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Volume 17, Number 9,
Issue of May 1, 1997
pp. 3002-3013
Copyright ©1997 Society for Neuroscience
Preferential Interaction of  -Conotoxins with Inactivated
N-type Ca2+ Channels
Jonathan W. Stocker1,
Laszlo Nadasdi2,
Richard W. Aldrich3, and
Richard W. Tsien1
1 Department of Molecular and Cellular
Physiology and 2 Howard Hughes Medical Institute, Stanford
University, Stanford, California 94305 and 3 Neurex
Corporation, Menlo Park, California 94025
 ABSTRACT
- INTRODUCTION
- MATERIALS AND METHODS
- RESULTS
- DISCUSSION
- FOOTNOTES
- REFERENCES
ABSTRACT 
The selective block of N-type Ca2+ channels by
 -conotoxins has been a hallmark of these channels, critical in
delineating their biological roles and molecular characteristics. Here
we report that the -conotoxin-channel interaction depends strongly
on channel gating. N-type channels ( 1B,
2, and  1) expressed in Xenopus oocytes were blocked with a variety of -conotoxins,
including -CTx-GVIA, -CTx-MVIIA, and SNX-331, a derivative of
-CTx-MVIIC. Changes in holding potential (HP) markedly altered the
severity of toxin block and the kinetics of its onset and removal.
Notably, strong hyperpolarization renders -conotoxin block
completely reversible. These effects could be accounted for by a
modulated receptor model, in which toxin dissociation from the
inactivated state is ~60-fold slower than from the resting state.
Because -conotoxins act exclusively outside cells, our results
suggest that voltage-dependent inactivation of Ca2+
channels must be associated with an externally detectable
conformational change.
Key words:
calcium channel;
-conotoxin;
inactivation;
voltage-dependent binding;
modulated receptor hypothesis;
N-type
INTRODUCTION
Specific interactions between peptide neurotoxins
and voltage-gated channels have been valuable in defining the
contributions of various channels to key physiological processes. For
example, the -conotoxins -CTx-GVIA and -CTx-MVIIA potently
block N-type Ca2+ channels (Kasai et al., 1987 ; McCleskey
et al., 1987; Plummer et al., 1989; Regan et al., 1991) and have been
critical for their biochemical isolation (McEnery et al., 1991;
Sakamoto and Campbell, 1991) and delineation of their diverse
functional roles (Nowycky et al., 1985; Hirning et al., 1988; Stanley
and Goping, 1991; Komuro and Rakic, 1992; Wheeler et al., 1994; Dunlap
et al., 1995). -CTx-GVIA is thought to bind to the outer mouth of
the N-type Ca2+ channel (McCleskey et al., 1987; Ellinor et
al., 1994). Because the three-dimensional structures of -CTx-GVIA
and other -conotoxins are known from nuclear magnetic resonance
(Pallaghy et al., 1993; Sevilla et al., 1993; Basus et al., 1995;
Farr-Jones et al., 1995; Nemoto et al., 1995), analysis of the
toxin-channel interaction might provide valuable information about the
outer vestibule of the Ca2+ channel.
As structural probes, peptide toxins have the potential of reporting
dynamic as well as static aspects of channel structure. Modulation of
block by changes in gating is well known for smaller molecules such as
quaternary ammonium compounds (Armstrong, 1969, 1971), local
anesthetics (Hille, 1977; Hondeghem and Katzung, 1977), organic
Ca2+ channel blockers (Lee and Tsien, 1983; Bean, 1984),
and sulfhydryl-reactive reagents (Yang and Horn, 1995; Larsson et al.,
1996; Liu et al., 1996). Peptide toxins offer advantages as probes of
voltage-dependent conformational changes because their effects are
potent and specific and their site of action is unequivocally
extracellular.
Inactivation is an important aspect of Ca2+ channel gating,
critical for governing the amount of Ca2+ entry during
repetitive firing (Carbone and Swandulla, 1990; Pelzer et al., 1990).
Although the kinetics of voltage-dependent inactivation differs
strikingly among Ca2+ channel types, it is not clear
whether Ca2+ channel inactivation involves internal or
external conformational changes or both. One possible internal
mechanism is the rapid occlusion of the open channel by a tethered
intracellular blocking group, as proposed for the N-terminal domain of
K+ channels (Zagotta et al., 1990) or the intracellular
III-IV loop in Na+ channels (Vassilev et al., 1988;
Stühmer et al., 1989; Catterall, 1993). Another possible
mechanism is a conformational change near the external mouth of the
Ca2+ channel pore, analogous to C-type inactivation of
K+ channels, which affects K+ channel
interactions with extracellular Cd2+,
tetraethylammonium+, and sulfhydryl modifiers (Hoshi et
al., 1990; Choi et al., 1991; López-Barneo et al., 1993; Yellen
et al., 1994; Baukrowitz and Yellen, 1995; Liu et al., 1996). Questions
about possible external conformational changes in Ca2+
channels are of further interest because subtype-specific differences in voltage-dependent inactivation kinetics have been traced to residues
in membrane-spanning segment IS6 and nearby extracellular and
cytoplasmic regions (Zhang et al., 1994). Here we explore such issues
by examining the effects of a series of -conotoxins, focusing on the
question of whether their potency is dependent on the gating state of
the N-type Ca2+ channel.
