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Vol. 285, Issue 3, 1051-1060, June 1998
Neurex Corporation, 3760 Haven Avenue, Menlo Park, California
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Abstract |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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The ability of voltage-gated potassium channel 
-subunits to form
heteromultimers has complicated efforts to use toxins to characterize
potassium channels in native cells. Here I investigate the effects of
subunit composition on toxin blocking affinity, using three members of
the Shaker subfamily of potassium channel -subunits (Kv1.1, Kv1.2
and Kv1.4), which are known to form heteromultimers in vivo,
in the Xenopus oocyte expression system. These subunits were
coexpressed as pairs in which one member was toxin-sensitive and the
other relatively insensitive. The blocking affinity of two dendrotoxins
(DTX-I and 
-DTX) and a scorpion toxin (tityustoxin-K) on the
resulting mixed population of channels was measured to evaluate three
models of toxin block. The single subunit model, in which a single
toxin-sensitive subunit renders the channel toxin sensitive, best
described all of the data for the two dendrotoxins and the block of
tityustoxin-K for a mixed population of channels composed of Kv1.1
and Kv1.2 subunits. However, with tityustoxin-K, the data for a
mixed population of Kv1.2 and Kv1.4 subunits was fit best by a model in
which the toxin interacts with all four subunits for high-affinity
block. The data suggest that subunit composition of potassium channels
can have a large effect on toxin block and that different toxins yield
strikingly diverse results with the same pair of subunits, even when
they are nearly identical in blocking affinity for the toxin-sensitive
subunit.
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Introduction |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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The
functional characterization of potassium channels in the CNS and
elsewhere will require a greater understanding of the correspondence
between potassium currents defined by biophysical and pharmacological
criteria and our expanding knowledge of the molecular composition of
potassium channels. This task has been complicated by the now
well-known ability of potassium channel subunits to coassemble with
members of the same molecularly defined subfamilies into
heterotetramers (Christie et al., 1990
; Isacoff et
al., 1990; Ruppersberg et al., 1990). Peptide toxins
from a variety of animal species have been used to inhibit components of potassium current in neurons and other cells (Benoit and Dubois, 1986; Halliwell et al., 1986; Hall et al., 1994;
Brew and Forsythe, 1995), and these toxins have been tested on a number
of cloned -subunits that form functional potassium channels in
heterologous expression systems (Stühmer et al., 1989;
Ramaswami et al., 1990; Grissmer et al., 1994;
Hopkins et al., 1994a). However, the molecular identity of
channels expressed in native cells has not been deduced with certainty
on the basis of the latter findings because of our incomplete
understanding of the subunit specificity of these toxins and of the
effect of subunit composition on toxin blocking affinity.
This study addresses the effects of voltage-gated potassium channel
subunit composition on toxin blocking affinity using the Xenopus oocyte expression system. I have focused on three
members of the Shaker subfamily of voltage-gated potassium channel
-subunits: Kv1.1, Kv1.2 and Kv1.4 (Tempel et al., 1988;
Chandy et al., 1990; Ramaswami et al., 1990).
These subunits are known to coassemble into heteromultimeric channels
in Xenopus oocytes (Ruppersberg et al., 1990;
Christie et al., 1990; Hopkins et al., 1994b) and in vivo (Wang et al., 1993; Sheng et
al., 1993; Rhodes et al., 1995). I have coexpressed
these subunits as pairs (Kv1.1/Kv1.2, Kv1.2/Kv1.4 and Kv1.1/Kv1.4) and
have used two dendrotoxins (DTX-I and -DTX) and tityustoxin-K to
evaluate three models of toxin binding to heteromultimeric potassium
channels. The subunit pairs and toxins were chosen such that one member
of the pair of subunits would be toxin-sensitive and the other
relatively insensitive. The results demonstrate that the model that
best describes the data can be both toxin- and subunit-specific. For
example, the blocking affinity of tityustoxin-K for a mixed
population of channels composed of Kv1.1 and Kv1.2 subunits could be
described by a model in which a single toxin-sensitive subunit renders
the channel toxin-sensitive (single subunit model). However, this model
did not describe the tityustoxin-K data for a mixed population of
Kv1.2 and Kv1.4 subunits. The single subunit model provided the closest
fit to the data for a mixed population of Kv1.2 and Kv1.4 subunits
using DTX-I. The blocking affinities of -DTX on mixed populations of
Kv1.1/Kv1.2 or Kv1.1/Kv1.4 subunits were best described by the single
subunit model. The data suggest that potassium channel subunit
composition can have a marked effect on a toxin's blocking affinity
and that the identity of the toxin is also an important factor.
