ABSTRACT
Tityustoxin-Ka (TSTX-Ka) is a 37 amino acid peptide component of
the venom of the scorpion, Tityus serrulatus, and blocks voltage-gated K
current (1) in highly specific channel types. TsTX-Ka is structurally similar to
charybdotoxin, agitoxin-2, and other scorpion toxins that bind to the
extracellular surface of the pore-forming region of Kvi-type (Shaker)
channels. The action of TsTX-Ka was studied on squid Kt channels using
the whole-cell patch clamp technique with dissociated giant fiber lobe (GFL)
neurons and with insect Sf9 cells infected with a baculovirus carrying the
CDNA for the corresponding channel (Brock et al., 1996). Under standard
conditions (12°0, pH 7.6, 20 mM external K*), TsTX-Ka reduces l for a
strong voltage step (+40 mV) in GFL neurons with an estimated Kp of 50
nM. Steady state voltage dependence and activation kinetics of residual ly
are minimally affected in 300 nM TSTX-Ka, but inactivation kinetics are
accelerated. Similar results were obtained in SF9 cells. Block of I, in GFL
neurons by TSTX-ka (300 nM) depends on external pH. Less block occurs
at low pH, with half-block occurring at pH 6.0, approximately the pK, of
histidine (6.02). This amino acid is located in the SgkviA channel (H351) at
a position homologous to F425 in Shaker B, a residue critical to binding of
charybdotoxin (Goldstein, et al., 1994) and agitoxin-2 (Gross & Mackinnon,
1996). These results suggest that H351 in the external mouth of the squid
SqkviA channel is also a determinant of TsTX-ka binding, and that
protonation of the side chain of this residue may decrease toxin sensitivity by
electrostatic repulsion.
INTRODUCTION
Kt channels are remarkably diverse. With more channel types than
any other ion channel studied, the literature has quickly grown confusing with
competing nomenclature and classification schemes (Rudy, 1988). The
ability to separate Kt currents on the basis of pharmacology, kinetics
genetics, and other methods has indicated the presence of many different K
channel types, often within a single cell. The process of identifying and
characterizing individual channel types has been aided by a molecular
biological approach. A single K“ channel gene can be expressed in a
number of model expression systems which lack background Kt channels in
order to record currents produced by a single defined channel type.
However, it remains difficult, if not impossible, to isolate macroscopic currents
of a single K“ channel type from a cell exhibiting many different K“ currents
without more specific pharmacological probes than are presently available
Certain toxins, however, may hold the key to this need for specificity.
Small peptide toxins from the venom of snakes, spiders, and scorpions
have been found to produce block at nanomolar concentrations of highly
specific K“ channel subtypes (both voltage-gated and Ca2t-activated)
(Dreyer, 1990; Strong, 1990). Many scorpion toxins, in particular, have been
found to exhibit substantial structural similarity with one another. In each of
the toxin sequences shown in Figure 1, there are 6 cysteine residues located
at highly conserved positions (Garcia et al., 1994), suggesting that each
peptide may share the same arrangement of disulfide bonds and, therefore
similar tertiary structures. Based on similarities of structure, these toxins may
also share a common mechanism of block. Several scorpion toxins have
been shown to bind to the extracellular surface of the ion conduction pore
and physically occlude ion flow like a cork in a bottle (Anderson et al., 1988:
Gross et al., 1994; Miller, 1988; Mackinnon and Miller, 1988, 1989:
Giangiacomo et al., 1992). Specific residues in a-subunits of Kvl (Shaker-
like) channels that are important for toxin binding have been localized to the
S5-S6 pore-forming region and, in particular, to those sites flanking the outer
mouth of the pore (Hartmann et al., 1991; Heginbotham et al., 1994; Gross et
al., 1994; Mackinnon and Yellen, 1990; Yellen et al., 1991; Yool and
Schwartz, 1991).
Thus far, no scorpion toxin studies have been reported on the delayed
rectifier K“ channel of the squid giant axon or in the parent cell bodies in the
giant fiber lobe (GFL) of the stellate ganglion. One of the goals of this study
was to identify which scorpion toxins exhibit potent block of K* currents in
squid GFL neurons. Öf those tested, only tityustoxin-Ka (TSTX-Ka)
exhibited significant block of GFL Ig. TSTX-Ka is a 37 amino acid peptide
from the venom of the Brazilian scorpion, Tityus serrulatus, previously shown
to block voltage-gated K“ current in the cloned Kv1.2 channel (Werkman et
al., 1993). Its effects were also tested in Sf9 insect cells expressing only the
SgkviA gene CDNA, recently identified and proposed to underlie the main
component of delayed rectifier K* current in GFL neurons and giant axons
(Rosenthal et al, 1996, in press).
