1Department of Anatomy and Neurobiology, University of Tennessee, Memphis, Tennessee 38163; and 2Department of Physiology, Northwestern University Medical School, Chicago, Illinois 60611
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ABSTRACT |
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Foehring, Robert C.,
Paul G. Mermelstein,
Wen-Jie Song,
Sasha Ulrich, and
D. James Surmeier.
Unique Properties of R-Type Calcium Currents in Neocortical and
Neostriatal Neurons.
J. Neurophysiol. 84: 2225-2236, 2000.
Whole cell recordings from acutely
dissociated neocortical pyramidal neurons and striatal medium spiny
neurons exhibited a calcium-channel current resistant to known blockers
of L-, N-, and P/Q-type Ca2+ channels. These
R-type currents were characterized as high-voltage-activated (HVA) by
their rapid deactivation kinetics, half-activation and half-inactivation voltages, and sensitivity to depolarized holding potentials. In both cell types, the R-type current activated at potentials relatively negative to other HVA currents in the same cell
type and inactivated rapidly compared with the other HVA currents. The
main difference between cell types was that R-type currents in
neocortical pyramidal neurons inactivated at more negative potentials
than R-type currents in medium spiny neurons. Ni2+ sensitivity was not diagnostic for R-type
currents in either cell type. Single-cell RT-PCR revealed that both
cell types expressed the 1E mRNA, consistent with this subunit being
associated with the R-type current.
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INTRODUCTION |
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Both pharmacological and
molecular techniques have indicated a diversity of
high-voltage-activated (HVA) Ca2+ channel types
in neurons. This diversity has been associated with differences in
biophysical properties, subcellular location, coupling to cellular
events and to differential modulation by neurotransmitters
(Hille 1994).
At least four HVA Ca2+ channel 1 subunits have
been identified in CNS neurons (
1A-D) (Birnbaumer et al.
1994
; Snutch and Reiner 1992
; Tsien et
al. 1995
). The
1B subunit has been identified with N-type
channels, which are blocked by
-conotoxin GVIA (Ctx GVIA)
(McCleskey et al. 1987
).
1C and
1D subunits
correspond to L-type channels, which are sensitive to dihydropyridines
(Bean 1989
; Tsien et al. 1988
). The
1A
subunit, associated with P- and Q-type channels (Bourinet et al.
1996
; Mermelstein et al. 1999
;
Piedras-Renteria and Tsien 1998
; Stea et al.
1994
), is sensitive to
-agatoxin IVA and Ctx-MVIIC
(Birnbaumer et al. 1994
; Mintz et al.
1992a
,b
; Randall and Tsien 1995
). Another
calcium channel
1 subunit (
1F) is only found in the retina and
presumed to form L-type channels because of sequence homology to
1C
and
1D (Bech-Hansen et al. 1998
; Strom et al.
1998
).
There has been some uncertainty, however, as to the type
of currents generated by channels possessing the 1E subunit. The
1E subunit was originally suggested to be associated with
low-voltage-activated, T-type currents (Bourinet et al.
1996
; Soong et al. 1993
), but may be associated
with HVA, R-type currents (Piedras-Renteria and Tsien
1998
; Schneider et al. 1994
). To further confuse
the issue, there are several reported similarities between R-type and
T-type currents. By definition, R-type currents are insensitive to the
aforementioned organic Ca2+ channel antagonists
(Birnbaumer et al. 1994
; Randall and Tsien 1995
); however, T-type currents are also generally not blocked by these peptides or dihydropyridines (Huguenard 1996
).
Further, both currents are reported to be highly sensitive to
Ni2+ block and, with strong depolarizations, to
inactivate rapidly (Huguenard 1996
; Randall and
Tsien 1995
).
Some reports suggest differences between R- and T-type
currents. First, in most cell types, T-type currents activate ~20-30 mV more negative than R-type currents. Second, R-type currents deactivate with time constants of a few hundred microseconds
(Randall and Tsien 1995, 1997
); 10 times
faster than T-type currents (Huguenard 1996
;
Matteson and Armstrong 1986
). Third, the inactivation
kinetics of T-type currents are steeply voltage dependent
(Carbone and Lux 1984
; Lee et al. 1999
;
Randall and Tsien 1997
), whereas those of R-type
currents are less so (Randall and Tsien 1997
). Last, because of the pronounced voltage-dependent inactivation of T-type currents, they are largely inactivated at holding potentials more positive than
50 mV (Huguenard 1996
).