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Materials and Methods |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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Oocyte expression.
Plasmid DNAs encoding mKv1.1
and mKv1.2 (kindly provided by Dr. Bruce Tempel at the University of
Washington) were linearized with EcoRI for in vitro
transcription of capped mRNAs (mCAP kit, Stratagene) using SP6 RNA
polymerase (Hopkins et al., 1994b). Plasmid DNAs encoding
hKv1.2 and hKv1.4 (kindly provided by Dr. Mark Tanouye at the
University of California, Berkeley), were linearized with NotI for
in vitro transcription of capped, polyadenylated mRNA using
T7 RNA polymerase (Ramaswami et al., 1990). Unless otherwise
noted, Kv1.1 refers to mKv1.1, Kv1.2 to hKv1.2 and Kv1.4 to hKv1.4.
Adult female Xenopus laevis were anesthetized with MS-222
for half an hour, followed by surgical removal of 2 to 4 ovarian lobes.
Pieces of ovary were washed in CFS containing 82.5 mM NaCl, 2 mM KCl, 1 mM MgCl2 and 5 mM HEPES (pH 7.6). Single oocytes free from
follicular cells and connective tissue were obtained by treatment with
1 mg ml
1 collagenase (Type A, Boehringer Mannheim,
Indianapolis, IN) in CFS at room temperature (22°C) for 3 hr. Healthy
oocytes at maturation stage V and VI were selected and transferred to
an incubation saline solution (ISS) containing 96 mM NaCl, 2 mM KCl, 1 mM MgCl2, 1.8 mM CaCl2, 0.01 mg
ml1 penicillin, 0.01 mg ml1 streptomycin
and 5 mM HEPES (pH 7.4). After 24 hr of incubation at 18°C, the
oocytes were injected with 50 nl of 3 to 1000 pg nl1 of
cRNA via a microinjector (Drummond, Broomall, PA). Oocytes were maintained in ISS at 18°C and tested for current expression 2 to
6 days after injection.
Electrophysiology and data analysis.
For whole-cell current
measurements, a two-electrode voltage clamp (Warner OC-725B) was used.
Both the voltage-recording and the current electrode were filled with 3 M KCl and had resistances of 0.5 to 2.0 M
. The oocytes were placed
in an 0.5-ml chamber and were perfused continuously at 5 ml
min1 with FRS consisting of 115 mM NaCl, 2.5 mM KCl, 1.8 mM CaCl2 and 10 mM HEPES (pH 7.2). Transient capacitive and
linear leakage currents were subtracted on-line by the P/4 procedure
(Armstrong and Bezanilla, 1974) built into the data acquisition
software (PClamp, Axon Instruments, Foster City, CA). The currents were filtered at 1 kHz and sampled at 5 kHz. The oocyte membrane was clamped
at 
70 mV, and membrane currents were elicited by 250-msec command
voltages from 70 to 50 mV. To obtain concentration-inhibition data
for an inhibitor, control data were obtained from a given oocyte in
FRS, and then, 5 min after perfusion of inhibitor, data were obtained
again. This procedure was repeated for an ascending series of inhibitor
concentrations. I have determined that, at the solution flow rate used,
5 min is an adequate time interval to obtain "steady-state" current
measurements for all inhibitors used. The peak potassium current at 50 mV was measured in the presence of each inhibitor concentration and
normalized to the current obtained in control FRS at the same voltage
(fractional inhibition). For a given concentration-inhibition curve,
each data-point represents the mean fractional inhibition value
obtained from the number of oocytes given in the figure legend for each curve. For the experiments in which toxins were applied sequentially to
the same oocyte, the membrane potential was held at 70 mV, and
100-msec command voltages were applied to 50 mV (0.2 Hz). I observed no
cumulative inactivation or rundown with any of the subunits tested with
this protocol. After a 10-pulse base-line period, a toxin was applied
directly to the bath solution. After the responses displayed a steady
level of block, another toxin was applied in the same manner, and the
experiment was continued until a new steady level of block was
achieved. For the fitting of drug-binding isotherms to
concentration-inhibition data, the adequacy of the fit was
statistically evaluated with the chi-square goodness-of-fit test with a
significance level of P < .05. All values are expressed as
mean ± S.E.M.
