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J Biol Chem, Vol. 274, Issue 36, 25237-25244, September 3, 1999
From the Department of Membrane Biochemistry and Biophysics, Merck
Research Laboratories, Rahway, New Jersey 07065
Correolide, a novel nortriterpene natural
product, potently inhibits the voltage-gated potassium channel,
Kv1.3, and [3H]dihydrocorreolide (diTC)
binds with high affinity (Kd ~ 10 nM)
to membranes from Chinese hamster ovary cells that express Kv1.3 (Felix, J. P., Bugianesi, R. M.,
Schmalhofer, W. A., Borris, R., Goetz, M. A., Hensens,
O. D., Bao, J.-M., Kayser, F., Parsons, W. H., Rupprecht, K.,
Garcia, M. L., Kaczorowski, G. J., and Slaughter, R. S. (1999) Biochemistry 38, 4922-4930). Mutagenesis studies were used to localize the diTC binding site and to design a high affinity receptor in the diTC-insensitive channel, Kv3.2.
Transferring the pore from Kv1.3 to Kv3.2
produces a chimera that binds peptidyl inhibitors of Kv1.3
with high affinity, but not diTC. Transfer of the S5 region
of Kv1.3 to Kv3.2 reconstitutes diTC binding at
4-fold lower affinity as compared with Kv1.3, whereas
transfer of the entire S5-S6 domain results in
a normal Kv1.3 phenotype. Substitutions in
S5-S6 of Kv1.3 with nonconserved
residues from Kv3.2 has identified two positions in
S5 and one in S6 that cause significant
alterations in diTC binding. High affinity diTC binding can be
conferred to Kv3.2 after substitution of these three
residues with the corresponding amino acids found in Kv1.3.
These results suggest that lack of sensitivity of Kv3.2 to
diTC is a consequence of the presence of Phe382 and
Ile387 in S5, and Met458 in
S6. Inspection of Kv1.1-1.6 channels indicates
that they all possess identical S5 and S6
domains. As expected, diTC binds with high affinity
(Kd values 7-21 nM) to each of these
homotetrameric channels. However, the kinetics of binding are fastest
with Kv1.3 and Kv1.4, suggesting that
conformations associated with C-type inactivation will facilitate entry
and exit of diTC at its binding site. Taken together, these findings
identify Kv1 channel regions necessary for high affinity
diTC binding, as well as, reveal a channel conformation that markedly
influences the rate of binding of this ligand.
Voltage-gated potassium channels participate in a number of
important cellular functions (1). Therefore, specific modulation of the
activity of these proteins may lead to the development of novel
therapeutic agents. Peptidyl blockers of potassium channels purified
from venom of different organisms have been useful in defining various
structural and functional properties of potassium channels (2). Two of
these peptides, margatoxin
(MgTX)1 and charybdotoxin
(ChTX), have been critical in identifying Kv1.3 as a target
for development of novel immunosuppressants (3-6). In the course of
screening for small molecule Kv1.3 inhibitors to support
these efforts, a novel nortriterpene, correolide, was isolated from the
plant Spachea correa (7) and shown to be a potent
Kv1.3 blocker (8). Correolide is a selective inhibitor of
the Kv1 family of potassium channels, displaying very low
affinity for other ion channels and membrane proteins. However, its
selectivity for Kv1.3 appears to be limited and in
functional assays it blocks other Kv1 channels with
4-20-fold lower potency. A tritium-labeled derivative of correolide,
[3H]dihydrocorreolide (diTC; Table I), binds reversibly
and saturably to Kv1.3; the stoichiometry of the binding
reaction suggests that one molecule of correolide binds per channel
tetramer; the binding sites for correolide and peptidyl blockers are
distinct (8). Because of the large structural diversity of
voltage-gated potassium channels (9-11), knowledge of specific
molecular determinants that determine high affinity binding of
modulators among different channel proteins is crucial for development
of potent and selective therapeutic agents.
In this study, we investigate the molecular basis for interaction of
diTC with Kv1.3 and design a high affinity receptor in the
diTC-insensitive channel, Kv3.2. Our results indicate that residues located in the S5 and S6 region are
responsible for the high affinity interaction of diTC with
Kv1.3 and that the lack of sensitivity of Kv3.2
to diTC is due to the presence of Phe382 and
Ile387 in S5, and Met458 in
S6. Although all Kv1 channels display conserved
residues in the S5 and S6 domains and diTC
binds with high affinity to all of these channels, the kinetics of
ligand association and dissociation are much faster with
Kv1.3 and Kv1.4 than with other Kv1
channels. The energy barrier for diTC to enter or leave its binding
site may be less for Kv1.3 and Kv1.4 as a
consequence of a change in channel conformation induced by C-type
inactivation, which is a common feature of these two channels. A
preliminary report of these findings has been made in abstract form
(12).