MATERIALS AND METHODS
Preparation of Xenopus oocytes and expression
of channels. Ovarian tissue was removed from anesthetized female
Xenopus laevis. Stage V-VI oocytes free of follicular
cells were obtained after incubation with shaking for 2 hr in a
Ca2+-free solution (82.5 mM NaCl, 2 mM KCl, 1 mM MgCl2, and 5 mM HEPES, pH 7.5) containing 1 mg/ml collagenase A as
described previously (Sather et al., 1993). The oocytes were washed and
transferred to a storage medium (96 mM NaCl, 2 mM KCl, 1.8 mM CaCl2, 1 mM MgCl2, and 5 mM HEPES, pH 7.6, with 2.5 mM sodium pyruvate and the antibiotics gentamycin,
penicillin, and streptomycin).
Selected oocytes were then stored at 18°C for several hours or
overnight before injection. Expression of calcium channels was achieved
by injection of 1b with 2/ and
1a cRNA that had been dissolved in water and mixed in
approximately equimolar ratios. The cRNAs were synthe-sized
in vitro using T7 or SP6 polymerase from corresponding
cDNAs, an 1b construct largely derived from human
hippocampal cDNA (Ellinor et al., 1994), 2/ (courtesy of Prof. T. Tanabe) and 1a (Ruth et al., 1989). Details
about the composition of these subunits are given in the earlier papers cited.
Electrophysiological recordings and toxin application.
Whole-cell Ba2+ currents were recorded using a
two-electrode voltage-clamp amplifier (model OC-725A, Warner
Instruments, Hamden, CT). Current-passing and voltage-measuring
electrodes were filled with 3 M KCl and typically had
resistances in the range of 1-5 M . The potential of the bath was
measured by a chlorided silver wire immersed in a reservoir filled with
3 M KCl connected to the chamber by a 3 M
KCl-agar bridge. A chlorided silver wire placed directly in the
recording chamber was used to measure the amount of current injected by
the current electrode.
Ba2+ current through the expressed Ca2+
channels was induced by stepping the holding potential (HP) to 0 mV for
test pulse with a 50 msec duration. Intrapulse duration was varied
between 5 sec and 1 min depending on the total time of a given
experimental run and the time resolution desired. The current signal
was filtered at 500 Hz (Frequency Devices, Haverhill, MA) and processed
using Axon Basic-based programs (Axon Instruments, Foster City, CA). Leak traces were taken using a  P/4 protocol and subtracted
off-line.
For current measurements, oocytes were placed in a perfusion solution
containing 5 mM Ba(OH)2, 2 mM KOH,
85 mM tetraethylammonium, and 5 mM HEPES, with
pH adjusted to 7.4 with methanesulfonic acid. In addition, all of the
solutions contained 0.1 mg/ml cytochrome c to saturate nonspecific
binding sites. During recording, the oocytes were perfused continuously
at a rate of ~0.5 ml/min. The -conotoxins GVIA (Peninsula
Laboratories), MVIIA (The Peptide Institute), MVIIC (SNX-230), TVIA
(SNX-185), and SNX-331 (Neurex) were all initially dissolved in water
at the stock concentration of 1.0 mM and stored at
20°C. Fresh dilutions of the peptides into the 5 mM
Ba2+ perfusion solution were made immediately before use.
Application of -conotoxins was achieved by complete perfusion of the
recording chamber with solution containing a given peptide.
RESULTS
Voltage-dependent block of N-type calcium channels by
an -conotoxin
During the course of characterizing a series of -conotoxins, we
found evidence that their ability to block N-type Ca2+
channels is strongly voltage-dependent. This was seen most dramatically with SNX-331, the Y13W derivative of the well known -conotoxin -CTx-MVIIC (Fig. 1A). The rapid
onset and recovery from block seen after application and washoff of
SNX-331 made it advantageous for use in this study. The availability of
N-type Ca2+ channels was assessed by monitoring the peak
current elicited by a brief test pulse to 0 mV. The kinetics of the
development and removal of block was strongly affected by the membrane
potential at which the oocyte was maintained (HP). At a strongly
hyperpolarized potential (HP = 120 mV), both the
onset and recovery from block appeared to follow simple exponential
time courses. In contrast, at a less negative potential
(HP = 70 mV), much more complex behavior was seen. In
this case, the onset of block by SNX-331 was strikingly biphasic,
consisting of an initial rapid phase, very much like that observed at
the strongly negative HP, followed by a much slower phase that required
at least 10 min of toxin exposure to approach completion. Likewise, the
removal of blockade after washoff of toxin at 70 mV was also complex.
A small fraction of current recovered rapidly, with a time course
similar to that found at 120 mV, but after completion of this phase,
a large portion of the overall current remained inhibited.
Fig. 1.
Ca2+ channel block by conotoxin is
highly voltage-dependent. A, The blocking
characteristics of SNX-331 (200 nM) applied to N-type
Ca2+ channels expressed in Xenopus oocytes
are shown for two different HPs: 120 mV and 70 mV.