While R-type Ca2+ currents have been
observed in a variety of neuronal populations, reports on their
biophysical properties have varied. This may be due to channel
heterogeneity or to contamination by other voltage-dependent calcium
currents that are not completely blocked under standard recording
conditions. To unequivocally determine the properties of R-type
currents in cortical and striatal neurons, we used three distinct
pharmacological regimens as well as a biophysical strategy. To provide
a complementary level of analysis, neurons also were subjected to
single-cell RT-PCR analysis of class E 1 subunit expression.
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METHODS |
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Acute isolation
Four- to 6-wk-old Sprague-Dawley rats were anesthetized with methoxyfluorane and then decapitated. The brains were extracted and placed in an oxygenated high sucrose solution that contained (in mM) 250 sucrose, 2.5 KCl, 1 NaH2PO4, 11 glucose, 4 MgSO4, 0.1 CaCl2, 1 kynurenic acid, 1 pyruvic acid, 0.1 nitro-arginine, 0.05 glutathione, and 15 HEPES (pH 7.3 adjusted with 1N NaOH; 300 mOsm/l). The tissue was then sliced into 400-µM sections using a vibrating tissue slicer (Cambden Instruments). The slices were held for a minimum of 1 h in a carboxygen (95% O2-5% CO2)-bubbled artificial cerebral spinal fluid (ACSF) that contained (in mM) 125 NaCl, 3 KCl, 2 CaCl2, 2 MgCl2, 1.25 NaH2PO4, 26 NaHCO3, 20 glucose, 1 kynurenic acid, 1 pyruvic acid, 0.1 nitroarginine, and 0.05 glutathione (pH 7.4 adjusted with 1 N NaOH; 310 mOsm/l). The primary motor and primary somatosensory cortex (hereafter referred to as sensorimotor cortex) or the dorsal striatum (posterior to anterior commisure) was then dissected from the slices with the aid of a stereo microscope.
The dissected tissue was incubated for 20-30 min in an oxygenated
ACSF-containing Pronase E (Sigma protease type XIV, 1.0 mg/ml at
32-35°C) (cf. Bargas et al. 1994; Lorenzon and
Foehring 1995
), 1 mM kynurenic acid, 1 mM pyruvic acid, 0.1 mM
nitro-arginine, and 0.05 mM glutathione. Following the incubation
period, the tissue was rinsed in a Na isethionate solution that
contained (in mM) 140 Na isethionate, 2 KCl, 1 MgCl2, 23 glucose, 15 HEPES, 1 kynurenic acid, 1 pyruvic acid, 0.1 nitro-arginine, and 0.05 glutathione (pH 7.3 adjusted
with 1 N NaOH; 310 mOsm/l). The tissue was then triturated in the same
solution using fire-polished Pasteur pipettes. The supernatant was
collected and then poured into a plastic Petri dish (Lux) positioned on
the stage of an inverted microscope (Nikon Diaphot 300). The cells were
allowed several minutes to adhere to the Petri dish, and then a
background flow of HEPES-buffered saline solution was initiated (~1
ml/min). This solution contained (in mM) 10 HEPES, 138 NaCl, 3 KCl, 1 MgCl2, and 2 CaCl2, pH 7.3 adjusted with 1 N NaOH, 300 mOsm/l.
Cultured cell preparation
For a few experiments, medium spiny cells from embryonic
day 19 (E19) rat embryos were cultured for 2 wk
according to the procedure outlined in Bargas et al.
(1991). The cells were maintained in 5%
CO2 at 37°C.
Recording solutions and pharmacological agents
The external recording solution used to isolate
Ca2+ channel currents consisted of (in mM) 125 NaCl, 20 CsCl, 1 MgCl2, 10 HEPES, 5 BaCl2, 0.001 TTX, and 10 glucose (pH 7.3 with
TEA-OH; 300-305 mOsm/l). The internal recording solution included the
following (in mM): 180 N-methyl-D-glucamine
(NMG), 4 MgCl2, 40 HEPES, 5-10 ethylene
glycol-bis(-aminoethyl ether)-N,N,N',N'-tetraacetic acid
(EGTA) or
bis-(o-aminophenoxy)-N,N,N',N'-tetraacetic acid (BAPTA), 0.1 leupeptin, 0.4 guanosine triphosphate (GTP), 2 ATP, and
0.01 phosphocreatine, pH 7.2 (adjusted with 0.1 N
H2SO4), 265-275 mOsm/l.
