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The Journal of Neuroscience, October 1, 2000, 20(19):7174-7182
Low-Affinity Blockade of Neuronal N-Type Ca Channels by the
Spider Toxin  -Agatoxin-IVA
Serguei S.
Sidach and
Isabelle M.
Mintz
Department of Pharmacology and Experimental Therapeutics, Boston
University Medical Center, Boston, Massachusetts 02118
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ABSTRACT |
The recognition of neuronal Ca channel diversity has led to
considerable efforts to identify useful classification criteria. Here,
we revisit the pharmacological definition of P- and Q-type Ca channels,
which is based on their respective high and low sensitivity to the
spider  -agatoxin-IVA (-Aga-IVA), using whole-cell recordings of
the Ca channel currents carried by 5 mM
Ba2+ in isolated rat subthalamic and sympathetic
neurons. In subthalamic neurons, -Aga-IVA (1 µM)
targeted multiple Ca channels. One population was blocked with high
potency. These channels carried 50.4 ± 3.4% (n = 5) of the control current and showed the same
inactivation kinetics and voltage-dependent high affinity for
-Aga-IVA as do prototypic P-type Ca channels. Other Ca channels were
targeted with weaker potency. This heterogeneous population contributed to 14.0 ± 1.7% (n = 5) of the control
current. It included N-type Ca channels as well as high-threshold Ca
channels that displayed the pharmacological signature of Q-type Ca
channels but resembled P-type Ca channels in their gating properties.
N-type Ca current block by -Aga-IVA (1 µM) was further
investigated in sympathetic neurons, which mainly express this Ca
channel type. Block was incomplete (~30% of the control current).
Its relief at positive potentials was consistent with -Aga-IVA
acting as a channel-gating modifier. These effects did not reflect a
complete loss of selectivity, because -Aga-IVA (1 µM)
had no effect on subthalamic Na and K currents or their T- and L-type
Ca currents. Our data confirm that -Aga-IVA is a selective P-type Ca
channel blocker. However, its diminished selectivity in the micromolar
range limits its usefulness for functional studies of Q-type Ca channels.
Key words:
spider toxin; -agatoxin; subthalamic neuron; sympathetic neuron; Purkinje neuron; N-type Ca channel; Q-type Ca
channel; P-type Ca channel
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INTRODUCTION |
Venoms of invertebrate and lower
vertebrate species are rich in toxins designed to produce rapid
immobilization of their victims (Olivera et al., 1985 ). Although
mammals are rarely the targeted prey, many venom toxins are
surprisingly potent blockers of ion channels found in mammalian CNS,
and those with the greatest selectivity are now widely used (Catterall,
1980; Rehm and Tempel, 1991; Adams et al., 1993).
Venom toxins are specially useful for studies of high-threshold
voltage-gated Ca channels whose identification mainly depends on
pharmacological criteria (McCleskey et al., 1987; Llinas et al., 1989;
Mintz et al., 1992; Sather et al., 1993; Newcomb et al., 1998). In
mammalian central neurons, high-threshold L-, N-, and P-type Ca
channels share the same electrophysiological signature at the single
channel (Plummer et al., 1989; Usowicz et al., 1992; Elmslie, 1997;
Dove et al., 1998) and macroscopic levels (Regan et al., 1991; Lorenzon
and Foehring, 1995). Still, they can be distinguished by their
respective sensitivity to dihydropyridines (DHPs) (Nowycky et al.,
1985; Fox et al., 1987; Cox and Dunlap, 1992), the snail toxin
-conotoxin GVIA (-CgTX) (Cox and Dunlap, 1992; Boland et al.,
1994), and the spider toxin -Agatoxin-IVA (-Aga-IVA) (Mintz et
al., 1992; Brown et al., 1994).
Pharmacological studies in expression systems have confirmed that DHPs,
-CgTX, and -Aga-IVA target distinct Ca channels (Dunlap et al.,
1995; Tsien et al., 1995). The match between structural identity and
pharmacology is now well established for L- and N-type Ca channels.
Class C and D genes encode the  1 subunit (1C and 1D) of the
dihydropyridine-sensitive L-type Ca channels (Mikami et al., 1989;
Williams et al., 1992b), whereas class B (or BIII) genes encode that
(1B) of the -CgTX-sensitive N-type Ca channels (Williams et al.,
1992a; Fujita et al., 1993). However, the diversity of
-Aga-IVA-sensitive Ca channel currents seen in mammalian neurons has
made it difficult to establish the precise relationship between the
class A gene products and their native counterparts. Both P-type Ca
channels, which are potently blocked by the toxin
(Kd, ~1 nM)
(Mintz et al., 1992), and Q-type Ca channels, which show weaker
sensitivity to the toxin (Randall and Tsien, 1995), have been related
to the 1A gene family (Mori et al., 1991; Sather et al., 1993;
Niidome et al., 1994; Berrow et al., 1996). Recent findings reconcile
these conflicting reports by suggesting that P- and Q-type Ca channels
are homologous. Their distinct phenotypes may reflect the differential
splicing of the 1A subunit (Bourinet et al., 1999) or its
association with different  subunits (Stea et al., 1994; Moreno et
al., 1997; Mermelstein et al., 1999).
The prototypic P- and Q-type Ca channel currents have been described in
cerebellar Purkinje neurons (Regan, 1991; Usowicz et al., 1992) and in
cultured cerebellar granule cells (Forti et al., 1994; Randall and
Tsien, 1995; Tottene et al., 1996). Because differences in toxin
sensitivity are often difficult to quantify experimentally (measures of
the steady-state current block by low toxin concentrations may be
confounded by current rundown, toxin nonspecific binding and, in brain
slice studies, poor access to the tissue), subtle differences in gating
have been used as a criterion to differentiate these two channel
populations. In cerebellar granule cells, Q-type Ca currents are
distinguished from the P-type Ca currents by their fast inactivation
kinetics (Randall and Tsien, 1995).