Materials--
Restriction enzymes and the pCI-neo vector were
bought from Promega. The pEGFP-N1 vector was from
CLONTECH, and Pfu DNA polymerase from
Stratagene. TsA-201 cell line, a subclone of the human embryonic kidney
cell line, HEK293, which expresses the SV40 T antigen, was a gift of
Dr. Robert DuBridge. All tissue culture media were from Life
Technologies, Inc., serum was from Hyclone, and the FuGENETM
transfection reagent was from Roche Molecular Biochemicals. HEK293
cells stably transfected with homotetrameric Kv1.1,
Kv1.3, Kv1.4, Kv1.5, and
Kv1.6 channels were obtained from Professor Olaf Pongs
(Zentrum für Molekulare Neurobiologie, Hamburg, Germany). HEK293
cells stably transfected with Kv1.2 were prepared as
described (8). Kv3.2 DNA was a generous gift of Dr.
Bernardo Rudy, New York University Medical Center. Correolide, diTC (29 Ci/mmol), and dihydrocorreolide (diHC) were prepared as described
previously (8). Hongotoxin1-A19Y/Y37F (HgTX1A19Y/Y37F) was
prepared and radioiodinated as described (13). Charybdotoxin and
stichodactyla toxin were purchased from Peninsula Laboratories. GF/C
glass fiber filters were obtained from Whatman, and polyethylenimine
was from Sigma. All other reagents were obtained from commercial
sources and were of the highest purity commercially available.
Mutant Channel Constructs--
A 9E10 c-Myc tag was introduced
at the C terminus of Kv1.3 and Kv3.2 using an
oligonucleotide cassette containing a HindIII and
NotI restriction site. In addition, the 5'-untranslated
region of Kv3.2 was replaced with the corresponding
sequence of Kv1.3 to improve channel expression. Chimeric
cDNAs of Kv1.3 and Kv3.2 were generated
using the gene splicing with overlap extension technique (14). Chimeras
were constructed as follows: in Kv3.2 (P..Kv1.3), the linker between transmembrane domain
S5 and S6 of Kv3.2 (referred to as
P) was replaced with the corresponding region from Kv1.3;
in Kv3.2 (S5, P..Kv1.3), both the
transmembrane domain S5 and P of Kv3.2 were
replaced with those from Kv1.3; in Kv3.2 (P,
S6..Kv1.3), both P and transmembrane domain
S6 of Kv3.2 were replaced with those from
Kv1.3; in Kv3.2
(S5-S6..Kv1.3) both P and
transmembrane domains S5 and S6 of
Kv3.2 were replaced with the corresponding segments from
Kv1.3. Site-directed mutagenesis was performed using the
"overlap extension" technique (15). Polymerase chain reaction was
carried out using proofreading Pfu DNA polymerase and the
integrity of all constructs was verified by nucleotide sequencing
(automated sequencer, ABI 377).
Transfection of TsA-201 Cells and Membrane Preparation--
The
procedures for handling TsA-201 cells, their transfection with
FuGENETM transfection reagent, and preparation of membrane vesicles
have been previously described (16). The final membrane pellet was
resuspended in 100 mM NaCl, 20 mM Hepes-NaOH,
pH 7.4. Aliquots were frozen in liquid N2 and stored at
125I-HgTX1A19Y/Y37F Binding--
The
interaction of 125I-HgTX1A19Y/Y37F with TsA-201
membranes was measured in a medium consisting of 50 mM
NaCl, 5 mM KCl, 20 mM Tris-HCl, pH 7.4, 0.1%
bovine serum albumin. For saturation experiments, membranes were
incubated with increasing concentrations of
125I-HgTX1-A19Y/Y37F in a total volume of 4 ml
for 20 h at room temperature. Separation of bound from free ligand
was achieved using filtration protocols as described (13).
diTC Binding--
Binding of diTC to membranes was carried out
in a medium consisting of 135 mM NaCl, 4.6 mM
KCl, 20 mM Tris-HCl, pH 7.4, 0.02% bovine serum albumin.
For saturation experiments, membranes were incubated in a total volume
of 0.2 ml with increasing concentrations of diTC for 20 h at room
temperature. To determine kinetics of ligand association, membranes
were incubated with diTC for different periods of time at room
temperature. Dissociation kinetics were initiated by addition of 10 µM diHC to membrane-bound diTC followed by incubation at
room temperature for different periods of time. Competition experiments
were carried out in a total volume of 1 ml with 1 nM diTC
in the absence or presence of increasing concentrations of diHC. Under
these conditions, IC50 values of inhibition are very close
to Ki values, because the concentration of diTC in
the incubation assay is ~5-fold below Kd.
