From the Department of Chemistry and Biochemistry, University of Notre Dame, Notre Dame, Indiana 46556
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ABSTRACT |
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The binding isotherms of the divalent metal
cations, Ca2+, Mg2+, and
Zn2+, to the synthetic -carboxyglutamic acid-containing
neuroactive peptides, conantokin-G (con-G) and conantokin-T (con-T),
have been determined by isothermal titration calorimetry (ITC) at
25 °C and pH 6.5. We have previously shown by potentiometric
measurements that con-G contains 2-3 equivalent Ca2+ sites
with an average Kd value of 2800 µM.
With Mg2+ as the ligand, two separate exothermic sites are
obtained by ITC, one of Kd = 46 µM
and another of Kd = 311 µM. Much
tighter binding of Zn2+ is observed for these latter two
sites (Kd values = 0.2 µM and
1.1 µM), and a third considerably weaker binding site is
observed, characterized by a Kd value of 286 µM and an endothermic enthalpy of binding. con-T
possesses a single exothermic tight binding site for Ca2+,
Mg2+, and Zn2+, with Kd
values of 428 µM, 10.2 µM, and 0.5 µM, respectively. Again, in the case of con-T, a weak
(Kd = 410 µM) endothermic binding
site is observed for Zn2+. The binding of these cations to
con-G and con-T result in an increase in the
-helical content of the
peptides. However, this helix is somewhat destabilized in both cases by
binding of Zn2+ to its weakest site.
Since the differences observed in binding affinities of these three cations to the peptides are substantially greater than their comparative Kd values to malonate, we conclude that the structure of the peptide and, most likely, the steric and geometric properties imposed on the cation site as a result of peptide folding greatly influence the strength of the interaction of cations with con-G and con-T. Further, since the Zn2+ concentrations released in the synaptic cleft during excitatory synaptic activity are sufficiently high relative to the Kd of Zn2+ for con-G and con-T, this cation along with Mg2+, are most likely the most significant metal ion ligands of these peptides in neuronal cells.
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INTRODUCTION |
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The N-methyl-D-aspartate (NMDA)1 subtype of glutamate receptor is a ligand-gated ion channel that displays high permeability for Ca2+. The marked excitotoxicity of glutamate is generally regarded as ascribable to its persistent interaction with the NMDA receptor (NMDAR), resulting in the establishment of neurodegenerative glutamatergic loops defined by uncontrolled elevations of intracellular Ca2+, followed by cell lysis and death. Because the Ca2+-mediated neuronal cell death, which is attendant to both acute (e.g. ischemia) and chronic (e.g. epilepsy, Parkinson's disease) neurodegenerative disorders, can be ameliorated by antagonists specific for the NMDAR (1-5), extensive biochemical characterization of drug-receptor interactions focused on this receptor is a widely studied topic.
Isolated from the venom ducts of predatory snails of the genus Conus, the conantokins-G (con-G) and -T (con-T) are potent and selective inhibitors of NMDAR function (6-8) and are the only peptide antagonists of this receptor subtype described to date. More specifically, this antagonism derives from a noncompetitive inhibitory effect of the polyamine agonist site of the receptor (6). The physiological responses elicited following intracranial injections in mice include a sleep-like state in neonatal mice and a hyperactive response in older animals (9).
An unusual feature of con-G and con-T is their high abundance of
-carboxyglutamic acid (Gla) (10, 11). Prior to its discovery in the
conantokins, the presence of this amino acid in polypeptides and
proteins had been noted only in certain bone proteins and in various
blood coagulation factors. In these contexts, the ability of Gla to
bind to Ca2+ plays an integral role in the adoption of
functional protein conformers (12). The interaction of Gla with
Ca2+ has been established for the conantokins, in which
cases it has been demonstrated that both con-G and con-T adopt a
significant degree of
-helicity in the presence of this cation (13).
