(Received for publication, May 5, 1995; and in revised form, July 17, 1995)
From the
-Dendrotoxin, a 59-amino acid basic peptide from the venom
of Dendroaspis angusticeps (green mamba snake), potently
blocks some but not all voltage-dependent potassium channels. Here we
have investigated the relative contribution of the individual
-subunits constituting functional Kv1.1 potassium channels to
-dendrotoxin binding. Three residues critical for
-dendrotoxin binding and located in the loop between domains S5
and S6 were mutated (A352P, E353S, and Y379H), and multimeric cDNAs
were constructed encoding homo- and heterotetrameric channels composed
of all possible ratios of wild-type and mutant
-subunits. Complete
mutant channels were about 200-fold less sensitive for the
-dendrotoxin block than complete wild-type channels, which is
attributable to a smaller association rate. Analysis of the bimolecular
reaction between
-dendrotoxin and the different homo- and
heteromeric channel constructs revealed that the association rate
depends on the number of wild-type
-subunits in the functional
channel. Furthermore, we observed a linear relationship between the
number of wild-type
-subunits in functional channels and the free
energy for
-dendrotoxin binding, providing evidence that all four
-subunits must interact with
-dendrotoxin to produce a high
affinity binding site.
Potassium channels of vertebrate neurons display a high degree of diversity that contributes to the complexity of function inherent to these cell types(1) . In the nervous system, potassium channels are crucial in controlling the resting membrane potential, repolarization of the action potential, and higher neuronal functions such as learning and memory(2) . The specific roles played by individual potassium channel types can be elucidated by taking advantage of differences in voltage-dependence, kinetics, and pharmacological specificity. Venom purification of certain snakes and scorpions has led to the isolation of peptide toxins, including potassium channel toxins, which are both selective and sensitive pharmacological tools to study the bountiful types of potassium channels (for a review, see (3) ).
First indications that
the venom from the Eastern green mamba Dendroaspis angusticeps contains a peptide toxin which facilitates neuromuscular
transmission came from studies of Barret and Harvey(4) .
Separation of the venom into several components resulted in a
particular interest of one polypeptide, named dendrotoxin, because it
enhanced the quantal acetylcholine release at vertebrate neuromuscular
junctions(5, 6) . The facilitation is most probably
the result of high affinity binding to voltage-dependent potassium
channels; in mammalian central neurones, dendrotoxin selectively blocks
an inactivating voltage-dependent potassium current that corresponds to
the 4-aminopyridine-sensitive ``A-type'' current(7) ,
and in mammalian peripheral neurons, dendrotoxin blocks slowly
inactivating or noninactivating potassium currents(8) .
Dendrotoxin constitutes about 2.5% of the total venom protein and is
composed of 59 amino acids with a molecular mass of 7077
Da(5, 9) . In 1988, Benishin et al.(10) isolated four different polypeptides from the venom
of the green mamba, designated as -,
-,
-, and
-dendrotoxin, and the amino acid composition of these four toxins
indicated that
-dendrotoxin is identical to dendrotoxin (called
DTX hereafter). (
)Two similar polypeptides, called toxin I
and K, have also been isolated from the venom of the black mamba Dendroaspis polylepis(11) , the former blocking
rapidly inactivating, voltage-dependent potassium current in the frog
node of Ranvier(12) . Although all of these toxins have a high
degree of sequence homology with several protease inhibitors, such as
the bovine pancreatic trypsin inhibitor, they do not share the
pharmacological properties of protease
inhibitors(13, 14, 15) . However, the
tertiary structure of DTX is very similar to that of bovine pancreatic
trypsin inhibitor, with two well defined regions of secondary
structure, a double-stranded antiparallel
-sheet and a short
-helical region at the amino terminus(16, 17) .
DTX is a basic polypeptide with 8 arginine, 6 lysine, 2 aspartate, and
3 glutamate residues. At neutral pH it carries an overall positive
charge, but the distribution of the charge is highly asymmetric. All
members of the DTX family have 6 conserved cysteines, which might
stabilize the structure through the formation of disulfide
bonds(18) .
Because DTX blocks some but not all potassium conductances with high affinity, it has not only been useful in the pharmacological classification of potassium channels(19) , but also in the purification and isolation of potassium channel proteins(20) . The variation in toxin sensitivity exhibited by the expressed potassium channels cloned from Drosophila(21) and mammals (22) has provided the opportunity to correlate primary sequences of potassium channels with toxin sensitivity. Hurst et al.(23) have shown that DTX binding occludes the potassium channel pore by binding at or near the external mouth of the Kv1 channel at residues located in the loop between transmembrane domains S5 and S6. In this loop, residues alanine (Ala-352), glutamate (Glu-353), and tyrosine (Tyr-379), of the RBK1 (Kv1.1) channel were capable of influencing DTX blockage of the channel. The same authors also noted that through-space electrostatic forces play a role in DTX binding and concluded that the bound DTX must be stabilized by a number of residue interactions.