Separation of bound from free ligand was achieved using a filtration
protocol as described (8). Triplicate samples were run for each
experimental point. Standard deviation of the mean was typically less
than 5%.
Data Analysis--
Data from saturation, ligand association and
dissociation experiments were analyzed as described (8, 16).
IC50 values for peptide and diHC inhibition were determined
using the equation: Beq = (Bmax Construction of Kv3.2/1.3 Channel
Chimeras--
Although diTC binds to Kv1.3 with high
affinity, this ligand does not interact with members from other
families of voltage-gated potassium channels such as Kv3.2
(8). Therefore, chimeras were constructed using regions of
Kv1.3 and Kv3.2 to identify those domains that
confer high affinity binding of diTC. This is an approach similar to
that used to identify determinants responsible for block of potassium
channels by peptide inhibitors (17, 18).
Kv1.3 channels are inhibited with high affinity by peptidyl
blockers such as ChTX (6, 19, 20), hongotoxin-1 (HgTX1) (13), MgTX (21), kaliotoxin (22), and stichodactyla toxin (23).
Furthermore, high affinity binding sites for radiolabeled ChTX (24),
MgTX (25), and HgTX1 (13) have been identified in membranes
prepared from cells containing Kv1.3 channels.
Kv3.2 channels, however, are not sensitive to any of the
identified peptidyl blockers of potassium channels (2). To introduce a marker into Kv3.2 that could be used to quantitate levels
of channel expression after transient transfection of TsA-201 cells, a
chimera was constructed in which the pore region of Kv1.3
was transferred to Kv3.2 (Fig.
1). Expression of this and other chimeras
was also monitored by immunoblotting membrane preparations using an
antibody against the c-Myc sequence that was added to the C terminus of these proteins. Such experiments indicate that wild type channels and
all chimeric channels analyzed in these studies give similar levels of
protein expression (data not shown).
To determine whether the chimeric proteins associate to form tetrameric
structures known to be required for binding of peptides, binding of
125I-HgTX1A19Y/Y37F was measured to membranes
derived from TsA-201 cells transiently transfected with either
Kv1.3 or Kv3.2(P..Kv1.3). Results
of these experiments are shown in Fig.
2A.
125I-HgTX1A19Y/Y37F binds to a single class of
sites that display Kd values of 0.29 and 3.8 pM for Kv1.3 and
Kv3.2(P..Kv1.3), respectively. Thus,
transferring the pore region of Kv1.3 yields a high
affinity receptor for HgTX1 in Kv3.2 that can
be used to monitor levels of channel expression. The differences in
HgTX1 affinity noted between Kv1.3 and this
chimera are similar to those found in studies where the pore of
Kv1.3 was transferred to Kv2.1 to confer
agitoxin 2 sensitivity to the later channel (18). In this study, we
have not investigated whether these differences are due to effects on
peptide association or dissociation kinetics.
Several other Kv3.2 chimeras were constructed (Fig. 1) and
characterized initially for functional channel tetramerization. In
these cases, either transmembrane domain S5,
S6, or the complete S5-S6 region of
Kv1.3 was transferred to the
Kv3.2(P..Kv1.3) chimera. Binding of
125I-HgTX1A19Y/Y37F was monitored to these
chimeras, and the results are shown in Fig. 2A. In all
instances, high affinity binding of
125I-HgTX1A19Y/Y37F occurred to a single class
of sites as indicated by the fact that the Hill coefficients of all
slopes shown in Fig. 2A are close to unity. The highest
affinity for peptide was observed with the chimera in which the
S5-S6 region of Kv1.3 had been
transferred. In fact, this chimera possesses the same affinity for
HgTX1 as does native Kv1.3. Binding of
125I-HgTX1A19Y/Y37F to Kv3.2/1.3
chimeras was further characterized. Peptidyl blockers of
Kv1.3 such as ChTX, MgTX, and stichodactyla toxin cause
concentration-dependent inhibition of
125I-HgTX1A19Y/Y37F binding (data not shown),
and inhibitory potencies of these peptides for the various chimeras
relative to the wild type channel scale with the ability of
125I-HgTX1A19Y/Y37F to bind to these proteins.
These data suggest that all Kv3.2 chimeras that were
constructed can assemble to form tetrameric structures and that the
molecular pharmacology of these chimeras with respect to the pore
resembles closely that of native Kv1.3 in terms of their
sensitivity to a number of different peptidyl inhibitors.
