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The Journal of Neuroscience, December 1, 2000, 20(23):8566-8571
The Status of Voltage-Dependent Calcium Channels in
 1E Knock-Out Mice
Scott M.
Wilson1,
Peter
T.
Toth2,
Seog Bae
Oh2,
Samantha E.
Gillard3,
Steven
Volsen4,
Dongjun
Ren2,
Louis H.
Philipson2,
E. Chiang
Lee1,
Colin F.
Fletcher1,
Lino
Tessarollo1,
Neal G.
Copeland1,
Nancy A.
Jenkins1, and
Richard J.
Miller2
1 Mouse Cancer Genetics Program, National Cancer
Institute-Frederick Cancer Research and Development Center, Frederick,
Maryland 21702, 2 Department of Neurobiology, Pharmacology,
and Physiology, The University of Chicago, Chicago, Illinois
60637, 3 Eli Lilly and Company, Lilly Corporate Center,
Indianapolis, Indiana 46285, and 4 Eli Lilly and Company,
Erl Wood, Manor, Windlesham, Surrey GU20 6PH, United Kingdom
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ABSTRACT |
It has been hypothesized that R-type Ca currents result from the
expression of the  1E gene. To test this hypothesis we
examined the properties of voltage-dependent Ca channels in mice in
which the 1E Ca channel subunit had been deleted.
Application of  -conotoxin GVIA, -agatoxin IVA, and
nimodipine to cultured cerebellar granule neurons from wild-type mice
inhibited components of the whole-cell Ba current, leaving a
"residual" R current with an amplitude of ~30% of the total Ba
current. A minor portion of this R current was inhibited by the
1E-selective toxin SNX-482, indicating that it
resulted from the expression of 1E. However, the
majority of the R current was not inhibited by SNX-482. The
SNX-482-sensitive portion of the granule cell R current was absent from
1E knock-out mice. We also identified a subpopulation of
dorsal root ganglion (DRG) neurons from wild-type mice that expressed
an SNX-482-sensitive component of the R current. However as with
granule cells, most of the DRG R current was not blocked by SNX-482. We
conclude that there exists a component of the R current that results
from the expression of the 1E Ca channel subunit but
that the majority of R currents must result from the expression of
other Ca channel subunits.
Key words:
dorsal root ganglia; cerebellar granule cells; pain; synaptic transmission; voltage-dependent calcium channels; 1E knock-out mice
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INTRODUCTION |
Voltage-sensitive Ca channels are of
great importance in coupling excitability to Ca-dependent events within
cells. It is clear that there are many different kinds of Ca channels.
The properties of these channels make them suitable for performing different tasks in neurons and other cells (Catterall, 1998 ). Voltage-sensitive Ca channels are multisubunit structures consisting of
a major pore-forming subunit (the 1 subunit)
and several ancillary subunits (Hoffman et al., 1999). At this time 10 different 1 subunits are known to exist (Ertel
et al., 2000). Seven of these (1A-1E,
1F, and 1S)
code for high-threshold Ca channels (Catterall, 1998; Hoffman et al.,
1999), whereas 1G-1I
code for low-threshold Ca channels (Cribbs et al., 1998; Perez-Reyes et
al., 1998; Lee et al., 1999). It has been important to try and
establish which 1 subunits are responsible for
the different types of Ca currents that can be recorded from particular
types of cells. However, there are many splice variants of the various
Ca channel subunits, and different combinations of these result in Ca
currents with a wide range of biophysical properties when they are
expressed in heterologous expression systems (Parent et al., 1997;
Pereverzev et al., 1998; Hoffman et al., 1999). Thus, the
potential diversity of Ca current types is very large. However, the
situation has been facilitated by the availability of a number of drugs
and toxins that selectively target different 1
subunits. Thus, -conotoxin GVIA is diagnostic for
1B, dihydropyridines are diagnostic for 1C, 1D, and
1S, -agatoxin IVA is diagnostic for
1A, etc. (Catterall, 1998; Hoffman et al.,
1999). One exception to this has been the Ca channel subunit encoded by
the 1E gene. Until very recently no specific
toxin or drug was known that targeted 1E Ca
currents when these were expressed in heterologous expression systems
(Newcombe et al., 1998). Consequently, the neuronal currents resulting
from the expression of 1E were not known with
certainty. There has been much speculation that neuronal Ca currents
that are insensitive to other drugs and toxins represent
1E currents (Hilaire et al., 1997;
Piedras-Renteria and Tsien, 1998; Tottene et al., 2000). Indeed, in
certain instances these "residual" or "R-type" Ca currents do
possess biophysical properties that resemble those resulting from the
expression of 1E subunits (Randall and Tsien,
1995; Tottene et al., 1996; Hilaire et al., 1997). Nevertheless, there
has been considerable controversy about the nature of
1E-based Ca currents in neurons, and very
little is known about their functions (Bourinet et al., 1996; Meir and
Dolphin, 1998). Recently, a toxin (SNX-482) has been described that
does selectively block the Ca currents that result from the expression
of the 1E subunit in heterologous expression
systems (Newcombe et al., 1998). Interestingly, this toxin was unable
to block neuronal R currents in several instances. On the other hand,
experiments using antisense knock-out of 1E
subunits in neurons have supported the idea that R currents do indeed
result from the expression of 1E subunits
(Piedras-Renteria and Tsien, 1998; Tottene et al., 2000).