1 at this voltage), i is the single
channel current amplitude for channels formed by each subunit type
(determined in separate patch-clamp experiments) and N is
the number of channels. This method might tend to underestimate
channels formed from hKv1.4 subunits, because they display rapid
N-type inactivation in contrast to the very slow, C-type
inactivation displayed by channels formed from the other subunits
(Hoshi et al., 1991; Ruppersberg et al., 1991;
López-Barneo et al., 1993). This potential source of
error should not affect any of the conclusions if it is small and
constant from experiment to experiment, because it would affect all
ratio estimates similarly. I could not simply coinject fixed ratios of
concentrations of cRNA, because for a given concentration of cRNA,
subunit expression levels were not equal. I chose not to transcribe
cDNA mixtures (Kavanaugh et al., 1992) because of the differences in translation initiation sequences for the different plasmid vectors used. These differences may cause differential translational efficiency, yielding inaccurate estimates of expression ratios based on cRNA or cDNA concentrations.
Modeling. The fraction of the total population of channels composed of a particular type of homomultimer or heteromultimer can be predicted with the binomial theorem if the subunit expression ratio is known:
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![I<SUB><UP>inhibitor</UP>[x]</SUB>/I<SUB><UP>control</UP></SUB>=<LIM><OP>∑</OP><LL>k<UP>=</UP>0</LL><UL>4</UL></LIM> <UP>Frac</UP><SUB>k</SUB>[1−(x<SUP><UP>Hill</UP>k</SUP>/(x<SUP><UP>Hill</UP>k</SUP>+<UP>IC</UP><SUB>50k</SUB><SUP><UP>Hill</UP>k</SUP>))]](/content/vol285/issue3/fulltext/1051/img002.gif)
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; Kavanaugh et al.,
1992). As the number of inhibitor-sensitive subunits is decreased,
IC50 increases exponentially because of an effect on the
binding free energy of the inhibitor with the channel:
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G° is Gibbs free-energy change,
R is the gas constant, T is absolute temperature
and Ki is the equilibrium dissociation constant.
If the binding energy provided by each subunit is additive to those
provided by other subunits, then
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G
and rearranging yield
![<UP>ln</UP>(K<SUB>i;<UP>heteromultimer with</UP> n <UP>subunit</UP> 1</SUB>)=n/4[<UP>ln</UP>(K<SUB>i;<UP>subunit</UP> 1</SUB>)−<UP>ln</UP>(K<SUB>i;<UP>subunit</UP> 2</SUB>)]+<UP>ln</UP>(K<SUB>i;<UP>subunit</UP> 2</SUB>)](/content/vol285/issue3/fulltext/1051/img006.gif)
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IC50, then
![<UP>ln</UP>(<UP>IC</UP><SUB>50;<UP>heteromultimer with</UP> n <UP>subunit</UP> 1</SUB>)=n/4[<UP>ln</UP>(<UP>IC</UP><SUB>50;<UP>subunit</UP> 1</SUB>)−<UP>ln</UP>(<UP>IC</UP><SUB>50;<UP>subunit</UP> 2</SUB>)]+<UP>ln</UP>(<UP>IC</UP><SUB>50;<UP>subunit</UP> 2</SUB>)](/content/vol285/issue3/fulltext/1051/img007.gif)
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).
DTX-I and -DTX were obtained from Alomone Labs (Jerusalem, Israel).
Tityustoxin-K was obtained from Dr. Mordecai Blaustein (University
of Maryland). TEA was obtained from Sigma (St. Louis, MO). The toxins
and TEA were diluted in FRS to obtain the final concentrations.
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Results |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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TEA blocking affinity described by energy additivity model.