Given that the
core of functional potassium channels is believed to be a tetramer
comprising four -subunits(24, 25, 26) ,
a mutation introduced in the cDNA coding for one
-subunit will
result in a 4-fold effect at the level of the functional channel
itself. When this mutation is critical for DTX binding and given that
the toxin interacts with all 4
-subunits, the macroscopic change
in binding affinity will be determined by the presence of these four
mutant subunits. So far, however, no data exist wherein the importance
of each of the
-subunits for DTX binding is assessed in potassium
channels. The present study reports experiments in which concatenated
homo- and heteromultimeric RCK1 channel
-subunits, with all
possible ratios of wild-type:mutant
-subunits (WT:MUT 4:0, 3:1,
2:2, 1:3, and 0:4), were expressed as functional potassium channels to
study the contribution of each subunit to DTX binding and to establish
whether all four
-subunits interact simultaneously with DTX.
Hurst et al.(23) have shown that mutation
of 3 residues in DTX-sensitive RBK1 channels, Ala-352, Glu-353, and
Tyr-379, to match the equivalent positions in DTX-insensitive RGK5
channels, proline (Pro-374), serine (Ser-375), and histidine (His-401),
causes a 150-fold decrease in DTX sensitivity. This provided a
molecular explanation for the differences in DTX sensitivity observed
among native potassium channels (Fig. 1A). Whether the
molecular footprint of DTX binding is mainly determined by the
interaction of the toxin with just one, or, in contrast, with all four
-subunits together, was not known. To investigate this we have
made the same mutations A352P, E353S, and Y379H in an
-subunit of
a RCK1 channel belonging to the Kv1.1 Shaker-type family. Next
we constructed concatenated multimeric cDNAs composed of different
combinations of WT and MUT
-subunits (Fig. 1B),
allowing us to discriminate between two models of binding wherein the
toxin either interacts with only one of the four
-subunits, or
interacts simultaneously with all four
-subunits (see also below).
Figure 1:
S5-S6
region of RBK1, RGK5, and RCK1. A, The S5-S6 region of
the -subunit of RBK1 beginning at residue Phe-346 and ending at
Lys-386, and of RGK5 beginning at Phe-368 and ending at Lys-408. DTX
sensitivity is crucially determined by residues Ala-352, Glu-353, and
Tyr-379 in RBK1, and Pro-374, Ser-375, and His-401 in RGK5. Dashed
horizontal lines represent identical residues. RBK1 is
DTX-sensitive (gray ovals), whereas RGK5 is DTX-insensitive (black ovals). B, the S5-S6 region of the
-subunit of RCK1 beginning at Phe-346 and ending at Lys-386. MUT
channels contain the A352P, E353S, and Y379H mutations. The Y379H
mutation is supposed to reside in the H5 or pore region. A pictorial of
concatenated homo- and heterotetrameric
-subunits is shown below,
with DTX-sensitive (gray) and DTX-insensitive (black)
-subunits forming functional potassium
channels.
Functional homo- and heteromeric RCK1 channels composed of all possible ratios of WT and MUT subunits revealed different pharmacological profiles for DTX block. Increasing concentrations of DTX were needed to achieve approximately the same extent of block, as WT subunits were substituted by MUT subunits (Fig. 2A). A constant concentration of 3 nM DTX blocked the currents through WT channels by about 80% and through channels composed of two WT and two MUT subunits by about 20%. The currents generated through complete MUT channels were practically unaffected at this toxin concentration (Fig. 2B).
Figure 2:
Macroscopic currents of homo- and
heteromeric channels. Current recordings obtained with the
two-microelectrode voltage clamp technique. Oocytes were held at
-90 mV and stepped to a test potential of 0 mV. A,
currents through channels comprising 4:0, 3:1, 2:2, 1:3, and 0:4 WT:MUT
-subunits are shown both before and after application of
increasing concentrations of DTX, leading to approximately the same
extent of blockage. B, currents through complete WT, 2:2
WT:MUT, and complete MUT channels are shown both before and after
application of 3 nM DTX. The current scale is 10 µA (Panel A) and 5 µA (Panel
B).
The functional expression
of the five different constructs, together with the fact that the
mutations did not alter the kinetics, gating and potassium selectivity
(V as a function of [K]
not
different between WT and mutant, data not shown), allowed us to test
two models of DTX binding, one with and one without energy additivity.