Test currents were evoked by stepping to 0 mV at 20 sec intervals, and
peak current values were plotted as a function of time. Sample traces
of the test current are shown at the times indicated by lower
case letters. Results from this exemplar oocyte are
representative of more than five experiments. B, A
partial recovery from block is observed after application and washout
of SNX-331 (5 µM) at the HP of 70 mV,
similar to that seen in A, but rapid relief of this
block occurs after hyperpolarizing the oocyte to an HP
of 120 mV. A second application of SNX-331 at the HP
of 120 mV gives a block, which after washoff of toxin yields rapid
and complete recovery of current. Illustrative results from this
exemplar oocyte are representative of seven experiments.
[View Larger Version of this Image (23K GIF file)]
The incompleteness of recovery was not caused by rundown or some
irreversible effect of the conotoxin, as illustrated by the protocol in
Figure 1B. After application and removal of SNX-331 (5 µM in this case) at the depolarized potential of 70
mV, a partial rapid recovery of current is observed similar to that in
Figure 1A. After the initial partial recovery, a
change of the HP to 120 mV yielded a dramatic and rapid
recovery from the remaining block. Relief of the block at the
hyperpolarized potential appears complete in that the larger peak
current level obtained at an HP of 120 mV can be accounted
for by the greater availability of channels in the resting (R) state at
that potential (Fig. 3A). As a further test of the voltage
dependence of block, reapplication of SNX-331 yielded a block that
could be rapidly and completely removed after toxin washoff in contrast
to the long-lived blocking behavior observed at the more depolarized
potential in the same oocyte.
Fig. 3.
Correlation between channel inactivation and
blocking characteristics of conotoxin. A, A steady-state
inactivation curve for the N-type Ca2+ channels expressed
in Xenopus oocytes was generated by measuring channel
availability as a function of HP. The holding voltage was stepped from
120 mV to 30 mV, and channels were held at each HP for 5 min before
giving the test pulse. The peak current at HP = 120 mV was normalized to 1 (i.e., 100% of the channels are estimated
to be in the R state), and the data were fit by a Boltzmann
relationship: I(V) = 1/(1 + exp((V V1/2)/k)), where V1/2 = 72.7 mV and k = 7.3 mV. The results shown are the average of five trials ± SD.
B, The blocking characteristics of SNX-331 (200 nM) were monitored at three different HPs. With test
currents being evoked every 10 sec, characteristic onset of and
recovery from block after application and washoff of SNX-331,
respectively, was observed for three different oocytes being held at
the HPs of 120, 100, and 70 mV.
[View Larger Version of this Image (22K GIF file)]
Voltage-dependent effects of -CTx-GVIA and
other conotoxins
This robust voltage-dependent interaction with the N-type calcium
channel was not only seen with SNX-331. A variety of toxins, including
the commonly used GVIA, MVIIA, TVIA, and MVIIC, were tested for their
voltage-dependent interaction with Ca2+ channels.
Application of each of the toxins at an HP of 80 mV gave a
rapid block of current (Fig. 2). After washoff, no or
very little recovery of current was observed for any of the four toxins (Fig. 2); however, after hyperpolarizing the oocyte to an HP
of 120 mV, the rate of recovery showed an immediate and dramatic increase. Although still slow, a nearly complete recovery of block was
observed for three of the four toxins tested after ~1 hr (Fig. 2).
Although the voltage dependence of recovery from block appears most
dramatic with SNX-331 because of its intrinsically rapid rate of block
and unblock, a wide variety of other -conotoxins appear to show some
degree of voltage-dependent interaction with N-type Ca2+
channels.
Fig. 2.
Voltage-dependent relief of block seen for GVIA,
MVIIA, TVIA, and MVIIC. GVIA (A), MVIIA
(B), TVIA (C), and MVIIC
(D) (all at 5 µM) were applied to oocytes
expressing the N-type Ca2+ channels initially held at 80
mV. After 7 min of toxin application, washout at the same HP gave very
little recovery of current. After an initial 7 min of washout at 80
mV, the HP of the oocyte was stepped to 120 mV, which
evoked a more rapid rate of current recovery. After what appeared to be
complete recovery of current, a second application of toxin was given
to ensure that this recovered current was still sensitive to block.
Results illustrate behavior observed in multiple oocytes
(n = 3 for each toxin).
[View Larger Version of this Image (28K GIF file)]
Correlation between degree of channel inactivation and
changes in toxin-blocking characteristics
The influence of membrane depolarization on the degree of block
might be interpreted in a number of ways. One possibility, based on
well described actions of local anesthetics on Na+ channels
and Ca2+ channel blockers on L-type Ca2+
channels, is that the strength of toxin binding depends on the state of
the channel, as in the classic modulated receptor hypothesis (Hille,
1977; Hondeghem and Katzung, 1977). In the simplest version of this
hypothesis (Fig. 3A, inset), toxin
binds more strongly to channels in the inactivated (I) state than to
those in the R state, thus giving rise to a voltage dependence of
block. As a test of this idea, we looked for a relationship between the degree of inactivation and the characteristics of the toxin block. The
voltage dependence of inactivation was determined using a conventional
protocol (Fig. 3A). As the HP was varied between 120 mV and 30 mV, the peak current during a test depolarization to
0 mV gradually diminished, indicating that an increasing proportion of
channels had become inactivated. A slight degree of inactivation was
observed at 90 mV, half inactivation was achieved at approximately 70 mV, and complete inactivation was obtained near 40 mV (Fig. 3A), in line with previous work on cloned N-type
Ca2+ channels (Williams et al., 1992; Fujita et al., 1993;
Ellinor et al., 1994; Bezprozvanny and Tsien, 1995).