This
-helical induction is particularly profound in the case of
con-G, which is essentially structureless in its apo form but assumes a
full end-to-end
-helical conformation when Ca2+ or
Mg2+ is fully bound to the peptide (13-15). In contrast,
con-T manifests appreciable
-helicity in the apo form which
increases slightly in the divalent cation-bound state (13, 16, 17).
Because the smaller population of conformers associated with the more structurally rigid metal-bound forms of these peptides would be expected to afford more discriminate binding to the NMDAR site, metal
complexation to these peptides may be necessary for full bioactivity.
Of the many Conus peptides currently characterized, all of
which appear to target neuronal or muscle cell receptors, only con-G
and con-T lack intramolecular disulfide bridges. This observation lends
support to the idea that conformational rigidity, either covalently or
noncovalently imposed, is an important element of receptor recognition
among members of the Conus-derived peptide family. In the
case of the conantokins, metal ions other than Ca2+ have
also been shown to effect
-helix induction. Of these,
Ca2+, Mg2+, and Zn2+ are the most
physiologically relevant metal cations in brain cells (14). Through
CD-monitored titrations of con-G, we have found that the affinity of
these divalent metal ions for the peptide is significantly greater for
Mg2+ and Zn2+ than for Ca2+ (14).
Although CD-monitored titrations represent a convenient approach for
assessing relative metal ion affinities, their corresponding Kd values cannot be accurately determined from this
method since the estimated Kd values fall in the
range of working peptide concentrations. This situation also
complicates determination of the stoichiometry of metal binding. To
gain a comprehensive quantitative assessment of these parameters, we
have employed isothermal titration calorimetry (ITC) for monitoring the
heat changes that accompany metal ion binding to the peptides. In
addition to Kd and stoichiometry, values for
H and
S can also be extracted from an ITC
profile. With these data in hand, the nature of metal ion binding to
both con-G and con-T can be more rigorously analyzed. The purpose of
the current communication is to elaborate the thermodynamic properties
of metal ion-conantokin binding.
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EXPERIMENTAL PROCEDURES |
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Peptide Synthesis--
The materials, synthetic protocols, and
purification procedures for obtaining con-G
(G-E--
-L-Q-
-N-Q-
-L-I-R-
-K-S-N-CONH2,
= Gla) and con-T
(G-E-
-
-Y-Q-K-M-L-
-N-L-R-
-A-E-V-K-K-N-A-CONH2,
= Gla) have been described earlier (13). Concentrations of peptide
stock solutions were determined by quantitative amino acid
analysis.
Isothermal Calorimetry-- The binding isotherms of Ca2+, Mg2+, and Zn2+ to the conantokins and malonate were determined by ITC measurements of the heat changes accompanying titration of the metal ions into solutions of the relevant sample. The titrations were performed with an OMEGA titration calorimeter (Microcal, Inc., Northhampton, MA) at 25 °C in a buffer containing 10 mM Mes, 100 mM NaCl, pH 6.5. Peptide samples ranging in concentrations from 0.23-1.0 mM in a total volume of 1.4 ml were placed in the reaction cell. After equilibration, an appropriate concentration of CaCl2, MgCl2, or Zn(OAc)2 (typically 30-50× higher in concentration than the peptide solution) in matching buffer was delivered at discrete intervals. The observed heat was measured after each injection. The total observed heat effects were corrected for the heat of dilution of ligand by performing control titrations in the absence of peptide. The resulting titration curves were deconvoluted for the best-fit model using the ORIGIN for ITC software package supplied by Microcal.
Circular Dichroism--
CD titrations of con-G and con-T as a
function of metal ion concentration were performed on an AVIV model
62DS spectrometer at 222 nm using a 0.1-cm path length cell
thermostatted at 25 °C. The peptides were dissolved in 10 mM Mes, 100 mM NaCl, pH 6.5, to a final
concentration of 0.5 mM. Mean residue ellipticities were
calculated by using a mean residue molecular mass of 133 Da for con-G
and 128 Da for con-T. The fractional -helical content was determined
from mean residue ellipticities at 222 nm using the empirical
relationship f(
-helix) = (
[
]222
2340)/30300 (18).
