(Received for publication, November 9, 1995; and in revised form, February 5, 1996)
From the
Anthopleurin B is a potent anemone toxin that binds with nanomolar affinity to the cardiac and neuronal isoforms of the voltage-gated sodium channel. A cationic cluster that includes Arg-12, Arg-14 and Lys-49 has been shown previously to be important in this interaction. In this study, we have used site-directed mutagenesis to determine the contribution to activity of two aliphatic residues, Leu-18 and Ile-43, that have previously been experimentally inaccessible. Leu-18, a residue proximal to the cationic cluster, plays a critical role in defining the high affinity of the toxin. In ion flux studies, this is exemplified by the several hundredfold loss in affinity (231-672-fold) observed for both L18A and L18V toxins on either isoform of the sodium channel. When analyzed electrophysiologically, L18A, the most severely compromised mutant, also displays a substantial loss in affinity (34-fold and 328-fold) for the neuronal and cardiac isoforms. This difference in affinities may reflect an increased preference of the L18A mutant for the closed state of the neuronal channel. In contrast, Ile-43, a residue distal to the cationic cluster, plays at most a very modest role in affinity toward both isoforms of the sodium channel. Only conservative substitutions are tolerated at this position, implying that it may contribute to an important structural component. Our results indicate that Leu-18 is the most significant single contributor to the high affinity of Anthopleurin B identified to date. These results have extended the binding site beyond the cationic cluster to include Leu-18 and broadened our emphasis from the basic residues to include the crucial role of hydrophobic residues in toxin-receptor interactions.
Voltage-sensitive sodium channels play a crucial role in the
transmission of electrical signals in excitable
cells(1, 2) . Several laboratories have used toxins
that alter channel function to advance our understanding of sodium
channel biology(3, 4) . These toxins have been
classified by Catterall (4) as binding to one of five different
receptor sites on the channel with consequent effects on either
activation, inactivation, or ion conductance. Of particular interest,
primarily because of their potential as cardiotonic agents, are anemone
toxins and -scorpion toxins that delay sodium channel
inactivation, resulting in enhanced sodium influx and ultimately giving
rise to an increase in the force of
contraction(5, 6, 7, 8) . The
classical drugs used to treat heart failure are the cardiac glycosides
ouabain and digoxin, which are potent inhibitors of the
Na
/K
-ATPase, and the
-adrenoreceptor agonists dopamine and
dobutamine(9, 10, 11) . The life threatening
toxicity associated with digoxin and the decreased effectiveness of the
-agonists as heart failure progresses underscore the importance of
identifying novel agents displaying cardiotonic activity(11) .
Sea anemones are a rich source of biologically active polypeptides
with diverse pharmacological activities. While most anemone toxins were
isolated as neurotoxins, Anthopleurin A and B (ApA and ApB) ()obtained from the sea anemone Anthopleura
xanthogrammica were originally isolated based on their displaying
cardiac stimulatory activity(8, 12) . In isolated
cardiac muscle, ApA is more potent than digoxin, being effective at
nanomolar (nM) concentrations(13) . More importantly,
studies using anesthetized dogs demonstrate that this activity is not
associated with adverse effects on heart rate or blood
pressure(13, 14) . ApB is even more potent than ApA,
displaying its maximal inotropic activity at 0.3
nM(8) . While their antigenicity and lack of oral
activity in animals preclude the use of these toxins as drugs in their
naturally occurring forms, understanding the molecular interactions
between these toxins and the sodium channel could form the basis for
rational design of new drugs displaying enhanced cardiotonic activity.
It is essential that the approach taken combine available structural
information with functional studies in order to define regions of the
molecule that contribute either to high affinity or selectivity for the
cardiac channel.
ApA and ApB are naturally occurring homologs that differ in only 7 out of 49 residues and are cross-linked by three disulfide bonds(8, 10) . Ion flux studies have established that ApB exhibits nanomolar affinity for both the cardiac and neuronal isoforms of the sodium channel, while ApA binds much less tightly to the latter(15) . Under voltage clamp conditions, both ApA and ApB bind preferentially to the cardiac channel, and ApB binding affinity is increased 100-fold in comparison with the values obtained by ion flux(16) . In contrast, ApB binding to the neuronal channel differs by only about 4-fold in these two assays. These results indicate that ApB binds both to the open and closed conformations of the channel and greatly prefers binding to the closed channel over other states.
