
s
, n =
2).
Figure 6:
Effects of dibucaine on the interaction of
carbamylcholine and lophotoxin with nicotinic receptors. Upper
panel, association of I-
-BTX to nicotinic
receptors was determined in the absence (
) and presence (
)
of 20 µM carbamylcholine. Association of
I-
-BTX was also determined in the presence of 20
µM carbamylcholine and 20 µM dibucaine
(
). Lines through the data were obtained by nonlinear
regression of numerically integrated differential equations describing
the association of two reversible ligands with the receptor. Middle
panel, irreversible inhibition of nicotinic receptors by 2
µM lophotoxin in the absence
(
-
) and presence (
- -
-
) of 20 µM dibucaine. Lines through
the data were obtained by nonlinear regression of a double first-order
exponential decay function (). Lower panel,
association of
I-
-BTX to nicotinic receptors was
determined in the absence (
-
) and
presence (
-
) of 50 µM lophotoxin. The association of
I-
-BTX was also
determined in the presence of 50 µM lophotoxin and 20
µM dibucaine (
- - -
). Lines through the data were obtained by nonlinear regression
of numerically integrated differential equations as described under
``Experimental Procedures.''
If the slow association rate of lophotoxin resulted from a slow
conformational transition of the receptor to a state similar to that
induced by agonists, then dibucaine should be expected to facilitate
this transition. However, dibucaine had little effect on the kinetic
parameters that describe the interaction of lophotoxin with the
nicotinic receptor. For example, dibucaine did not significantly effect
the apparent rates of irreversible inhibition (k and k
)
by lophotoxin (Fig. 6, middle panel). In addition, the
association of
I-
-BTX in the presence of lophotoxin
was unaffected by the presence of dibucaine (Fig. 6, lower
panel). Thus, no significant changes in k
, k
, or k
were observed for
lophotoxin in the presence of dibucaine, suggesting that the binding of
lophotoxin does not induce a slow conformational change in the receptor
similar to that induced by agonists.
Bipinnatin-A, -B, and -C are converted from inactive
protoxins into effective irreversible inhibitors of nicotinic
acetylcholine receptors after preincubation in buffer (Groebe et
al., 1994). In contrast to the bipinnatins, the apparent rate of
irreversible inhibition by lophotoxin did not increase after
preincubation in buffer for up to 8 h. Thus, although its structure is
similar to the bipinnatins, lophotoxin did not appear to undergo
activation to a more effective inhibitor. In addition, the k for lophotoxin did not decrease significantly
after preincubation in buffer, suggesting that lophotoxin is a
relatively stable irreversible inhibitor of nicotinic receptors. These
results are consistent with the observation that the longer lophotoxin
is incubated with nicotinic receptors, the lower its observed IC
(Abramson et al., 1991).
Comparison of the structure
of lophotoxin with the bipinnatins suggests that the greater stability
of lophotoxin results from different substituents at positions R and R
(Fig. 1). At position R
,
lophotoxin lacks an acetate ester that is present in the bipinnatins.
It is possible that spontaneous, non-enzymatic hydrolysis of the
acetate ester at position R
may be an initiating event in
activation of the bipinnatins. Alternatively, it is possible that the
presence of an aldehyde at position R
in lophotoxin is
responsible for the increased activity of lophotoxin and for its
greater stability.
Although the ability of lophotoxin to
discriminate between the two acetylcholine-binding sites is relatively
small, it is sufficient to demonstrate that lophotoxin preferentially
inhibits the binding site that has lower affinity for metocurine
(Culver et al., 1984). Additional investigations have shown
that the higher affinity site for lophotoxin is the
acetylcholine-binding site located near the /
-subunit
interface (Sine and Claudio, 1991a). In this study, unique values for
the reversible affinity (K
) and the rate of
irreversible inhibition (k
) were determined for
each acetylcholine-binding site. Preferential inhibition of the site at
the
/
-subunit interface was shown to result from both a
higher affinity and a faster rate of irreversible inhibition at this
site. The ratio of k
/K
defines a bimolecular reaction constant (k
) for
the interaction of lophotoxin with each binding site of the receptor
(Main, 1964; Berman and Leonard, 1989; Groebe et al., 1994).
There was an approximate 4-fold difference in the bimolecular reaction
constants for the two acetylcholine-binding sites, reflecting the
extent to which lophotoxin preferentially inhibits the binding site at
the
/
-subunit interface. Of the four lophotoxins for which
the extent of discrimination between the two binding sites on the
receptor is known, lophotoxin is the least able to distinguish between
the two sites (Groebe et al., 1994). Apparently, the
substituent at R3 is important for determining the extent of site
discrimination by the different lophotoxins.
Association (k) and dissociation (k
) rate
constants for reversible inhibitors can be determined directly if the
inhibitor is radiolabeled. In addition to these rate constants, the
rate of irreversible inhibition (k
) can also be
determined directly for radiolabeled irreversible inhibitors. For
example, the kinetic rate constants for the reversible and irreversible
inhibition of µ opioid receptors by the irreversible antagonist
-funaltrexamine were measured directly from the specific binding
of [
H]
- funaltrexamine to bovine striatal
membrane preparations (Liu-Chen et al., 1990). The association (k
) and dissociation (k
) rate
constants of unlabeled reversible inhibitors are difficult to obtain,
but they can be determined indirectly from analysis of the extent to
which the unlabeled inhibitor reduces the association rate of a
radiolabeled ligand (Motulsky and Mahan, 1984; Contreras et
al., 1986; Hawkinson and Casida, 1992). This indirect method
results in valid estimates of k
and k
for unlabeled competitors only if measurable
accumulation of bound radiolabeled ligand occurs before the unlabeled
competitor reaches equilibrium (Contreras et al., 1986).
