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Vol. 54, Issue 6, 1080-1087, December 1998
Department of Neurobiology, Harvard Medical School, Boston, Massachusetts 02115
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Summary |
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Top
Summary
Introduction
Materials & Methods
Results
Discussion
References
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The antihypertensive agent mibefradil completely and reversibly
inhibited T-type calcium channels in freshly isolated rat cerebellar
Purkinje neurons. The potency of mibefradil was increased at less
hyperpolarized holding potentials, and the apparent affinity was
correlated with the degree of channel inactivation. At 35°, the
apparent dissociation constant Kapp was 1 µM at a holding voltage of 
110 mV (corresponding to
noninactivated channels) and 83 nM at a holding voltage of
70 mV (corresponding to 65% inactivation). The increased affinity
was attributable mainly to a decreased off-rate. Mibefradil also
inhibited P-type calcium channels in Purkinje neurons, but inhibition
was much less potent. At a holding potential of 70 mV, the
Kapp for mibefradil inhibition of P-type
channels was ~200-fold higher than that for inhibition of T-type
channels. Mibefradil should be a useful compound for distinguishing
T-type channels from high voltage-activated calcium channels in neurons
studied in vitro.
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Introduction |
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Top
Summary
Introduction
Materials & Methods
Results
Discussion
References
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Voltage-dependent
calcium channels in neurons have diverse functions, including
crucial roles in excitability (Huguenard, 1996
), neurotransmission
(Dunlap et al., 1995), activation of intracellular signaling
pathways, and regulation of gene expression (Ghosh and Greenberg,
1995). Multiple types of voltage-gated calcium channels in neurons have
been distinguished by kinetics, voltage dependence, single-channel
properties, and pharmacological characteristics (Tsien et
al., 1995). Some types of calcium channels distinguished in native
cells have been convincingly identified with particular gene products,
namely N-type channels with 
1B subunits, L-type channels
with 1C and 1D subunits, and P-type and Q-type channels (which
have many similarities) with 1A gene products (reviewed by Hofmann
et al., 1994; Mori, 1994; Dunlap et al., 1995;
Randall, 1998). The 1E gene is proposed to encode the
"R-type" current in central neurons (Zhang et al., 1993;
Wakamori et al., 1994; Randall and Tsien, 1997), but the
correspondence is less certain because of a lack of distinguishing
pharmacological agents.
Of native calcium channels, T-type (low-voltage-activated) channels are
the most distinctive in terms of voltage dependence, kinetics, and
single-channel properties (Tsien et al., 1995; Huguenard, 1996; Ertel and Ertel, 1997). Such channels, which are characterized by
rapid inactivation kinetics, inactivation at moderately depolarized holding potentials, slow deactivation kinetics, and small
single-channel conductances, are present in a wide range of excitable
cells, including neurons, neuroendocrine cells, cardiac muscle, and
smooth muscle, as well as some nonexcitable cells (reviewed by Ertel et al., 1997). Recently, Perez-Reyes and colleagues
(1998) discovered a new calcium channel 1 subunit, termed
1G, that seems to correspond to T-type channels. Although no
pharmacological correspondence has been established, currents from the
cloned channels have the distinctive fast inactivation, slow
deactivation, and small single-channel conductance characteristic of
native T-type channels.
The identification of the new clone will bring renewed attention to
T-type channels, including investigations of their pharmacological characteristics. There is currently no peptide toxin for T-type channels comparable to those targeting N-type or P-type channels, and
there are no organic molecules with potency and selectivity comparable
to those of dihydropyridines for L-type channels.
Amiloride, which is mainly known as a blocker of epithelial sodium
channels, is somewhat selective for T-type channels over other types of calcium channels but requires concentrations of ~1 mM for
complete inhibition (Tang et al., 1988; McCobb et
al., 1989; Takahashi et al., 1989). Nickel is
somewhat selective for T-type current in some cell types (Fox et
al., 1987) but has little selectivity in others (Regan,
1991; Huguenard, 1996).