This laboratory has previously cloned a synthetic gene for ApB and produced the recombinant protein using a bacterial expression system (17, 18) . Using site-directed mutagenesis, we were able to specifically target residues and determine their contribution to activity by measuring sodium uptake in tissue culture cells. These studies have emphasized cationic residues that are either unique to ApB or conserved among anemone toxins(15, 19) . Studies on the unique cationic residues in ApB indicated that the polar side chain of Arg-12 is important in activity(15) . In contrast, mutating the conserved positively charged residues Arg-14 and Lys-48 establishes that these residues play a smaller role in activity(19) . A set of three mutant toxins containing pairwise replacements of the cationic side chains were not completely inactivated although their apparent affinities were significantly diminished raising the possibility of compensatory effects upon replacement of single cationic sites(16) . Although our previous studies have identified residues which contribute 1-2 kcal/mol of binding energy, it is clear that additional sites of interaction remain unidentified(15) .
The solution structures of both ApA and
ApB have recently been solved by multidimensional NMR and reveal a
four-stranded anti-parallel -sheet structure common to both
polypeptides(20, 21) . In a recently published model,
the cationic side chains of Arg-12, Arg-14, and Lys-49 of ApB are
located close together, and the solution structure, while not
unambiguous, is generally consistent with this
model(16, 21) . The three-dimensional structures of
homologous scorpion toxins that interact with the sodium channel and
affect either activation or inactivation are highly similar to each
other although unrelated to those of anemone
toxins(22, 23, 24) . A notable conserved
structural similarity among scorpion toxins is a surface-exposed
hydrophobic region(22, 23, 25) . Based on
sequence analogies and chemical modification studies, Fontecilla-Camps et al.(26) proposed that this hydrophobic region is
either directly involved in channel binding or helps align other
residues that are important for both binding and specificity. Although
ApB lacks an analogous surface hydrophobic face, a number of its
hydrophobic side chains are at least partially exposed, including a
subset of those found in proximity to the cationic cluster mentioned
above(8, 16, 21) . The present study was
initiated in order to ascertain the extent to which these exposed
hydrophobic residues in ApB might participate in the toxin-channel
interaction. Sequence comparisons among homologous toxins and
three-dimensional structural information lead us to target Ile-43 and
Leu-18 to determine their role in toxin activity. Our results indicate
that only highly conservative substitutions, resulting in modest
changes in affinity, are tolerated for Ile-43, a residue which is
distal to the proposed cationic cluster. In striking contrast, Leu-18,
a proximal residue, contributes significantly to the high affinity of
ApB for both isoforms of the sodium channel, thus further delineating
the binding surface presented by these toxins. This study represents
the first direct demonstration that hydrophobic residues play an
essential role in sodium channel neurotoxin function.
where k was the inverse of the fitted
of modification time course and [T] the toxin
concentration.
Figure 1:
Veratridine-dependent Na
uptake by N1E-115 cells. Dose-response curves for ApB (
), L18A
(
), L18V (
), I43L (
), and I43V (
) were
determined as described under ``Experimental Procedures.''
These data have been corrected for basal uptake due to veratridine. The solid lines are theoretical curves determined as described by
Cleland(32) , and the points represent the
experimental data. The neuronal K
(nM)
obtained are: ApB (22 ± 3) and L18A (7897 ± 985), L18V
(5087 ± 1751), I43L (52 ± 9), and I43V (173 ± 25).
Based on these data, the maximal levels of uptake detected for the
mutants relative to ApB were L18A (0.7), L18V (0.4), I43L (1.1), and
I43V (1.0).
Figure 2:
Veratridine-dependent Na
uptake by RT4-B cells. Dose-response curves for ApB (
), L18A
(
), L18V (
), I43L (
), and I43V (
) were
determined as described for Fig. 1. These data have been
corrected for basal uptake due to veratridine. The cardiac K
(nM) obtained are: ApB (9 ± 3)
and L18A (6051 ± 795), L18V (2884 ± 906), I43L (19
± 4), and I43V (25 ± 4). Based on these data, the maximal
levels of uptake detected for the mutants relative to ApB were L18A
(0.7), L18V (0.6), I43L (0.6), and I43V
(0.9).
Figure 3: Comparison of toxin-channel interactions by ion flux and voltage clamp. Affinities were estimated in N1E-115 (N) and RT4-B (C) cell lines as described under ``Experimental Procedures,'' and the results (Fig. 1, Fig. 2, and Table 3) were compared on a log scale ± S.E. of the estimate.