The model without energy additivity includes the following main
features. (i) DTX binds on one of the
-subunits and occludes
thereby the pore. (ii) There are four statistically distinguishable
configurations available for a bound DTX molecule. (iii) When the
channel has four identical subunits, such as the WT and MUT
homotetramers, these four configurations will be energetically
identical. (iv) When the channel has different sets of subunits, like
the heterotetramers, then the interaction energy may be different for
each configuration. (v) Channel inhibition can be described by a
Langmuir adsorption isotherm. (vi) K
values for
the heterotetrameric channels can be predicted in terms of the observed K
values of homotetrameric WT and MUT channels,
according to
with K values as microscopic binding
constants. The model with energy additivity is characterized by the
following main features. (i) DTX interacts simultaneously with all four
-subunits. (ii) A linear relationship exists between the free
energy for DTX binding and the number of WT subunits. (iii) Channel
inhibition can be described by a Langmuir adsorption isotherm. (iv) K
values for heterotetramers channels can again be
predicted in terms of the observed K
values for
homotetrameric WT and MUT channels, and is given by
The model without energy additivity predicts that, as long as at
least one WT subunit is present, the channel will principally remain
sensitive for DTX block. The model with energy additivity predicts
equal spacing for the K values on a logarithmic x axis for all the different channel constructs. Fitted K
values for block by extracellularly applied DTX
of homomultimeric WT and MUT channels were 1.1 nM (n = 17 oocytes) and 199.3 nM (n =
25), respectively (Fig. 3A). Fitted K
values for heteromultimeric channels with 3:1, 2:2, and 1:3
WT:MUT subunits were 5.1 nM (n = 5), 17.6
nM (n = 26), and 84.4 nM (n = 7), respectively. These values correlate very well with
the predictions of the model which includes energy additivity, as
opposed to the model without energy additivity (Fig. 3B). A summary of the K
values, as fitted from the observed data and as predicted in
models with or without energy additivity, is shown in Fig. 4.
The semilogarithmic plot of the K
values as a
function of the number of WT
-subunits reveals a linear
relationship, as expected from the equal spacing of the K
values in Fig. 3B (right
plot). The same linear relationship is also found on a linear plot
between the free energy for DTX binding and the number of WT subunits (Fig. 4, right ordinate). Comparison between complete
WT and complete MUT channels revealed that the mutations, A352P, E353S,
and Y379H, result in channels that have a 200-fold decreased
sensitivity for DTX, corresponding to a change in binding energy
equivalent to more than 3 kcal/mol.
Figure 3:
DTX
sensitivity of multimeric constructs. A, dose-response curves
for complete WT and MUT channels, with K values of 1.1 and 199.3 nM, respectively (Hill
coefficient of 1). Points are mean ± S.E. from 17 (WT) and 25
(MUT) oocytes. B, dose-response curves for complete WT and MUT
channels as in Panel A (full lines), together with
predicted dose-response curves (dashed lines) in a model
without energy additivity (left) and with energy additivity (right). A superior prediction of the data is obtained in a
model which includes energy additivity. Data points are mean ±
S.E. from 5 (3:1 WT:MUT), 26 (2:2 WT:MUT), and 7 (1:3 WT:MUT) oocytes.
For 2:2 WT:MUT channels, results obtained from tetrameric and dimeric
cDNAs were pooled.
Figure 4:
K values and changes in binding energy. Semilogarithmic plot
of K
values (left ordinate) and
linear plot of mutant-induced changes in binding energy normalized to
complete WT channels (
G; right ordinate),
as a function of the number of mutant
-subunits in the constructs. Open squares, predictions without energy additivity; open
circles, predictions with energy additivity; filled
circles, observations. The change in binding energy is given by
G = -RT ln(1 M/K
). Lines were fitted by eye
to the data.
Blockage induced by DTX showed
no voltage-dependence, as the degree of block was not different in the
range of test potentials from -30 to +40 mV (Fig. 5, A-C). The same extent of current inhibition was observed
whether increasing concentrations of DTX were applied sequentially or
cumulatively. Furthermore, DTX binding did not alter channel gating;
the maximal conductance (g) curve in control and
1 nM DTX conditions was characterized by a V
value of -16.0 ± 3.1 mV (n = 10) and
-16.5 ± 4.2 mV (n = 8), respectively,
which is not significantly different (Fig. 5D). The
steady-state inactivation was also not shifted by 1 nM DTX:
-27.9 ± 1.4 mV (n = 5) and -28.9
± 2.8 mV (n = 5) in control and DTX conditions,
respectively. The block by DTX occurred rapidly and binding was
reversible (Fig. 6A). The rapid onset and offset of the DTX
action indicates an extracellular site of action.