MATERIALS AND METHODS
Giant Fiber Lobe Neurons.
Cell bodies were dissociated from the posterior tip of the GFL-stellate
ganglion of Loligo opalescens and maintained in primary culture at 16°0 in L¬
15 medium containing supplemental salts to match the composition of sea
water (Gilly et al., 1990). Cells were normally used within one week of
isolation.
Production of Recombinant Baculovirus.
A recombinant baculovirus carrying the SgkV1A CDNA was created
by Brock et al. (1996). Briefly, the coding region of SgkviA, spliced to the 5
and 3' untranslated regions of the Xenopus B-globin gene, was subcloned
into the baculovirus expression vector pVL1392 (Stratagene, La Jolla, CA).
Recombinant baculovirus was isolated and purified by standard procedures
(Summers and Smith, 1987). Sf9 cells were cotransfected with the SgkviA
expression vector and linearized, wild-type AcMNPV DNA. Occlusion¬
negative plaques were purified and amplified by two rounds of plaque
purification. Viral stocks were prepared from final supernatants, titered, and
stored at 4°
Maintenance and Infection of Sf9 Cells.
Sf9 cells were maintained as suspension cultures in complete
TNM:FH media (TNM:FH media + 10% fetal bovine serum, 10 mgm
streptomycin, 100 units/ml penicillin, and 5 mg/ml fungizone) at 27°0
(Summers and Smith, 1987). For infections, log-phase cells were seeded in
culture dishes at 10' cells/cm2 and allowed to settle for 30-60 minutes. The
media was then removed, and fresh media was added. Infected cells were
maintained at 18°0
Electrophysiology.
Recordings from Sf9 cells at 18°0 and GFL neurons at 12°0 were
made using standard whole-cell patch clamp techniques. All recordings
were made from cells at a holding potential of -80 mV, with linear capacity
and ionic currents subtracted online using a standard P/-4 technique.
Recordings were sampled at 2 kHz or 20 kHz and filtered at 10 kHz. The
recording solutions had the following compositions (in mM): 150K Sf9
Intemal, 20 KCI, 50 KF, 80 K-Glutamate, 10 Lysine-Hepes, 1 EGTA, 1
EDTA, 26.2 Glycine, 85 Sucrose, 4 Mg-ATP, 4.12 TMA-OH, pH 7.0; 20K
Sf9 external, 20 KCI, 168 Nacl, 10 Cacl,, 10 MgCl, 10 MgSO,, 5 Hepes,
1.6 NaÖH, pH 7.2; 150K GFL intemal, 20 KCI, 50 KF, 80 K-Glutamate, 10
Lysine-Hepes, 1 EGTA, 1 EDTA, 381 Glycine, 291 Sucrose, 4 Mg-ATP, 5
TMA-OH, pH 7.0; 20K GEL exteral, 20 KCI, 480 Nacl, 10 Cacl,, 10
MgCl», 10 MgSO,, 5 Hepes, pH 7.66. In addition, 200 nM tetrodotoxin was
used to block Na’ current, and in some cells 0.8 uM CdCI, was used to
block Ca“ current.
Scorpion Toxins.
Tityustoxin-Ka, a-dendrotoxin, and pandinustoxin were kindly provided by
Dr. M. P. Blaustein and D. R. Matteson, Dept. of Physiology, Univ. of
Maryland. Stock solutions of toxin (50-250 uM) in H,O were stored in plastic
microtubes at 4°0. Toxins were diluted in external solution and slowly
perfused (1-2 min for complete solution change) into the recording chamber
after stable control currents were recorded.
RESULTS
Sensitivity of the SgKvi A Channel.
The interaction of scorpion toxins with ion channel receptors is highly
specific to ion channel subtypes (Adams and Olivera, 1994; Eccles et al,
1994; Rogowski et al., 1994). Several scorpion toxins were examined for
their ability to block K“ current in GFL neurons. 250 nM pandinustoxin and
50 nM a-dendrotoxin, known to be potent blockers of voltage-gated K'
current in other receptor types, did not significantly affect K+ current («10%
change in peak l amplitude at +40 mV) in GFL cells. Tityustoxin-Ka,
however, did appreciably block GFL Ig in the nanomolar concentration range.