Sedimentation Equilibrium--
Experiments were conducted using
a Beckman XL-I analytical ultracentrifuge operating at 20 °C in
absorbance mode at 280 nm at rotor speeds of 45,000 and 52,000 rpm. The
buffer used was 10 mM Mes, 100 mM NaCl, pH 6.5. The partial specific volume of con-T (0.72 ml/g) was calculated from
its amino acid composition by assigning the Gla residue the
of glutamate (0.66 ml/g). The density of the buffer
was determined to be 1.005 g/ml. Sedimentation data were analyzed using
the single ideal species model included in the Beckman XL-I software.
Baseline offset values were constrained to zero for all data sets.
Calculated molecular weights represent the average of the results from
three separate scans.
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RESULTS |
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Calorimetric titrations of con-G, con-T, and malonate with various
divalent metal cations were performed at 25 °C, pH 6.5. Examples of
the heat changes accompanying the binding of incremental additions of
Zn2+ to con-G and con-T are shown in the top
panels of Fig. 1. The binding
isotherm corresponding to a plot of integrated heats as a function of
the molar ratio of Zn2+/peptide is displayed in the
lower panels. These Zn2+ titrations
represent the more complicated isotherms of the Ca2+,
Mg2+, and Zn2+ data sets with 3 and 2 enthalpic
transitions resulting from the complexation of Zn2+ with
con-G and con-T, respectively. The deconvolution of the data for con-G
was achieved by fixing a multiple site model to an n = 3, thereby allowing only Kd and H
values to float during the iterative nonlinear least squares
minimization. For con-T, n1 was constrained to
be equal to n2. The same constraint was employed
in the case of Mg2+ binding to con-G, wherein an apparent
stoichiometry of 2 was obtained. For those titrations involving an
n of approximately 1, all parameters (n,
Kd,
H) were allowed to float during the minimization process. Excellent fits were obtained for
stoichiometries corresponding to integer or near-integer values. This
strongly suggests that neither metal-induced aggregation of peptide
monomer nor metal-induced dissociation of an apo aggregate occurred
under the conditions of the calorimetric titrations. An exception to this general observation occurred in the case of the Zn2+
titration for con-T. For these data, a second acceptable model was
generated corresponding to Kd1 = 60 nM, n1 = 0.45;
Kd2 = 760 nM,
n2 = 0.53;
Kd3 = 440 µM,
n3 = 0.95. Insofar as these parameters
might indicate Zn2+-induced peptide aggregation, further
exploration of this possibility was pursued, as described below.
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In addition to the additional peptide binding sites that can be occupied by Zn2+, the thermodynamic signature of Zn2+ binding to the conantokins was also unique compared with Mg2+ and Ca2+ in that a late endothermic transition occurs in the titration of both peptides. This is significantly more pronounced in the case of con-T (Fig. 1, Table I). As can be seen from the summary of the data in Table I, all other experimentally determined enthalpic changes for both peptides are exothermic. Also to be noted from Table I are the positive entropies attending occupation of each titratable site, with the exception of the entropy associated with the weaker of the two Mg2+ sites in con-G.
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Comparison of the Kd values for Ca2+,
Mg2+, and Zn2+ for con-G and con-T shows a
dramatic increase in affinity for Zn2+ when compared with
Mg2+ which, in turn, binds to the peptides much tighter
than Ca2+. For con-G, the tightest of the 3 Zn2+ sites (Kd = 0.2 µM)
displays 200-fold more avid binding than the tightest of the 2 Mg2+ sites (Kd = 46 µM)
and a 12,000-fold increase in affinity relative to the 2-3 eq
Ca2+ sites. For con-T, this discrimination is somewhat less
pronounced, with the strength of the interaction for the tight
Zn2+ site being approximately 40- and 1000-fold greater
than that associated with the Mg2+ and Ca2+
sites, respectively. In contrast, malonate manifests only a very modest
selectivity for Zn2+ as compared with Mg2+ and
Ca2+ (2.3- and 6.5-fold, respectively). In addition, the
absolute Kd values that characterize the binding of
these cations to malonate range from 1 to 4 orders of magnitude higher
than the same Kd values associated with their
complexation to the conantokins (Table I). Both H and
S values for metal ion binding to malonate are positive.
