1Departments of Entomology and Neuroscience, University of California at Riverside, Riverside, California 92521; and 2Department of Botany, Faculty of Life Sciences, Tel-Aviv University, Ramat-Aviv 69978, Tel-Aviv, Israel
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ABSTRACT |
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Lee, Daewoo,
Michael Gurevitz, and
Michael E. Adams.
Modification of Synaptic Transmission and Sodium Channel
Inactivation by the Insect-Selective Scorpion Toxin LqhIT.
J. Neurophysiol. 83: 1181-1187, 2000.
The peptide
Lqh
IT is an
-scorpion toxin that shows significant selectivity
for insect sodium channels over mammalian channels. We examined the
symptoms of Lqh
IT-induced paralysis and its neurophysiological correlates in the house fly (Musca domestica). Injection
of Lqh
IT into fly larvae produced hyperactivity characterized by
continuous, irregular muscle twitching throughout the body. These
symptoms were correlated with elevated excitability in motor units
caused by two physiological effects of the toxin: 1)
increased transmitter release and 2) repetitive action
potentials in motor nerves. Increased transmitter release was evident
as augmentation of neurally evoked synaptic current, and this was
correlated with an increased duration of action potential-associated
current (APAC) in loose patch recordings from nerve terminals.
Repetitive APACs were observed to invade nerve endings. The toxin
produced marked inhibition of sodium current inactivation in fly
central neurons, which can account for increased duration of the APAC
and elevated neurotransmitter release at the neuromuscular junction.
Steady-state inactivation was shifted significantly to more positive
potentials, whereas voltage-dependent activation of the channels was
not affected. The shift in steady-state inactivation provides a
mechanism for inducing repetitive activity in motoneurons. The effects
of Lqh
IT on sodium channel inactivation in motor nerve endings can
account both for increased transmitter release and repetitive activity leading to hyperactivity in affected insects.
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INTRODUCTION |
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Scorpions have evolved a diversity of
peptide toxins comprising an effective biochemical strategy for prey
capture. These toxins modify ion channels in nerve membranes, producing
physiological changes leading to various types of excitatory or flaccid
paralysis. A primary target of scorpion venom toxins is the
voltage-sensitive sodium channel, and an extensive literature is
available on their modification of mammalian (Couraud and Jover
1984; Martin-Eauclaire and Couraud 1995
;
Strichartz et al. 1987
) and insect channels (Zlotkin et al. 1994
).
-Scorpion toxins were some of
the first to be described in detail, both with respect to biochemical
characteristics and neurophysiological actions. To a rather surprising
degree, the
-scorpion toxins are characterized by their specific
modification of vertebrate sodium channels, despite the fact that
scorpions prey primarily on insects. Indeed, the binding site for
-scorpion toxins (site 3) (Catterall et al. 1992
) is
defined exclusively by characteristic epitopes on the mammalian sodium
channel (Rogers et al. 1996
).
A variety of scorpion toxins specific for insect sodium channels
have been identified (Zlotkin et al. 1971,
1994
), but only recently was an
-scorpion toxin-like
peptide with potency against insects discovered. This toxin, Lqh
IT
has high sequence-similarity to
-scorpion toxins and prolongs action
potentials in a manner characteristic of this class of peptides
(Adam et al. 1966
; Eitan et al. 1990
;
Wang and Strichartz 1985
). This effect results from inhibition of sodium channel inactivation (Catterall et al.
1992
; Strichartz et al. 1987
). Although also
active against mammalian sodium channels, Lqh
IT differs from
previously described
-scorpion toxins in that it shows preference
for insect channels (Eitan et al. 1990
; Gordon et
al. 1996
; Gordon and Zlotkin 1993
). Structural correlates of the toxin responsible for insect activity recently have
been defined (Zilberberg et al. 1996
,
1997
).