To further characterize the -Aga-IVA-sensitive Ca channels, we have
recorded voltage-gated Ca channel currents in freshly isolated rat
subthalamic neurons. These neurons constitute a homogeneous population
of extrinsic glutamatergic neurons (Iribe et al., 1999), with little or
no contamination from interneurons (Hammond and Yelnik, 1983). They are
robust and easily dissected from brain slices. In addition, they
display a complex repertoire of voltage-gated Ca channels, all of which
made them well suited for this study. Recently, they have attracted
special attention after reports that their hyperactivity contributes to
the symptoms of Parkinson's disease (Bergman et al., 1990).
We found that nanomolar concentrations (50-100 nM) of the
toxin -Aga-IVA target subthalamic Ca channels that are remarkably similar to P-type Ca channels in cerebellar Purkinje and granule neurons (Tottene et al., 1996). In contrast, high toxin concentrations affect a heterogeneous channel population: -Aga-IVA (1 µM) blocked -CgTX-sensitive N-type Ca channels and
high-threshold Ca channels that display some but not all the
characteristics of cerebellar granule cell Q-type Ca channels.
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MATERIALS AND METHODS |
Enzymatic dissociation of subthalamic, Purkinje, and
sympathetic neurons. Subthalamic neurons were freshly dissociated
with enzyme (Mintz et al., 1992). Briefly, 400-µm-thick coronal
slices were cut, in ice-cold Ringer's solution, from brains of 9- to 13-d-old rats. The subthalamic nucleus was immediately dissected out
under 400× magnification. It was then incubated for 7 min in a
solution maintained at 35-36°C, which contained (in mM): 81.4 Na2S04, 30 K2SO4, 5.8 MgCl2, 10 Na-HEPES, 20.4 glucose, and 0.5%
phenol red, pH 7.4 with NaOH, plus 3 mg/ml protease type XXIII. All
chemicals were purchased from Sigma (St. Louis, MO) unless mentioned
otherwise. After the incubation with enzyme, the brain tissue was
rinsed in a minimum essential medium (MEM) solution (Life Technologies,
Grand Island, NY; reference 11090-073) which contained Earle's salts
with 10 mM Na-HEPES, 15 mM glucose, 1 mg/ml
bovine serum albumin, and 1 mg/ml trypsin inhibitor (36°C, pH 7.4 with NaOH). The Earle's salt solution had no glutamine. Cells were
dissociated in the same MEM solution by gentle trituration through a
fire-polished glass pipette. This protocol yields a homogeneous
population of neurons that retain enough of their primary dendrites for
the typical morphology of subthalamic neurons to be recognized (Hammond
and Yelnick, 1983).
Purkinje neurons were isolated form the cerebellar vermis of 9- to
11-d-old rats. The cerebellum was dissected out in ice-cold Ringer's
solution. The vermis was divided into three or four pieces (~1
mm3), which were incubated for 8 min in
the enzyme-containing solution described above. The subsequent steps in
the procedure were then identical to those followed to prepare
subthalamic neurons (see above).
Sympathetic neurons were prepared from 9- to 13-d-old rats (Boland et
al., 1994; McDonough et al., 1997a). The ganglia were dissected out in
ice-cold oxygenated Leibovitz's L-15 medium solution (Life
Technologies; reference 11415-064). Each ganglion was cut into two or
three pieces before being incubated for 20 min, at 35°C, in a
calcium-free Tyrode's solution that contained (in mM): 150 NaCl, 4 KCl, 2 MgCl2, 10 glucose, and 10 Na-HEPES, plus 25 U/ml papain (Worthington Biochemicals, Lakewood, NJ),
0.5 mM EDTA, and 2 mM L-cysteine,
pH 7.4 with NaOH. After this first incubation, the ganglia were
transferred into a calcium-free Tyrode's solution containing 2 mg/ml
collagenase (type I) and 8 mg/ml dispase (Boehringer Mannheim,
Indianapolis, IN). This incubation was performed at 35°C for 40 min.
The ganglia were then rinsed, and the cells were released in the MEM
solution, which contained 10 mM Na-HEPES, 15 mM
glucose, 1 mg/ml bovine serum albumin, and 1 mg/ml trypsin inhibitor
(36°C, pH 7.4 with NaOH).
Isolated neurons were stored at room temperature (18-22°C) and
remained viable for 5-8 hr.
Voltage-clamp recordings. Patch-clamp recordings of Ca
channel currents were performed in the whole-cell configuration using Ba2+ ions (5 mM) as the charge
carrier. Patch pipettes (~2 M ) were pulled from borosilicate glass
capillaries (Fisher Scientific, Suwane, GA), coated with Sylgard, and
fire-polished. In all the recordings used in this report, the
capacitance and the access series resistance were compensated to
minimize the voltage errors to <5 mV. Three to 10 G seals were
routinely obtained, allowing the data to be presented without leak
correction. Measurements of current block by the different toxins were
performed without correcting for current rundown. In most experiments,
at least 10 min elapsed between the onset of the whole-cell recording
and the beginning of the experiment to allow the initial current
rundown to stabilize. After stabilization, current rundown was
typically <10% over the complete course of the experiment (usually
1-2 hr).
Whole-cell currents were recorded using an Axopatch 200B amplifier
(Axon Instruments, Foster City, CA). Voltage step commands and data
acquisition were controlled using the XOP Pulse Control (Herrington and
Bookman, 1994; http://chroma.med.miami.edu/cap) in IGOR (WaveMetrics,
Lake Oswego, OR) and an ITC16 analog-to-digital converter (Instrutech
Corp., Great Neck, NY). The currents were digitized every 100 µsec
and filtered at 2 kHz. All potentials were corrected for a liquid
junction potential of  10 mV between the internal recording solution
and the Tyrode's solution in which the pipette current was zeroed
before establishing the seal.
Data analysis and illustration were performed with IGOR. Statistics are
given as mean ± SEM.