Figure 5:
Effect of DTX on voltage-dependence and
gating. A, current recordings in control and 1 and 10 nM DTX conditions. Oocytes were held at -90 mV and stepped to
test potentials varying between -80 and +40 mV. B,
corresponding current-voltage (I-V) relationship,
characterized by outward rectification typical for RCK1 channels. C, percentage current left over in 1 and 10 nM DTX
conditions, as a function of V
. No voltage-dependence
was observed. D, corresponding maximal conductance-voltage (g
-V
) relationship. No
DTX-induced shift in the half maximal potential of activation
(V
) was observed (vertical dashed
lines).
Figure 6:
Bimolecular kinetics of DTX inhibition. A, an oocyte expressing WT channels was depolarized to 0 mV
for 1 s from a holding potential of -90 mV every 5 s, both in the
absence and presence of 3 nM DTX (control, open
triangles; wash-in, open triangles plus black
bar; wash-out, open circles). Values for and
in this experiment were 14.7 and 72.3 s,
respectively. B, bimolecular reaction scheme with C = channel, C:DTX = channel with bound DTX, k
and k
the apparent
first-order association and first-order dissociation rate constants,
respectively,
and
the second- and first-order rate
constants of association and dissociation, respectively,
and
the time constants for approach to
equilibrium upon wash-in and wash-out,
respectively.
Since DTX binding
was reversible and did not alter channel gating, we next investigated
whether DTX blockade followed a kinetic behavior of a simple
bimolecular reaction. Current inhibition upon DTX application and
recovery upon DTX removal followed a single exponential time course
compatible with a bimolecular reaction scheme (Fig. 6B;
for details, see legend). The effects of increasing DTX concentrations
on the kinetics of block on a WT RCK1 channel are shown in Fig. 7A. As required by a bimolecular scheme, the
apparent first-order association rate constant (k) increased linearly with toxin concentration,
whereas the first-order dissociation rate constant (k
) remained constant. Fig. 7B summarizes the second-order association rate constants (
) and
the first-order dissociation rate constants (
= k
) of all the different channel constructs we
have expressed (with ratios WT:MUT
-subunits ranging from 4:0 to
0:4). Gradually exchanging the four WT
-subunits by four MUT
-subunits, resulting in channels which become more and more
insensitive for DTX block, can be attributed to a gradual decrease of
the association rate constant. In contrast, the
values remain
constant for all channel constructs. Dividing the
values by the
values for all the different channel constructs correlates well
with the observed K
values (Fig. 4), which
is in accordance with the equation K
=
/
(Fig. 6B).
Figure 7:
Association and dissociation rate
constants. A, the apparent first-order rate constant of
association (k) and first-order rate constant of
dissociation (k
) for DTX on a WT channel were
calculated using the equations in 6B and plotted as a function
of the DTX concentration. Each symbol represents an independent
experiment. B, from similar measurements as those in Panel
A, the second (
)- and first (
)-order rate constants of
association and dissociation were then determined for all the possible
channel constructs (n
3) and plotted as a function of the
number of WT
-subunits in the channel
constructs.
Next, through-space
electrostatic interactions between charged residues in RCK1 and DTX
were examined by measuring the effectiveness of DTX to inhibit current
in solutions of different ionic strength. For complete WT channels, the
fitted K was 1.1 nM (n =
17 oocytes) in normal ND-96 solution and 0.64 nM (n = 5) in an iso-osmotic solution containing only 48 mM NaCl (sucrose substitution). For complete MUT channels, the K
was, respectively, 199.3 nM (n = 25) and 43.6 nM (n = 6). This
increase in sensitivity to DTX suggests that through-space
electrostatic forces play a role in DTX binding, corroborating the
results obtained by Hurst et al.(23) .