The modulated receptor hypothesis in its simplest form predicts that
the characteristics of toxin block should be governed entirely by the
gating properties of the channel. Accordingly, the time course and
extent of block should remain unaltered over a voltage range in which
the gating state is not changed. We tested this by examining properties
of toxin block at potentials at which the occupancy of the R state had
reached saturation: 100 mV and 120 mV (Fig. 3A). As
predicted, the characteristics of onset and removal of toxin block were
indistinguishable at these levels of HP (Fig. 3B). In
contrast, at an HP of 70 mV, in which ~50% of the
channels are in the I state, a dramatic difference was observed in both
the blocking and unblocking characteristics. The same concentration of
SNX-331 caused a significantly greater degree of inhibition, and the
block displayed a biphasic onset and only partial recovery. Thus,
changes in the balance between the R and I states are correlated with
differences in toxin block.
The absence of a significant difference between the blocking behavior
at 100 mV and 120 mV runs counter to a scenario in which the
membrane electric field exerts a direct effect on the local
concentration of toxin near its receptor site. This interpretation seems unlikely in any case, because, if anything, depolarizations should decrease the local concentration of the positively charged -conotoxin, thereby diminishing its blocking effect.
Affinity of SNX-331 for the R state of the channel
As a further test of the modulated receptor scheme, we set out to
characterize the interaction of the toxin with R and I states of the
channel and to determine to what extent these were different. Determination of the affinity of the toxin for the R state was relatively straightforward. As a standard procedure, the membrane potential was held at 120 mV, at which the vast majority of the channels are in the R state, and the blocking effect of SNX-331 was
monitored over a wide range of concentrations (Fig. 4).
After application of the toxin, the amplitude of the peak current
decreased with an exponential time course toward a lower, steady level
(Fig. 4A). The percentage of current remaining at
various toxin concentrations conformed reasonably well to a
dose-response relationship describing one-to-one block (Fig.
4B). Half-block was attained at 180 nM toxin, giving an approximate estimate of the dissociation constant to
the R state (KR). The approach to steady state
was relatively quick at the negative HP. The rate constants
(  1) for toxin block showed a linear dependence on
toxin concentration, as expected for a first-order reaction (Fig.
4C). The slope of this dependence provided an estimate of
the on-rate for toxin block (kon), 4 × 104 M1 · sec1,
and the y-intercept gives an off-rate
(koff) of 2.8 × 102 sec.
Fig. 4.
Simple and rapid blockade of current by SNX-331 at
an HP of 120 mV. A, Onset and degree of
block by a variety of SNX-331 concentrations covering a 3000-fold range
was measured for oocytes held at HP = 120 mV.
Onset of block at each concentration could be fit well by a
single-exponential time course. The steady-state degree of block was
determined from the fit to a single-exponential plus a constant.
B, The amount of current after steady-state block had
been reached was determined as in A, averaged
(n = 5, ±SD), and plotted as a function of
toxin concentration. A good fit to the data was achieved using the
functional form for a one-to-one binding relationship: (1/(1 + ([Tx]/KD))), where
KD = 180 nM. C,
Rate constants for the exponential time course of the onset of block at
different toxin concentrations were determined from the data in
A. The averaged rate constants at four of the
intermediate toxin concentrations were plotted as a function of
concentration (n = 5, ±SD) and were fit well
by the relationship ()1 = kon[Tx] + koff,
where kon = 4.0 × 104
M1 · sec1, and
koff = 2.8 × 102 · sec1.
[View Larger Version of this Image (25K GIF file)]
Affinity of SNX-331 for the I state of the channel
Determination of the toxin's affinity for the I state of the
channel cannot be made as directly because complete inactivation of the
channels leaves no current to be measured. However, it is possible to
characterize the toxin's affinity for channels under less extreme
conditions and extract an estimate of the dissociation constant for the
I state (KI). According to the modulated
receptor hypothesis, the apparent affinity of toxin at any given HP
should be the weighted sum of the affinities for the various states
(Bean et al., 1983). Considering only R and I states for the
moment,
|
(1)
|
where Kapp(V) is
the KD of toxin for channel, and
h (V) is the fraction of available
channels. Because KR and
h(V) were determined
previously, a measurement of Kapp is sufficient to determine KI. We chose to do this with an
HP of 70 mV, where h(V)  0.5.
The potency of the toxin was clearly greater at 70 mV than at
120 mV (Fig. 5A). For example, 1 nM SNX-331 reduced the peak current by at least one-third
at the depolarized potential, whereas it had no detectable effect at
the more hyperpolarized potential (Fig. 4A).