Mibefradil (Ro 40-5967; Posicor) is an antihypertensive drug that is
structurally different from other classes of calcium channel blockers
(Clozel et al., 1991, 1997; Triggle, 1996). The antihypertensive action of mibefradil probably reflects vasodilating effects (Osterrieder and Holck, 1989), which are hypothesized to result
from block of calcium channels in vascular smooth muscle cells (Bian
and Hermsmeyer, 1993). In both vascular smooth muscle cells and cardiac
muscle cells, mibefradil is more potent in blocking T-type calcium
channels than L-type calcium channels (Mishra and Hermsmeyer, 1994a; Bénardeau and Ertel, 1998). The half-maximal blocking concentration (EC50) for T-type channels
in vascular muscle is ~100 nM (Mishra and Hermsmeyer,
1994a). In contrast, mibefradil inhibition of cloned 1A, 1B,
1C, and 1E calcium channels and native L-type
channels is much weaker (EC50 = 3-21 µM) (Mishra and Hermsmeyer, 1994b; Bezprozvanny and
Tsien, 1995; Bénardeau and Ertel, 1998). Mibefradil has not yet
been tested with 1G channels.
Mibefradil has been found to inhibit T-type calcium channels in several
neuronal preparations, including neuroblastoma cells (Randall and
Tsien, 1997), sensory neurons (Todorovic and Lingle, 1998), and spinal
motoneurons (Viana et al., 1997). In neurons, unlike
vascular or cardiac muscle, block of T-type channels seems to be no
more potent than that of various high-threshold calcium channels
(Randall and Tsien, 1997; Viana et al., 1997). However, comparisons were made either across cell types or on overall calcium currents, where distinction among components may be difficult.
We investigated the action of mibefradil on cerebellar Purkinje neurons under conditions in which homogeneous T-type calcium currents could be obtained. We found that mibefradil inhibited T-type calcium channels in Purkinje neurons with greater potency, compared with all other calcium currents examined, including T-type currents in vascular muscle. The potency of mibefradil depended strongly on the holding potential; at physiological resting potentials, mibefradil eliminated T-type current with minimal effects on P-type current. Thus, mibefradil should be a useful tool for analyzing the cellular function of neuronal T-type calcium currents and for correlating expressed cDNA with native T-type currents.
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Materials and Methods |
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Top
Summary
Introduction
Materials & Methods
Results
Discussion
References
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Preparation of Purkinje neurons.
Cerebellar Purkinje neurons
were isolated from the brains of 9-16-day-old Long-Evans rats with a
slight modification of previously described procedures (Mintz et
al., 1992; McDonough et al., 1996). Rats were
anesthetized with methoxyflurane, and the hearts were perfused with
ice-cold Ca2+-free Tyrode's solution (150 mM NaCl, 4 mM KCl, 2 mM
MgCl2, 10 mM HEPES, 10 mM
glucose, pH adjusted to 7.4 with NaOH) to rapidly cool the brain
tissue. Cerebellar chunks were removed with fine scissors and minced
with a razor blade in cold oxygenated dissociation solution (82 mM Na2SO4, 30 mM K2SO4, 5 mM MgCl2, 10 mM HEPES, 10 mM glucose, 0.001% phenol red, pH adjusted to 7.4 with
NaOH). Tissue was then transferred into dissociation solution with 3 mg/ml protease XXIII (Sigma) (pH readjusted to 7.4 with NaOH) and
incubated at 35° for 7-8 min, under a continual stream of pure
oxygen. Tissue was then transferred to dissociation solution with 1 mg/ml trypsin inhibitor (Sigma) and 1 mg/ml bovine serum albumin
(Sigma) (pH adjusted to 7.4 with NaOH) and was allowed to cool to room
temperature. Tissue was maintained in this solution or in Tyrode's
solution (composition as described above but with 2 mM
CaCl2) for up to 8 hr, with light oxygenation.
Tissue chunks were triturated in Tyrode's solution as needed, and
Purkinje neurons were identified morphologically (Regan, 1991).
Recording of calcium channel currents.
Calcium channel
currents were recorded using the whole-cell configuration of the
patch-clamp technique, using patch pipettes made from borosilicate
glass tubing (100-µl Boralex capillaries; Dynalab, Rochester, NY) and
coated with Sylgard (Dow Corning Corp., Midland, MI). Pipettes had
resistances of 1-3 M
when filled with internal solution. After a
stable whole-cell recording in Tyrode's solution was obtained, the
cell was lifted off the bottom of the dish and positioned directly in
front of a gravity-fed array of 12 perfusion tubes made of 250-µm
(i.d.) quartz tubing connected (with Teflon tubing) to glass reservoirs.
110 mV, where it was a depolarizing step.