Solutions. The solution in the recording patch pipette
contained (in mM): 108 cesium methanesulfonate, 4 MgCl2, 9 EGTA, 9 HEPES (acid), 4 Mg-ATP, 0.3 mM GTP (Tris salt), and 14 creatine phosphate (Tris salt),
pH 7.4 with CsOH. For studies of K channel currents,
Cs+ was replaced with
K+.
The extracellular solution contained (in mM): 160 tetraethylammonium (TEA)-Cl, 5 BaCl2, 10 HEPES, and 0.1 EGTA, pH 7.4 with TEAOH, and 1 mg/ml cytochrome
c. For studies of Na and K channel currents, the
extracellular solution was regular Tyrode's solution, which contained
(in mM): 150 NaCl, 4 KCl, 2 CaCl2, 2 MgCl2, and 10 glucose, pH 7.4 with NaOH, as well as 1 mg/ml cytochrome
c.
Stock solutions of (100 µM) nimodipine [Research
Biochemicals International (RBI); Sigma] and Bay K 8644 (RBI) were
prepared in DMSO and stored in the dark. A 0.5 mM solution
of -CgTX and a 100 µM stock solution of -Aga-IVA
(Peptide Institute, Osaka, Japan; or free samples kindly provided by
Pfizer Inc., Groton, CT) were prepared in distilled water. The stock
solution with -Aga-IVA was aliquoted in 10 µl samples to minimize
the number of thawing and freezing cycles for a single vial. These
aliquots were stored at 80°C. The peptide -Aga-IVA obtained from
the Peptide Institute or Pfizer showed identical potency and selectivity.
Concentrated 10× solutions, to be added to the recording mini-chamber,
were prepared by diluting the stock solutions in the standard Tyrode's
solution (for Na or K current recordings) or in the
Ba2+ (5 mM)-containing TEA
solution (for Ca channel current recordings). All 10× solutions also
contained 1 mg/ml cytochrome c.
The pipette tips, the recording chamber, and the vials that contained
the toxin stock and 10× solutions were all siliconized to prevent
toxin loss through nonspecific binding.
Drug application. All cells were recorded in a 100 µl
mini-chamber. Small volumes of concentrated (10×) toxin solutions were pipetted into the mini-chamber to expose the recorded cell to high
concentrations with minimal amounts of the toxins being used.
A small inlet and outlet were used to exchange the external solution in
the mini-chamber when recovery from toxin block was studied. When
compared with more conventional application techniques (such as the use
of arrays of "sewer" pipes), this experimental procedure leads to
accurate measurements of steady-state current block. However, the time
course of current block observed when toxins are applied directly into
the recording chamber was too variable to provide useful kinetic information.
All experiments were done at room temperature (18-22°C).
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RESULTS |
-Aga-IVA block of multiple Ca current components
In subthalamic neurons, the spider toxin -Aga-IVA blocked more
than one Ca channel current (Fig. 1). In
the experiment of Figure 1, Ca channel currents carried by 5 mM Ba2+ were elicited every 10 sec by a 30 msec voltage step from 80 to 20 mV. Within 10 min of
-Aga-IVA application (50 nM final concentration), the
control current was reduced by 51%. Raising the toxin concentration to
100 nM had little if any effect, indicating a saturating
block of this current component by 50 nM -Aga-IVA (n = 3). In the same cell, further increase of
-Aga-IVA concentration from 100 nM to 1 µM produced an additional current reduction, which amounted to ~16% of the control current. In five similar experiments, 50-100 nM -Aga-IVA inhibited
50.4 ± 3.4% of the control current, whereas its subsequent
application at 1 µM blocked 14.0 ± 1.7%
of the total current. These data suggest that -Aga-IVA targets at
least two populations of Ca channels, blocking one with high potency
and the other with lower potency.
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Figure 1.
Subthalamic neuron Ca channel currents blocked by
increasing concentrations of the spider toxin -Aga-IVA. Each
data point represents the peak current elicited by a
test pulse from 80 to 20 mV at 10 sec intervals.
Inset, Representative current traces in control
conditions (1) and in the presence of 50 nM (2), 100 nM
(3), and 1 µM
(4) -Aga-IVA.
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P-type current block in Purkinje and subthalamic neurons
Figure 2 compares the basic
properties of the subthalamic current targeted by nanomolar
concentrations of -Aga-IVA (50-200 nM) with those of
the P-type Ca current recorded in Purkinje neurons.
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Figure 2.
Characteristics of the Ca channel current blocked
by 100-200 nM -Aga-IVA in subthalamic neurons.
A, Current-voltage relationships of the Ca channel
current sensitive to 100 nM -Aga-IVA in subthalamic
( ) and Purkinje ( ) neurons. The data points
represent the mean values ± SEM, normalized to the peak current
of the I-V curve for 9 subthalamic and 10 Purkinje
neurons. The current values illustrated for subthalamic neurons were
obtained by subtracting currents recorded in the presence of 2.5 µM -CgTX, 2.5 µM nimodipine, and 100 nM -Aga-IVA from those measured in the presence of 2.5 µM -CgTX and 2.5 µM nimodipine. The
I-V curves of Purkinje neurons were obtained by
subtracting the currents unaffected by 100 nM -Aga-IVA
from the control recordings. B, Voltage-dependent relief
of current block by -Aga-IVA (100 nM) in a subthalamic
neuron. Currents were elicited by a test pulse from 80 to 20 mV
before and 5 msec after a train of 36 depolarizing steps from 80 to
+150 mV (15 msec duration), applied at 50 Hz, in control conditions
(left panel) and in the presence of 100 nM -Aga-IVA (right panel).