Likewise, inhibition by 10 nM toxin was >60% at 70 mV
and <10% at 120 mV. Figure 5B shows the dose dependence
of the fractional current remaining 20 min after beginning the
application of SNX-331. This duration of toxin exposure allowed block
to come reasonably close to steady state even at the lowest toxin
concentrations but avoided the rundown seen in control experiments over
much longer periods. The percentage of block at various conotoxin
concentrations was roughly fit with a theoretical binding curve,
yielding a value of Kapp(70) of 6 nM. Using Equation 1 and taking h = 0.5 and KR = 180 nM, the
calculated value of KI is ~3 nM.
This represents an ~60-fold increase in affinity relative to the R
state. The estimated change in affinity must be regarded as a lower
limit, because the toxin-channel interaction may fall short of
equilibrium even after 20 min.
Fig. 5.
Increase in potency of blockade of current
observed at depolarized potentials. A, Blockade of
current at an HP of 70 mV was monitored by applying a
variety of SNX-331 concentrations for 20 min. Whereas the onset of
block at 70 mV occurred with a much slower time course than that
observed at an HP = 120 mV, the degree of block
at low dosages was also much greater than that observed at 120 mV,
indicating a dramatic increase in the efficacy of block at the
depolarized potential. B, Averaged values of the percentage of current remaining after a 20 min application of SNX-331
were plotted as a function of toxin concentration
(n = 4, ±SD). The data were fit to the
functional form (1/(1 + ([Tx]/KD))) to
place an upper limit on the estimate of the apparent affinity of
SNX-331 for the channel at an HP of 70 mV.
[View Larger Version of this Image (25K GIF file)]
Varying residency in the open state has no detectable
effect on toxin block
Having found a large difference between R and I states with
respect to the apparent affinity of SNX-331, we went on to consider the
possibility of preferential interactions with the channel's open
state. A series of experiments was conducted to see whether toxin
blocking characteristics might be altered as a result of modifying the
proportion of time the channels spent in the open state (Fig.
6). Test-pulse durations of 25, 50, or 250 msec were used during application of SNX-331 (1 µM), with the cycle
time fixed at 5 sec. Because inactivation was incomplete even after a
250 msec depolarization, this procedure resulted in a large variation
in the fraction of time spent in the open state (Fig. 6,
inset). There was no appreciable difference in the time
course of onset of block or the steady-state degree of block as the
test-pulse duration was lengthened 10-fold. Likewise, these properties
remained unchanged when the frequency of pulses was varied
between once every 30 sec and once every 5 sec (data not shown). The
process of channel inactivation put practical restrictions on the
percentage of the overall duty cycle that could be spent at the test
potential level. Within these limitations, it is clear that differences in toxin affinity for R and open states of the channel could not be
detected under the present experimental conditions.
Fig. 6.
Variation of the total amount of time spent in the
open state has no effect on toxin block. Blockade of current after
application of SNX-331 (1 µM) was monitored while varying
test-pulse duration. The HP during the duration of the
experiment was 120 mV, and all test pulses were to 0 mV. The cycle
time between pulses was kept at a constant 5 sec, and the duration of
pulses was varied between 25, 50, and 250 msec. Each point represents
the average of data from four experiments.
[View Larger Version of this Image (20K GIF file)]
Simulation of voltage-dependent block by a modulated
receptor scheme
A critical test of the applicability of the modulated receptor
hypothesis is to determine whether it can simulate the kinetics of the
toxin-channel interaction in a realistic manner (Fig.
7). We attempted this using the simplest modulated
receptor scheme containing only two gating states, R and I, and the
corresponding toxin-bound states, RT and IT. Our objective was to see
whether this minimal model could predict the major characteristics of block and unblock of -conotoxins at both hyperpolarized
(HP = 120 mV) and depolarized (HP = 70 mV) potentials. Inasmuch as possible, the kinetic parameters of
the scheme were determined experimentally. kon
and koff, the rate constants for toxin binding to and dissociation from the the R state, were based on data obtained at HP = 120 mV (Fig. 4). kon
was extracted from the slope of the relationship between
1 and toxin concentration; koff
was estimated as
kon · KR, where KR was obtained from the observed dose
dependence (Fig. 4B). The equilibrium between R and I
states was described conventionally by the voltage-dependent rate
constants h and h for development of and
recovery from inactivation, respectively. h and
h were constrained to be equal at HP = 70 mV, where h = h/(h + h) = 0.5 (Fig.
3A). At this HP, the rate of equilibration
between R and I states in Xenopus oocytes was found to be
exceedingly slow ( ~ 100 sec) [J. W. Stocker and R. W. Tsien,
unpublished data; also seen in native channels in dissociated neurons
(Jones and Marks, 1989)], and the values of h and
h were adjusted accordingly. At HP = 120 mV, h was set at a value 1000-fold greater than h to describe an equilibrium greatly in favor of the R
state.
Fig. 7.