The intracellular (pipette) solution contained 56 mM CsCl,
68 mM CsF, 2.2 mM MgCl2,
4.5 mM EGTA, 9 mM HEPES, 4 mM
MgATP, 14 mM creatine phosphate (Tris salt), and 0.3 mM GTP (Tris salt) (pH adjusted to 7.4 with CsOH). The
external solution contained 160 mM tetraethylammonium
chloride, 10 mM HEPES, 5 mM
BaCl2, 600 nM tetrodotoxin, and 1 mg/ml cytochrome c (from horse heart; Sigma). Standard
external solution also contained 1 µM nimodipine (3-5 µM nimodipine in a few experiments) to block
L-type currents and, for isolation of T-type currents, 10 µM 
-conotoxin-MVIIC (Bachem California, Torrance, CA)
to block P-type currents (McDonough et al., 1996). External
solutions were exchanged in <1 sec by moving the cell between
continuously flowing solutions from the perfusion tubes. Reported
potentials were not corrected for a junction potential of 2 mV
between the pipette solution and the Tyrode's solution in which the
offset potential was zeroed before seal formation.
Most experiments were performed at 35°, at which T-type currents were
consistently larger than at room temperature. Some of the increased
current amplitude at holding potentials of 80 to 90 mV was the
result of a temperature dependence of steady state inactivation
(Boltzmann fit parameters for inactivation curves: 23°,
V1/2 = 81 ± 0.3 mV, k = 9.5 ± 1 mV, n = 8; 35°,
V1/2 = 74 ± 0.6 mV, k = 7.4 ± 0.4 mV, n = 6) (see Fig. 5). In addition, maximal currents from strongly hyperpolarized holding potentials were
also consistently larger at 35° than at room temperature.
Mibefradil was the kind gift of Dr. Jean-Paul Clozel and Dr. Eric Ertel
(F. Hoffmann-La Roche, Basel, Switzerland). Mibefradil was prepared as
a 10 mM stock solution in water. The potency of mibefradil
(tested on T-type channels in Purkinje neurons) in this stock solution
changed little in approximately 1 month with storage at 4°. However,
after 4-12 months of storage the potency of the drug was greatly
diminished (by at least a factor of 5 after 12 months). Loss of potency
occurred for powder stored at room temperature for 4 months, for powder
stored at 20° for 12 months (in a sealed vial inside a desiccator),
and for a 10 mM stock solution in distilled water stored at
4°. All of the experiments reported here were performed with drug
used within 5 weeks of receipt.
Determination of the dose-response relationship for mibefradil at 35°
was complicated by run-down of calcium currents. As evident from Fig.
2, there was often a decline of ~10% in the magnitude of T-type
current over several minutes. We estimate the resulting errors in
fitted half-blocking concentrations as <10%. Values are
reported as mean ± standard error.
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Results |
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Top
Summary
Introduction
Materials & Methods
Results
Discussion
References
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Inhibition of T-type channels by mibefradil.
Cerebellar
Purkinje neurons were previously found to exhibit rapidly inactivating,
T-type calcium currents (Kaneda et al., 1990; Regan, 1991;
Mouginot et al., 1997), as well as more slowly inactivating,
higher-threshold calcium currents that are mainly (85-100%)
attributable to P-type channels, with small contributions from
L-type channels and N-type channels (Mintz et
al., 1992). To record currents from T-type channels in isolation,
we used a combination of the L-type channel blocker
nimodipine and the peptide toxin -conotoxin-MVIIC, which blocks both
P-type and N-type currents (Hillyard et al., 1992; McDonough
et al., 1996). As shown in Fig.
1, 10 µM
-conotoxin-MVIIC completely blocked the slowly inactivating
component of current (in the presence of 5 µM
nimodipine), leaving in isolation a rapidly inactivating component of
current. Such currents were present in 70 of 70 Purkinje neurons tested
under these conditions (5 mM Ba2+ as
charge carrier, 35°). These currents exhibited all of the characteristics expected of T-type currents, including rapid
inactivation, slow deactivation, and high sensitivity to inactivation
by moderately depolarized holding potentials (complete inactivation at
a holding potential of 50 mV).
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80 mV to 30 mV (in the continuous
presence of nimodipine and -conotoxin-MVIIC). Inhibition developed
within seconds and reversed within a few minutes. T-type current was
inhibited equally well at test potentials from 60 mV to 0 mV (Fig.
3).
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Inhibition of T-type channels at different holding potentials.
The potency of mibefradil depended strongly on the holding potential
(Fang and Osterrieder, 1991; Bezprozvanny and Tsien, 1995). Fig.
4 shows the dose dependence of mibefradil
block of T-type current at holding potentials of 110 mV and 70 mV.