Tityustoxin-Ka causes potent block of lg in GFL neurons.
When applied to GFL neurons, 300 nM TSTX-Ka blocked 86% of
peak GFL K“ current activated by a strong depolarization to +40mV (Figure
2). A Kp was estimated using the equation:
Ko
fractional block =
300 nM + Kp
Requiring such a relatively high concentration, GFL Ig is considerably less
sensitive to TsTX-Ka than is lg in other cell types. For example, in cloned
Kv1.2 channels expressed in B82 fibroblast cells, half-block occurs at
0.21 nM, 2.4 orders of magnitude less than in SgkviA (Werkman et al,
1993). The time course of toxin effect appeared to be very slow, with half¬
block occurring after 3-4 minutes and maximum block after 33 minutes. A
single exponential (t = 8.9 min) fit these data reasonably well. Reversal
was slow (220 min) and incomplete, but these effects were not studied in
detail. A similar slowly developing effect of toxin at this concentration also
occurs with lg in perfused squid giant axons (Gilly, personal communication).
TSTX-Ka did not significantly affect activation properties (Figure 3).
Residual lg in 300 nM TSTX-Ka could be scaled such that the time course of
activation closely resembled that in control recordings (Figure 3A).
Deactivation kinetics, measured by fitting a single exponential to the tail
current evoked after a prepulse to +40mV, were not substantially altered by
TsTX-Ka (Figure 3B). Similarly, the voltage dependence of l activation was
largely unaffected (Figure 30).
TsTX-Ka did, however, tend to make inactivation faster and more
complete, particularly with increasing toxin exposure time. Ig records taken
during the developing block by TsTX-Ka are scaled to the same peak in
Figure 4 in order to permit comparison of inactivation kinetics. Single
exponential fits of the inactivating phase resulted in the indicated time
constant (t) values. In addition, inactivation of GFL K+ channels was
substantially more complete in TSTX-Ka. Some of this apparent increase in
the degree of completion of inactivation is probably due to small, residual
Ca“ current, even though the experiment in Figure 4 was carried out in the
presence of 0.8 mM CdCl, in the external solution, which blocks most of the
Ca2t current.
TsTX-Ka block in Sf9 cells is similar to that in GFL neurons.
Although maximum block in Sf9 cells expressing only SgkviA
channel CDNA is less than in GFL (45% block vs. 86% in GFL), the kinetics
of TsTX-Ka block is similar, with half block occurring after =2-3 minutes (20
K+ ext., pH 7.2, 18°0) (Figure 5A). Similarly, inactivation was found to be
faster in TsTX-Ka (t= 45 ms vs. 58 ms in control) (Figure 5B), but this effect
was not as strong as in GFL neurons. Degree (i.e. completeness) of
inactivation is not much affected by TSTX-Ka. Since Sf9 cells express only
the SgkviA channel type, the similarity to TSTX-Ka block of GFL L
suggests that its effects are specific to a single channel type in squid GFL
neurons.
Tityustoxin-Ka block of GFL Ig is pH-dependent.
300 nM TSTX-Ka blocked peak Kt current in GFL neurons in a pH¬
dependent manner with 90% block at pH 9.6, 25% block at pH 5.6, and half
block at pH 6.0 (Figure 6). Fractional block values vs. pH were sigmoidal,
similar to a 1:1 binding curve centered at pH 6.0 and scaled to 0.86 in order
to reflect maximum block achieved at this dose. Control experiments were
also performed to assess the effect of pH itself in this range on ly evoked by
test pulses to +40 mV (Figure 7). pH effects in the absence of toxin were
minor and did not greatly alter the peak current (Figure 7A) or activation and
inactivation rates (Figure 7B).
DISCUSSION
300 nM Tityustoxin-Ka, a concentration z6 times greater than its
estimated Kp of 50 nM in GFL neurons, was found to block Kt current in
both GFL neurons and in Sf9 cells expressing the SgkviA channel,
presumably the major component of K“ current in GFL cells (Rosenthal et al.,
1996, in press). However, this concentration is 2.4 orders of magnitude
greater than that needed to block cloned Kv1.2 channels in B82 fibroblast
cells (Werkman et al., 1993). This difference in sensitivity is typical of the
exquisite specificity of scorpion toxins for their receptors.