Simulation of voltage-dependent block by a
modulated receptor scheme. An attempt to model the voltage dependence
of block (A) using the modulated receptor scheme
diagrammed in C yielded the results shown in
B. The data in A showing the voltage
dependence of toxin-blocking characteristics are identical to that
shown in Figure 1A. Good agreement is observed
between a comparison of data (A) with the predictions
made by the model (B). The modulated receptor scheme
used to generate the predicted blocking behavior includes four states
of the channel; resting (R), inactivated (I), resting with toxin bound
(RT), and inactivated with toxin bound
(IT). Appropriate differential equations were
solved numerically with Matlab (fourth and fifth order Runge-Kutta
formulas for integration). The parameters shown in
C were given the following values:
kon = 4 × 104
M1 · sec1,
koff = 8 × 103
sec1, k on = 1.6 × 105 M1 · sec1,
koff = 1.368 × 104
sec1, h(70 mV) = h(70
mV) = 0.01 sec1, h(120 mV) = 2 × 104 sec1, h(120 mV) = 0.2 sec1, h(70 mV) = 4 × 104 sec1,
h(70 mV) = 1.71 × 106
sec1, h(120 mV) = 8 × 103 sec1, and h(120 mV) = 3.42 × 102 sec1. The fractional
populations of the four states of the model are plotted separately in
D for the case in which toxin is applied and washed out
at the depolarized potential of 70 mV. The rapid buildup of a large
population of channels in the inactivated toxin-bound state
(IT) during application of toxin can be easily
seen.
[View Larger Version of this Image (27K GIF file)]
The higher affinity of toxin for the I state was embodied in the
parameters kon and
koff, which were adjusted to allow simulations of onset of block to approximate the experimental data at 70 mV (Fig.
5A). For toxin-bound forms of the channel, the rate
constants for inactivation and recovery from inactivation were
h and h. By the principle of
microscopic reversibility, the ability of the gating state to affect
the toxin interaction must be accompanied by a corresponding influence
of bound toxin on the gating transition. Accordingly, the scaling that
was used to modify the I  IT transition was also applied to the
IT RT reaction, and the steepness of the voltage dependence of
h  h/(h + h) was kept the same as that of
h (Fig. 7, legend). We found that it was
necessary at depolarized potentials to set h and
h to values considerably slower than h
and h to account for the dynamic and steady-state
characteristics of the toxin block.
With parameters assigned in this way, the minimal modulated receptor
scheme generates kinetics of channel block and unblock (Fig.
7B) that agree reasonably well with the experimental
observations at both HPs (Fig. 7A). The simple behavior at
120 mV results from toxin association and dissociation from the R
state only. The more complex kinetics at HP = 70 mV
can be understood in terms of the changing occupancy of the four states
(Fig. 7D). Both resting and inactivated toxin-bound states
(RT and IT) undergo an initial rapid increase in occupancy after toxin
application at the expense of R and I, accounting for the early rapid
decrease in current observed experimentally. During sustained exposure to toxin, channels accumulate in the IT state, causing a continual depletion of channels from all other states, including the R state (Fig. 7D), thereby causing the slow second phase of toxin
block observed experimentally (Fig. 7A). After removal of
toxin, recovery from the IT state occurs only very slowly because the
dissociation of the toxin (off-rate, koff) and
the rate of recovery from inactivation (h) are both
quite slow. A small component of rapid recovery is observed,
corresponding to the small proportion of channels in the RT state that
can quickly make a direct transition to the R state.
Buildup and recovery of slowly recovering fraction are both
strongly voltage-dependent
Until now, we have focused on experiments in which the same
level of HP was maintained during toxin exposure and washout. These
protocols leave open questions about whether the trapping of channels
in a long-lasting nonconducting form depends on the gating state of the
channels during toxin association, toxin dissociation, or both. To
distinguish between these possibilities, we turned to more complex
protocols in which toxin was applied at one level of HP and removed at
a sharply different HP. Figure 8A
illustrated an experiment in which the HP was switched between 80 mV
and 120 mV in various possible combinations in the presence and
absence of applied SNX-331. When toxin application was carried out at the relatively depolarized potential but removed at the strongly negative potential (trial 1), a rapid and complete recovery of current
ensued (compare with recovery after trial 4). Likewise, when toxin was
applied at a hyperpolarized potential followed by washoff at a
depolarized potential (trial 2), the current after washoff returned to
its pretoxin, depolarized HP level (i.e., before trial 1) without
indication of trapping. These results are in sharp contrast to the
prominent trapping observed when toxin application and washoff were
both performed at HP = 80 mV (trial 3).
Fig. 8.
Both buildup and relief of block from the slowly
recovering fraction of channels are strongly voltage-dependent.
A, A protocol was used to investigate the blocking
characteristics of toxin when application and washout occur at
different HPs. First, SNX-331 (1 µM) was applied at an
HP = 80 mV. Washoff of toxin occurred with a
concomitant change in the HP to 120 mV. After recovery of current, SNX-331 was applied again, but now with the oocyte held at
120 mV, and washoff after this application occurred at an
HP of 80 mV. In the last two segments of this
protocol, the same HP is maintained throughout toxin application and
washoff. Toxin application and washoff is first done at an
HP of 80 mV. Full recovery of current is then achieved
by hyperpolarizing the cell to 120 mV, and application and washoff of
toxin at this hyperpolarized potential follows. B, Using
the same modulated receptor scheme and parameters as in Figure 7, a
model of the expected peak current levels during the above given
protocol was generated. C, Plot of the fractional
population of the I state of the channel with toxin-bound
(IT) as predicted by the modulated receptor
scheme.