The concentration required for half-maximal block was ~2
µM when current was elicited from 110 mV but only
~0.1 µM when current was elicited from 70 mV.
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70 mV, where
current is approximately 70% inactivated) than to closed resting
states. To distinguish between these possibilities, we quantitatively
compared the potency of inhibition and the voltage dependence of
inactivation. The potency of inhibition was determined for a range of
potentials from 110 mV to 70 mV, using concentrations of mibefradil
of 20 nM to 10 µM. The dose-response
relationship for inhibition could be fit reasonably well by the
equation 1/(1 + [mibefradil]/Kapp), the
relationship expected for 1:1 binding of drug to receptor (Fig.
5). Values for
Kapp increased with more hyperpolarized
holding voltages and approached saturation at holding voltages of 100
mV (849 nM) and 110 mV (1000 nM).
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h)/KI, where h is
the fraction of channels in the resting state and 1 h is the fraction of channels in the inactivated state (Bean et al., 1983). Fig. 6 examines
whether the relationship between Kapp and
holding potential matches this prediction. We determined the voltage
dependence of inactivation of T-type channels under the same
experimental conditions as those under which
Kapp was determined (Fig. 6A). Inactivation
determined with the holding potential established for 4 sec could be
fit well by a Boltzmann function (half-maximal voltage
V1/2 = 75 mV, slope k = 7.3 mV) (Fig. 6A). Fig. 6B shows that, with h defined by this
function, the voltage dependence of Kapp
can be fit well assuming KR = 1.2 µM and KI =77
nM. The correspondence between the voltage
dependence of inactivation and the voltage dependence of mibefradil
affinity supports the hypothesis that the increased potency of
mibefradil at more depolarized holding potentials is the result of a
higher affinity of the drug for inactivated channels.
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Voltage dependence of mibefradil kinetics.
The increased
mibefradil affinity for inactivated states, compared with resting
states, could reflect a faster on-rate of drug binding, a slower
off-rate, or some combination of the two. To investigate possible
state-dependent differences in mibefradil kinetics, we examined the
onset and reversal of inhibition at different holding potentials. The
rate of inhibition was not obviously different at different voltages
(data not shown). However, the rate of inhibition may not reflect only
the interaction between mibefradil and the channel, because as the
mibefradil concentration was increased 100-fold, from 500 nM to 5 µM, the time constant of inhibition
decreased only modestly, from 8.2 to 2.7 sec (n = 3;
tested at a holding potential of 100 mV), and the relationship between the rate of inhibition and the drug concentration was highly
nonlinear. One possibility is that the onset of mibefradil inhibition
is limited by a step such as mibefradil partitioning into the membrane,
rather than interaction with the channel. Thus, we cannot rule out the
possibility that the on-rate for drug binding to the channel is
voltage-dependent.
100 mV (
= 19 sec) than at 80 mV ( = 42 sec). In combined
results from multiple cells measured with the same protocol (Fig. 7C),
the effective rate constant for recovery increased approximately 8-fold
from 70 mV (0.009 ± 0.003 sec
1) to
110 mV (0.07 ± 0.005 sec1). This
suggests faster unbinding of drug from resting channels than from
inactivated channels. The 8-fold change in the off-rate can account for
most of the change in the apparent dissociation constant between 70
and 110 mV (~12-fold) (Fig. 5). The effective rate constant for
recovery from block probably does not accurately measure the actual
unbinding of drug molecules, because the rate of recovery depended
somewhat on the mibefradil concentration and the length of exposure.
For example, in one cell the rate of recovery exhibited predominant
time constants of 31, 46, and 53 sec after application of 5 µM mibefradil for 4, 14, or 34 sec (measured at 90 mV).
Also, for the longest application, washout was clearly nonexponential.
Reversal after prolonged mibefradil application may be slowed because
of accumulation of mibefradil within the membrane or the cell. For this
reason, the results in Fig. 7 were determined with short (~4-sec)
exposures.
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Selectivity for T-type over P-type channels in Purkinje
neurons.
We directly compared the potency of mibefradil at T-type
channels and P-type calcium channels. Fig.
8 shows combined T-type and P-type
currents recorded from a Purkinje neuron (with no -conotoxin-MVIIC in the external solution) at 35°, with a holding voltage of 70 mV.
Physiologically, the resting potential of Purkinje neurons is typically
positive to 70 mV (Raman and Bean, 1997). The transient current, corresponding to T-type current, was abolished by 500 nM mibefradil, but the steady state current, corresponding
to P-type current, was inhibited only ~7% at a test voltage of 20 mV. The effects of mibefradil were fully reversible (data not shown).