The pH dependence of TsTX-Ka block of GFL 1g indicates a pH
sensitivity of those residues in the toxin or in the Kt channel closely involved
in binding. Histidine is the only amino acid capable of buffering in the 5.0 to
8.0 pH range (Lehninger, 1970). The amino acid sequence of TSTX-Ka
(Figure 1) contains no histidine residues, therefore the presence of a histidine
residue in a position in the Kt channel important for toxin binding might
mediate pH dependence of TsTX-Ka block in GFL neurons. Mutagenesis
studies have identified specific residues in the Shaker B K“ channel critically
important for binding charybdotoxin, a scorpion toxin similar to TSTX-Ka
(Figure 1) (Goldstein and Miller, 1992; Mackinnon and Miller, 1989;
Mackinnon et al., 1990). One of these residues (F425), when mutated to
glycine, led to a 2000-fold increase in toxin affinity, indicating this residue's
importance in mediating toxin binding (Goldstein et al., 1994).
The amino acid sequence of the Shaker Kt channel is similar to that of
SqkviA in the pore-forming region, except at 3 positions (Rosenthal et al.,
1996, in press). One of these is histidine (H351 in SgkviA) instead of the
F425 of Shaker. It is very plausible that this residue mediates pH
dependence of TsTX-Ka block by the degree to which the amino side chains
are protonated according to the model:
a-subunit - His + Ht
a-subunit - His
(inaccessible to toxin binding)
The sigmoidal curve in Figure 6 is practically identical to the titration curve for
histidine with a pK, of 6.02 (Lehninger, 1970). Therefore, as the proportion of
protonated histidine residues increases with decreasing pH, the amount of
block decreases by the same amount.
Interactions of charged residues with toxin binding at position F425 in
Shaker have been previously identified. Gross and Mackinnon (1996) have
shown that a single molecule of the peptide agitoxin-2, when bound to a
single a-subunit of the Shaker K+ channel, is sufficient to completely block
the channel. If this is also true in the case in the TsTX-Ka and the SgkviA
channel in GFL neurons, protonation of 1, 2, or 3 of the 4 a-subunit H351
residues in a given channel would not necessarily prevent block, since at
least 1 subunit would remain available for toxin binding. It would instead
reduce the probability of toxin binding by reducing the number of available
site
Although other scorpion toxins are characterized as having simple
mechanisms of block (Goldstein et al., 1994), TSTX-Ka exhibits properties of
a more complicated blocking mechanism. TsTX-Ka block in both GFL and
Sf9 preparations is accompanied by faster and more complete inactivation
of the remaining K“ current. Whether TsTX-Ka is selectively sparing
channels with this type of inactivation or whether, allosteric effects act to
change inactivation properties is unclear. Inactivation properties are
extremely sensitive to the species of amino acid at position 449 in Shaker
(threonine) which in SgkviA is a serine. T449 in Shaker, like F425, is critical
to charybdotoxin and agitoxin-2 binding. Another interesting puzzle is the
slow time course of TsTX-Ka block. Further studies are clearly needed to
confirm and further analyze this effect. Also needed is a study using Shaker
Kt channels which lack the histidine residue at position 425 to confirm that
TsTX-Ka block of this channel, if it occurs at all, is not pH dependent.
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Biochem. Pharmacol. 115: 93-136.
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Giangiacomo K. M., Garcia M. L., and McManus O. B. (1992). Mechanism
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receptor of a Shaker Kt channel: peptide and channel residues
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Gross A., Abramson T., and Mackinnon R. (1994). Transfer of the scorpion
toxin receptor to an insensitive potassium channel. Neuron 13: 961¬
966.
Gross A., and Mackinnon R. (1996). Agitoxin footprinting the Shaker
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Brown A.M. (1991). Exchange of conduction pathways between
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Heginbotham L., Lu Z., Abramson T., and Mackinnon R. (1994). Mutations
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receptor site for charybdotoxin, a pore-blocking potassium channel
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Miller C. (1988). Competition for block of a Ca2t-activated K“ channel by
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Tityustoxin Ka blocks voltage-gated noninactivating K“ channels and
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identification of Sgkv1A: a candidate for the delayed rectifier K channel
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FIGURE LEGENDS
Figure 1 Several families of scorpion toxins share substantial structural
similarity. All 9 toxins listed contain 37 - 39 amino acids. 6 cysteine residues
are located in the same position in each toxin, suggesting a common tertiary
structure. X's are spacers for alignment.