[View Larger Version of this Image (22K GIF file)]
These experiments provided an additional opportunity for testing the
validity of the modulated receptor hypothesis with regard to conotoxin
block. As shown in Figure 8B, the minimal model
correctly simulates all the essential features of the experimental
results. In the context of the model, strong hyperpolarization is able to alleviate trapping of channels in the IT state because it greatly speeds the recovery from IT RTR. On the other hand, the
application of toxin at a hyperpolarized potential allows only a very
small population of channels to ever reach the IT state, so that little trapping is found even if the washout is performed at the relatively depolarized potential.
DISCUSSION
The discovery of a marked potential dependence of -conotoxin
block of N-type Ca2+ channels, not anticipated from earlier
studies, has important implications. As we discuss below, it reflects
the ability of the -conotoxin to discriminate between resting and
inactivated states of the channel, a feature not previously reported
for a peptide toxin and a Ca2+ channel. The enhancement of
toxin affinity after steady depolarization bears relevance to
biochemical and cell biological experiments in which -conotoxins
serve as labels of N-type Ca2+ channels, and to
physiological studies in which the toxin is used to dissect their
contributions. Finding that a peptide toxin can interact preferentially
with a voltage-gated Ca2+ channel after its inactivation
provides a fresh perspective on the structural changes that accompany
this form of gating.
Importance of voltage-dependent interaction for studies
using -conotoxins
Membrane depolarization strongly affected the interaction
between N-type Ca2+ channels and -conotoxins. Although
this was observed most clearly in the case of SNX-331 because of its
relatively rapid rate of dissociation, a marked voltage dependence was
a general finding for other members of the -conotoxin family,
including -CTx-GVIA, -CTx-MVIIA, -CTx-MVIIC and -CTx-TVIA
(Fig. 2). In all cases, membrane hyperpolarization to strongly negative
HPs sharply accelerated the removal of block after washout of
-conotoxin, allowing a complete recovery. This is a striking result,
because the blocking effects of toxins such as -CTx-GVIA have been
considered largely or completely irreversible on the time scale of
electrophysiological experiments. Variations in voltage protocol may
have contributed to the partial recovery in those few cases in which it
has been reported (Aosaki and Kasai, 1989; Plummer et al., 1989;
Ellinor et al., 1994).
Our results open up new possibilities for experiments using
-conotoxins such as -CTx-GVIA. The ability to remove
-conotoxin inhibition at strongly hyperpolarized potentials could be
very useful in experiments in which reversibility of blockade of N-type channels is important. An equilibration between toxin and channel can
be characterized on the time scale of electrophysiological recordings,
making quantitative studies of structural relationships between mutant
channels and variant toxins possible (cf. Hidalgo and MacKinnon, 1995).
However, our experiments also reveal the need for caution in the
interpretation of experiments that rely on -conotoxins to define
properties of N-type channels or their physiological contributions.
Special care must be taken when comparing experimental results acquired
under conditions in which membrane potential varies or is undetermined.
For example, toxin block in cells with negative resting potentials will
show properties very different from characteristics of toxin binding to
depolarized membrane fragments, thus explaining previous discrepancies
between radioligand binding (picomolar dissociation constants) and
electrophysiology (nanomolar IC50s) (for an analogous case,
see Bean, 1984).
Interpretation of the voltage dependence of blockade
The mechanism of the potential-dependence of -conotoxin block
was of considerable interest. It cannot be explained by a movement of
the toxin's positive charge along the membrane electric field, in
which case hyperpolarization would promote blockade. The relief of
-conotoxin inhibition by hyperpolarization that was observed contrasts with the voltage dependence of -scorpion toxin binding to
neuronal Na+ channels, which greatly increases in affinity
the more negative the membrane potential (Catterall, 1977), and that of
tetrodotoxin and saxitoxin block of skeletal muscle Na+
channels, which is accentuated at negative potentials in proportion to
toxin charge (Satin et al., 1994).
Another scenario for voltage dependence is an electrostatic interaction
between the cationic toxin and the positive charges of the
voltage-sensing machinery. However, the predicted effect would be a
relief of block with increasing depolarization, once again in conflict
with the observed voltage dependence. Thus, blockade of N-type channels
by -conotoxins differs from blockade of P-type Ca2+
channels by -Aga-IVA, which is relieved by very strong
depolarizations, presumably reflecting antagonism between toxin binding
and activation (Mintz et al., 1992; Randall and Tsien, 1995).
Similarly, in Na+ channels, a partially blocking form of
µ-conotoxin can oppose voltage-dependent activation (French et al.,
1996).
Our experiments demonstrated that the characteristics of toxin
block changed in accordance with the degree of inactivation. The
potency of block was greatly enhanced at depolarized HP levels that
promote inactivation (Fig. 5); at the other extreme, over a voltage
range in which inactivation was completely removed, the characteristics
of block showed no detectable voltage dependence (Fig. 3). These
observations led to the proposal that the conotoxin has greater
affinity for inactivated channels than for those at rest.