The values of k and k
for
lophotoxin were determined by numerical integration of the series of
differential equations that describe the binding of lophotoxin and
I-
-BTX coupled with nonlinear least-squares
regression (Chandler et al., 1972; Williams et al.,
1979; Zimmerle and Frieden, 1989; Frieden, 1993). The kinetic rate constants k
and k
should be independent of lophotoxin concentration, and, as
expected, the values for k
and k
determined using this method did not vary systematically with
toxin concentrations between 20 and 100 µM. In addition,
the average K
for lophotoxin determined using this
method (k
/k
) was similar to
the average K
determined independently from the
irreversible inhibition of receptors by lophotoxin. These observations
support the validity of this indirect approach for the determination of
kinetic rate constants for unlabeled irreversible receptor antagonists.
The apparent association rate constant of lophotoxin was
approximately 10-fold slower than expected for a
diffusion-limited bimolecular interaction. While the apparent values of k
and k
depend upon the model
chosen, simple steric hindrance of the binding of lophotoxin to the
nicotinic receptor () is sufficient to account for the low
value observed for k
. However, a more complicated
kinetic model that would also be consistent with the slow apparent
association rate constant of lophotoxin involves an initial rapid
association followed by a slow reversible conversion of the receptor
from a conformation with low affinity for lophotoxin to a conformation
with higher affinity (Vauquelin et al., 1980; Contreras et
al., 1986; Lejczak et al., 1989). For example, agonists
of the nicotinic receptor can be considered slow binding ligands
because they promote a slow conversion of the receptor from a low
affinity state to a higher affinity state (Weiland et al.,
1976, 1977; Boyd and Cohen, 1980; Morrison and Walsh, 1988). A similar
model has also been proposed for agonists and some antagonists of
-adrenergic receptors (Contreras et al., 1986; Samama et al., 1994). In fact, this may be a general mechanism by
which the function of receptors is properly regulated (Karlin, 1967;
Jackson, 1989).
In the absence of agonist, there is no significant
accumulation of desensitized receptors at the concentration of
dibucaine used in these investigations (Sine and Taylor, 1982).
However, in the presence of agonist, it is thought that dibucaine
facilitates transition of the nicotinic receptor from a state with low
affinity for agonists to a desensitized state with higher affinity for
agonists (Weiland et al., 1977). This is demonstrated in the
observation that the presence of dibucaine resulted in a substantial
increase in the ability of carbamylcholine to slow the apparent initial
association rate of I-
-BTX to the nicotinic
receptor. If the apparently low value of k
observed for lophotoxin was due to a slow conformational change
in the receptor similar to that induced by agonists, then dibucaine
would be expected to increase the apparent rate of irreversible
inhibition by lophotoxin. However, dibucaine did not significantly
alter the kinetic rate constants for lophotoxin. This indicates that
lophotoxin does not induce a transition of the nicotinic receptor to a
desensitized state as is observed for agonists. In addition, the
kinetic rate constants do not describe lophotoxin's affinity for
a desensitized receptor. These results, however, do not rule out the
possibility that the binding of lophotoxin stabilizes an as yet
uncharacterized conformation of the receptor that has a higher affinity
for the toxin and that this conformation may facilitate receptor
inhibition (Samama et al., 1994). Since lophotoxin reacts
covalently with the receptor, such a mechanism would be consistent with
the concept of a quiescent affinity label as described by Krantz et
al.(1991).
Another possible explanation for the apparently low
value of k observed for lophotoxin is that the
actual concentration of active toxin is significantly lower than
expected. For example, fluoromethyl ketones are slow binding inhibitors
of acetylcholinesterase that undergo a reversible conversion in aqueous
solution to an inactive hydrated species (Allen and Abeles, 1989). The
equilibrium between the inactive hydrate and active ketone species
greatly favors the inactive hydrate form, and it is the unexpectedly
low concentration of the active ketone that results in the slow
apparent association rates of these inhibitors. Indeed, the actual
association rate constants are calculated to be nearly diffusion
limited when the true concentrations of the active ketone species are
considered (Allen and Abeles, 1989). A slow reversible interconversion
between an inactive and active form of lophotoxin, as exemplified by
the fluoromethyl ketones, cannot be ruled out. In addition, although
lophotoxin does not appear to undergo significant irreversible
hydrolysis in buffer to either an inactive or a more active form over
the time scale investigated in this study, it is possible that
lophotoxin (L`) is slowly and irreversibly converted (t
> 20 h) into an active inhibitor (L), which either reacts rapidly with the receptor or degrades
rapidly into an inactive form (L*) as described by .
Under these conditions, a low steady-state concentration of active lophotoxin (<1 nM) could be rapidly produced. If this were the case, then lophotoxin would appear to be a relatively stable, slow binding inhibitor.
A complete understanding of the
mechanism for the irreversible inhibition of nicotinic receptors by the
lophotoxins would necessarily need to account for observed differences
in the stabilities and biological activities of the different
lophotoxins. It appears that structural differences at positions
R and R
contribute to the relative stability of
the lophotoxins while differences at position R
contribute
to toxin selectivity for the two acetylcholine-binding sites. However,
the effects of these structural differences may only serve to obscure
similarities in the mechanism by which the different lophotoxins
irreversibly inhibit nicotinic receptors. For example, is
an attractive model for the irreversible inhibition of nicotinic
receptors because it is consistent with the kinetic data for both
lophotoxin and the bipinnatins (Groebe et al., 1994).