TEA blocking affinity for heteromultimeric K+ channels
composed of TEA-sensitive Kv1.1 and mutant TEA-insensitive Kv1.1
subunits is well described by the energy additivity model (Heginbotham and MacKinnon, 1992; Kavanaugh et al., 1992). This is also
true for heteromultimeric channels composed of Kv1.1 and relatively TEA-insensitive Kv1.2 subunits (Hopkins et al., 1994b). I
attempted to reproduce this result as a check on the methods used here. Figure 2A depicts an experiment in which
TEA-sensitive Kv1.1 was coinjected with TEA-insensitive Kv1.2 with a
fractional expression of 0.6 for Kv1.1. The concentration-inhibition
curve for the resultant mixed population of channels was well described
by the energy additivity model, but not by the single subunit model or
the dominant negative model. Figure 2B shows that as the fraction of
Kv1.1 subunits was varied in three separate experiments, the
IC50 values for the mixed population of channels was well
fit by the line predicted for the energy additivity model. The single
subunit and dominant negative models did not provide adequate fits to the data. The dominant negative model was found to provide very poor
fits to any of the data sets; it will not be further depicted.
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-DTX, DTX-I and tityustoxin-K block of homomultimeric
K+ channels.
-DTX is a dendrotoxin isolated from
the venom of Dendroaspis angusticeps that blocks some
voltage-gated K+ channels (Benishin et al.,
1988; Hall et al., 1994). Whereas Kv1.1 was very sensitive
to -DTX, Kv1.2 was much less sensitive, and Kv1.4 was completely
insensitive to this toxin (table 1; figs.
3 and 4).
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; Harvey and Karlsson, 1982).
Both Kv1.1 and Kv1.2 are sensitive to DTX-I, Kv1.2 being somewhat more
sensitive to the toxin (table 1; fig. 7).
As with -DTX, Kv1.4 was insensitive to DTX-I (table 1; fig. 7).
Tityustoxin-K is a peptide toxin isolated from the Brazilian
scorpion Tityus serrulatus. It blocks voltage-gated
potassium currents in some preparations (Blaustein et al.,
1991; Eccles et al., 1994; Rogowski et al., 1994)
and can block Kv1.2 with high affinity (Werkman et al.,
1993). I have also found that tityustoxin-K can inhibit Kv1.2
(IC50 = 105 pM for mKv1.2; IC50 = 550 pM for hKv1.2) and that Kv1.1 and Kv1.4 are insensitive to tityustoxin-K (table 1; figs. 5A and
6).
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-DTX blocking affinity for Kv1.1/Kv1.2 and Kv1.1/Kv1.4
heteromultimers described by single subunit model.
When Kv1.1 and
Kv1.2 were coexpressed in a 1:1 ratio (Kv1.1 fractional expression
0.5), the resulting channels had high sensitivity to -DTX (fig. 3A,
and B). The data from the mixed population of channels were well
described by the single subunit model (fig. 3B). When the fraction of
Kv1.1 subunits was varied in three separate experiments, the
IC50 values for the mixed population of channels were well
fit by the line predicted for the single subunit model. The energy
additivity model provided a poor fit to the data (fig. 3C).
-DTX-insensitive Kv1.4 subunits were
overexpressed relative to Kv1.1 subunits (Kv1.1 fractional expression
0.24). The mixed population of channels displayed an IC50
of 634 pM, and the IC50 values predicted by the single
subunit and energy additivity models for this expression ratio were 1 and 404 nM, respectively.
Tityustoxin-K blocking affinity for Kv1.2/Kv1.1 heteromultimers
described by single subunit model.
When tityustoxin-K-sensitive
Kv1.2 and tityustoxin-K-insensitive Kv1.1 were coexpressed (Kv1.2
fractional expression 0.22), the resulting channels were very sensitive
to tityustoxin-K (fig. 5A). The single subunit model provided the
best fit to the data. In three experiments using different expression
ratios, the IC50 values for the mixed population of
channels were well fit by the line predicted for the single subunit
model and were not well fit by the line predicted for the energy
additivity model (fig. 5B).
Tityustoxin-K blocking affinity for Kv1.2/Kv1.4 heteromultimers
not described by single subunit model.
When
tityustoxin-K-sensitive Kv1.2 and tityustoxin-K-insensitive Kv1.4
were coexpressed (Kv1.2 fractional expression 0.71), the sensitivity of
the resulting channels to tityustoxin-K was much lower than
predicted for the single subunit model and was well fit by the energy
additivity model (fig. 6). The single subunit model predicted that the
sensitivity of the mixed population of channels to tityustoxin-K
should be nearly identical to that for Kv1.2 homomultimers in this
experiment, because I estimated that 71% of the expressed subunits
were toxin-sensitive. The single subunit model predicted an
IC50 of about 800 pM for the mixed channel data, whereas
the energy additivity model predicted an IC50 of about 7 nM. The IC50 for the mixed subunit data was 7 nM.