The apparent binding affinities of wild-type ApB for
the neuronal and cardiac isoforms of the sodium channel, as estimated
by ion flux measurements, are 22 nM and 9 nM,
respectively (15, 18, 19) . Thus, the
apparent neuronal K for the L18A mutant (7.9
µM) represents at least a 359-fold reduction in affinity.
Similarly, a 231-fold reduction is observed for L18V, displaying a
neuronal K
of 5.1 µM. The same
trend is evident in RT4-B cells, with 672- and 320-fold decreases in
apparent affinity for the L18A and L18V toxins, respectively.
Comparison of affinities in the two cell types yields a discrimination
index for L18A of 0.5, indicating that its ability to preferentially
bind to the cardiac channel is compromised. L18V also has a reduced
discrimination index of 0.7. The highest velocity observed for the
L18A/L18V mutants is between 40 and 70% that of wild-type ApB in both
cell lines and appears not to represent a V
.
These results are consistent with the essential nature of the contact
made between Leu-18 and both the cardiac and neuronal isoforms of the
sodium channel.
Fig. 1and Fig. 2also show the
considerably smaller changes in apparent binding affinity observed for
the Ile-43 mutants. K values for I43L were only
2-fold different from that of wild type ApB in both cell types assayed.
A comparable reduction in apparent affinity of 2.8-fold was obtained
for the I43V substitution on the cardiac channel. Surprisingly, while
the maximal uptake values obtained for I43V in both cell types and for
I43L in the neuronal line are comparable to ApB, the I43L mutant
displays a reduced relative V
of 0.6 in the
RT4-B line (Fig. 2).
This study targets hydrophobic residues in the toxin ApB by
site-directed mutagenesis to establish their role in activity. Previous
experiments demonstrated the importance of a cationic cluster including
Arg-12, Arg-14, and Lys-49 to toxin
activity(15, 16, 19) . Using a model
structure of ApB described by Khera et al.(16) , we
have identified residues based on proximity to this basic region and
assessed their contribution to biological activity. In this model, the
CD1 carbon of Leu-18 is within 10 Å of the NH1 and NH of Arg-14 while Ile-43 is clearly on the opposite face of the
molecule. Selecting Ile-43, distal to the cluster, and Leu-18, a
residue proximal to the cationic region, has enabled us to to determine
the nature of the potential intermolecular contacts made by two
distinct surfaces of the toxin.
Using a PCR-based approach with a wobble containing primer, we generated two panels of mutants, of which a subset was expressed and characterized. Only highly conservative substitutions to leucine or valine were tolerated at position 43. We interpret these results as supporting the hypothesis that Ile-43 is involved in ApB folding, consistent with our model suggesting that it packs tightly against the Phe-24 and Tyr-25 side chains(16) . We suggest that the I43A and I43G mutants disrupt the resulting hydrophobic region, preventing the protein from folding to a form allowing correct pairing of the three disulfide bonds. Since the ApB coordinates are not accessible in the Brookhaven data base, we are presently unable to verify this prediction.
Structural
characterization of the mutants includes amino acid analysis and
circular dichroism studies. While the amino acid compositions are
overall in good agreement with that of wild-type ApB, the glutamic acid
contents unexpectedly ranged from 0.6-1.1 residues per mol.
Previously, we attributed this to a system artifact(16) .
Recently, however, characterization by mass spectrometry and N-terminal
sequencing of ApB expressed from pKB13 revealed the presence of two
forms, one having a residue of glutamate at the amino terminus. ()This extra glutamate is found only in ApB expressed from
pKB13, in which the toxin sequence is preceded by five consecutive
glutamates. Glu-ApB, like an N-terminally extended form we
characterized earlier(18) , is functionally identical with the
wild-type toxin. Furthermore, all the mutants retained as their
predominant secondary structural motif the
-sheet as assessed by
circular dichroism. Thermal denaturation profiles for all mutants were
essentially unchanged, confirming the structural stability of the
toxins.