Numerical simulations were carried out to see whether the voltage and
time dependence of -conotoxin block could be accounted for by a
classical modulated receptor scheme (Figs. 7, 8). We focused mainly on
a four-state kinetic scheme comprised of the normal R and I states and
their toxin-bound counterparts (RT and IT). The omission of an open
state was justified by the observed lack of dependence of
blockade on residency in the open state under our experimental
conditions (Fig. 6). The treatment of inactivation as a one-step
R I reaction is undoubtedly a simplification, given repeated
observations of multiexponential time courses for both channel
inactivation and recovery from inactivation (Bezprozvanny et al., 1995;
see below). The four-state scheme is merely a minimal model, with the
advantage of simplicity.
The rate constants interconnecting the four states were based as
closely as possible on direct experimental observations. Data on the
voltage and time dependence of inactivation were used to model the
equilibrium between R and I states; the observed kinetics of toxin
block at 120 mV, at which inactivation was negligible, was used to
describe the R RT transition. Data obtained at 70 mV provided
constraints on properties of the I IT transition. Parameters were
given values that maintained the microscopic reversibility of the
overall system at any given potential. Within the bounds of these
extensive constraints, the minimal four-state model was able to
simulate the essential features of block and unblock by SNX-331 at both
strongly negative and depolarized HPs (Fig. 7) and at various toxin
concentrations (simulations not shown). In particular, the scheme was
successful in accounting for the observed kinetic behavior at
depolarized potentials the biphasic onset of block and the partial
recovery from block after removal of toxin. Both characteristics were
explained by the trapping of channels in the inactivated, toxin-bound
(IT) state (Fig. 7D). In the simulations, trapping is
relieved at strongly hyperpolarized potentials because of the steep
voltage dependence of h. The overall conclusion of the
modeling is that the voltage dependence of block can be satisfactorily
explained by preferential binding to the I state, within the framework
of the modulated receptor hypothesis.
Implications for the mechanism of
voltage-dependent inactivation
Peptide toxins have been widely used as probes of channel
structure, most extensively in studies of the outer vestibule of K+ channels (Stampe et al., 1994; Aiyar et al., 1995;
Ranganathan et al., 1996). It is particularly interesting to find a
clear case in which a peptide toxin interacts preferentially with a voltage-gated channel in its inactivated state (cf. Liu et al., 1996).
The -conotoxins are large, highly charged molecules effective as
blockers when applied outside cells (McCleskey et al., 1987), but not
when delivered inside (Feldman et al., 1987). Because toxin blockade
can occur only through interaction with an extracellular aspect of the
channel, a preferential interaction with the inactivated channel must
mean that inactivation is accompanied by an externally detectable
conformational change. The locus of this structural change can be
narrowed further if -conotoxin binds near the mouth of the pore, as
suggested by several lines of evidence. All four motifs of the N-type
Ca2+ channel contribute to its interaction with
-CTx-GVIA, but especially motifs I and III, which are believed to
occupy positions diametrically opposite each other across the pore
mouth (Ellinor et al., 1994). These results suggest that the toxin
straddles the external mouth of the pore while engaged in favorable
interactions with residues in extracellular loops near the putative
pore-forming region (e.g., IIIS5) (Ellinor et al., 1994). Furthermore,
increases in external Ba2+ greatly slow -conotoxin
block, consistent with the notion that the toxin comes close to
divalent cations bound at the locus of selectivity (Boland et al.,
1994; McDonough et al., 1996). Taken together, these observations
suggest that the toxin interacts with the channel near the
extracellular pore mouth, suggesting that this region harbors a
conformational change associated with inactivation. This is
reminiscent of the mechanism proposed for C-type inactivation in
K+ channels (Choi et al., 1991; López-Barneo et al.,
1993; Yellen et al., 1994; Baukrowitz and Yellen, 1995, 1996).
An important aim for future experiments is to utilize the favorable
properties of peptide toxins (rigid, well characterized backbone
structure, many derivatives with extensively modified side chains) to
obtain a clearer picture of the structural basis of inactivation.
Residues near S6 segments, adjacent to the pore-forming region, are a
logical place to start because they appear to be important in
controlling the rate of voltage-dependent inactivation in
Ca2+ channels such as doe-1 and
1A (Zhang et al., 1994; Hering et al., 1996). Although
toxin interactions can be most readily studied with the large
collection of natural and synthetic -conotoxins known to affect
N-type Ca2+ channels, any structural insights about
inactivation are likely to apply across the entire family of
voltage-gated Ca2+ channels.
FOOTNOTES
Received Nov. 28, 1996; revised Feb. 18, 1997; accepted Feb. 20, 1997.
This research was supported by grants from National Institutes of
Health (R.W.T., R.W.A.) and a training grant in cardiac electrophysiology (National Institutes of Health) (J.W.S.). R.W.A. is
an investigator of the Howard Hughes Medical Institute. We are grateful
to P. T. Ellinor, W. A. Horne, and T. Tanabe, V. Flockerzi, and F. Hofmann for cDNAs for 1B subunits, 2/
subunits, and 1 subunits, respectively. We thank Ilya
Bezprozvanny and Xiao-Hua Chen for helpful discussions and Rebecca Agin
for skilled technical support.
Correspondence should be addressed to Dr. Richard W. Tsien, Department
of Molecular and Cellular Physiology, Beckman Center, B105A, Stanford,
CA 94305-5426.
Dr. Stocker's present address: ICAgen, Inc., 4222 Emperor Boulevard,
Durham, NC 27703.
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