The stock solutions of the calcium channel antagonists (with the
exception of nifedipine) were dissolved in water. Stock solutions of
the calcium channel antagonists
-conotoxin-GVIA (Ctx-GVIA: 500 µM),
-conotoxin-MVIIC (Ctx-MVIIC: 500 µM), and
-agatoxin-IVA
(AgTX: 100 µM; all from Bachem, Torrance, CA) were aliquoted and
frozen. Each of the stocks was diluted to the appropriate concentrations in the external recording solution immediately prior to
the experiment. Nifedipine (RBI, Nattick, MA) was first dissolved in
95% ethanol before being added to the external solution resulting in a
final ethanol concentration of <0.05%. This concentration of ethanol
had no effect on these cells (Bargas et al. 1994
;
Lorenzon and Foehring 1995
). Nifedipine was protected
from ambient light. Cytochrome C was combined with solutions containing
AgTX to prevent nonspecific binding of AgTX to glass and plastic
(Bargas et al. 1994
; Lorenzon and Foehring
1995
).
Single-cell RT-PCR
The methods utilized for the single-cell RT-PCR were similar to
those described previously (Surmeier et al. 1996;
Yan and Surmeier 1996
). Electrodes contained ~5 µl
of sterile recording solution (see above). Some cells were harvested
without recording, with electrodes filled with water. The capillary
glass used for making electrodes had previously been heated to 200°C
for 4 h. Sterile gloves were worn during the procedure to minimize
RNase contamination.
After aspiration, the electrode was broken and contents ejected into a
presiliconized, 0.5-ml Eppendorf tube containing 5 µl diethyl
pyrocarbonate (DEPC)-treated water, 0.5 µl RNAsin (28,000 U/ml), and
0.5 µl dithiothreitol (DTT; 0.1 M). One microliter of either oligo dT
(0.5 mg/ml) or random hexanucleotides (50 ng/ml) was added and mixed
before the mixture was heated at 70°C for 10 min and incubated on ice
for more than 1 min. Single-strand cDNA was synthesized from the
cellular mRNA by adding SuperScript II RT (1 µl, 200 U/ml), ×10 PCR
buffer [2 µl, 200 mM TrisCl (pH 8.4)], 500 mM KCl,
MgCl2 (2 µl, 25 mM), RNAsin (0.5 ml, 28,000 U/ml), DTT (1.5 µl, 0.1 M), and mixed dNTPs (1 µl, 10 mM). The reaction mixture (20 µl) was incubated at 42°C for 50 min. The reaction was terminated by heating the mixture to 70°C for 15 min and
then icing. The RNA strand in the RNA-DNA hybrid was then removed by
adding 1 µl RNase H (2 U/ml) and incubating for 20 min at 37°C. All
reagents except RNAsin (Promega, Madison, WI) were obtained from GIBCO
BRL (Grand Island, NY). The cDNA from the reverse transcription (RT) of
RNA in single cortical neurons was subjected to polymerase chain
reactions (PCR) to detect the expression of mRNAs coding for
Ca2+ channel 1 subunits.
Conventional PCR was carried out with a thermal cycler (MJ Research,
Watertown, MA) and thin-walled plastic tubes (Perkin Elmer, Norwalk,
CT). PCR primers were developed from GenBank sequences with the
commercially available software OLIGO (National Biosciences, Plymouth, MN) and have been described previously (Mermelstein et
al. 1999). To detect individual mRNAs, 2.5 µl of the
single-cell cDNA was used as a template for conventional PCR
amplification. Reaction mixtures contained 2-2.5 mM
MgCl2, 0.5 mM of each of the dNTPs, 1-µM
primers, 2.5 U Taq DNA polymerase and buffer (Promega). The
thermal cycling program was 94°C for 1 min, 58°C for 1 min and
72°C for 1.5 min for 45 cycles.
PCR products were separated by electrophoresis in 1.5-2% agarose gels and visualized by staining with ethidium bromide. In representative cases, amplicons were purified from the gel (QIAquick Gel Extraction Kit, QIAGEN, Hilden, Germany) and sequenced by the University of Tennessee Molecular Resource Center or St. Jude Children's Research Hospital Molecular Resource Center. These sequences were found to match published sequences.
PCR reactions were carried out following procedures designed to
minimize the chances of cross-contamination (Cimino et al. 1990). Negative controls for contamination from extraneous and genomic DNA were run for every batch of neurons. To ensure that genomic
DNA did not contribute to the PCR products, neurons were aspirated and
processed in the normal manner except that the reverse transcriptase
was omitted. Contamination from extraneous sources was checked by
replacing the cellular template with water. Both controls were
consistently negative in these experiments.
