©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Lophotoxin Is a Slow Binding Irreversible Inhibitor of Nicotinic Acetylcholine Receptors (*)

(Received for publication, May 19, 1994; and in revised form, October 26, 1994)

Duncan R. Groebe Stewart N. Abramson (§)

From the Department of Pharmacology, School of Medicine, University of Pittsburgh, Pittsburgh, Pennsylvania 15261

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

Lophotoxin and the bipinnatins are members of the lophotoxin family of marine neurotoxins, which covalently react with Tyr in the alpha-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-alpha-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 10^6-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.


INTRODUCTION

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 (alpha(2)beta) in a circular arrangement spanning the membrane bilayer (Unwin, 1993). The two acetylcholine-binding sites of the receptor are located near the alpha/- and alpha/-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 alpha-subunits (alphaTyr) 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 alphaTyr 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 alphaTyr, 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 alpha-subunits of the nicotinic receptor, and 3) react with alphaTyr but not with an adjacent alphaTyr (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 alpha/-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(3)H-1 cells by lophotoxin.


EXPERIMENTAL PROCEDURES

Materials

Lophotoxin was purified as previously described and stored as a dry powder at -20 °C (Abramson et al., 1991). Stocks of lophotoxin were resuspended to 10 mM in 100% dimethyl sulfoxide and stored at -20 °C. Iodinated alpha-bungarotoxin (I-alpha-BTX, (^1)10-20 µCi/µg) was purchased from DuPont NEN. All other materials, chemicals, and media were purchased from Fisher, Life Technologies, Inc., Sigma, and VWR.

Maintenance of BC(3)H-1 Cells

BC(3)H-1 cells were maintained in growth medium, and multiwell plates were treated with denatured porcine skin gelatin as previously described (Groebe et al., 1994). For all experiments, cells in growth medium were seeded into 24-well plates at a density of 8-12 times 10^3 cells/well. The growth medium was changed upon confluency (day 4 or 5) and changed once more prior to the experiment (days 7-10). The cells were used in experiments 2-3 days following the second medium change. For every experimental protocol, multiwell plates of cells were equilibrated to room temperature for 30 min, washed once with 1.0 ml of assay buffer (140 mM KCl, 25 mM HEPES, 5.4 mM NaCl, 1.8 mM CaCl(2), 1.7 mM MgSO(4), 0.06 mg/ml bovine serum albumin, pH 7.4), and equilibrated for 20 min in 1.0 ml of fresh assay buffer. The binding site density for I-alpha-BTX was 219 ± 10.2 fmol/well (n = 29).

Irreversible Inhibition of Nicotinic Receptors by Lophotoxin

Stocks of lophotoxin (10 mM in 100% dimethyl sulfoxide) were diluted to the indicated toxin concentrations and 1% dimethyl sulfoxide in assay buffer. Irreversible inhibition of nicotinic receptors by lophotoxin was performed at room temperature as described by Groebe et al.(1994). Briefly, the assay buffer was replaced with 250 µl of the diluted lophotoxin preparation. At each time point, the cells were washed twice with 2.0 ml of assay buffer and then incubated for 1 h in 250 µl of a saturating concentration of I-alpha-BTX (15-20 nM). The cells were washed twice with 2.0 ml of assay buffer and resuspended in two 0.5-ml washes of 1% Triton X-100, and the amount of bound I-alpha-BTX was measured. Individual time points were performed in triplicate. Total I-alpha-BTX binding was determined from cells incubated for 1 h with I-alpha-BTX. Nonspecific binding of I-alpha-BTX was determined from cells incubated for 30 min with 100 nM alpha-BTX prior to 1 h of incubation with I-alpha-BTX.