Binding of diTC to Kv3.2/1.3 Channel Chimeras--
To
determine whether the high affinity interaction of diTC can be
conferred to the correolide-insensitive channel, Kv3.2, diTC binding was evaluated in membranes prepared from the same Kv3.2/1.3 chimeras. diTC binds to a single class of sites
present in this Kv1.3 membrane preparation with a
Kd of 9.4 nM (Fig. 2B). This
value is nearly identical to the Kd of diTC measured
in other types of membranes where Kv1.3 is expressed (see
Ref. 8 and this study). diTC binding cannot be detected with the
Kv3.2(P..Kv1.3) chimera at a ligand
concentration of 20 nM (data not shown), indicating that
some other channel domain(s), and not the pore region, is necessary for
the high affinity interaction of diTC with Kv1.3. In
addition, the Kv3.2 chimera containing only the
S6 region from Kv1.3 did not display any
detectable diTC binding (data not shown). Because the number of
tetrameric channels present in these membranes can be determined
independently from maximal levels of
125I-HgTX1A19Y/Y37F binding, it is possible to
estimate the Kd of diTC for these constructs to be
>1 µM. However, the Kv3.2 chimeras containing either the S5 or the
S5-S6 region of Kv1.3 did bind diTC
potently with Kd values of 48.8 and 18 nM, respectively (Fig. 2B). Thus, the presence
of S5 from Kv1.3 appears to be necessary for
conferring a high affinity interaction of diTC with Kv3.2. Attempts to demonstrate a loss of diTC binding in Kv1.3 by
transferring S5 from Kv3.2, however, were
unsuccessful. The resulting construct did not yield any detectable
125I-HgTX1A19Y/Y37F binding, despite the fact
that expression of the protein could be easily detected in Western
blots. It is interesting that the chimera that contains the
S5-S6 region of Kv1.3 displays about 4-fold higher affinity for diTC than when S5 is
transferred to Kv3.2 by itself. This finding is consistent
with the observation from competition binding experiments that a 4-fold
loss in diHC affinity is observed when S6 from
Kv3.2 is transferred to Kv1.3 (data not shown),
and suggests that differences in S6 between the two
channels are not the major contributor to correolide sensitivity.
Comparison of the sequences of Kv1.3 and Kv3.2
in S5 and S6 indicate that 16 nonidentical
residues are present; 11 in S5 and 5 in S6
(Fig. 3). To determine whether any of
these residues contribute to the differences observed in diTC binding,
individual mutations were introduced in Kv1.3 to
incorporate those amino acids present in Kv3.2. In these
studies, binding parameters were determined with each mutant for both
125I-HgTX1A19Y/Y37F and diTC, and the results
are presented in Fig. 4. In these plots,
a ratio of 1 indicates that the behavior of the mutant is identical to
wild type, whereas a ratio of >1 reflects decreased binding as a
result of the amino acid substitution. All mutants give similar levels
of channel expression, as indicated by the maximum density of
125I-HgTX1A19Y/Y37F binding sites, and all but
one, V364I, displayed an identical affinity for
125I-HgTX1A19Y/Y37F as wild type (Fig.
4A). These data indicate that, with the V364I exception, no
major structural changes were conferred in the pore of the channel as a
consequence of these mutations. In marked contrast, three mutations,
L346 F and F351I, both in S5, and L422M in
S6 had significant effects on diTC binding, causing 25-, 3-, or 5-fold decreases in ligand affinity, respectively (Fig.
4B). There is also a modest effect of a 2-fold increase in
Kd with the mutation S362T. These data are
consistent with previous observations regarding the
Kv3.2/1.3 chimeras, and suggest that lack of high affinity
interaction of diTC with Kv3.2 could be due to the combined
effect of 3 nonconserved residues in Kv1.3, 2 located in
S5 and 1 in S6.
To test this idea further, two Kv3.2 mutants,
Kv3.2F382L/M458L, and Kv3.2F382L/I387F/M458L
were constructed so that the corresponding residues in
Kv1.3 were introduced into Kv3.2, and their
diTC binding properties were compared with those of Kv1.3.
Results of these experiments are presented in Fig.
5. diTC binds to a single class of sites
that display Kd values of 5.7, 20.9, and 10.2 nM for Kv1.3, Kv3.2F382L/M458L, and
Kv3.2F382L/I387F/M458L, respectively (Fig. 5A).
Thus, introduction of Leu, Phe, and Leu into Kv3.2 at
positions 382, 387, and 458, respectively, reconstitutes a high
affinity receptor for diTC; in the absence of these mutations, the
Kd of Kv3.2 for diTC is >1
µM. As suggested from mutagenesis experiments with
Kv1.3, the double mutant Kv3.2F382L/M458L has a
4-fold lower affinity for diTC than does Kv1.3. These
results were also confirmed in competition experiments (Fig.