In some respects, the -scorpion toxins are among the most
well-characterized elements of the scorpion venom cocktail. Many electrophysiological studies have documented their inhibition of
mammalian sodium channel inactivation (Adam et al. 1966
;
Martin-Eauclaire and Couraud 1995
; Wang and
Strichartz 1985
), and the characteristic
-scorpion toxin
binding site (site 3) has been pinpointed through site-directed
mutagenesis and photoaffinity labeling (Rogers et al.
1996
). However, few studies have documented the symptoms
associated with
-scorpion toxin action in the intact animals, and
little has been done to relate these to their effects on synapses. It is interesting to note that
-scorpion toxins do not alter the sodium
channel activation mechanism, nor are they associated with persistent
depolarization of nerve membranes (Catterall 1980
). Nevertheless, Lqh
IT is reported to cause hyperexcitation in treated insects, the natural prey animals of scorpions (Eitan et al.
1990
).
Here we provide a detailed examination of -scorpion toxin
action on an insect model at three levels: behavioral, synaptic, and
sodium channel modification. We find that hyperexcitability produced in
treated fly larvae is likely caused by two consequences of toxin on
nerve terminals: increased transmitter release and repetitive firing of
action potentials. These effects in turn can be attributed to
prolongation of inward sodium current in nerve terminals and a shift of
steady-state sodium channel inactivation to more positive potentials.
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METHODS |
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Scorpion toxin
In most experiments, native LqhIT (MW = 7,258) purified
from the venom of Leiurus quinquestriatus hebraeus was used
(Eitan et al. 1990
). In some experiments (Figs. 4 and
5), the recombinant toxin produced in Escherichia coli was
employed (Zilberberg et al. 1996
).
Bioassay
House flies (Musca domestica; NAIDM strain) were
obtained from laboratory colonies maintained in the Department of
Entomology, University of California, Riverside. For toxicity assays,
third instar house fly larvae were injected with various concentrations of LqhIT dissolved in insect saline through an abdominal
intersegmental membrane. All larvae used in this bioassay weighed ~20 mg.
Intracellular recording
House fly prepupae were dissected, pinned in silicone elastomer
(Sylgard) dishes, and flooded with physiological saline. The saline
formulation is (in mM) 135 NaCl, 5 KCl, 0.75 CaCl2, 1 MgCl2, 5 NaHCO3, and 5 HEPES, adjusted to pH 7.2. Muscles
for recordings were obtained from the longitudinal ventrolateral
muscles 6A and 7A of house fly (Bindokas and Adams 1989;
Irving and Miller 1980
). Excitatory junctional
potentials (EJPs) were evoked by a suction electrode attached to the
cut ends of motor nerves. Isolated voltage pulses at a rate of 0.4 Hz
were generated by a Grass S88 stimulator. EJPs were measured
intracellularly with glass microelectrodes (5-10 M
) filled with 3 M
KCl. Voltages were amplified with an Axoclamp 2A (Axon Instruments)
amplifier, and signals were digitized and processed with Data 6000A
waveform analyzer (Analogic Instruments, Woburn, MA).
Two electrode voltage clamp
Synaptic currents from house fly muscles were evoked by
stimulation of a motor nerve via the suction electrode and recorded with an Axoclamp 2A in two-electrode voltage-clamp mode. Tip resistance of current passing and recording electrodes were around 2 and 5-10
M, respectively. Output signals were filtered with a Warner Instruments LPF-100 low-pass filter at 0.5 kHz.
Loose patch-clamp recordings
Miniature excitatory junctional currents (MEJCs), excitatory
junctional currents (EJCs), and action potential-associated currents (APACs) were measured extracellularly from house fly neuromuscular junctions by patch pipettes that were pulled and fire-polished to
achieve an internal tip diameter of 10-15 µm as previously described
(Bindokas and Adams 1989). The saline formulation for loose patch-clamp recordings is the same as that for intracellular recordings except for the addition of 4 mM MgCl2
to minimize minute muscle movements. Saline-filled pipettes were placed
over neuromuscular junctions, located by manipulation of pipette tips
to sites producing maximum amplitude records after nerve stimulation.