Whole cell recordings
Whole cell recordings were acquired using a DAGAN 8900 or an
Axopatch 200B electrometer. The recordings were monitored and controlled by pCLAMP6 (Axon Instruments) installed on a 486 computer. The electrodes were pulled from 7052 glass (Garner) and fire polished. Typically, series resistance compensation of 70-90% was employed. Cells were not included in the comparisons of biophysical properties if
the estimated series resistance-related voltage error (peak I * uncompensated series resistance) was >5 mV. Voltage
control was also assessed by observing tail currents after brief
voltage steps (see Bargas et al. 1994; Lorenzon
and Foehring 1995
). Cells were discarded if tail currents were
broad or unstable. The liquid junction potential was ~8 mV and was
not subtracted in the presented data. A gravity-fed parallel array of
glass tubes was used to apply the drugs to the cell being studied.
SYSTAT (SYSTAT, Evanston, IL) was used to carry out all statistical
calculations. The population data are represented as median, mean ± SEM, or box plots (Tukey 1977). In the box
plots, the internal line represents the median while the outer edges of
the box represent the inner quartiles of the data set. The bars
extending from the box depict the two outer quartiles of the data set.
Data points greater than two times the inner quartiles were considered
outliers and indicated as asterisks on the plots. Statistical
differences were determined with the Kruskal-Wallace (comparisons of
multiple populations) or Mann-Whitney U test (
0.05), unless otherwise noted.
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RESULTS |
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R-type currents
We used whole cell patch-clamp recordings from acutely dissociated
pyramidal cells (sensorimotor cortex) or medium spiny neurons (dorsal
striatum) to test whether there was a component of the current
resistant to block by organic Ca2+ channel
antagonists. Currents were evoked by repeated (every 5 s) voltage
steps (from 80 to 0 mV) or voltage ramps (0.3 mV/ms) from
80 to +60
mV (Bargas et al. 1994
; Lorenzon and Foehring 1995
).
In pyramidal cells, we employed three different methods to isolate the
R-type current. Two protocols involved sequential addition of specific
blockers of L-type (nifedipine: 5-10 µM), N-type (Ctx-GVIA: 1 µM),
and P/Q-type channel blockers (Fig. 1). In the first method, AgTx (100 nM) was added (in the presence of nifedipine and Ctx-GVIA) to block P-
and some Q-type current. Subsequently, Ctx-MVIIC (1-3 µM) was added
to nifedipine, GVIA, and AgTx to block Q-type currents (Fig.
1, A-C). The block of Q-type
current by Ctx-MVIIC was slow ( ~ 106 s)
(Mermelstein et al. 1999
), requiring ~10 min to reach a quasi steady-state level in most neurons (Foehring and
Armstrong 1996
; McDonough et al. 1996
;
Mermelstein et al. 1999
). With this combination of
blockers, 21 ± 3% of the initial current remained (median: 16%;
n = 18 cells; Fig. 1C). In most cells, this
component inactivated rapidly (Fig. 1B). Also, it was
completely blocked by 400 µM Cd2+ (Fig.
1A), indicating that it was carried through calcium
channels.
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In a separate group of cells (n = 16), L- and N- type
currents were blocked with nifedipine and Ctx-GVIA, P-type currents were blocked with 25 nM AgTx (Randall and Tsien 1995)
and Q-type currents with 1 µM AgTx (Randall and Tsien
1995
) (Fig. 1, D-F). The block of Q-type
current with µM AgTx had a
of ~45 s (Mermelstein et al.
1999
). Under these conditions, 21 ± 3% of the initial
current was unblocked (median: 20%; Fig. 1, C and
D). This current also inactivated rapidly (Fig.
1D) and was blocked completely with 400 µM
Cd2+ (Fig. 1D). We found no
significant differences in percentage of current remaining or its
biophysical properties (see Properties of R-type
current) using these two methods, so we combined data for
further analyses.
Dissociated medium spiny neurons were also tested with both isolation protocols, with no significant differences in the results, so the data were combined. Sequential application of the organic antagonists for >10 min left 18 ± 2% of the original current (median: 17%; n = 19; Fig. 2). In 8 of 10 cells tested with voltage steps, the resistant current inactivated rapidly. In the remaining two cells, the resistant current inactivated slowly (e.g., Fig. 2C).
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In a few cortical cells, we isolated the residual
current by preincubating the slices for at least 2 h in Ctx-MVIIC
(2 µM) and then recorded in the presence of 10 µM nifedipine, 1 µM Ctx-GVIA, 1-3 µM Ctx-MVIIC, and 100 nM AgTx (cf., Fig. 4). The
remaining current was small (median = 269 pA; n = 9 cells), but comparable in amplitude to the current isolated by
sequential addition of blockers (median: 201 pA; n = 31 cells). In cultured medium spiny neurons (see METHODS), a
component of the current was found to be insensitive to 12-h
preincubation in Ctx-MVIIC (1 µM) and recording in the presence of
Ctx-MVIIC plus nifedipine (5 µM; n = 9 cells; data
not shown). These data suggest that all three protocols effectively isolated R-type currents and that this current represents a substantial fraction of the somatodendritic Ca2+ current in
both pyramidal neurons and medium spiny neurons (see also
Churchill and MacVicar 1998; Eliot and Johnston
1994
; Foehring and Armstrong 1996
; Magee
and Johnston 1995
; Magnelli et al. 1998
; McDonough et al. 1996
; Mermelstein et al.