We have made two replacements to evaluate the contribution
of the hydrophobic residue Leu-18. Substitution with alanine represents
a side chain truncation in which all interactions made by atoms beyond
the -carbon are removed. Replacement with valine should restore
some of the hydrophobicity. Characterization of both mutants by ion
flux demonstrates a pivotal role for Leu-18 in toxin activity as
exemplified by the several hundredfold (231-672-fold) loss in
activity observed in both model systems analyzed. By comparison, double
neutralization mutants in the cationic cluster reduce apparent
affinities by a maximum of 72-fold(16) .
The estimated
maximal level of uptake obtained for the Leu-18 mutants is only
40-70% of that seen at saturating levels of ApB. Either we have
not saturated ApB binding sites even at concentrations of 10-25
µM mutant toxin, or the ability of the bound mutants to
stabilize the open conformation is compromised. Based on the results
depicted in Fig. 1and Fig. 2, we favor the former
explanation, which suggests that the K values
are even higher than those presented here. Because the range of
concentrations we are able to assay is restricted by the amount of
protein we can produce, we are unable to confirm this prediction.
Nonetheless, the key role of Leu-18 is amply supported by the data
presented.
In order to more closely assess the role of Leu-18 in
activity, we assayed the most severely impaired mutant, L18A, by whole
cell patch clamp. There are important differences in the two assay
systems used. Under ion flux conditions, the channel is maintained in
the open conformation due to the presence of subsaturating
concentrations of the alkaloid veratridine. In contrast, in the
electrophysiological assays, the cells are clamped at a potential
sufficient to ensure the maintenance of channels in the closed
conformation during toxin binding. Substantial losses (34- and
328-fold) in binding affinity are seen for the L18A mutant in both cell
types although the loss of affinity in N1E-115 cells is less dramatic (Table 3). This disparity between the effects of L18A on
electrophysiological versus ion flux parameters in N1E-115
cells led us to a more general consideration of the role of
electrostatic interactions in toxin affinity. Our previous
characterization of an R12S/K49Q charge neutralization mutant pointed
to electrostatics as a more important determinant of binding to
neuronal than cardiac channels(16) . In order to test this
hypothesis, we first increased the extracellular calcium concentration
from 0.5 to 2 mM. Under these conditions, the K for ApB is unaffected in RT4B cells (n = 2) but
increases 9-fold from 5 to 45 nM (n = 5) in
the N1E-115 line. Reducing the ionic strength by one-half (with sucrose
replacement) has no effect on the K
in RT4B cells (n = 4) but increases the affinity for ApB in N1E-115
cells to 27 nM (n = 3). These results are
consistent with the hypothesis that electrostatics are more important
for binding of ApB to the neuronal than to the cardiac isoform of the
sodium channel and underscore the contribution of hydrophobic
interactions in the latter system. It is important to emphasize that
the reductions in apparent affinity estimated for L18A by either method
represent the greatest changes detected to date for either single or
double site substitutions. This study has therefore extended the limits
of the binding site to include Leu-18 as a residue making a vital
contribution to apparent binding affinity. Changes observed for the
Ile-43 mutants are more modest, ranging from 2-8-fold, and are
consistent with the nonessentiality of Ile-43.
Hydrophobic forces are generally considered to be important in protein-protein interactions(33) . Computational studies by Miyamoto and Kollman (34, 35) emphasize the importance of hydrophobic interactions between ligand and protein. In an elegant study on the human growth hormone (hGH):receptor (hGHR) interface, Clackson and Wells (36) used alanine scanning mutagenesis to identify functionally important residues on the hGHR. While the greatest losses in binding affinity (4.5 kcal/mol) were observed when tryptophan residues were replaced, large effects (1.5-3.5 kcal/mol) were also detected when the hydrophobic groups Ile and Pro were substituted. In contrast, generally smaller effects (1-2 kcal/mol) were seen with charged residues(36) . They concluded that a central hydrophobic region, surrounded by less important contact sites that are frequently hydrophilic in nature, forms a functional epitope accounting for three-quarters of the binding free energy(36) . We propose that in our system the hydrophobic residue Leu-18 is of primary importance in binding, while the cationic residues identified previously may be analogous to the less important contacts (16) .
Identification of Leu-18, as a critical determinant of high affinity channel binding, represents a potential starting point in drug design. We have successfully extended the currently available functional map beyond the cationic cluster to include Leu-18. We have also learned that while Ile-43 has minor effects on binding it is involved in folding and is probably an important structural determinant. Beyond the scope of this study, but an interesting direction for the future, will be the identification of complementary residues on the sodium channel with which defined sites of ApB interact.