Once a large amplitude synaptic current was obtained, slight downward
force was applied to produce approximate seal resistance of ~0.5
M
. The loose seal permitted relatively rapid access to bath-applied toxins. Preparations were allowed to equilibrate for at least 20 min
after positioning the pipette to minimize the effects of mechanical
disturbance. To monitor APACs at high gain, saline containing 2-5 mM
CoCl2 or CdCl2 (see figure
captions) was perfused to block EJCs. MEJCs, EJCs, and/or APACs were
amplified with an Axopatch 1B (Axon Instruments), and output was
filtered at 1 kHz through a four-pole low-pass Bessel filter and
processed with a Data 6000A. Recordings usually were made in an
AC-coupled, "auto-track" mode, which often caused a small amount of
overshoot in current traces.
Preparation of house fly central neurons
Neurons from adult house fly thoracic and abdominal ganglia were
dissociated and cultured for 1-2 days. Short-term culturing promotes
expression of functional sodium channels in neuronal cell bodies that
are normally inexcitable due to low expression of these channels.
Ganglia were dissected in ice-cold house fly saline containing (in
mg/100 ml) 800 NaCl, 20 KCl, 5 NaH2PO4, 100 NaHCO3, and 100 glucose, pH 7.2 (Wu et al.
1983). Ganglia were treated for 1 h at room temperature
with 1 mg/ml collagenase (Type IA, Sigma). After washing, ganglia were
gently triturated in Schneider's Drosophila medium (GIBCO)
supplemented with 15% fetal bovine serum, 50 mg/ml streptomycin, 50 unit/ml penicillin, and 0.05 mg/ml insulin (O'Dowd and Aldrich
1988
; Wu et al. 1983
). Dissociated neuronal cell
bodies were plated onto poly-D-lysine-coated dishes and
incubated for 1-2 days at room temperature.
Whole cell recordings of neuronal sodium currents
Neuronal sodium currents were recorded in whole cell
configuration (Hamill et al. 1981) using 1-2 M
Sylgard-coated patch pipettes, manufactured from borosilicate glass
(Boralex glass; Dynalab). Pipettes were filled with (in mM) 70 CsF, 70 CsCl, 2 MgCl2, 0.1 CaCl2,
1.1 EGTA, and 10 HEPES (pH 7.2), whereas neurons were bathed in a
recording solution containing (in mM) 140 NaCl, 3 KCl, 4 MgCl2, 2 CaCl2, 1 CoCl2, 20 TEA-Cl, 1 4-aminopyridine (4-AP), and 5 HEPES (pH 7.2). Currents were recorded using an Axopatch 200A amplifier
(Axon Instruments) and filtered at 2 kHz. Neurons were clamped at a
holding potential of
105 mV. Currents were evoked by brief
depolarizing steps to test potentials
(VT). Eighty-five percent of series
resistance was compensated, and leak currents were on-line subtracted
by using a P/4 procedure (Bezanilla and Armstrong 1977
).
Data were compensated for liquid junctional potentials of +5 mV on
average. Cell stimulation and data acquisition were performed using
pCLAMP 5.5.1 software (Axon Instruments) in Dell 466/MX personal computer.
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RESULTS |
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LqhIT induces an excitatory paralysis
Injection of house fly larvae with microgram doses of LqhIT
caused hyperactivity leading to paralysis and death. Larvae (~20 mg
body wt) injected with 1.5 µg Lqh
IT exhibited moderate sporadic contractions; local twitching and rolling of the body occurred within
1-2 min of injection. Within 10 min intense hyperactivity, manifested
as uncoordinated and continuous muscle twitches throughout the insect
body, was observed. In the dose range 0.15-1.5 µg/larva, hyperactivity persisted for several days until death occurred. In
contrast, larvae injected with an equivalent volume (1 µl) of
distilled water (n = 3) or physiological saline
(n = 7) showed none of the symptoms mentioned above.