Association of I-alpha-BTX to Nicotinic Receptors in the Presence of Carbamylcholine or Lophotoxin

To determine the association rate of I-alpha-BTX, 250 µl of I-alpha-BTX (15-20 nM) was added to multiwell plates of BC(3)H-1 cells. At the indicated times, the cells were washed twice with 2.0 ml of assay buffer, and the amount of bound I-alpha-BTX was measured. To determine the association and dissociation rates of unlabeled competitors, lophotoxin or carbamylcholine were simultaneously added with I-alpha-BTX. At the indicated times, the cells were washed as before, and the amount of bound I-alpha-BTX measured. To investigate the effect of dibucaine on the association and dissociation rates of lophotoxin and carbamylcholine, cells were equilibrated with 250 µl of dibucaine (20 µM) in assay buffer for 30 min. Simultaneous incubation of lophotoxin or carbamylcholine with I-alpha-BTX was then performed in the continued presence of dibucaine.

Data Analysis, Nonlinear Regression, and Calculation of Kinetic Constants

An equation describing a double exponential decay as a function of time () was fit by nonlinear regression to data obtained from irreversible inhibition by lophotoxin. Nonlinear regression was performed on a Northgate 386 personal computer using InPlot 4.0 (GraphPad).

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, LbulletR represents an initial reversible complex, L-R represents an irreversible complex, K(d) represents the dissociation constant for formation of the LbulletR complex, and k(3) represents the rate constant for formation of the L-R complex (Kitz and Wilson, 1962; Culver et al., 1984; Rakitzis, 1984). A plot of kversus lophotoxin concentration describes a hyperbolic curve in which the maximum k is equal to k(3), and the concentration of toxin ([L]) at half-maximal k is equal to K(d) ().

The covalent reaction between lophotoxin and alphaTyr prevents the binding of I-alpha-BTX. If I-alpha-BTX does not bind to the receptor when lophotoxin reversibly occupies the acetylcholine-binding site, then the simultaneous interaction of both I-alpha-BTX and lophotoxin with the nicotinic receptor can be described by ,

where L, R, LbulletR, L-R, and k(3) are as defined for , B represents I-alpha-BTX, BbulletR represents a reversible complex between I-alpha-BTX and the receptor, k(1) and k(2) represent the association and dissociation rate constants, respectively, for lophotoxin, and k(4) and k(5) represent the association and dissociation rate constants, respectively, for I-alpha-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(T)], [L(T)], and [R(T)] represent total concentrations of I-alpha-BTX, lophotoxin, and receptor, respectively. The value for k(4) was experimentally determined from the association of I-alpha-BTX in the absence of competing drugs (5.49 ± 0.430 times 10^4M s, n = 4). The value for k(5) was previously determined to be 3.3 times 10 s (Sine and Claudio, 1991b). The value for k(3) 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(3) was used in the analysis. Thus, the only unknown parameters in are k(1) and k(2). 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(1) and k(2). 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.).


RESULTS

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 (-bullet-) or with 20 µM lophotoxin that had been allowed to pre-incubate in assay buffer for 2 h (- -circle- -) or 8 h (- -times- -). The value of log (100*R/R) is the log of the percentage of specifically bound I-alpha-BTX at each time point (R) to the specific binding of I-alpha-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 alpha/-subunit interface in nicotinic receptors from BCH-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 alpha/-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 (bullet), 1.0 µM (circle), 2.0 µM (times), 5.0 µM (), 10.0 µM (), 20.0 µM (box), and 50 µM (up triangle) 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-alpha-BTX at each time point (R) to the specific binding of I-alpha-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-alpha-BTX association (Weiland et al., 1977). For example, if it is assumed that the association of lophotoxin is diffusion limited (e.g.k(1) geq 1 times 10^7M s), then 100 µM lophotoxin (K(d) = 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 nMI-alpha-BTX (k(1) = 5.49 times 10^4M s) requires at least 50 min to occupy the same number of sites. Thus, if 100 µM lophotoxin and 20 nMI-alpha-BTX are simultaneously added to receptors, lophotoxin would be expected to reversibly occupy almost all of the acetylcholine-binding sites before I-alpha-BTX was bound to any significant extent. The result would be a marked reduction in the apparent initial association rate of I-alpha-BTX. Surprisingly, however, 100 µM lophotoxin had little, if any, effect on the apparent initial association rate of I-alpha-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-alpha-BTX to nicotinic receptors in the presence and absence of lophotoxin. Association of I-alpha-BTX (20 nM) to nicotinic receptors was determined in the absence of lophotoxin (bullet) or after simultaneous addition of I-alpha-BTX and 20 µM (circle) or 100 µM (times) lophotoxin. Lines through the data were obtained by nonlinear regression of numerically integrated differential equations as described under ``Experimental Procedures.''