Figure 2 300 nM TSTX-Ka (pH 7.6) produces potent block of Kt current in
GFL neurons. (A) Current activated with depolarizing voltage steps from
-80 mV to +40 mV was reduced 86%. A long 250 ms pulse (left) and a
short 25 ms pulse (right) are shown. (B) 300 nM TSTX-Ka blocked K-
current via a prolonged time course. Following control measurements, TSTX¬
Ka was applied (thick horizontal bar) in external solution perfused into the
recording chamber. Washout was slow and incomplete. Points represent
measurements of peak current elicited by a voltage step to +40 mV.
Figure 3 300 nM TSTX-Ka (pH 7.6) does not markedly affect activation or
deactivation kinetics, or voltage dependence of conduction. (A) A 25 ms
voltage step from -80 mV to +40 mV elicited K“ current in toxin which could
be scaled by a factor of 3.2 to match peak control current (right). The
positive-going slope in toxin is largely unaffected and scales well to match
the slope in control recordings. (B) Tail currents were elicited by a
depolarizing prepulse to +40 mV followed by a test pulse between -130 mV
and -50 mV. A single exponential fit to the resulting current gives time
constants (t) which are largely unchanged in toxin. (C) Conduction-voltage
16
properties were ascertained by measuring the change in current evoked
upon repolarization (to -80 mV) from a series of voltage steps to test
potentials between -50 mV and +60 mV and dividing by the voltage change
(dI/dV). All measurements were normalized to the maximum conductance
value under each condition. Conductances are not markedly changed in
toxin, indicating that the voltage dependence of the channels were
unaffected by toxin.
Figure 4 Inactivation is faster and more complete in 300 nM TSTX-Ka (pH
7.6). Kt current recordings are shown after 0, 11, 20, and 40 minutes in
toxin. Current was activated by 250 ms depolarizing voltage steps from
-80 mV to +40 mV. Each sweep is scaled to match the peak current level in
the control recording. Scaling factors are shown in parenthesis. The time
constant (t) decreases from 125 ms in control solution, to 68.9 ms after 40
min in 300 nM TSTX-Ka. In addition to the rate of inactivation, the amount of
current at any time point is less than that in control, indicating that more
current inactivates in toxin.
Figure 5 A delayed time course of block and faster inactivation kinetics are
also seen in Sf9 insect cells expressing SgkviA channels, the main
component of Kt current in GFL neurons. (A) 300 nM TSTX-Ka applied at
time zero blocks K“ current in Sf9 cells to a lesser extent than in GFL
neurons. However, the slow rate of toxin effect seen in GFL cells is also
exhibited in Sf9 cells. The half-time to block is 2-3 min in Sf9 (3-4 min in
GFL). Peak Kt current was elicited using depolarizing voltage steps from
-80 mV to +40 mV. (B) Inactivation is faster in Sf9 cells (t-45 ms ys. 58
ms in control) but not more complete as seen in GFL Ig. Actual current traces
are shown in control solution and in 300 nM TSTX-Ka (scaled by a factor of
1.86 to match peak control current). Kt current was elicited as in (A).
Figure 6 TsTX-Ka block in GFL neurons is pH dependent. The maximum
block attained (30 - 40 min after application) with 300 nM TSTX-Ka at pH
values between 5.6 and 9.6 are shown. At high pH block is maximal; at
low pH block is minimal. A 1:1 binding curve is overlaid, centered at pH 6.0
and scaled by 0.86 to match the maximum block achieved at this dose. The
dotted vertical line indicates the pH at which half of the blockable current is
blocked (pH 6.0). Peak currents in toxin were measured at each pH
following depolarizing 25 ms voltage steps from -80 mV to +40 mV and
expressed as a percentage of control current.
Figure 7 pH effects alone do not account for differential block at varying pH.
(A) Actual current traces from the same cell, elicited by depolarizing voltage
steps (from -80 mV to +40 mV) are shown at pH 5.6, 7.6, and 9.6. Peak
current increases with increasing pH, however the difference between pH 5.6
and pH 7.6 (control) is less than 10%. (B) When peak Kt currents are
scaled to match control, inactivation properties are largely similar at varying
pH.
FIGURES
O

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GOHINO %) INJaano


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GOAINO %) ZONVIORGNOS
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0.6-
0.4-
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FIGURE 5

10
TIME (min)
CONTROL


300 nM TSTX-Ka
(SCALED)

15

5 nA
50 ms
20
c
1.0-
0.6 -
0.4 -
0.2-
0.0


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FIGURE 6
10
.
A
B
(SCALED)
FIGURE 7
pH 9.6
S
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pH 5.6
ktaav-
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pH 5.6
pH 9.6

—
10 nA
50 ms