1999
; Pennington and Fox 1995
; Randall
and Tsien 1995
; Yu and Shinnick-Gallagher 1997
). We next characterized the biophysical and pharmacological properties of
the residual (R-type) current in both cell types.
Properties of R-type current
NEOCORTICAL PYRAMIDAL NEURONS.
We first measured the kinetics (time-to-peak;
activation) and steady-state voltage
dependence of current activation and the kinetics of deactivation (at
70 mV; Fig. 3). The average
time-to-peak (TTP) at 0 mV was 5.5 ± 0.3 ms (median: 5.1 ms;
n = 40 cells; Fig. 3, A and B),
significantly shorter than the TTP for N-, L-, or P-type currents in
these cells (Lorenzon and Foehring 1995
) (Table
1). The
activation was best fit by assuming
third-order kinetics and fitting the initial current trajectory with an
exponential function: I = Io * (exp
t/
)3 + b, where
I is current, Io is the
initial baseline, t is time, and b is the maximum
current. Mean
activation was 3.6 ± 0.4 ms at 0 mV (median: 3.2 ms; n = 15). Deactivation time
constants were determined by fitting an exponential to the tail current generated by stepping from 0 to
70 mV (Fig. 3A). The
average time constant was 221 ± 66 µs (median: 241 µs;
n = 24; Fig.
4B). This is similar to other
HVA subtypes in these cells (e.g., L-type: 0.4 ± 0.2 ms; N-type:
0.4 ± 0.2 ms) (Lorenzon and Foehring 1995
). This
feature clearly distinguished this resistant current from a T-type
current (Randall and Tsien 1995
, 1997
).
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Neostriatal medium spiny neurons (acutely dissociated)
As with pyramidal neurons, the current time-to-peak (at 0 mV) was
short (6.9 ± 0.5 ms; n = 6; Fig.
6A). The
activation (fit as in cortical neurons) was
5.1 ± 0.5 ms (n = 6) and
deactivation was 275 ± 47 µs
(n = 4; Fig. 6B), suggesting that the
residual current is an HVA current. The steady-state voltage dependence of activation was tested with voltage ramps (see above; Fig. 6, C and D). The mean
Vh was
15 ± 1.2 mV
(n = 10), and Vc was
7 ± 0.2 ms (n = 10).
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Most medium spiny neurons tested (8/10) had both rapid and slow components to inactivation at 0 mV (Fig. 7A). In these eight cells, the fast component had an inactivation time constant that averaged 66 ± 7 ms (53 ± 5% of inactivation); the slower component had a time constant that averaged 1,404 ± 176 ms (47 ± 5%). During a 400-ms step to 0 mV, the R-type current declined to 53 ± 4% (n = 8) of its original amplitude. In a fraction of the medium spiny neurons (2/10), the R current decayed with only a single time constant of intermediate rate (677, 899 ms).
|
We tested the voltage dependence of inactivation in medium spiny
neurons using a protocol similar to that described above for pyramidal
cells (Fig. 7C). The half-inactivation voltage averaged 33 ± 4 mV, with a slope factor of 17 ± 1 mV
(n = 6).
Ni2+ sensitivity
The R-type current in neurons (Churchill and MacVicar
1998; Eliot and Johnston 1994
; Forti et
al. 1994
; Magee and Johnston 1995
;
Randall and Tsien 1995
), as well as
1E-associated
current in expression systems (Soong et al. 1993
;
Williams et al. 1994
; Zamponi et al.
1996
) have been reported to be more sensitive to block by
Ni2+ than other HVA Ca2+
channels. To test this hypothesis, Ni2+
dose-response relationships were constructed for neocortical pyramidal
and striatal medium spiny neurons.
We first asked whether the current blocked by a low dose of Ni2+ (10-20 µM) inactivated rapidly, like R-type current. Of five neocortical pyramidal cells tested, the percent inactivation of the Ni2+-sensitive current at 400 ms was 23, 70, 0, 0, and 43%; these results suggest that Ni2+ did not selectively block the R current (data not shown). Similarly, in medium spiny neurons, Ni2+ (10 µM) blocked a current that was indistinguishable in inactivation rate from the total current (n = 5; data not shown).