The stoichiometry of approximately 0.6 determined for the association
of Zn2+ with malonate suggests that some degree of
dimerization (or possibly higher order aggregation) may accompany
binding of this particular metal ion to malonate.
In an attempt to address the phenomena underlying the endothermic
transitions observed for con-G and con-T, CD-monitored titrations of
these peptides with Zn2+ were performed under conditions
that paralleled those implemented in the ITC experiments. As can be
seen for con-G (Fig. 2A), an essentially linear increase in -helicity attends the addition of
Zn2+, up to an apparent plateau occurring at a
metal/peptide ratio of 2:1 (m:m). The linear phase of this titration,
with a midpoint of 1:1 (m:m), reflects extremely tight binding up to 2 eq of Zn2+, such that virtually all added metal ion exists
in the peptide-bound form. The inset of Fig. 2A
reveals that above a Zn2+/peptide molar ratio of 2, a small
but defined decrease in
-helicity is induced. This phenomenon was
not noted at similar metal/peptide ratios of Ca2+ and
Mg2+ (data not shown). The CD-monitored titration of con-T
with Zn2+ is illustrated in Fig. 2B. High
affinity binding of Zn2+, as with con-G, is seen in the
early portion of the profile, while a significant decrease in
-helicity accompanies occupation of the second Zn2+
site. Again, this decrease in
-helical content was not observed in
similarly conducted Ca2+ and Mg2+ titrations.
Because the CD-titratable diminutions in
-helical content correlate
closely with the endothermic phases of the ITC profiles, it appears
that occupancy of the weakest Zn2+ site of both con-G and
con-T effects a partial unwinding of the
-helix, which is
considerably more pronounced for the latter peptide.
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As indicated above, calorimetric analysis of the Zn2+
titration profile of con-T was suggestive of a more complex model of
metal binding involving peptide association. Zn2+-induced
peptide dimerization (or higher order aggregation) could also account
for the decrease in con-T and con-G -helicity observed at higher
Zn2+/peptide molar ratios. To address this issue,
sedimentation equilibrium analysis of con-T was performed in both the
apo state and at various Zn2+ concentrations. As shown in
Fig. 3, the apparent molecular weight of
con-T increases from 3160 in its uncomplexed state to 3390 at a
Zn2+/peptide ratio of 1:1 (m:m), and 3650 at a
cation/peptide ration of 5.6:1 (m:m). Although these results imply some
degree of self-association, the residuals for the fitted data do not
display any systematic deviation (Fig. 3). Furthermore, the molecular
weights determined at 0.65 mM and 0.9 mM con-T
in the presence of 5 mM Zn2+ were calculated to
be 3710 and 3510, respectively, opposite to the trend expected if the
peptide were undergoing self-association.