5B). diHC inhibits binding of diTC to these channels with
Ki values of 6, 19.2, and 9.4 nM for
Kv1.3, Kv3.2F382L/M458L, and
Kv3.2F382L/I387F/M458L, respectively. All these data, taken
together, strongly suggest that lack of sensitivity of
Kv3.2 to diTC is due to the presence of only three amino
acids; Phe382 and Ile387 in S5, and
Met458 in S6.
Interaction of DiTC with Kv1 Channels--
Because the
S5 and S6 domains are conserved among all
Kv1 channels, it might be expected that diTC would bind to
these proteins with similar Kd values. Previously
reported data indicate that diTC binds with high affinity to a single
class of sites in human and rat brain membranes, a receptor most likely
comprised of heteromultimers of Kv1.1/1.2/1.4 channels (8,
13), and that Kd values determined from either
equilibrium binding or ratio of rate constants are equivalent for both
brain and Kv1.3 membranes. However, there are marked
differences in the kinetics of ligand binding. Thus, rates of ligand
association and dissociation are about 20-fold faster for
Kv1.3 than with brain synaptic membranes. Because brain
membranes do not contain a homogeneous population of any given
Kv1 channel, the properties of diTC binding to HEK293 membranes containing either Kv1.1, Kv1.2,
Kv1.3, Kv1.4, Kv1.5, or
Kv1.6 homotetrameric channels were investigated next. Under equilibrium conditions, diTC binds in a saturable fashion and with
similar Kd values (7-21 nM) to a single
class of sites present in each of these membrane preparations (Table
I). These findings are consistent with
the postulate that high affinity diTC binding would be nearly
equivalent for all members of the Kv1 channel family.
The kinetics of diTC association to homomultimeric Kv1
channels were investigated next. At a ligand concentration close to Kd, diTC associates with channels in each of these
membrane preparations in a time-dependent fashion; binding
kinetics are pseudo-first order (Fig. 6).
However, the association rate constant (k+1),
varies greatly depending on the Kv1 channel under consideration. Thus, association rates of diTC with either
Kv1.3 or Kv1.4 channels are about 10-50-fold
faster than those determined for the other Kv1 channels
(Table I). The kinetics of ligand dissociation were also determined
from each of these channels. In all cases, dissociation of diTC is a
time-dependent event that follows monoexponential kinetics,
indicative of a first-order reaction (Fig.
7). Dissociation rate constant values
(k Kinetics of diTC Binding to Kv1.3 Channel Mutants with
Altered C-type Inactivation--
The idea that C-type inactivation
induces a conformational change in Kv1.3 that modifies the
accessibility of diTC to its binding site was tested by investigating
two channel mutants, Kv1.3/G375Q and
Kv1.3/H399N, that display altered inactivation kinetics. In
Kv1.3, residues Gly375 and His399
are located in the external vestibule of the channel and appear to be
directly involved in the conformational change that leads to channel
inhibition (27). The mutant Kv1.3/G375Q is 8-fold slower in
achieving C-type inactivation than wild type Kv1.3, whereas
Kv1.3/H399N is 50-fold faster in this respect. Importantly, under equilibrium conditions, both Kv1.3 mutants and the
wild type channel bind diTC with identical affinities when the reaction is monitored in membranes prepared from TsA-201 cells transiently expressing these proteins (data not shown). However, kinetics of diTC
dissociation are markedly different among these various channels (Fig.
8). Ligand dissociation follows
first-order kinetics with t1/2 values of 59, 18, and 305 min for Kv1.3, Kv1.3/H399N, and
Kv1.3/G375Q, respectively. Thus, diTC dissociation kinetics are 5-fold slower from a protein where the rate of C-type inactivation is diminished, whereas it is 3-fold faster from a Kv1.3
mutant displaying enhanced inactivation. Because equilibrium binding studies demonstrate that the affinity of diTC for all these
Kv1.3 channels is the same, it is predicted that the
kinetics of ligand association will also be modified accordingly. These
data are consistent with the idea that diTC accessibility to and from
its binding site is influenced by the conformational change eliciting C-type inactivation.
Correolide is the first potent, small molecule, natural product
inhibitor of a voltage-gated potassium channel to be described. It
displays remarkable specificity among ion channels in that it only
interacts with Kv1 family channels. The results presented in this manuscript address two important aspects concerning the interaction of diTC with potassium channels. The first deals with the
molecular basis for high affinity interaction of diTC with Kv1 channels, and in this context it has been possible to
confer high affinity diTC binding to the diTC-insensitive channel,
Kv3.2. Second, with respect to Kv1 channels,
which possess a conserved diTC binding domain and to which diTC binding
is of equivalent high affinity, the kinetics of ligand binding track
with the ease by which these channels enter the C-type inactivated state.