Augmentation of synaptic responses and repetitive activity caused
by LqhIT
We observed the effects of LqhIT on neuromuscular responses
after bath application of toxin to the larval musculature in vitro.
Body wall muscles of house fly prepupae typically respond to nerve
stimulation with a single EJP, but do not contract (Fig. 1, dotted lines). At the lowest
concentrations of Lqh
IT (300 pM), evoked synaptic responses
increased in size (Fig. 1A), and visual inspection revealed
appearance of a twitch contraction in the postsynaptic cell. A second
effect that emerged at higher toxin concentrations (1.5 nM) was
repetitive synaptic responses to individual stimuli (Fig.
1B). This resulted in even larger twitch contractions. This
repetitive activity in response to toxin exposure only occurred after
nerve stimulation; spontaneous repetitive activity in the absence of
applied stimuli was not observed in any of the experiments conducted
(n = 11). Both the increased amplitude of synaptic
responses and repetitive activity resulting from Lqh
IT exposure are
indicative of increased excitability in house fly motor units and
provide a physiological basis for the symptoms observed in vivo.
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LqhIT prolongs the duration of the presynaptic action
potential
We investigated further the physiological basis of increased
excitability by measuring synaptic currents. Using two electrode voltage clamp, we measured evoked synaptic currents in muscles 6A and
7A (Fig. 2A, trace a). As
shown in Fig. 2A, trace b, application of 5 nM LqhIT
caused a significant increase in the amplitude of the evoked EJC after
5 min of exposure. After 10 min., the EJC was increased by threefold,
and repetitive responses were registered (Fig. 2A, trace c).
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Because -scorpion toxins are known to affect neuronal sodium
channels, we hypothesized that the increase in EJC amplitude resulted
from a presynaptic action of Lqh
IT, probably through elevated
transmitter release. However, augmented EJC amplitudes also could
result from increased sensitivity of the postsynaptic cell to released
neurotransmitter, a postsynaptic effect. The latter possibility would
be reflected as larger amplitudes of spontaneous MEJCs after toxin
exposure. In loose patch-clamp recordings from junctional areas under
control conditions, we observed MEJCs, the amplitudes of which remained
unchanged after exposure to the toxin (Fig. 2B). We averaged
a total population of 224 MEJCs accumulated from 4 independent
experiments before and after application of 2.5 nM Lqh
IT and found
that the amplitudes remained unchanged. These data indicate that
postsynaptic membrane sensitivity to released transmitter was unaltered
by the toxin. We also observed no significant difference in the
frequency of spontaneous MEJCs before and after toxin exposure (Fig.
2C).
We next analyzed the effect of LqhIT on nerve terminal action
potentials. Simultaneous measurements of presynaptic currents and
postsynaptic potentials are shown in Fig.
3. Under normal conditions, nerve
stimulation led to single EJCs and EJPs. Preceding each EJC was a small
current that appeared in all-or-none fashion as stimulus intensity was
increased. This current, which we refer to as APAC, appeared abruptly
at the same stimulus intensity as the EJC and EJP. After application of
Lqh
IT (10 nM), two obvious changes in synaptic responses were
observed: increased EJC and EJP amplitude, and repetitive activity.
Notably, each EJC was preceded by an APAC, suggesting that repetitive
postsynaptic events originated presynaptically.
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To confirm that the APAC indeed represented the presynaptic action potential, we first blocked the EJC by application of 2 mM cadmium (Fig. 4A). No effect on the APAC was evident. Subsequent addition of tetrodotoxin (2 µM) to the bath abolished the APAC, confirming its dependence on voltage-activated sodium channels. We conclude from this experiment that the APAC represents the presynaptic action potential as it invades the nerve terminal.