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DISCUSSION |
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The multiple metal cation sites detected for con-G (Table I) are consistent with the multiple cation sites proposed from modeling by a genetic algorithm/molecular dynamics simulation method using the coordinates provided from the NMR-derived structures of the Ca2+- and Mg2+-bound peptides (15, 19). The NMR structure of the Mg2+-complexed form is notable for the spatial proximity of the side chain carboxylates of Gla10 and Gla14, which are optimally positioned for metal ion coordination. This proximity would be unlikely in the absence of a stabilizing cationic bridge. A cluster comprised of Gla3, Gla4, and Gla7 represents a second potential coordination site. The viability of these side chains as metal binding loci was supported by the simulated docking of Mg2+ to the Mg2+-loaded NMR-derived structure using the genetic algorithm/molecular dynamics simulation method (15). This approach also identified Gla7 as another likely site for Mg2+ in con-G. ITC experiments failed to detect this putative third locale which may bind Mg2+ too weakly to be amenable to this calorimetric approach. However, a third, albeit relatively weak site, was detected during the Zn2+ titration of con-G and may reflect binding at this proposed Gla7 site. Synthetic peptide variants containing individual Gla to Ala substitutions strongly support a model wherein the tight Mg2+ site is maintained by Gla10 and Gla14, with the weaker site being comprised of Gla3, Gla4, and Gla7 (15). The orientation of Gla10 and Gla14 as determined from the Ca2+-bound NMR solution structure of con-T is strikingly similar to that found in con-G and, by analogy, can be considered a likely binding locus for metal ions (16). Docking of Ca2+ into this structure using the aforementioned genetic algorithm/molecular dynamics simulation yielded a lowest energy structure showing the carboxylates of Gla10 and Gla14 as coordination site donors. A second site appears maintained by the two carboxylates of Gla3 and the side chain amide carbonyl of Gln6. A second binding site was not experimentally detected for Ca2+ and Mg2+, but was observed with Zn2+, a more avid ligand. Titrations with Zn2+ of individual [Gla10Ala]con-T and [Gla14Ala]con-T variants suggest that the high affinity site of con-T includes these residues.2
Previous studies have demonstrated that no detectable higher order
species are present upon Ca2+ loading of either con-G or
con-T (13). In the presence of saturating concentrations of
Mg2+, the monomeric molecularity of both peptides has also
been established.3 However,
with Zn2+, the calorimetric titration of con-T yielded data
for which a fit consistent with peptide dimerization could be
generated. In addition, from the CD titration data, one interpretation
of the unusual trend of decreasing con-T helicity coinciding with
increasing Zn2+ concentration could include
Zn2+-induced peptide aggregation upon weak-site occupancy,
which leads to helix unwinding. To test the possibility of
Zn2+-induced con-T aggregation under the conditions
employed in both the calorimetry and CD experiments, we opted for
sedimentation equilibrium analysis of the apo and
Zn2+-complexed peptide states (Fig. 3). Inspection of the
residuals of the fits to the single ideal species model for uncomplexed and Zn2+-chelated con-T reveals the random distribution of
points diagnostic of ideal solute behavior. Insofar as the distribution
of residuals is considered a rigorous indicator of analyte ideality,
the data are consistent with strictly monomeric solution behavior of
con-T. However, when compared with the calculated sequence-based
molecular weight of 2680, the higher apparent weight-average molecular
weight of this peptide determined from sedimentation equilibrium (3160) is suggestive of association. Furthermore, the increase in apparent molecular weight in the presence of Zn2+ also appears to be
inconsistent with the contention of solute ideality. We contend that
these disparate results can be addressed in terms of an artificially
high partial specific volume of the peptide (0.72 ml/mg) that
was employed in the molecular weight determination. Because the value
of
for Gla has not been determined, we assigned to it the
glutamate value of 0.66 ml/mg in calculating the
of con-T.
This is almost certainly an overestimate of the
for this
residue when considering that the presence of an additional carboxylate
in Gla should effectively lower the
associated with this
residue by virtue of its increased hydrophilicity and capacity for
hydration. In addition, in the presence of Zn2+ the
intimate and preferential interaction of the metal ion with the Gla
side chains may profoundly perturb the
of Gla. When considering the
relatively high weight percentage of Gla in con-T, its contribution to
the
value of the peptide is significant. The observation that
an increase in molecular weight does not attend increased peptide
concentration at high Zn2+ concentrations is additional
compelling evidence in support of the nonassociative behavior of
con-T.