DTX-I blocking affinity for Kv1.2/Kv1.4 heteromultimers described
by single subunit model.
To determine whether the results with
tityustoxin-K on Kv1.2/Kv1.4 heteromultimers was a general property
of toxin binding to this subunit pair, I performed a similar mixing
experiment but instead used DTX-I. This toxin was chosen because its
blocking affinity for Kv1.2 homomultimers was nearly identical to that of tityustoxin-K (table 1) and, like tityustoxin-K, it displayed low blocking affinity for Kv1.4 (fig. 7).
In contrast to the results with tityustoxin-K, I found that the
blocking affinity for DTX-I on Kv1.2/Kv1.4 heteromultimers was more
closely described by the single subunit model than by the energy
additivity model (fig. 7).
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Sequential application of tityustoxin-K and DTX-I to oocytes
coexpressing Kv1.2/Kv1.4 heteromultimers.
To obtain further
evidence that the blocking affinity of tityustoxin-K and DTX-I for a
mixed population of Kv1.2 and Kv1.4 subunits cannot be described by the
same blocking affinity models, I performed experiments in which each
toxin was applied sequentially at the same concentration to the same
oocyte. Two such experiments are shown in figure
8A and B. Regardless of the order in
which the toxins were applied, DTX-I displayed significantly greater potency against the mixed population of channels than did
tityustoxin-K, in spite of their nearly identical potency against
Kv1.2 homomultimers (table 1). Results from 16 oocytes are shown in
figure 8C. DTX-I (1 nM) blocked about 30% of the potassium current on
average, whereas 1 nM tityustoxin-K blocked about 10% of the
current, and this difference was statistically significant. Figure 8D
shows the toxin-binding isotherms for DTX-I and tityustoxin-K on
Kv1.2 and Kv1.4 and the predicted toxin-binding isotherms for the
single subunit and energy additivity models for the measured fractional expression (Kv1.2: 0.19) in this batch of oocytes. These results predict that a toxin whose blocking affinity is described by the single
subunit model under these conditions should block about 25% of the
current, whereas for the energy additivity model, the toxin should
block about 5% of the current. These results strongly suggest that for
a mixture of Kv1.2 and Kv1.4 potassium channel subunits coexpressed in
oocytes, the blocking affinity of DTX-I can be described by the single
subunit model but that of tityustoxin-K cannot.
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Discussion |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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I have characterized the blocking affinity of three peptide toxins
on three cloned potassium channel subunits either expressed alone as
homomultimers or coexpressed as a mixed population of channels
(homomultimers and heteromultimers) in Xenopus oocytes. For
two subunit combinations (Kv1.1/Kv1.2 and Kv1.1/Kv1.4) -DTX blocking
affinity for the mixed population of channels was well described by the
single subunit model. The blocking affinity of DTX-I for Kv1.2/Kv1.4
subunits was also described by this model. In contrast, the results
with tityustoxin-K were dependent on subunit composition. For
Kv1.1/Kv1.2 subunits, the data with tityustoxin-K were consistent
with the single subunit model, whereas for Kv1.2/Kv1.4 subunits, the
single subunit model did not fit the data.
The methodology and its assumptions.
Other studies addressing
similar questions have used tandem cDNA constructs of potassium channel
subunits to constrain the stoichiometry of the channels (Isacoff
et al., 1990; Liman et al., 1992; Heginbotham and
MacKinnon, 1992; Kavanaugh et al., 1992). Although this
approach has yielded important information, it is difficult to know
that the stoichiometry has been constrained in the manner intended
(McCormack et al., 1992) and that heteromultimeric channels
constructed in this manner are functionally equivalent to channels with
identical stoichiometry that coassemble. I have chosen the mixing
approach of unaltered subunits because the expressed channels more
closely approximate their "native" state. The mixing approach
complements these other methods.