This study has identified that all four individual subunits
of the RCK1 channel must interact simultaneously with a DTX molecule to
produce a high affinity binding site. A similar mechanism of additive
contributions from four tyrosine residues (Tyr-449) in the four
subunits of Shaker-type channels has also been shown for the
external TEA binding(26, 28) . The observation that
each subunit in a symmetrical tetrameric channel is equally involved in
the process of toxin interaction might be difficult to reconcile with
the asymmetrical structure of a DTX peptide. However, the following
points can be raised in accordance with our observations. If the
overall dimensions of the toxin are much smaller than the outer channel
vestibule, the binding process may be governed by a single toxin
residue, annulling the importance of asymmetry of the peptide and
interactions with other channel residues. The observed high affinity
binding of DTX results then from the likelihood that a DTX molecule has
ideally approached its binding site on the channel, as opposed to the
frequent occurrence of collisions without binding (which have very
large rate constants) between DTX and the potassium channel due to an
inapt surface contact. For charybdotoxin (CTX), Garcia-Calvo et al.(29) have shown that the target size for the rat brain
Kv1.3 CTX receptor is 253 kDa, which is about 4 times the size of a
single pore-forming -subunit of 58 kDa. The target size of 253 kDa
implies that the toxin-binding site on Kv1.3 channels is lost as the
channel tetramer is destroyed. This indicates a simultaneous
interaction of CTX with all four subunits, similar to our results for
DTX on Kv1.1 channels. In marked contrast with the foregoing, the
CTX-binding site on the maxi-potassium channel activated by calcium
seems associated with only a single
-subunit
complex(29) . The finding of energy additivity of the four
-subunits for DTX binding does not allow us to conclude what type
of molecular interaction is taking place. The observed free energy of
DTX binding can be decomposed into a coulombic and a sterical term. As
long as the three-dimensional structure of WT and MUT pore regions is
not known, we can merely speculate which of the two terms is
determinant. Additionally, interpretations of the free energy of toxin
binding may be confounded by unknowable dehydration energies for
residues that become dehydrated upon toxin binding, i.e. for
toxin or channel residues that are buried in the region of intimate
channel-toxin contact(30) .
This is the first report
analyzing the contribution of each of the individual -subunits to
the DTX binding site in a functional potassium channel, based on the
expression of multimeric cDNAs as opposed to co-injection experiments.
Qualitatively, the use of homo- and heteromultimeric cDNA constructs is
superior, since each type of tetramer encodes a homogeneous population
of channels, at the same time controlling the ratio of WT:MUT monomers
in the functional channel (see also (26) ). Based on
co-injection experiments of WT and MUT Shaker potassium
channels, MacKinnon (25) has demonstrated that the sensitivity
for CTX, a 37-amino acid toxin from the scorpion Leiurus
quinquestriatus quinquestriatus, depends on the ratio of WT:MUT
subunits, and that the channels that include one or more MUT subunits
have an intermediate CTX sensitivity, but that the WT CTX-sensitive
phenotype is dominant. A recent report by Russel et
al.(31) , however, suggests also that in heterotetramers
from 2 Kv1 class potassium channels (Kv1.2 and Kv1.5), the
CTX-insensitive monomer dominates the CTX pharmacology of the channel.
From this we conclude that it is unlikely that the mechanism of
blockage by DTX and CTX is the same (see also (32) ). The
destabilized binding of DTX in MUT channels is expressed in a decreased
association rate (
). Together with the finding that the four
-subunits interact simultaneously with a DTX molecule, this means
that the mutations are expressed with equal energies in both the bound
and transition states.
Although multimeric channel constructs have
proven very useful in our previous studies (e.g. see (26) ), we performed an additional test for the validity of our
multimeric constructs by screening the sensitivity of the hybrid
channels for block by external TEA. The logarithm of the K values of the homo- and heteromultimeric
channels varied linearly with the number of WT subunits, indicating
that also for the mutants in this study a TEA molecule is energetically
stabilized in the pore of the channel by additive contributions from
the four
-subunits(26, 28) . Furthermore, the
currents generated through tetrameric 2:2 WT:MUT cDNA channel
constructs were phenotypically indistinguishable from the currents
through dimeric WT:MUT channel constructs, including their
pharmacological profile.
Recombinant toxins and their associated large number of possible mutants constitute important new tools to delineate the site by which toxins recognize their multi-subunit targets. All residues of CTX have already been submitted to individual mutagenesis and it was found that the affinity changed dramatically when mutations were made at eight positions, among which three are positively charged residues, three hydrophobic, and two with hydrogen bonding capacity(33, 34) . Since CTX competes with TEA and DTX in various assays(3) , it was suggested that positively charged residues on DTX may also govern its binding. It was specifically tempting to anticipate that the lysine triplet (Lys-28, Lys-29, and Lys-30) on DTX may be associated with its specific binding, since residue Lys-27 on CTX appears to be located in the center of symmetry of the Shaker channel, playing a crucial role in blocking its pore(30) . However, using recombinant mutant DTX, Danse et al.(32) have provided evidence that the lysine triplet is unlikely to constitute a major element for the functional properties of DTX. Therefore, new DTX mutants are needed to clearly identify the residues by which DTX establishes intimate contact with RCK1 channels.