The exotherms associated with peptide-metal binding correlate in the
expected fashion with the degree of conformational change effected by
metal binding. In the case of Mg2+ complexation to
con-G, wherein the peptide undergoes a dramatic change in -helicity
upon Mg2+ binding (14), the total change in the
H for binding of 2 eq of ligand is
8.7 kcal/mol. The
enthalpic change for con-T, which undergoes a distinctly smaller
increase in
-helicity upon saturation of its metal ion sites with
Mg2+, is
4.4 kcal/mol. Clearly, bond formation occurs to
a greater extent in the con-G metal-induced transition than in the
con-T system. It seems unlikely that electrostatic components
contribute to these favorable
H values since metal ion
binding to malonate, which occurs strictly through electrostatic
interactions, possesses an unfavorable
H (Table I),
leaving entropic forces to drive the binding event. The effective
neutralization of clustered, destabilizing negative charge that occurs
upon chelation of metal ions to the Gla head groups of the peptide may
simply allow favorable intrapeptide hydrogen bonds to prevail.
The positive, albeit small, entropies that accompany the exothermic
transitions are somewhat surprising considering the significant degree
of order that metal ion binding imposes on these peptides. Factors with
favorable entropies, such as release of the metal ion from its
coordination sphere and release of structured water surrounding polar
and non-polar residues, are likely contributing in the peptide
systems. From the positive TS values for metal complexation to malonate, it is probable that such contributions take
entropic precedence, despite the absence of some elements of rotational
bond freedom.
The positive H values observed for occupancy by
Zn2+ of the weak sites of both con-G and con-T correlate
with a small degree of
-helix unwinding as implied from parallel
CD-monitored titrations (Fig. 2). The relatively large positive entropy
values linked to weak site Zn2+ binding for both peptides
is also consistent with the notion of increased structural disorder.
The nature of this destabilization in con-G is difficult to state with
certainty because the results of simulated Mg2+ docking to
con-G (15) point toward Gla7 as a third binding locus.
Because all five Gla residues of con-G exist on the same face of the
helix in the metal-loaded structure (15), inclusion of an additional
divalent cation would be expected to impart increased stabilization to
the peptide. We expect that ITC titrations with Zn2+
employing various Gla to Ala variants of con-G will address the nature
of this third site and these studies are currently in progress. For
con-T, we contend that the greater degree of
-helix unwinding that
occurs upon Zn2+ loading can be explained in terms of the
capping potential of Gla4. The NMR solution structures of
both apo- and Ca2+-loaded con-T indicate that the side
chain of Gla4 is an apparent capping residue in both
structures. This contention is supported by CD data on the
[Gla4Ala]con-T variant which displays compromised
-helical content in both its apo- and Ca2+-loaded forms
(17). In the presence of high Zn2+ concentrations, we
suggest that Gla4 is recruited from its side-chain to
main-chain interaction role to share with Gla3 in the
maintenance of a weak metal-binding site, thus destabilizing the
-helix in the N-terminal vicinity.