Studies with chimeric channels constructed from domains of
Kv1.3 and the diTC-insensitive channel, Kv3.2,
indicate that it is possible to confer correolide sensitivity to
Kv3.2 by transferring only the S5 region of
Kv1.3. These data suggest that differences between
Kv1.3 and Kv3.2 in S5 are a major
determinant in the lack of sensitivity of the latter channel to this
inhibitor. Site-directed mutagenesis studies have identified three
nonconserved residues that may account for the lack of sensitivity of
Kv3.2 to diTC. High affinity interaction of diTC with
Kv3.2 has been achieved by mutating these residues to the
corresponding amino acids found in Kv1.3. Two of these
residues are located in S5, whereas the third is in
S6. Interestingly, differences in the S6
regions of these two channels appear to play a minor role in diTC
binding, despite the fact that residues of S6 have been
shown to contribute to formation of the pore (28, 29). Two important
questions arise from these studies. Is the binding domain for diTC
restricted primarily to the S5 region? Alternatively, are
residues of S5 critical for conferring a special
conformation to a binding domain that is located in another region of
the channel? Comparison of the S5 and S6
regions of all Kv1 channels indicates that there are no
differences among any of these proteins. However, ligand binding
kinetics are markedly different between channels possessing C-type
inactivation and those channels that do not display this property. It
appears that conformational changes induced by C-type inactivation make
drug access to the diTC binding domain easier. Thus, it is possible
that the specific residues that contribute to the binding site for diTC
are common to many channels, but that the three-dimensional structure
of the receptor differs between these channels. On the other hand, the
pathway for drug entering its receptor site could be the
rate-determining step for binding. For some Kv1 channels,
this energy barrier represents an additional 2 kcal/mol, but for other
channels, such as Kv3.2, it could be much larger so as to
prevent drug binding at all. Site-directed mutagenesis studies along
S5 and S6 in Kv1.3 would be
required to identify those specific residues that contribute to the
diTC binding pocket. With use of the recently disclosed high resolution structure of the KcsA channel (28), it may be possible to model the
structure of Kv1.3. Such a model could be useful in general for probing the molecular determinants that control diTC binding to
potassium channels.
Correolide and several structural analogs were isolated from the plant
Spachea correa (7) based on their ability to block Kv1.3 potently in a functional efflux assay (8). These
compounds also inhibit lymphocyte activation mediated by
Ca2+-dependent stimulation of T cells in
in vitro assays, as well as in a mini-pig in vivo
delayed-type hypersensitivity
test.2 The mechanism by which
correolide and its analogs suppress T cell activation is directly
related to blockade of Kv1.3. Like peptide inhibitors,
correolide partially depolarizes T cells (8). This depolarization leads
to diminution of the Ca2+ signaling that occurs upon
activation of the T cell receptor complex. As expected, the properties
of correolide and its analogs resemble those of peptidyl inhibitors of
Kv1.3, such as MgTX, in immunological assays (4, 5). Given
these observations, correolide-derived agents might be candidates for
development as novel immunosuppressant drugs for treatment of graft
rejection and autoimmune diseases.
The development of a safe immunosuppressant requires a minimum side
effect profile. For agents targeting voltage-gated potassium channels,
this is a monumental task given the large diversity of these proteins
and their wide distribution throughout the body. Peptidyl inhibitors of
Kv1 channels are not state-dependent channel blockers and are generally neurotoxic. Although the safety profile of
correolide and its analogs requires further investigation, preliminary
results from evaluation of two correolide analogs that, after acute
administration, block a delayed-type hypersensitivity reaction in
mini-pigs, do not reveal a significant toxic side effect
profile.2 Perhaps correolide achieves functional
selectivity in vivo as a blocker of T cell Kv1.3
channels. Such functional selectivity may be related to the mechanism
by which correolide blocks Kv1.3. Preliminary studies
indicate that this agent interacts with high affinity with either open
or C-type inactivated, but not with the closed state of the channel (8,
30). Kv1.3 exists primarily in the inactivated state in T
cells with a resting potential of approximately We thank Dr. Bernardo Rudy for his gift of
Kv3.2, and Dr. David Melillo for radiochemical synthesis support.
*
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: Dept. of Membrane
Biochemistry and Biophysics, Merck Research Laboratories, P.O. Box
2000, Rahway, NJ 07065. Tel.: 732-594-7564; Fax: 732-594-3925; E-mail:
maria_garcia@merck.com.