The association (k(1)) and dissociation (k(2)) 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-alpha-BTX in the presence of lophotoxin to determine k(1) and k(2) for lophotoxin (Fig. 5). As expected, the values obtained for k(1) (1580 ± 255 M min, n = 6) and k(2) (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(1) and k(2) at both sites could not be determined. However, the average K(d) calculated from the ratio of k(2)/k(1) (16.6 ± 4.6 µM) was similar to the average K(d) (7.6 µM) of the two individual binding sites determined independently (Table 1).

The observations that 1) the apparent initial association rate of I-alpha-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 10^6-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-alpha-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-alpha-BTX (Fig. 6, upper panel). In the absence of carbamylcholine, dibucaine did not affect the association rate constant of I-alpha-BTX with the receptor (k(1) = 5.48 times 10^4 ± 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-alpha-BTX to nicotinic receptors was determined in the absence (bullet) and presence (circle) of 20 µM carbamylcholine. Association of I-alpha-BTX was also determined in the presence of 20 µM carbamylcholine and 20 µM dibucaine (times). 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 (bullet-bullet) and presence (circle- - -circle) 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-alpha-BTX to nicotinic receptors was determined in the absence (bullet-bullet) and presence (circle-circle) of 50 µM lophotoxin. The association of I-alpha-BTX was also determined in the presence of 50 µM lophotoxin and 20 µM dibucaine (times- - -times). 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-alpha-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.


DISCUSSION

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(1) and R(2) (Fig. 1). At position R(2), 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(2) may be an initiating event in activation of the bipinnatins. Alternatively, it is possible that the presence of an aldehyde at position R(1) 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 alpha/-subunit interface (Sine and Claudio, 1991a). In this study, unique values for the reversible affinity (K(d)) and the rate of irreversible inhibition (k(3)) were determined for each acetylcholine-binding site. Preferential inhibition of the site at the alpha/-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(3)/K(d) 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 alpha/-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(1)) and dissociation (k(2)) 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(3)) 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 beta-funaltrexamine were measured directly from the specific binding of [^3H]beta- funaltrexamine to bovine striatal membrane preparations (Liu-Chen et al., 1990). The association (k(1)) and dissociation (k(2)) 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(1) and k(2) 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(1) and k(2) for lophotoxin were determined by numerical integration of the series of differential equations that describe the binding of lophotoxin and I-alpha-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(1) and k(2) should be independent of lophotoxin concentration, and, as expected, the values for k(1) and k(2) determined using this method did not vary systematically with toxin concentrations between 20 and 100 µM. In addition, the average K(d) for lophotoxin determined using this method (k(2)/k(1)) was similar to the average K(d) 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 10^6-fold slower than expected for a diffusion-limited bimolecular interaction. While the apparent values of k(1) and k(2) 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(1). 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 beta-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-alpha-BTX to the nicotinic receptor. If the apparently low value of k(1) 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(1) 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(1) and R(2) contribute to the relative stability of the lophotoxins while differences at position R(3) 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).


FOOTNOTES

*
This work was supported in part by National Institutes of Health Grant NS29951, the Smokeless Tobacco Research Council Grant 0280, and the Pharmaceutical Manufacturers Association Foundation (Faculty Development Award). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed. Tel.: 412-648-9751; Fax: 412-648-1945; sna{at}prophet.pharm.pitt.edu.

(^1)
The abbreviation used is: alpha-BTX, alpha-bungarotoxin.


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