) and against (Taglialatela et al., 1994) the first assumption. If like subunits are more likely to coassemble than unlike ones, then there would be more homomultimers and
3:1 heteromultimers, and fewer 2:2 heteromultimers, than would be
predicted by chance. It might be expected that this type of preferential assembly would yield biphasic concentration-inhibition curves. This was not observed. The converse case, that nonidentical subunits preferentially coassemble, would result in enrichment in
heteromultimers at the expense of homomultimers. This case would
predict that when toxin-insensitive subunits were in greater abundance
than toxin-sensitive ones, the toxin sensitivity of the channels would
be greater than predicted, because there would be fewer
toxin-insensitive homomultimers. I observed this effect (compare fig.
5A), although not consistently. Overall, there were no consistent
features in the data that suggested significant deviations from random
coassembly of subunits. For the second assumption, I have insufficient
information to model all possible heteromultimers with these or any
other models. However, there is evidence that this assumption is not
unreasonable (Naranjo and Miller, 1996).
To check the modeling methods, I used TEA binding to channels composed
of TEA-sensitive Kv1.1 and TEA-insensitive Kv1.2 subunits, which was
described by the energy additivity model in a previous study (Hopkins
et al., 1994b) and when Kv1.1 was coexpressed with TEA-insensitive mutant Kv1.1 subunits in two other studies (Heginbotham and MacKinnon, 1992; Kavanaugh et al., 1992). The data were
well fit by the energy additivity model only (fig. 2, A and B). This suggests that any systematic errors or violations of the modeling assumptions were not large enough to yield misleading results.
Additionally, I obtained a Hill coefficient of 0.95 for the TEA data
for Kv1.1 (table 1), which has been shown to display a Hill coefficient
of about 1 for the rat homolog of this subunit in a previous study
(Christie et al., 1990). This suggests that Hill
coefficients less than 1 obtained for the three toxins were not due to
the specific methods used to obtain the concentration-inhibition data,
because they were the same for both TEA and the toxins.
-DTX and DTX-I blocking affinities described by the single
subunit model.
In mixing experiments with Kv1.1 and Kv1.2
subunits, the blocking affinity of -DTX was best described by the
single subunit model for several expression ratios of the two subunits
(fig. 3). This suggests that heteromultimeric channels composed of
these subunits would display blocking affinities very similar to those of Kv1.1 homomultimers. Similar results were obtained when Kv1.1 and
Kv1.4 were coexpressed (fig. 4).
). When
Kv1.2 and the DTX-I-insensitive mutant Kv1.1 were coexpressed in
oocytes, the DTX-I data for the resulting channels were well fit by the
single subunit model (table 2; Hopkins
et al., 1994b). Therefore, DTX-I blocking affinity for
heteromultimeric channels in which one type of subunit was
toxin-sensitive and the other was not were always best described by the
single subunit model.
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-DTX display blocking affinity consistent with the
single subunit model raises the question of whether this is a general
property of all dendrotoxins. Using tandem tetramers with wild-type and
toxin-insensitive mutant subunits, Tytgat et al. (1995)
found that the blocking affinity of -DTX was best described by the
energy additivity model. Their data suggest that the data presented
here may not be representative of all dendrotoxins.
The model describing blocking affinity of tityustoxin-K is
subunit-specific.