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70 mV to inactivate most T-type currents and were measured near the
end of test pulses to avoid the small remaining T-type currents.
Sequential applications of increasing concentrations of mibefradil to
P-type channels at a holding potential of 70 mV showed a
half-blocking concentration of ~3 µM (Fig.
9A). For comparison, the dose-response
relationship for inhibition of T-type currents was determined under
identical conditions (except with 10 µM
-conotoxin-MVIIC included in the external solution). Mibefradil potency for T-type currents was somewhat higher at room temperature, with a half-blocking concentration of ~20 nM, than at
35° (half-block by ~85 nM). Fig.
10 shows combined dose-response results
for block of P-type current and T-type current at 70 mV at room
temperature. Curves were fitted to the equation 1/(1 + [mibefradil]/Kapp); best fits gave
Kapp values of 3 µM for
mibefradil at P-type channels and 14 nM for mibefradil at
T-type channels. Thus, the selectivity of mibefradil for T-type over
P-type channels was ~200-fold, when currents were measured under the
same conditions.
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Discussion |
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Top
Summary
Introduction
Materials & Methods
Results
Discussion
References
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Voltage dependence and kinetics. Our results show that mibefradil produces potent and selective block of T-type calcium channels in cerebellar Purkinje neurons. Any quantitative consideration of the potency or selectivity of mibefradil must take into account the pronounced voltage dependence of its action, whereby inhibition is more powerful at less hyperpolarized holding voltages. This voltage dependence seems to reflect voltage-dependent gating of the channel and preferential binding of drug molecules to particular gating states. In particular, the voltage dependence of drug potency closely mirrors the voltage dependence of inactivation, suggesting high affinity binding to inactivated states of the channel.
The strong voltage dependence of mibefradil potency in Purkinje neurons contrasts with the mild voltage dependence (and weaker block) of T-type channels in neuroblastoma cells (Randall and Tsien, 1997) and other
cell lines (Mehrke et al., 1994). Interestingly, little or
no voltage dependence for block of calcium channels was also reported
for vascular muscle (Mishra and Hermsmeyer, 1994b). In contrast, there
was strong voltage dependence for inhibition of cardiac T-type channels
(Bénardeau and Ertel, 1998), similar to our results. Further
characterization of the molecular biological characteristics of T-type
channels should reveal whether the differences in the voltage
dependence of block among various cell types are correlated with
expression of different gene products or splice variants. The 15-fold
difference in affinity for inactivated versus resting channels
suggested by our data (Fig. 7C) can be compared with the 30-70-fold
differences seen for mibefradil block of cloned 1A, 1B, 1C,
and 1E channels (Bezprozvanny and Tsien, 1995), although the
absolute potency of the drug at those channels was 1 order of
magnitude lower than that at T-type channels in Purkinje neurons.
The tighter binding of drug to inactivated channels is at least partly
attributable to a slower rate of dissociation from inactivated
channels, because the apparent off-rate was 8-fold faster with a
holding voltage of 110 mV (resting channels) than with a holding
voltage of 70 mV (mostly inactivated channels). A similar difference
in the apparent off-rate was found for cloned 1A, 1B, 1C, and
1E channels (Bezprozvanny and Tsien, 1995), consistent with
fundamental similarities in the basis of the voltage dependence of
mibefradil. In our experiments, the weak dependence of the apparent
on-rate on the mibefradil concentration and the concentration
dependence of the apparent off-rate suggest that the situation is more
complicated than direct bimolecular interaction of mibefradil molecules
in the extracellular solution with binding sites. Mibefradil might gain
access to its binding site by partitioning into the cell membrane. This
could explain the complexities of the on and off kinetics. Given these
complexities, the results are probably consistent with the voltage
dependence of drug binding arising entirely from changes in the rate of unbinding.
Potency.