We then tested the response of the whole Ba2+
current to doses of Ni2+ between 100 nM and 2 mM.
The data were obtained with either steps to 10 mV (30 ms) or with
voltage ramps. We plotted relative peak current
(I/Imax) as a function of
log dose of Ni2+. For pyramidal neurons, the
resulting curve was well fit by a single Langmuir isotherm of the form
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To test the Ni2+ sensitivity of the R-type current directly, we preincubated neocortical slices in 2 µM Ctx-MVIIC for at least 2 h and then recorded in the presence of 100 nM AgTx, 1 µM Ctx-GVIA, 10 µM nifedipine, and 2 µM Ctx-MVIIC (see METHODS). These data were also well fit by a single Langmuir isotherm, with an EC50 of ~48 µM (not shown).
Single-cell RT-PCR
Single-cell RT-PCR revealed that most neocortical pyramidal
neurons express detectable levels of mRNAs for all five HVA
Ca2+ channel 1 subunits tested (
1A-E; data
not shown). Detectable levels of class E
1 subunit mRNA were found
in six of seven identified pyramidal neurons (Fig.
8A). In addition, nearly every
cell expressed detectable levels of
1A (4/5 cells) (see
Mermelstein et al. 1999
) and
1B mRNA (4/5). All cells
expressed detectable levels of either
1C (5/5) or
1D (3/5) mRNA
(data not shown). Both
1C and
1D mRNA were co-expressed in three
of five of cells tested.
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Detectable levels of mRNA for 1E were also found in nearly every
striatal medium spiny neuron tested (14/15; Fig. 8B). The majority of medium spiny neurons express detectable levels of mRNA for
1A,
1B, and
1C, or
1D (Bargas et al. 1994
;
Mermelstein et al. 1999
; Song and
Surmeier 1996
).
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DISCUSSION |
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We have previously provided pharmacological evidence for the
expression of L-, N-, P-, and Q-type channels in neocortical pyramidal
and striatal medium spiny cells (Bargas et al. 1994; Lorenzon and Foehring 1995
; Mermelstein et al.
1999
). Here we demonstrate that a component of the HVA
Ca2+ current in both cell types is resistant to
block by organic antagonists (R-type). We characterized the biophysical
properties and Ni2+ sensitivity of these R-type
currents. Single-cell RT-PCR was used to test for the expression of
1E subunits in these cells.
The data presented here and our previous work (Bargas et al.
1994; Mermelstein et al. 1999
) demonstrate that
neocortical pyramidal neurons and striatal medium spiny neurons express
mRNAs for at least five different
1 subunits:
1A, B, C, D, and E. The
1E mRNA was ubiquitously expressed in both pyramidal and medium
spiny neurons, in agreement with previous in situ hybridization studies reporting the presence of
1E mRNA in cortex and dorsal striatum (Ludwig et al. 1997
; Plant et al. 1998
;
Soong et al. 1993
; Tanaka et al. 1995
;
Williams et al. 1994
; Yokayama et al.
1995
). Considering that
1A-D subunits are associated with
P-, Q-, N-, and L-type currents (Mermelstein et al.
1999
; Piedras-Renteria and Tsien 1998
;
Stea et al. 1994
), these data are also consistent with
1E subunits being associated with R-type. The association between
1E subunits and R-type currents has been suggested on the basis of
sequence homology between the
1E subunit and other HVA subunits (Williams et al. 1994
). Also, macroscopic current
properties are similar in
1E-associated channels expressed in
oocytes (Williams et al. 1994
) or cell lines
(Page et al. 1997
; Stephens et al. 1997
;
Williams et al. 1994
) to R-type currents in cerebellar
granule cells (Randall and Tsien 1995
). More recently,
antisense experiments have strengthened the link between R-type
currents and
1E subunits in cerebellar granule cells
(Piedras-Renteria and Tsien 1998
).
R-type versus T-type current
R-type current was first described in cerebellar granule neurons,
where relative to other HVA Ca2+ currents, R-type
currents inactivate more rapidly and at more negative potentials
(Randall and Tsien 1995, 1997
). In these
respects, R-type currents are similar to T-type currents. Granule cell
R-type currents (Randall and Tsien 1995
,
1997
) typically inactivate more slowly and activate and
inactivate at more positive potentials than T-type currents. Typical
T-type currents have half-activation voltages of approximately
50 to
60 mV and half-inactivation voltages of approximately
80 mV,
although a wide range of values has been reported (Huguenard
1996
; Lee et al. 1999
).