Finally, the current study raises important issues concerning the identity of the metal ion that predominates in the bioactive conformation of these peptides. It has been established in numerous studies with Gla-containing bone and blood proteins that Ca2+ mediates their functional aspects. In the case of prothrombin, factor IX, and protein C, which contain 10, 12, and 9 Gla residues, respectively, in their N-terminal domains, no major preference for Mg2+ versus Ca2+ has been noted from intrinsic fluorescence titration experiments (20-23). Only a slight (2.5-fold) preference is observed for Mg2+ over Ca2+ in terms of their Kd values for malonate (Table I). Despite the smaller ionic radius for Mg2+ (86 pm) as compared with Ca2+ (114 pm), this disparate charge to radius ratio fails to impart appreciable selectivity differences in these cases. However, for con-G, the Mg2+ tight site and weak site affinities in comparison with Ca2+ are 175- and 9-fold, greater, respectively. For con-T, Mg2+ displays a 42-fold increase in binding strength compared with Ca2+. Both con-G and con-T display distinctly lower Kd values for Zn2+ than for either Ca2+ or Mg2+. A pronounced (ca. 10,000-fold) greater affinity for Zn2+ over Ca2+ has recently been reported for a Gla-containing de novo designed peptide, which undergoes a metal-induced helical transition (24). These investigators maintain that the greater covalent character of electrostatic interactions involving Zn2+, ascribable to its filled 3d orbitals as well as higher charge density for this cation, are the major contributors to this preferential binding. However, the primacy of these factors is not borne out by our malonate data, which indicate that Zn2+ manifests only a modest (6.5-fold) increase in affinity for malonate compared with Ca2+. This would suggest that the binding sites of those peptides, which avidly bind Zn2+, are particularly amenable to this smaller metal ion simply because their induced binding site cation pockets are intrinsically small. Hence, the strength of metal-ion binding in these molecules may rely more on proper steric and geometric fit than on purely electrostatic considerations.
The consideration that Zn2+ is the dominant metal ion effector of conantokin function is supported by studies that indicate that transiently high local concentrations of Zn2+ (ca. 300 µM) can be attained in the synaptic cleft during excitatory synaptic activity (25, 26). Intracerebral injections of the conantokins into pre-2-week-old mice have been shown to induce a prolonged sleep-like state in these subjects at an estimated final brain concentration of 15 µM (13). Assuming a free Zn2+ concentration of 300 µM, this would result in essentially complete occupancy of the two tight Zn2+ sites in con-G and approximately 50% occupancy of the weak site. In the case of con-T, saturation of the high affinity Zn2+ site and 40% occupancy of the low affinity Zn2+ site would be attained at these peptide and ligand concentrations. At a cerebrospinal fluid Mg2+ concentration of 1.5 mM, tight and weak site loading of con-G would be 97% and 80%, respectively. For con-T, essentially 100% of the peptide molecules would be complexed with Mg2+. At a cerebrospinal fluid Ca2+ concentration of 1.5 mM, Ca2+ loading would be considerably less than Mg2+ and Zn2+, with 30% and 77% occupancy of the metal sites in con-G and con-T, respectively. We therefore propose that Mg2+, as well as Zn2+, are more plausible candidates for the role of in vivo metal ions for the conantokins. However, it should be noted that the high coordination numbers and irregular coordination geometry displayed by Ca2+ confer upon Ca2+-bound proteins the ability to bridge phospholipid membranes and/or other proteins. This particular feature of Ca2+-complexed biomolecules may mediate the functionality of the conantokins, despite the compromised binding capacity displayed by Ca2+ compared with Mg2+ and Zn2+. Dissecting the roles of these metal ions in the conantokins with respect to the NMDAR is complicated by their involvement in receptor function, namely the Ca2+ permeability associated with the receptor (see above), voltage-dependent Mg2+ blockage of the receptor (27, 28), and allosteric voltage-insensitive and -sensitive blocks by Zn2+ (29, 30). Elucidating the precise role of these metal ions in terms of conantokin antagonism of the receptor is essential for a full pharmacological profile of the these peptides and represents one of the major investigative challenges associated with future work in this area.
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FOOTNOTES |
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* This work was supported by Grant HL-19982 from the National Institutes of Health (to F. J. C.) and the Kleiderer-Pezold family endowed professorship (to F. J. C.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed. Tel.: 219-631-6456;
Fax: 219-631-8017; E-mail: castellino.1{at}nd.edu.
1
The abbreviations used are: NMDA,
N-methyl-D-aspartate; NMDAR, NMDA receptor;
con-G, conantokin-G; con-T, conantokin-T; Gla, -carboxyglutamic
acid; ITC, isothermal titration calorimetry; Mes,
4-morpholineethanesulfonic acid.
2 S. E. Warder, unpublished data.
3 M. Prorok, unpublished data.
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REFERENCES |
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