The potency of mibefradil in inhibiting T-type
channels in Purkinje neurons is greater than previously reported for
other types of calcium channels, including T-type channels in other cell types. In previous studies of T-type currents in various neuronal
cells, the concentration of mibefradil producing 40-60% block was 1 µM in neuroblastoma cells (Randall and Tsien, 1997), 3 µM in rat sensory neurons (Todorovic and Lingle, 1998),
and 1 µM in spinal motoneurons (Viana et al.,
1997); only mild voltage dependence was reported in these cases. We
found half-block with concentrations ranging from 14 nM to
1 µM, depending on the holding potential and the
temperature. Interestingly, this high potency is closer to that
observed in vascular smooth muscle (half-block by ~100 nM
at a holding potential of 80 mV) (Mishra and Hermsmeyer, 1994a) than
to potencies observed in other neuronal preparations. These results
seem to indicate that the T-type channels in Purkinje neurons may be
different from those in neuroblastoma cells, sensory neurons, or spinal
motoneurons. However, in making these comparisons, the loss of potency
that we noted when the drug had been stored for many months at either
room temperature or 20° must be taken into consideration. It was
important that we made our measurements within approximately 1 month of
receiving fresh drug; the condition of the drug may be an important
variable in different studies.
Selectivity.
Our results strongly support the idea that
mibefradil is a selective blocker of T-type calcium channels, compared
with other calcium channels (Clozel et al., 1997; Ertel
et al., 1997; Hermsmeyer et al., 1997), and they
indicate that this selectivity is also found in neurons. The
direct comparison of block of T-type channels and P-type channels at
the same physiological holding potential showed that concentrations of
200-500 nM could produce essentially complete inhibition
of T-type channels, with <5% inhibition of P-type channels. We did
not attempt to quantify the sensitivity of L-type or N-type
currents, which each contribute <5-10% of the overall high-threshold
current in Purkinje neurons. However, because Bezprozvanny and Tsien
(1995) found that cloned 1A and 1B channels showed similar
sensitivities (both similar to our measurements of native P-type
channels) and that 1C channels were considerably less sensitive,
there is no reason to expect that native N-type or L-type
channels would be any more sensitive than P-type channels. Consistent
with this, Viana et al. (1997) found relatively low
sensitivity (EC50 ~ 3 µM) of
L-type currents in spinal motoneurons.
Utility. Mibefradil is the first pharmacological agent with enough selectivity to inhibit neuronal T-type currents without significantly affecting other types of calcium channels. It should be useful for studies of the functional roles of neuronal T-type channels, which are still incompletely understood.
The lack of a high affinity ligand for T-type channels is one reason why no biochemical studies of these channel proteins have been possible. Because channels are presumably maximally inactivated in membrane preparations, they would be in the high affinity state in such preparations. Our results suggest a dissociation constant at least as low as ~15-50 nM, possibly near the range allowing radioligand studies. Mibefradil does not cross-the blood-brain barrier, so no effects on central neurons should occur with oral administration. If mibefradil or a membrane-permeant analog did gain access to the central nervous system, it is conceivable that potent block of T-type channels might produce anticonvulsant effects similar to those of ethosuximide (Coulter et al., 1990; Huguenard, 1996). In fact, mibefradil
block of neuronal T-type channels is much more potent than that by
ethosuximide (or any other known blocker), and the weak activity
against other calcium channel types should leave their functions
(including normal neurotransmission) unperturbed.
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Acknowledgments |
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We thank Chinfei Chen and Abraha Taddese for helpful discussions and comments on the manuscript.
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Footnotes |
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Received May 26, 1998; Accepted August 25, 1998
This work was supported by National Institutes of Health Grant NS36855.
Send reprint requests to: Dr. Bruce P. Bean, Department of Neurobiology, Harvard Medical School, 220 Longwood Ave., Boston, MA 02115. E-mail: bbean{at}warren.med.harvard.edu
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Abbreviations |
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HEPES, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid;
EGTA, ethylene
glycol bis(
-aminoethyl
ether)-N,N,N',N'-tetraacetic
acid.
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References |
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Top
Summary
Introduction
Materials & Methods
Results
Discussion
References
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-conotoxin MVIIC.
J Neurosci
16:
2612-2623 subunits and its modulation by Ro 40-5967.