C, Time-dependent inactivation of the
-Aga-IVA-sensitive Ca channel currents elicited by a 1 sec test
depolarization from 80 to 10 mV in a Purkinje neuron (left
panel) and a subthalamic neuron (right
panel). Each current trace was obtained by subtracting
the currents recorded after addition of 100 nM -Aga-IVA
from those recorded in control conditions. D1, D2,
Steady-state inactivation of the -Aga-IVA-sensitive Ca channel
currents recorded in a Purkinje neuron (D1) and a
subthalamic neuron (D2). Currents were elicited by
voltage steps applied every 10 sec to 30 mV (D1) or
15 mV (D2) while the cell was maintained at a 80 mV
(filled symbols) or 65 mV (open
symbols) holding potential (HP). The effect of
200 nM -Aga-VA was quantified at each holding potential.
Complete relief from current block was induced 20 sec after termination
of the first toxin application by the administration of a train of 36 depolarizations (each 15 msec long) from 80 to +130 mV at 50 Hz
(arrows). The current traces illustrated represent the
current sensitive to 200 nM -Aga-IVA at each holding
potential. These traces were obtained by subtracting currents recorded
after addition of the toxin from the control currents.
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Voltage-dependent activation
Figure 2A illustrates the current-voltage
relationships of the -Aga-IVA-sensitive current in Purkinje and
subthalamic neurons. Currents were elicited by 20 msec depolarization
steps applied from 80 mV to different voltages. For each test pulse,
the -Aga-IVA-sensitive current was determined by subtracting the
-Aga-IVA-resistant current from the control recordings and measuring
its peak. The data points represent averages ± SEM of such
measurements for 8 Purkinje cells and 10 subthalamic neurons. The
P-type Ca current in Purkinje neurons activated at more negative
potentials than did its counterpart in subthalamic neurons. Consistent
with a previous report (Regan, 1991), the I-V curve in
Purkinje neurons peaked at approximately 20 mV. In contrast, in
subthalamic neurons, the current affected by 100 nM -Aga-IVA was maximal at approximately 10 mV.
Voltage-dependent unblock
A strong voltage dependence of the off rate characterizes the
high-affinity binding of -Aga-IVA to P-type Ca channels (Mintz et
al., 1992; McDonough et al., 1997). This property results in the
complete unblock of P-type Ca current at positive potentials (>120
mV). In Figure 2B, control currents were elicited, in
a subthalamic neuron, by a 80 to 20 mV test pulse. They were
unaffected by trains of large depolarizations (Fig. 2B,
left panel). After the administration of 100 nM -Aga-IVA and the resulting 43% current reduction, the application of the identical train of depolarizations restored the test current back to its control value (Fig.
2B, right panel).
Time-dependent inactivation
A characteristic of P-type Ca current in Purkinje neurons is its
slow inactivation during prolonged test pulses (Regan, 1991; Usowicz et
al., 1992; Dove et al., 1998). We compared the kinetics of
-Aga-IVA-sensitive currents in a Purkinje cell (Fig. 2C,
left, 200 nM -Aga-IVA) and in a
subthalamic neuron (Fig. 2C, right, 50 nM -Aga-IVA). In both cases, currents were
elicited by a 1 sec test pulse applied from 80 to 10 mV. Once their
amplitude were normalized, the current traces were superimposable.
Steady-state inactivation
P-type Ca channels in cerebellar granule cells display dramatic
steady-state inactivation (Tottene et al., 1996). Using the same
approach, we have investigated the inactivation properties of the
-Aga-IVA-sensitive current in subthalamic neurons (n = 3) and in Purkinje cells (n = 4). Representative
recordings are illustrated in Figure 2, D1 (Purkinje
neuron) and D2 (subthalamic neuron). In both cases,
-Aga-IVA was first tested on currents elicited by 30 msec pulses
applied from 80 to 30 mV (Purkinje neuron) or 15 mV (subthalamic
neuron). The application was maintained for >6 min to ensure
steady-state current block. The toxin was then washed out of the
mini-chamber, and we applied large depolarizations to facilitate
current recovery to its control value. The cell holding potential was
then changed from 80 to 65 mV, and current was again elicited by 30 msec steps to 30 mV (Purkinje neuron) or 15 mV (subthalamic
neuron). Pronounced current inactivation occurred within 6-8 min of
changing the holding potential. -Aga-IVA was then reapplied to
quantify the fraction of P-type Ca current that escaped inactivation.
In both cell types, we found a significant reduction in the amplitude
of the current affected by -Aga-IVA. A change in holding potential
from 80 to 65 mV inactivated between 40 and 80% of the P-type Ca
current in Purkinje neurons (n = 4) and between 45 and
75% of the P-type Ca current in subthalamic neurons (n = 4).
We were unable to complete the inactivation curve of P-type Ca channels
in Purkinje and subthalamic neurons. In both cell types, it was
difficult to reverse steady-state current inactivation. Moreover,
current runup in Purkinje neurons and current rundown in subthalamic
neurons often interfered with a precise estimate of inactivation.
Overall, we found that the subthalamic current inhibited by nanomolar
concentrations of -Aga-IVA was very similar to Purkinje neuron
P-type Ca current. Except for a slight difference in their voltage-dependent activation, both currents were identical. They displayed little time-dependent inactivation during a 1-sec-long test
depolarization (Fig. 2C) but showed considerable
steady-state inactivation at holding potentials as negative as 65 mV
(Fig. 2D1,D2). In subthalamic as in Purkinje neurons,
the efficacy of toxin block was voltage-dependent (Fig.
2B). On the basis of these similarities, despite the
absence of structural data, we now refer to the subthalamic Ca current
affected by -Aga-IVA (100 nM) as the
subthalamic P-type Ca current.
Subsequent experiments investigate the current component targeted by
micromolar concentrations of -Aga-IVA.
-Aga-IVA selectivity
To verify the toxin selectivity when it is used in the micromolar
range, we tested its effects on a variety of identified voltage-gated
ion currents.