R- and T-type channels can most clearly be distinguished by their
deactivation kinetics. Whereas R-type currents in granule cells
deactivate with time constants of a few hundred microseconds at
negative potentials (Randall and Tsien 1995) (~240
µs at
80 mV), T-type currents deactivate with time constants of a
few milliseconds (Huguenard 1996
; Matteson and
Armstrong 1986
; Randall and Tsien 1997
). T-type
currents (but not R-type) also typically show "criss-crossing" traces in I-V protocols (Randall and Tsien
1997
). T-type and
1E channels can also be distinguished by
their single channel conductance (Schneider et al. 1994
;
Wakamori et al. 1994
; but see Meir and Dolphin
1998
). T-type channels are also generally more permeable to
Ca2+ than Ba2+
(Huguenard 1996
). There is some controversy as to
whether R-type and
1E channels are more permeable to
Ca2+ than Ba2+ (e.g.,
Bourinet et al. 1996
) or vice versa (e.g.,
Magnelli et al. 1998
; Williams et al.
1994
).
PYRAMIDAL AND MEDIUM SPINY NEURONS.
T-type 1 subunits (primarily
1G) (Talley et al.
1999
) and low-voltage-activated (LVA = T-type) currents
are expressed in neocortical pyramidal neurons (Sayer et al.
1990
; Tarasenko et al. 1998
; Ye and
Akaike 1993
). The LVA current inactivated with a
of ~24
ms (Tarasenko et al. 1998
) and the half-inactivation voltage was approximately
90 mV (Sayer et al. 1990
;
Tarasenko et al. 1998
; Ye and Akaike
1993
). T-type, but not R-type currents are completely
inactivated at holding potentials of approximately
50 mV in cortical
pyramidal cells (Sayer et al. 1990
; Ye and Akaike
1993
) and in other cell types (Huguenard 1996
).
When recording with Ba2+ as the charge carrier,
we find T-type to be nearly undetectable in most acutely dissociated
cells. When present, T-type currents are typically <50 pA in amplitude
(Lorenzon and Foehring 1995
; unpublished observations).
This was also true for medium spiny cells (Bargas et al.
1994
), although T-type currents are more prominent in embryonic
or cultured medium spiny neurons (Bargas et al. 1991
;
Hoehn et al. 1993
). The resistant current studied here
(both cell types) is clearly identified as HVA on the basis of
deactivation kinetics, lack of "criss-cross" in the I-V
curve, and relative insensitivity to depolarized holding potentials
(
50 mV). In addition, no current was observed to activate negative to
approximately
50 mV. The half-activation and inactivation voltages
for R-type current reported here (both cell types) were also more
positive than published values for T-type currents in cortical and
striatal neurons (Bargas et al. 1991
; Hoehn et
al. 1993
; Sayer et al. 1990
; Tarasenko et
al. 1998
; Ye and Akaike 1993
).
BIOPHYSICS OF R-TYPE CURRENTS.
The R-type current comprised ~18% of the whole cell
Ba2+ current in pyramidal neurons and 17% in
medium spiny neurons. This compares to ~15-20% in cerebellar
granule neurons (Randall and Tsien 1995), 32% in N. accumbens (Churchill and MacVicar 1998
), and ~40% in
embryonic motoneurons (Magnelli et al. 1998
). We
isolated the R-type current with combinations of organic blockers for
L- (nifedipine), N- (Ctx-GVIA), and P/Q-type channels (AgTx and
Ctx-MVIIC). We used three different isolation procedures, including
preincubation for several hours in MVIIC, to rule out the possibility
that the residual current reflects insufficient time for toxins to
exert their effects. The deactivation time constant was rapid in
pyramidal neurons (~221 µs at 0 mV) and medium spiny neurons
(~270 µs), similar to values reported for R-type current in
cerebellar granule cells (deactivation
was ~210 µs)
(Randall and Tsien 1997
), spinal motoneurons (330 µs)
(Magnelli et al. 1998
), and CA1 pyramidal cell dendrites
(~200 µs) (Kavalali et al. 1997
).
PHARMACOLOGY OF R-TYPE CURRENTS.
There was no clear evidence for a high affinity
Ni2+ block of the whole cell
Ba2+ current in either pyramidal cells or medium
spiny cells. The current sensitive to low doses (10-50 µM) of
Ni2+ was not rapidly inactivating in these cells.
In pyramidal cells, the small difference in Ni2+
affinity for the isolated R-type current versus the whole current (EC50s 48 vs. 85 µM) is of limited diagnostic
utility. Our data are consistent with studies on antagonist-resistant
currents in cerebellar granule cells (R: 66 µM: Zhang et al.