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Exposure of the preparation to LqhIT (20 nM) led within 1 min to
repetitive EJCs, each preceded by an APAC (Fig.
5, A-C). After addition of
cadmium to the bath, repetitive APACs could be observed in the absence
of EJCs. Again, APACs were abolished after TTX application (Fig.
5D). Under these conditions, we noted a significant increase
in duration of the APAC. Further analysis of the APAC at high gain
showed that Lqh
IT increased APAC duration by more than twofold
(n = 5; Fig. 6). In some
experiments, variability in the amplitude of the APAC was observed,
probably due to slight changes in the position of the loose patch
pipette caused by minute muscle movements. For this reason, APACs often
were normalized for comparison. These results indicate that action
potentials invading nerve terminals are prolonged after exposure to
Lqh
IT. To verify that these changes originate at the level of the
sodium channel, we recorded whole cell currents from dissociated
central neurons.
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Slowing of sodium channel inactivation by LqhIT
We recorded voltage-dependent sodium currents from dissociated
central neurons (20-30 µm diam) of M. domestica using
whole cell patch-clamp recordings. Because recordings from freshly
dissociated fly neurons produced only minute sodium currents, a
protocol was developed for short-term cultured central neurons
dissociated from thoracic and abdominal ganglia (Fig.
7A). Robust, voltage-dependent sodium currents were measured in cultured neurons within 1-2 days (Fig. 7B). Currents activated in the range of 30 and
40
mV and inactivated rapidly. Peak currents occurred at around
5 mV
(Fig. 8, A and D).
All currents were uniformly sensitive to TTX, and we observed no
evidence of biophysical or pharmacological heterogeneity in any of the
neurons sampled.
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LqhIT (20 nM) produced a large increase in steady-state sodium
current without changing the rising phase of the current (Figs. 7C and 8B). Higher concentrations (40 nM) of
Lqh
IT produced no further change (n = 3), indicating
that the effect of the toxin at 20 nM was complete within 1 min of
application (Fig. 7D). All neurons sampled had TTX-sensitive
sodium currents, confirmed by application of this toxin at the
conclusion of the experiment. Voltage-dependent activation was
unaffected, but peak sodium currents were slightly augmented (Figs.
8D and 9A). Test
potentials to activate 50% of sodium channels, were
19.6 ± 0.2 mV before and 20.8 ± 0.6 mV after application of the toxin,
respectively. No changes in the reversal potential were evident.
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Modification of sodium channel inactivation by LqhIT was
particularly obvious in comparisons of steady-state inactivation (Fig.
9B). For these curves, a double-pulse protocol was employed, consisting of 100-ms prepulses to various voltages followed by the test
potential (
10 mV; Fig. 9B). Peak sodium currents
evoked by the test potential were normalized to the maximum peak
current and plotted as a function of prepulse potentials. The h
curve was fitted by the Boltzman equation: h
= ((1
C)/{1 + exp[(VP
V1/2)/k]}) + C, where V1/2 is the voltage
at half-inactivation, VP and
k are the prepulse potential and the slope factor,
respectively, and C is the noninactivated fraction.
Lqh
IT (20 nM) shifted the h
curve to more positive potentials and
produced a small percentage of steady-state current (~10%) that
remained activated (Fig. 9B). We note in particular
that, under normal conditions, a
20-mV prepulse inactivated virtually
all available sodium channels in the absence of the toxin. After
Lqh
IT treatment, ~40% of the current remained at this prepulse potential.
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DISCUSSION |
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We have shown that the behavioral effects of LqhIT in the house
fly are associated with modification of neuromuscular transmission. The
early effects of Lqh
IT are characterized by local twitching of the
body wall and rolling of the whole insect. Subsequently, the body wall
musculature throughout the insect becomes progressively more
hyperactive, culminating in lethality. Our observations indicate that
these symptoms can be explained by increased excitability of efferent
motor units through two main mechanisms: 1) elevation of
evoked neurotransmitter release and 2) repetitive firing of motor axons.