2
G. Koo, J. T. Blake, K. Shah, M. J. Staruch, F. Dumont, D. Wunderler, M. Sanchez, O. B. McManus, A. Sintina-Meisher, R. C. Boltz, M. A. Goetz, R. Baker, J. Bao, F. Kayser, K. M. Rupprecht, W. H. Parsons, X.-C. Tong, I. E. Ita, J. Pivnichny, S. Vincent, P. Cunningham, D. Hora, Jr., W. Feeney, G. Kaczonwski, and M. S.,
Springer, submitted for publication.
The abbreviations used are:
ChTX, charybdotoxin;
Kv, voltage-gated potassium channel;
diTC, [3H]dihydrocorreolide;
diHC, dihydrocorreolide;
HgTX1, hongotoxin-1;
125I-HgTX1A19Y/Y37F, monoiodotyrosine-HgTX1A19Y/Y37F;
MgTX, margatoxin;
Kd, equilibrium dissociation constant;
k
Binding of Correolide to Kv1 Family Potassium
Channels
MAPPING THE DOMAINS OF HIGH AFFINITY INTERACTION*
,
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
70 °C.
Bmin)/(1+(I/IC50)nH) + Bmin, where Beq is the
degree of binding at the ligand concentration tested with no inhibitors
present, Bmin is the minimum amount of ligand
bound at higher concentrations of inhibitor where the binding curve has
leveled off, I is the inhibitor concentration, and
IC50, the inhibition constant. For the data presented,
Bmax was usually around 100% and
Bmin was 0%.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (28K):
[in a new window]
Fig. 1.
Kv3.2/1.3 chimeras.
Schematic representation of the different channels or chimeras used in
transient transfection of TsA-201 cells. Although not indicated in the
figures, all subunits contain a c-Myc sequence added to their C
termini.
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Fig. 2.
Binding of
125I-HgTX1A19Y/Y37F and diTC to
Kv3.2/1.3 chimeras. A, membranes prepared
from TsA-201 cells transiently transfected with either
Kv1.3 ( 
), Kv3.2(P..Kv1.3) (
),
Kv3.2(S5, P.. Kv1.3) (
),
Kv3.2(P, S6..Kv1.3) (
), or
Kv3.2(S5-S6..Kv1.3)
(
), were incubated with increasing concentrations of
125I-HgTX1A19Y/Y37F until equilibrium was
achieved. Other experimental conditions are given under "Experimental
Procedures." Specific binding data are presented relative to the
maximum receptor occupancy. Data were fit as described under
"Experimental Procedures." (), Kd = 0.29 pM, nH = 0.96; (), Kd = 3.8 pM, nH = 1.0; (), Kd = 1.03 pM, nH = 0.90; (),
Kd = 1.33 pM, nH = 0.90;
(), Kd = 0.28 pM, nH = 1.05. B, membranes prepared from TsA-201 cells transiently
transfected with either Kv1.3 (),
Kv3.2(S5, P..Kv1.3) (), or
Kv3.2(S5-S6..Kv1.3)
(), were incubated with increasing concentrations of diTC as
indicated under "Experimental Procedures." Nonspecific binding was
determined in the presence of 10 µM diHC. Specific
binding data are presented relative to the maximum receptor occupancy.
Data were fit as described under "Experimental Procedures." (),
Kd = 9.4 nM, nH = 0.90;
(), Kd = 48.8 nM, nH = 1.0; (), Kd = 18 nM, nH = 1.0.
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Fig. 3.
Comparison of amino acid sequences in the
S5 and S6
domains of Kv1.3 and
Kv3.2. The sequences of the S5 and
S6 region of Kv1.3 (GenBankTM
accession no. L23499) and Kv3.2 (accession no. M34052) are
presented.
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Fig. 4.
Binding of
125I-HgTX1A19Y/Y37F and DiTC to
Kv1.3 mutants. A, membranes prepared from
TsA-201 cells transiently transfected with either Kv1.3 or
the corresponding Kv1.3 mutant were incubated with
increasing concentrations of
125I-HgTX1A19Y/Y37F until equilibrium was
achieved. Other experimental conditions are given under "Experimental
Procedures." Data are presented as the ratio of Kd
values for wild type and mutant Kv1.3 versus the
corresponding residue mutated. B, membranes prepared as
above were incubated with 1 nM diTC in the absence or
presence of increasing concentrations of diHC until equilibrium was
achieved. Data are presented as the ratio of Ki
values for wild type and mutant Kv1.3 versus the
corresponding residue mutated and represent the mean ± S.E. of at
least two different transfection experiments.
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[in a new window]
Fig. 5.