1993), dentate granule cells (Eliot and Johnston
1994
), raphe neurons (~50 µM: Pennington and Fox 1995
) and
expressed
1E channels (27 µM: Williams et al. 1994
;
28 µM: Soong et al. 1993
) or doe-1 channels (33 µM: Elinor et al. 1993
). In spinal motoneurons, the R-type
current was not especially Ni2+-sensitive
(Magnelli et al. 1998
). Zamponi et al.
(1996)
reported that in addition to channel block,
Ni2+ causes a shift in the voltage dependence of
gating that is greater in
1E channels than other channel types. In
oocytes, this leads to an apparent Ki at
10 mV
of 21 µM, which shifted to 144 µM at +10 mV. Our data were obtained
at
10 mV.
DIVERSITY OF R-TYPE CURRENTS. We have discussed several examples of variability in the biophysical properties of R-type currents in various cell types. The R-type currents in neocortical pyramidal neurons and striatal medium spiny neurons were rapidly activating, deactivating, and inactivating. In both cell types, activation occurred at relatively hyperpolarized potentials. The primary difference between these two cell types was in the voltage dependence of inactivation, which occurred at much more hyperpolarized potentials in pyramidal cells than in medium spiny neurons. Variability was evident in a single cell type as well, especially in the kinetics of inactivation. In medium spiny neurons, some cells only expressed a slowly inactivating current, whereas most cells had a prominent rapidly inactivating component as well. What could be the basis for the biophysical differences between resistant currents in different cell types?
R-type currents may be attributable to activation of more than one type of channel. Pietrobon and colleagues (Forti et al. 1994Functional consequences
The hyperpolarized activation voltage range of R-type current in
neocortical pyramidal cells and medium spiny neurons may facilitate a
role in synaptic integration in the subthreshold voltage range in those
cells. Consistent with a role in synaptic integration, these channels
are common in the dendrites of hippocampal pyramidal neurons
(Kavalali et al. 1997; Magee and Johnston
1995
; Yokayama et al. 1995
). R-type
currents contribute to Ca2+ action potentials in
cultured cerebellar granule neurons (D'Angelo et al.
1997
) and have also been shown to contribute to action potential-induced Ca2+ entry in calyx type
terminals in the rat medial trapezoid body (Wu et al.
1998
). R-type channels may also contribute to synaptic transmission from cerebellar inhibitory interneurons to Purkinje neurons (Doroshenko et al. 1997
). Because of
their hyperpolarized inactivation range and rapid kinetics of
inactivation, R-type currents would be more sensitive to sustained
depolarizations than other HVA current subtypes in pyramidal neurons
and thus may play a lesser role during sustained activity. Due to the
more depolarized half-inactivation voltage in medium spiny neurons, R-type current in these cells may be less inactivated during sustained depolarizations.
In expression systems, 1E-associated channels are generally reported
to be poorly modulated by a variety of signaling pathways (Page
et al. 1997
; Shekter et al. 1997
; Simen
and Miller 1998
; Toth et al. 1996
), although
there are exceptions (Meza and Adams 1998
; Yassin
et al. 1996
). The modulation may be sensitive to the
relationship between the
1E subunit and particular
subunits (Shekter et al. 1997
). In contrast, the
1E subunit
appears to be a substrate for phosphorylation by several kinases
(Yokayama et al. 1995
). In the calyx synapse in the
medial trapezoid body, metabotropic glutamate and
GABAB receptors inhibited R-type current (Wu et al. 1998
). As of yet, we have not found evidence
for modulations of R-type current in either neocortical pyramidal or
striatal medium spiny neurons. N- and P-type channels are substrates
for several pathways in these cells (Foehring 1996
;
Howe and Surmeier 1995
; Stewart et al.
1999
; Surmeier et al. 1995
).
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ACKNOWLEDGMENTS |
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Excellent technical assistance was provided by B. Harms and C. Windham.
This work was supported by National Institute of Neurological Disorders and Stroke Grants NS-33579 to R. C. Foehring, NS-34696 to D. J. Surmeier, and NS-10028 to P. G. Mermelstein.
Present addresses: P. G. Mermelstein, Dept. of Neuroscience, University of Minnesota, 4-140 Jackson Hall, 321 Church St. SE, Minneapolis, MN 55455; W.-J. Song, Division of Biophysical Engineering, Osaka University, Toyonaka, Osaka 560, Japan.
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FOOTNOTES |
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Address for reprint requests: R. C. Foehring, Dept. of Anatomy and Neurobiology, University of Tennessee, 855 Monroe Ave., Memphis, TN 38163 (E-mail: foehring{at}nb.utmem.edu).
Received 19 April 2000; accepted in final form 18 July 2000.
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REFERENCES |
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