Elevated levels of transmitter release, evidenced here as increases in
EJP and EJC amplitudes, are likely the result of a prolonged action
potential duration in motor neurons. Evidence for this comes from the
increased durations of action potential-associated current invading
motor nerve terminals. Sustained depolarization of the nerve terminal
allows for increased Ca2+ influx and hence
increased neurotransmitter release (Katz and Miledi
1967). The progressive increase in transmitter release caused
by the toxin accounts for the gradual increases in twitch contractions
observed in affected animals.
Repetitive activity caused by LqhIT was evident both presynaptically
in nerve terminals (APAC, EJCs) and postsynaptically in body wall
muscles (EJPs). This repetitive activity originates in the nerve
terminal because it persists in the presence of extracellular cobalt,
which abolishes postsynaptic responses through blockade of transmitter
release. Furthermore, each postsynaptic event was found to be preceded
by an action potential-associated current. Repetitive motor-unit
activity combined with the appearance of muscle action potentials lead
to intense and prolonged muscle contraction observed at the behavioral level.
The neuromuscular effects of LqhIT can be understood at the level of
the sodium channel as a modification of the sodium channel inactivation
process. We found that steady-state inactivation in house fly neurons
is increased after exposure to 20 nM Lqh
IT. Furthermore, the
steady-state inactivation curve is shifted markedly to more positive
potentials and is incomplete even at test potentials greater than or
equal to +20 mV. Similar concentrations of the toxin also inhibit
inactivation of sodium channels in cockroach giant axons, mammalian
muscle fibers (Eitan et al. 1990
), and mammalian
peripheral nerves (Martin-Eauclaire and Couraud 1995
; Strichartz et al. 1987
; Wang and Strichartz
1982
, 1985
). Such effects of the toxin are not
observed in insect muscle, due to the absence of voltage-activated
sodium current. Instead, calcium channels are responsible for
excitatory electrogenesis in insect muscle.
We have shown that, under normal conditions, a significant portion of
the h curve for housefly central neurons lies to the left of the
m
curve. However, after toxin exposure, the steady-state inactivation curve shifted to the right of the m
curve, providing a
plausible explanation for the appearance of repetitive firing in house
fly motoneurons. The consequences of decreased steady-state sodium
channel inactivation include changes in threshold and tendency to fire
repetitively (Vallbo 1964
). We have shown that the
specific effects of Lqh
IT on house fly motor units include
inhibition of sodium channel inactivation, prolonged action potential
duration, and increased transmitter release. These observations provide a physiological basis for the in vivo effects of Lqh
IT.
Although LqhIT cannot be characterized as an "insect-specific"
neurotoxin, it is reported to be more selective for insects (Eitan et al. 1990
). These workers cited a 20-fold
higher toxicity to insects over mammals. Data presented here show that
20 nM Lqh
IT produces maximal inhibition of sodium channel
inactivation in house fly neurons. Much higher concentrations (>200
nM) are needed to elicit similar effects on sodium channels in rat
dorsal root ganglion neurons (Norris et al. 1995
;
Zilberberg et al. 1996
). In combination, these data
provide a molecular basis for the selective toxicity of Lqh
IT in
insects versus mammals.
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ACKNOWLEDGMENTS |
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We thank Dr. David N. Mbungu for valuable assistance during the early stages of this project.
This work was supported by Binational Agricultural Research and Development Fund Grants IS-1982-91 and IS-2486-94C.
Present address of D. Lee: Dept. of Anatomy and Neurobiology, University of California, Irvine, CA 92697.
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FOOTNOTES |
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Address for reprint requests: M. E. Adams, Dept. of Neuroscience, 5419 Boyce Hall, University of California, Riverside, CA 92521.
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.
Received 4 August 1999; accepted in final form 1 November 1999.
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REFERENCES |
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