In subthalamic neurons, -Aga-IVA (1 µM) had no effect
voltage-gated Na currents (n = 4). As shown in Figure
3A, the Na current elicited by
a voltage step from 80 to 30 mV was identical before and 6 min
after toxin application. It did not affect voltage-gated K currents
either (n = 4). In Figure 3B, using another
subthalamic neuron, a similar K current was recorded during a 80 to
10 mV voltage-step before and after toxin application (1 µM for 1 min).
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Figure 3.
Insensitivity of subthalamic Na, K, and Ca (T- and
L-type) currents to 1 µM -Aga-IVA. A,
-Aga-IVA (1 µM) effect on the Na current elicited by a
test depolarization from 80 to 30 mV. Extracellular
Cd2+ (0.5 mM) was present throughout the
recordings to suppress Ca channel currents. Addition of 1 µM TTX, at the end of the experiment, abolished all
voltage-gated inward currents (data not illustrated). B,
-Aga-IVA (1 µM) effect on the K current elicited by a
test pulse from 80 to +60 mV. The extracellular solution contained 1 µM TTX and 0.5 mM Cd2+ to
block Na and Ca currents. C, -Aga-IVA (1 µM) effect on low-threshold T-type Ca currents. A small
test pulse from 100 to 55 mV elicited a low-threshold, slowly
deactivating T-type current that was unaffected by exposure to 1 µM -Aga-IVA (left panel).
Right panel, Effect, in the same cell, of 1 µM -Aga-IVA on high-threshold Ca channel currents
elicited by a larger depolarization from 80 to 30 mV.
D, -Aga-IVA (1 µM) effect on Bay K
8644-enhanced L-type Ca currents. The currents illustrated in the
left panel were elicited by a small depolarization
applied from 90 to 50 mV in control conditions, after addition of
Bay K 8644 (3 µM), and in the presence of Bay K 8644 (3 µM) plus -Aga-IVA (1 µM). In the same
cell (right panel), high-threshold Ca currents
elicited by a 80 to 20 mV step depolarization were inhibited by 1 µM -Aga-IVA. The data presented were obtained in
different subthalamic neurons.
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We then tested the effects of -Aga-IVA (1 µM) on
identified Ca currents.
The application of -Aga-IVA (1 µM) had no effect on
the low-threshold T-type currents recorded in subthalamic neurons
(n = 3). In Figure 3C, left, -Aga-IVA (1 µM) spared the low-threshold current activated
by a 100 to 55 mV voltage-step and the slowly deactivating T-type
tail current. In the same cell, it reduced the high-threshold currents
elicited from a less negative holding potential by 56% (Fig. 3C,
right).
L-type currents enhanced with the dihydropyridine agonist Bay K 8644 (3 µM) were equally insensitive to -Aga-IVA (1 µM; n = 5). After treatment with Bay K
8644 (3 µM), the threshold for activation of
L-type Ca channels is decreased, and their mean open time is increased,
resulting in the appearance of a low-threshold current and slowly
deactivating tail currents (Fig. 3D, left). These currents,
exclusively carried through L-type Ca channels, did not change after a
6 min exposure to -Aga-IVA (1 µM). In the
same cell, the reduction of other high-threshold currents confirmed the
efficacy of the toxin application (Fig. 3D, right).
These data suggest that the toxin -Aga-IVA remains selective in the
micromolar range. However, our subsequent investigation of its effect
on N-type Ca currents demonstrated that this selectivity is not complete.
-Aga-IVA block of N-type Ca currents
Sympathetic neurons
We tested the block of N-type Ca channels in rat sympathetic
neurons, in which up to 90% of the Ca channel current is carried through -CgTX-sensitive N-type channels. As shown previously (Mintz
et al., 1992), 200 nM -Aga-IVA had no effect on the Ca channel current recorded in these cells. In Figure
4A, the small reduction
of the current elicited by a voltage step from 80 to 5 mV was
indistinguishable from current rundown (n = 4). In
contrast, significant current reduction was observed after applications of -Aga-IVA (1 µM) (Fig.
4B). In this experiment, the small L-type current
component (<2% of the control current) was blocked with nimodipine
(2.5 µM). The subsequent application of
-Aga-IVA (1 µM) produced a slow and
significant current inhibition. On average, a 10 min application of the
toxin reduced the control current by 28.2 ± 2.2%
(n = 11). This effect was completely reversible. The Ba
current returned to its control value within 1 min of toxin washout;
its sensitivity to -CgTX confirmed its identification as an N-type
Ca channel current (Fig. 4C).
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Figure 4.
Block of N-type Ca channel current by 1 µM -Aga-IVA in sympathetic neurons. A,
Lack of effect of 200 nM -Aga-IVA on the Ca channel
current recorded in a sympathetic neuron. Time course of the peak
current elicited by voltage steps applied from 80 to 5 mV every 10 sec (left panel) and representative current
traces (right panel) are shown. B,
Ca channel current block produced by 1 µM -Aga-IVA.
Time course (left panel) and representative
current traces (right panel) elicited by
voltage-steps from 80 to 15 mV at 10 sec intervals are shown.
Nimodipine (2.5 µM) was present throughout the
experiment. Arrows (left panel)
indicate the application of a 50 Hz train of 70 depolarizing steps from
80 to +120 mV (each 15 msec duration). C,
Reversibility of N-type current block by 1 µM
-Aga-IVA. Left panel, Current traces elicited by a
voltage step from 80 to 15 mV in control conditions and 15 min
after addition of 1 µM Aga-IVA. Right
panel, Current recordings were elicited by similar test pulses
5 min after wash of Aga-IVA and 2 min after subsequent addition of
2.5 µM CgTX. The experiments were performed in
different sympathetic neurons.