Pflügers Arch
429:
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M. A. Diana, Y. Otsu, G. Maton, T. Collin, M. Chat, and S. Dieudonne T-Type and L-Type Ca2+ Conductances Define and Encode the Bimodal Firing Pattern of Vestibulocerebellar Unipolar Brush Cells J. Neurosci., April 4, 2007; 27(14): 3823 - 3838. [Abstract] [Full Text] [PDF]
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Y. Kim and L. O. Trussell Ion Channels Generating Complex Spikes in Cartwheel Cells of the Dorsal Cochlear Nucleus J Neurophysiol, February 1, 2007; 97(2): 1705 - 1725. [Abstract] [Full Text] [PDF]
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M. A. Castro-Alamancos, P. Rigas, and Y. Tawara-Hirata Resonance (~10 Hz) of excitatory networks in motor cortex: effects of voltage-dependent ion channel blockers J. Physiol., January 1, 2007; 578(1): 173 - 191. [Abstract] [Full Text] [PDF]
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M. L. Molineux, F. R. Fernandez, W. H. Mehaffey, and R. W. Turner A-Type and T-Type Currents Interact to Produce a Novel Spike Latency-Voltage Relationship in Cerebellar Stellate Cells J. Neurosci., November 23, 2005; 25(47): 10863 - 10873. [Abstract] [Full Text] [PDF]
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B. E McKay and R. W Turner Physiological and morphological development of the rat cerebellar Purkinje cell J. Physiol., September 15, 2005; 567(3): 829 - 850. [Abstract] [Full Text] [PDF]
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A. M. Swensen and B. P. Bean Robustness of Burst Firing in Dissociated Purkinje Neurons with Acute or Long-Term Reductions in Sodium Conductance J. Neurosci., April 6, 2005; 25(14): 3509 - 3520. [Abstract] [Full Text] [PDF]
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 M. M. McNulty and D. A. Hanck State-Dependent Mibefradil Block of Na+ Channels Mol. Pharmacol., December 1, 2004; 66(6): 1652 - 1661. [Abstract] [Full Text] [PDF]
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C. S. Chan, R. Shigemoto, J. N. Mercer, and D. J. Surmeier HCN2 and HCN1 Channels Govern the Regularity of Autonomous Pacemaking and Synaptic Resetting in Globus Pallidus Neurons J. Neurosci., November 3, 2004; 24(44): 9921 - 9932. [Abstract] [Full Text] [PDF]
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M. D. Womack, C. Chevez, and K. Khodakhah Calcium-Activated Potassium Channels Are Selectively Coupled to P/Q-Type Calcium Channels in Cerebellar Purkinje Neurons J. Neurosci., October 6, 2004; 24(40): 8818 - 8822. [Abstract] [Full Text] [PDF]
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M. D. Womack and K. Khodakhah Dendritic Control of Spontaneous Bursting in Cerebellar Purkinje Cells J. Neurosci., April 7, 2004; 24(14): 3511 - 3521. [Abstract] [Full Text] [PDF]
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L. Huang, B. M. Keyser, T. M. Tagmose, J. B. Hansen, J. T. Taylor, H. Zhuang, M. Zhang, D. S. Ragsdale, and M. Li NNC 55-0396 [(1S,2S)-2-(2-(N-[(3-Benzimidazol-2-yl)propyl]-N-methylamino)ethyl)-6-fluoro-1,2,3,4-tetrahydro-1-isopropyl-2-naphtyl cyclopropanecarboxylate dihydrochloride]: A New Selective Inhibitor of T-Type Calcium Channels J. Pharmacol. Exp. Ther., April 1, 2004; 309(1): 193 - 199. [Abstract] [Full Text]
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M. Cataldi, A. Gaudino, V. Lariccia, M. Russo, S. Amoroso, G. di Renzo, and L. Annunziato Imatinib-Mesylate Blocks Recombinant T-Type Calcium Channels Expressed in Human Embryonic Kidney-293 Cells by a Protein Tyrosine Kinase-Independent Mechanism J. Pharmacol. Exp. Ther., April 1, 2004; 309(1): 208 - 215. [Abstract] [Full Text]
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G. Pinato and J. Midtgaard Regulation of Granule Cell Excitability by a Low-Threshold Calcium Spike in Turtle Olfactory Bulb J Neurophysiol, November 1, 2003; 90(5): 3341 - 3351. [Abstract] [Full Text] [PDF]
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A. M. Swensen and B. P. Bean Ionic Mechanisms of Burst Firing in Dissociated Purkinje Neurons J. Neurosci., October 22, 2003; 23(29): 9650 - 9663. [Abstract] [Full Text] [PDF]
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C. J. Habermann, B. J. O'Brien, H. Wassle, and D. A. Protti AII Amacrine Cells Express L-Type Calcium Channels at Their Output Synapses J. Neurosci., July 30, 2003; 23(17): 6904 - 6913. [Abstract] [Full Text] [PDF]