The blocking affinity of tityustoxin-K for
Kv1.1/Kv1.2 channels was well described by the single subunit model,
whereas for Kv1.2/Kv1.4 channels, the data were well fit by the energy
additivity model (figs. 6 and 8). However, I favor the idea that some
other model is more appropriate to describe these data for the
following reasons. The energy additivity model describes the binding of an inhibitor that interacts with four subunits simultaneously. That
tityustoxin-K binding to Kv1.2 can be described by the single subunit model when coexpressed with Kv1.1 suggests that a single Kv1.2
subunit provides an adequate high-affinity binding site and this is
incompatible with the energy additivity model when Kv1.2 is coexpressed
with another subunit (Kv1.4). Another model that could describe these
data would invoke electrostatic repulsion between Kv1.4 subunits and
the toxin. Thus Kv1.4 subunits would not be a neutral influence on the
binding of tityustoxin-K to the heteromultimeric channels that were
also composed of toxin-sensitive Kv1.2 subunits but would instead
contribute electrostatic repulsion. The net result would be decreased
blocking affinity of the toxin for the channel. Tityustoxin-K is a
basic peptide (Rogowski et al., 1994), and Kv1.4 has a basic
residue (lysine) at a position in the pore-forming region that is
occupied by a valine in Kv1.2. If tityustoxin-K binds in the
pore-forming region, then this site is an obvious candidate for an
electrostatic repulsive interaction between the toxin and Kv1.4
subunits. Support for this hypothesis comes from Gross and MacKinnon
(1996), who used lysine-scanning mutagenesis of the agitoxin binding
site of Shaker potassium channels. Many of these lysine point mutations
decreased the affinity of agitoxin for the channel even when it was
present in only one subunit of the tetramer. The quantitative effects
of these mutations were much closer to what would be predicted by the
energy additivity than to what the single subunit model would predict
(compare table 1; figs. 2 and 3 of Gross and MacKinnon). This is
interesting because another nonlysine point mutation in the
pore-forming region of Shaker (D431N), which renders it insensitive to
agitoxin, yields data that are well described by the single subunit
model when this mutant subunit is mixed with wild-type subunits in
Xenopus oocytes (MacKinnon, 1991). Another study has shown
that the binding free energy of a charged ligand to a protein is
influenced predictably by the number of positive or negative charges on
the protein that are remote from the binding site (Gao et
al., 1996). Thus electrostatic interactions between a peptide
toxin with a net positive charge and positive residue(s) on a channel
subunit could decrease the net binding free energy of the toxin to a
channel with toxin-sensitive subunits. That the charge distribution on
the toxin is also important is suggested by the toxin specificity
(tityustoxin-K but not DTX-I) of this effect.
Implications for the use of toxins to characterize potassium
channels in native cells.
Peptide toxins are used to block
potassium currents in neurons and other cells, and in some cases a
degree of selectivity for components of current has been achieved
(Benoit and Dubois, 1986; Halliwell et al., 1986). However,
the molecular identity of the inhibited channels cannot be inferred
from data from cloned channel subunits. First, the subunits that are
constituents of channels under consideration may not have been cloned
and characterized. Second, few if any of the toxins are highly
selective for one subunit (Hopkins et al., 1996). Third, the
data presented here suggest that the subunit composition of the channel
can dramatically affect toxin blocking affinity, even for a toxin whose
blocking affinity can be described by the single subunit model under
some conditions. Finally, other factors peculiar to the cells under study may affect toxin binding independent of the intrinsic affinity of
the toxin for the channels.
; Garcia-Calvo et al., 1993). In the context
of molecular and functional characterization of potassium channels
in situ, a toxin would be most useful when used in
conjunction with other toxins and experimental techniques. Toxins will
continue to be useful in discerning the roles of potassium current
components that shape spike firing patterns and other facets of
cellular physiologic activity (Brew and Forsythe, 1995).
| |
Acknowledgments |
|---|
I thank Drs. Richard Aldrich, George Miljanich and Stephen M. Smith for comments on the manuscript, Dr. Bruce Tempel for mKv1.1 and mKv1.2, Dr. Mark Tanouye for hKv1.2 and hKv1.4 and Beth Sampson for technical assistance.
| |
Footnotes |
|---|
Accepted for publication February 23, 1998.
Received for publication November 5, 1997.
Send reprint requests to: William F. Hopkins, Neurex Corporation, 3760 Haven Ave., Menlo Park, CA 94025.
| |
Abbreviations |
|---|
-DTX, -dendrotoxin;
CFS, calcium-free
solution;
-DTX, -dendrotoxin;
DTX-I, dendrotoxin-I;
FRS, frog
Ringer solution;
IC50, compound concentration inhibiting
50% of potassium current;
ISS, incubation saline solution;
tityustoxin-K, scorpion toxin.
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References |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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, from scorpion venom, blocks voltage-gated, non-inactivating potassium current in cultured central neurons.
Neuropharmacology
33:
1523-1528[Medline].- and 
-subunit polypeptides in rat brain.
J Neurosci
15:
5360-5371[Abstract]. blocks voltage-gated noninactivating K+ channels and unblocks inactivating K+ channels blocked by -dendrotoxin in synaptosomes.
Proc Natl Acad Sci USA
91:
1475-1479-dendrotoxin footprint on a mammalian potassium channel.
J Biol Chem
270:
24776-24781, a structurally novel and highly potent K+ channel peptide toxin, interacts with the -dentrotoxin binding site on the cloned Kv1.2 K+ channel.
Mol Pharmacol
44:
430-436[Abstract].
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