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We tested the voltage dependence of -Aga-IVA N-type current block
using trains of 70 pulses to +150 mV as they produced maximal unblock
of the subthalamic current fraction inhibited by 100 nM -Aga-IVA. There was considerable variability in the results. Such
trains had almost no effect in some sympathetic neurons but produced
significant unblock in others. Reliable unblock of the sympathetic
N-type current consistently required stronger and more numerous
depolarizations than necessary for the relief of P-type Ca current
block in subthalamic neurons. Typically (Fig. 4B,
arrows), partial unblock of the N-type current was best
demonstrated after relatively mild depolarizations (here 70 pulses to
+120 mV). Larger depolarizations elicited significant inactivation of
the control current, which then obscured the relief of the current blockade.
Subthalamic neurons
Because the pharmacology of Ca channels in peripheral and central
neurons may differ, we then investigated the effect of -Aga-IVA (1 µM) on subthalamic neuron N-type Ca current. To test for
overlap in the Ca current components blocked by -Aga-IVA (1 µM) and -CgTX (2.5 µM), two sets of
experiments were performed in alternate recordings of subthalamic
neurons (n = 20). In one set, we quantified the effect
of -Aga-IVA (1 µM) first and then measured
the additional current block produced by -CgTX (2.5 µM) plus -Aga-IVA (1 µM). In the other set, toxins were applied in
reverse order. We measured the effect of -CgTX (2.5 µM) first and the effect of coapplication of
the toxins second.
In Figure 5, A and
B, the data points illustrate the time course of the Ba
current recorded in control conditions, during the application of
-Aga-IVA alone (Fig. 5A) or -CgTX alone (Fig. 5B), and during the coapplication of -Aga-IVA (1 µM) and -CgTX (2.5 µM). The corresponding current traces are
depicted in the insets. In both experiments, the Ba current was
activated every 10 sec by a 80 to 15 mV voltage step. Because
-CgTX-sensitive Ca currents are more susceptible to rundown than
other voltage-gated Ca currents (our unpublished data),
drug applications were timed to ensure that the -CgTX effect was
measured after the same length of recordings in both experimental
conditions (~6 min after time 0).
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Figure 5.
Block of N-type Ca channel current by 1 µM -Aga-IVA in subthalamic neurons. A,
Ca channel current block by 1 µM -Aga-IVA followed by
1 µM -Aga-IVA plus 2.5 µM -CgTX.
B, In another rat subthalamic neuron, Ca channel current
block by 2.5 µM -CgTX followed by 2.5 µM
-CgTX plus 1 µM -Aga-IVA. In A and
B, the data points represent the time
course of the peak current elicited every 10 sec by a 20 msec test
pulse from 80 to 15 mV. Insets, Corresponding
current traces. C, Average currents blocked by -CgTX
and -Aga-IVA in 12 experiments performed as in A and
8 experiments performed as in B. The top two
bars illustrate the current fractions blocked by 1 µM -Aga-IVA applied alone (white bar)
or after preblock of N-type Ca channels with 2.5 µM
-CgTX (gray bar). The bottom two
bars represent the current fractions blocked by 2.5 µM -CgTX applied alone (black bar) or
after application of 1 µM -Aga-IVA (striped
bar).
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On average, -Aga-IVA blocked 55.9 ± 2.5% of the control
current. This effect was reduced to 40.7 ± 2.1% when it was
measured after N-type current block with -CgTX (Fig. 5C),
suggesting that ~27% of the -Aga-IVA-sensitive current is also
sensitive to -CgTX. The toxin -Aga-IVA interfered in a similar
manner with -CgTX block of the N-type current component. Applied
alone, -CgTX inhibited 47.6 ± 1.8% of the control current.
This -CgTX-sensitive current fraction was reduced to 33.1 ± 1.7% of the control current when -CgTX was tested after the
application of -Aga-IVA (1 µM). Such overlap
between the -Aga-IVA- and -CgTX-sensitive current components suggests that -Aga-IVA (1 µM) also targets
N-type Ca channels in subthalamic neurons. Our numbers indicate that
-Aga-IVA (1 µM) blocked ~30% of the
-CgTX-sensitive N-type Ca channel current in subthalamic neurons, a
value remarkably similar to the magnitude of its effect on N-type
current in sympathetic neurons.
Block of other Ca channels by micromolar concentrations
of -Aga-IVA
In some subthalamic neurons (n = 14), a large
enough current remained unaffected after blockade of L-, N-, and P-type
Ca currents for its sensitivity to micromolar concentrations of
-Aga-IVA to be assessed.
Figure 6A illustrates
the time course of the Ba currents elicited by a 80 to 25 mV step
depolarization applied every 10 sec. The recordings were performed in
the continuous presence of nimodipine (2.5 µM)
and -CgTX (2.5 µM). A 100 nM -Aga-IVA was first applied to produce
saturating block of the P-type current. Subsequent increase of the
toxin concentration to 1 µM resulted in
additional current block, which represented 20.0 ± 2.9% of the
current recorded in the presence of nimodipine and -CgTX (n = 14). There was considerable variability in the
amplitude of this current component, which did not correlate with that
of the P-type Ca current. This current could be large in cells endowed with a relatively small P-type current component and vice versa, suggesting that different Ca channel types underlie these two currents.
We conclude that subthalamic neurons express a high-threshold current,
which is distinct from the P-type current component and which displays
the pharmacological properties of the Q-type Ca current in cerebellar
granule cells.
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Figure 6.
Ca channel current inhibited by 1 µM
-Aga-IVA after block of L-, N-, and P-type Ca channel currents in
subthalamic neurons. A, Each data point
represents the peak current elicited by a voltage-step from 80 to
25 mV at 10 sec intervals. The current traces illustrated in the
left panel were recorded in the presence of 2.5 µM nimodipine and 2.5 µM -CgTX after
addition of 100 nM -Aga-IVA and after subsequent
increase of -Aga-IVA concentration to 1 µM.
B, Comparison of the current components affected by 100 nM -Aga-IVA (left) and 1 µM
-Aga-IVA (right) in a similar experiment performed,
on another subthalamic neuron, in the continuous presence of 2.5 µM nimodipine and 2.5 µM -CgTX. Currents
were elicited by a 100 msec depolarizing step from 100 to 10 mV.