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Z. M. Khaliq, N. W. Gouwens, and I. M. Raman The Contribution of Resurgent Sodium Current to High-Frequency Firing in Purkinje Neurons: An Experimental and Modeling Study J. Neurosci., June 15, 2003; 23(12): 4899 - 4912. [Abstract] [Full Text] [PDF]
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R. K. Cloues and W. A. Sather Afterhyperpolarization Regulates Firing Rate in Neurons of the Suprachiasmatic Nucleus J. Neurosci., March 1, 2003; 23(5): 1593 - 1604. [Abstract] [Full Text] [PDF]
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 E. Perez-Reyes Molecular Physiology of Low-Voltage-Activated T-type Calcium Channels Physiol Rev, January 1, 2003; 83(1): 117 - 161. [Abstract] [Full Text] [PDF]
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P. Benquet, J. Le Guen, Y. Pichon, and F. Tiaho Differential Involvement of Ca2+ Channels in Survival and Neurite Outgrowth of Cultured Embryonic Cockroach Brain Neurons J Neurophysiol, September 1, 2002; 88(3): 1475 - 1490. [Abstract] [Full Text] [PDF]
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 P. Mariot, K. Vanoverberghe, N. Lalevee, M. F. Rossier, and N. Prevarskaya Overexpression of an alpha 1H (Cav3.2) T-type Calcium Channel during Neuroendocrine Differentiation of Human Prostate Cancer Cells J. Biol. Chem., March 22, 2002; 277(13): 10824 - 10833. [Abstract] [Full Text] [PDF]
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S. I. McDonough, L. M. Boland, I. M. Mintz, and B. P. Bean Interactions among Toxins That Inhibit N-type and P-type Calcium Channels J. Gen. Physiol., March 12, 2002; 119(4): 313 - 328. [Abstract] [Full Text] [PDF]
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R Bournaud, J Hidalgo, H Yu, E Jaimovich, and T Shimahara Low threshold T-type calcium current in rat embryonic chromaffin cells J. Physiol., November 15, 2001; 537(1): 35 - 44. [Abstract] [Full Text] [PDF]
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D. G Placantonakis and J. P Welsh Two distinct oscillatory states determined by the NMDA receptor in rat inferior olive J. Physiol., July 1, 2001; 534(1): 123 - 140. [Abstract] [Full Text] [PDF]
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I. M. Raman, A. E. Gustafson, and D. Padgett Ionic Currents and Spontaneous Firing in Neurons Isolated from the Cerebellar Nuclei J. Neurosci., December 15, 2000; 20(24): 9004 - 9016. [Abstract] [Full Text] [PDF]
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R. L. Martin, J.-H. Lee, L. L. Cribbs, E. Perez-Reyes, and D. A. Hanck Mibefradil Block of Cloned T-Type Calcium Channels J. Pharmacol. Exp. Ther., October 1, 2000; 295(1): 302 - 308. [Abstract] [Full Text]
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 W.-Y. Son, J.-H. Lee, J.-H. Lee, and C.-T. Han Acrosome reaction of human spermatozoa is mainly mediated by {alpha}1H T-type calcium channels Mol. Hum. Reprod., October 1, 2000; 6(10): 893 - 897. [Abstract] [Full Text] [PDF]
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F Pouille, P Cavelier, T Desplantez, H Beekenkamp, P J Craig, R E Beattie, S G Volsen, and J L Bossu Dendro-somatic distribution of calcium-mediated electrogenesis in Purkinje cells from rat cerebellar slice cultures J. Physiol., September 1, 2000; 527(2): 265 - 282. [Abstract] [Full Text] [PDF]
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J. C. Gomora, L. Xu, J. A. Enyeart, and J. J. Enyeart Effect of Mibefradil on Voltage-Dependent Gating and Kinetics of T-Type Ca2+ Channels in Cortisol-Secreting Cells J. Pharmacol. Exp. Ther., January 1, 2000; 292(1): 96 - 103. [Abstract] [Full Text]
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J. C. Gomora, J. A. Enyeart, and J. J. Enyeart Mibefradil Potently Blocks ATP-Activated K+ Channels in Adrenal Cells Mol. Pharmacol., December 1, 1999; 56(6): 1192 - 1197. [Abstract] [Full Text]
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I. M. Raman and B. P. Bean Ionic Currents Underlying Spontaneous Action Potentials in Isolated Cerebellar Purkinje Neurons J. Neurosci., March 1, 1999; 19(5): 1663 - 1674. [Abstract] [Full Text] [PDF]
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 P. B. Hansen, B. L. Jensen, D. Andreasen, and O. Skott Differential Expression of T- and L-Type Voltage-Dependent Calcium Channels in Renal Resistance Vessels Circ. Res., September 28, 2001; 89(7): 630 - 638. [Abstract] [Full Text] [PDF]
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