Binding of diTC to Kv1.3,
Kv3.2F382L/M458L, and
Kv3.2F382L/I387F/M458L. A, saturation
studies. Membranes prepared from TsA-201 cells transiently transfected
with either Kv1.3 ( ), Kv3.2F382L/M458L
(), or Kv3.2F382L/I387F/M458L (), were incubated with
increasing concentrations of diTC as indicated under "Experimental
Procedures." Nonspecific binding was determined in the presence of 10 µM diHC. Specific binding data are presented relative to
the maximum receptor occupancy. Data were fit as described under
"Experimental Procedures." , Kd = 5.7 nM, nH = 0.96; , Kd = 20.9 nM, nH = 0.96; , Kd = 10.2 nM, nH = 1.03. B, competition
experiments. Membranes prepared as above were incubated with 1.2 nM diTC in the absence or presence of increasing
concentrations of diHC until equilibrium was achieved. Inhibition of
binding was assessed relative to an untreated control. ,
IC50 = 7.3 nM, nH = 1.0, Ki = 6 nM; , IC50 = 20.3 nM, nH = 1.0, Ki = 19.2 nM; , IC50 = 10.5 nM,
nH = 1.0, Ki = 9.4 nM.
The structure of diTC
1 for diTC
binding to homotetrameric Kv1 channels were calculated as
described in the text. Kd values for diTC were also
determined under equilibrium binding conditions as described. These
values are the average of 2-4 determinations carried out with
different membrane
preparations.
1) for the different Kv1
channels are listed in Table I. Kd values determined
from the ratio of k1 versus
k1 are similar to those determined under
equilibrium binding conditions, suggesting that diTC binds to
homotetrameric Kv1 channels through a simple bimolecular
reaction. As observed for k1, Kv1.3
and Kv1.4 are the channels that display the fastest
dissociation kinetics. Differences in k1
values between these and other Kv1 channels are about 5-25-fold. Thus, the affinity of diTC for all Kv1
channels, as determined under equilibrium binding conditions, is
similar because relative differences in association and dissociation
rate constants cancel each other. These data suggest that with some
Kv1 channels, ligand must overcome a much larger energy
barrier than is present in either Kv1.3 or
Kv1.4 during binding and unbinding reactions. Interestingly, Kv1.3 and Kv1.4 are the only
channels in the Kv1 family that undergo significant C-type
inactivation, a process involving a conformational change of some
residues in the outer vestibule of the channel that constricts the pore
and causes blockade of the ion conduction pathway (26). It is possible
that accessibility to the binding site for diTC becomes more facile
after C-type inactivation occurs.
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Fig. 6.
Binding of diTC to Kv1 channels:
association kinetics. Membranes prepared from HEK293 cells stably
transfected with either Kv1.1, Kv1.2,
Kv1.3, Kv1.4, Kv1.5, or
Kv1.6 channels were incubated with diTC for the indicated
periods of time at room temperature. Nonspecific binding, determined in
the presence of 10 µM diHC, is time-invariant and has
been subtracted from the experimental points. Specific binding data
have been fit to the pseudo-first order association reaction as
described under "Experimental Procedures."
View larger version (28K):
[in a new window]
Fig. 7.
Binding of diTC to Kv1 channels:
dissociation kinetics. Membranes prepared from HEK293 cells stably
transfected with either Kv1.1, Kv1.2,
Kv1.3, Kv1.4, Kv1.5, or
Kv1.6 channels were incubated with 10 nM diTC.
Dissociation kinetics were initiated by adding 10 µM diHC
and incubating at room temperature for different periods of time.
Specific binding data have been fit to a single monoexponential curve
corresponding to a first-order reaction.
View larger version (26K):
[in a new window]
Fig. 8.
Binding of diTC to mutant Kv1.3
channels. Membranes prepared from TsA-201 cells transiently
transfected with either Kv1.3 ( ),
Kv1.3/H399N (), or Kv1.3/G375Q () were
incubated with 10 nM diTC. Dissociation kinetics were
initiated by addition of 10 µM diHC followed by
incubation at room temperature for different periods of time. Specific
binding data have been fit to a single monoexponential decay
corresponding to a first-order reaction.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
50 mV (4). If
correolide would preferentially associate with the inactivated form of
the channel, it may be possible to shift the channel equilibrium to the
drug-bound form that is nonconductive. In firing neurons, however, the
drug-sensitive inactivated form of the channel may be present for only
a very brief period of time; drug binding might be negligible under
these conditions, given the slow onset of correolide action observed with brain native channels (8). Although more detailed studies are
necessary to discern the mechanism by which correolide interacts with
potassium channels, it is tempting to speculate that the state-dependent interaction of this agent, together with
its kinetics of binding, may be important for limiting toxicity
in vivo.
ACKNOWLEDGEMENTS
FOOTNOTES
Recipient of Erwin Schrödinger Fellowships J-01108-MED and
J-01460-MED of the Austrian Research Foundation.
ABBREVIATIONS
1, dissociation rate constant;
k1, association rate constant.
REFERENCES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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