The current traces illustrated here were obtained by subtraction.
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|
Rapid inactivation is a characteristic of the prototypic Q-type Ca
current described in cerebellar granule cells (Randall and Tsien,
1995). In the experiment illustrated in Figure 6B, we
evaluated this parameter for the -Aga-IVA-sensitive currents of
subthalamic neurons. As in Figure 6A but in a
different subthalamic neuron, -CgTX (1 µM)
and nimodipine (2.5 µM) were present throughout the experiment to ensure blockade of the N- and L-type Ca currents. To
maximize the transient components, we used a very negative holding
potential (100 mV) and longer (100 msec duration) test pulses. The
currents were studied in control conditions, after addition of 100 nM -Aga-IVA, and after further increase of the toxin concentration to 1 µM. Both currents,
determined by subtraction, showed little time-dependent inactivation
during test depolarizations. After proper scaling, they were nearly superimposable.
| |
DISCUSSION |
Toxin selectivity
Our data confirm the initial description of the toxin -Aga-IVA
as a potent and selective blocker of P-type Ca channels. As documented
in other cell types (Bargas et al., 1994; Brown et al., 1994; Eliot and
Johnston, 1994; Tottene et al., 1996), 50-100 nM toxin
concentration produced saturating blockade of the P-type Ca current. At
concentrations <100 nM, the toxin had no effect on
identified subthalamic T-, L-, and N-type Ca current. These data are
consistent with occlusion studies, performed in other neuronal types,
which demonstrated a high selectivity of the toxin when used in the
nanomolar range (Brown et al., 1994; Foehring and Scroggs, 1994; Fisher
and Bourque, 1995; Desmadryl et al., 1997; Churchill and Macvicar,
1998; Connor and Christie, 1998).
The toxin -Aga-IVA retained a high degree of selectivity when used
in the micromolar range. It had no effect on voltage-gated Na and K
channels and on L- and T-type Ca channels. However, its selectivity was
not complete, as demonstrated by its significant reduction of
-CgTX-sensitive N-type Ca channel currents.
The block of N-type Ca channel current by 1 µM
-Aga-IVA was readily assessed in sympathetic neurons, in which up to
90% of the Ca current flows through -CgTX-sensitive N-type Ca
channels (Boland et al., 1994). In subthalamic neurons, the sensitivity of N-type Ca channels to -Aga-IVA was inferred from the partial occlusion between -Aga-IVA (1 µM) and -CgTX (2.5 µM) blocking effects. A similar overlap in the current
components targeted by 1 µM -Aga-IVA and -CgTX has
been documented in other neuronal types (Yu and Shinnick-Gallagher,
1997), suggesting that other mammalian central neurons may express
N-type Ca channels that are weakly sensitive to -Aga-IVA.
Another high-threshold Ca current was also targeted by -Aga-IVA (1 µM) in subthalamic neurons. The current that remained in
the presence of -CgTX, nimodipine, and 100-200 nM
-Aga-IVA, a combination of antagonists designed to selectively
inhibit N-, L-, and P-type Ca channels, was partially blocked by an
increase in -Aga-IVA concentration from 100 nM to 1 µM. In contrast to the transient Q-type Ca current
described in some neurons (Randall and Tsien, 1995; Desmadryl et al.,
1997; Mermelstein et al., 1999), it resembled the -Aga-IVA-sensitive
P-type Ca current in its activation and inactivation properties.
Voltage-dependent unblock suggests a common mechanism of block
Previous studies in Purkinje neurons have demonstrated that
-Aga-IVA is a channel-gating modifier that shows striking voltage dependence in its affinity for P-type Ca channels (McDonough et al., 1997b). Our data indicate that a similar mechanism is engaged by
the high-affinity blockade of subthalamic neuron P-type Ca channels. In
these cells as in Purkinje neurons, trains of large depolarizations
completely recovered the current blocked by 50-100 nM
-Aga-IVA. Consistent with a complete unbinding of the toxin, current
reblock occurred after termination of the large depolarizations with a
time course identical to that seen during the first exposure to the toxin.
For practical reasons, we investigated the N-type Ca current block by
-Aga-IVA in sympathetic neurons. With protocols adapted to minimize
current inactivation, voltage-dependent relief of N-type Ca current
block was evident. However, it was quantitatively different from the
relief of the P-type current block in subthalamic and Purkinje neurons,
because it was less complete and kinetically more complex. Current
reblock, after termination of the large depolarizations, showed a
multiexponential time course. It was too rapid to represent the toxin
binding to the channels, suggesting a relatively stable association of
-Aga-IVA to unblocked channels.
Although it is likely, we were unable to demonstrate the
voltage-dependent unblock of the additional current component affected by 1 µM -Aga-IVA. This current fraction was too small,
and current reblock in the presence of 1 µM toxin was too
fast to allow a precise measurement of the current amplitude after
trains of large depolarizations. Still, our data on P- and N-type Ca
channel currents are consistent with a general mechanism for
-Aga-IVA action as a gating modifier, whose binding to low- or
high-affinity sites likely interferes with the voltage sensors of these channels.
Structural correlates of -Aga-IVA-sensitive Ca channels
Gating modifiers of voltage-gated ion channels recognize common
features in the extracellular S3-S4 linker that flanks the channel S4
transmembrane voltage sensor (Li-Smerin and Swartz, 1998). In the case
of the 1A subunit, a high-affinity binding site for -Aga-IVA has
been located to the S3-S4 linker of domain IV (Bourinet et al., 1999).
Alternate splicing in this region produces two isoforms, 1A-a and
1A-b, which differ by the insertion of two amino acids (NP) in
positions 1605 and 1606. These splice variants show respectively high
and low affinity for -Aga-IVA, and they may account for the native
P- and Q-type Ca currents recorded in mammalian central neurons.
P-type Ca channels
Our data demonstrate that P-type Ca channels in subthalamic and
cerebellar Purkinje neurons are very similar to the
-Aga-IVA-sensitive G1 Ca channels that have been extensively
characterized in cerebellar granule cells (Tottene et al., 1996). They
displayed the same kinetics during test depolarization and the same
dramatic steady state inactivation (V1/2,
approximately 65 mV). These channels are not identical though.
Subthalamic P-type Ca channels and cerebellar G1 Ca channels did
activate with comparable voltage dependence (both currents carried by 5 mM Ba2+ peaked at
approximately 10 mV). However, the P-type Ca channels in Purkinje
neurons showed their typical more negative range for activation (Regan,
1991), with a peak of the I-V curve located at
approximately 25 mV.
Altogether, these findings are consistent with the hypothesis that
neuronal P-type Ca channels, defined by their sensitivity to nanomolar
amounts of the toxin -Aga-IVA, belong to the same class A gene
family. They may display minor structural differences, but their 1
subunits are likely to be very similar to the splice variant 1A-a,
the only 1A isoform identified so far that carries a high-affinity
binding site for -Aga-IVA (Bourinet et al., 1999).
N-type Ca channels
N- and P-type Ca channels are closely related, with extensive
homology between their respective 1B and 1A subunits (Tsien et
al., 1995). They share high-affinity binding sites to the Ca channel
antagonists -conotoxin-MVIIC and grammatoxin (McDonough et al.,
1996, 1997a). They display similar gating properties, and, except for
their different sensitivity to selective blockers, they are nearly
indistinguishable (Plummer et al., 1989; Usowicz et al., 1992;
Rittenhouse and Hess, 1994; Dove et al., 1998). It is thus not
surprising that N-type Ca channels showed weak sensitivity to the
P-type Ca channel blocker -Aga-IVA.
In sympathetic neurons, we observed a partial blockade (~30%) of the
N-type current after exposure to -Aga-IVA at micromolar concentrations, suggesting that a large fraction of the
-CgTX-sensitive N-type Ca current is insensitive to -Aga-IVA.
Such heterogeneity is consistent with the diversity of the 1B
subunits that is generated by differential splicing in these cells (Lin
et al., 1997; Lü and Dunlap, 1999). Interestingly, some variants
differ in the S3-S4 linkers of domains III and IV, which are possible
binding sites for -Aga-IVA (Lin et al., 1997). It will be
interesting to see whether -Aga-IVA discriminates between these
different N-type Ca channel subtypes.
In subthalamic neurons, approximately the same 30% fraction of the
-CgTX-sensitive Ca current was blocked by -Aga-IVA. We do not
know whether this partial block represents incomplete or maximal effect
of -Aga-IVA, because we did not perform occlusion experiments with
toxin concentrations greater than 1 µM.
Q-type and other high-threshold Ca channels
In the absence of structural data, weak sensitivity to the toxin
-Aga-IVA (1 µM) has been the main criterion to define
Q-type Ca channels (Sather et al., 1994, Randall and Tsien, 1995).
These channels, whose structural identity was recently established
(Bourinet et al., 1999), have been challenging objects of study.
Unlike L-, N-, and P-type Ca channels whose investigation is greatly
facilitated by their predominant expression in some excitable cells,
Q-type Ca channels account for a relatively small component of the
whole-cell Ca current recorded in cerebellar granule cells (Randall and
Tsien, 1995). Because these cells express a significant P-type Ca
current, the pharmacological separation of the two current components
is difficult. Despite the widespread expression of 1A genes in
mammalian CNS (Westenbroek et al., 1995; Sakurai et al., 1996), so far,
no favorable experimental system has been identified for studies of
native Q-type 1A Ca channels.
Our finding that more than one Ca channel type show weak but
significant sensitivity to 1 µM -Aga-IVA underlies the
difficulty in relating the current blocked by 1 µM
-Aga-IVA to 1A Q-type Ca channels. This composite current is
carried through -CgTX-sensitive 1B N-type Ca channels and other
Ca channel types, which may include 1A Q-type Ca channels and
possibly 1E R-type Ca channels. The latter are expressed in
subthalamic neurons (Yokoyama et al., 1995), and their
pharmacology in expression systems suggest that some (Soong et al.,
1993; Stephens et al., 1997), but not all (Ellinor et al., 1993;
Wakamori et al., 1994), are blocked by 1 µM -Aga-IVA.
Considering the high degree of homology of DHP-insensitive Ca channels,
it is likely that -Aga-IVA (1 µM) targets variants in
each class of this family. Weak sensitivity to -Aga-IVA and gating
properties (which are highly variable depending on the subunit
associated with the 1A subunit) are thus incomplete criteria of
identification for 1A Q-type Ca channels. The full characterization
of these channels in neurons will require a combined approach with
molecular, pharmacological, and electrophysiological tools, as already
begun in cortical and neostriatal neurons (Mermelstein et al.,
1999).
The toxin -Aga-IVA retains its usefulness as a selective and potent
blocker of neuronal P-type Ca channels. It is not a selective blocker
of 1A Ca channels because, like many other Ca channel antagonists
(Hillyard et al., 1992; McDonough et al., 1996, 1997a), it affects a
variety of DHP-insensitive Ca channels when used in the micromolar
range. Notwithstanding this limitation, it will continue to be an
important tool to relate the properties of Ca channel clones to the
complexity of their native counterparts.
| |
FOOTNOTES |
Received Feb. 7, 2000; revised July 5, 2000; accepted July 14, 2000.
This work was supported by National Institutes of Health Grant NS-34550
to I.M.M. We thank Dr. Bruce P. Bean for helpful comments.
Correspondence should be addressed to Isabelle M. Mintz, Department of
Pharmacology and Experimental Therapeutics, Boston University Medical
Center, 80 East Concord Street, Boston, MA 02115. E-mail:
imintz{at}bu.edu.
| |
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