To answer questions further concerning the nature and functions of
1E Ca currents, we have generated mice in
which the 1E gene has been deleted. We
demonstrate that R currents are heterogeneous, consisting of elements
that result from the expression of 1E as well
as other non-1E-dependent components.
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MATERIALS AND METHODS |
Construction of a Cacna1e null mutation. A
bacterial artificial chromosome (BAC) containing the Cacna1e
gene was isolated by screening the Research Genetics CITB 129/Sv
library with a Cacna1e cDNA. By the use of a probe specific
for sequences encoding domain II of the Cacna1e protein, an
~18 kb BamHI fragment was identified and cloned in the
yeast shuttle vector pRS314. This vector had been modified
previously to contain the thymidine kinase expression cassette. The DNA
sequence of this fragment was determined and revealed to contain 11 exons that encode all of domain II.
The plasmid containing the 18 kb Cacna1e fragment was
transformed into the yeast strain H1515 by LiCl. Yeast containing the Cacna1e plasmid were transformed with a PCR fragment that
had been generated using a template containing the Neo and URA3
expression cassettes. The PCR fragment was generated using chimeric
primers that contain 50 bp of intron sequences of Cacna1e
and 20 bp that correspond to the 5' or 3' ends of the selection
cassette. The fragment produced encodes both selection cassettes and 50 bp arms, which are homologous to intron 4 and intron 8. After selection on growth plates lacking uracil, DNA was isolated from uracil prototrophs and transformed into bacterial strain DH10B. Plasmids were
sequenced to confirm that the PCR fragment had integrated into the
desired site.
The Cacna1e targeting vector was linearized using
NotI and electroporated into CJ7 ES cells. After
selection with G418/FIAU, DNA was prepared from resistant clones
and analyzed by genomic blot hybridization. An external 3' probe was
used to screen the NdeI-digested DNA for the presence of a
rearranged 6.6 kb fragment and 6 kb endogenous fragment. Clones that
exhibited the correct rearrangement were then analyzed using a 5'
internal probe to screen EcoRI-digested DNA for the presence
of a 12 kb rearranged fragment and 8.7 kb endogenous fragment. Two
clones were selected for blastocyst injection and produced several
highly chimeric founder mice. Founders were then bred to C57BL/6J females.
Western blotting. Wild-type and 1E-deficient
mice were decapitated, and the brains were removed on ice. The
cerebellum was separated from the rest of the brain, and the brain
regions were processed as follows. Tissue was homogenized for 20 strokes in an ice-cold buffer containing 0.32 M sucrose, 5 mM Tris, 5 µg/ml aprotinin, 5 µg/ml pepstatin, 5 µg/ml leupeptin, and 1 mM 4-(2 aminoethyl)-benzenesulfonyl-fluoride, pH 7.4. Homogenates were centrifuged at 1000 × g for 10 min. The resulting
supernatant was diluted to 15 ml in sucrose buffer and centrifuged for
15 min at 20,000 × g. The supernatant was discarded,
and the outer membrane portion of the pellet was gently washed and
resuspended in 1 ml of sucrose buffer leaving the inner mitochondrial
pellet intact. The suspended pellet was centrifuged in a bench top
centrifuge at 14,000 rpm for 10 min at 4°C to remove the sucrose, and
the resulting pellet was resuspended in 5 mM Tris
and 2 mM EDTA, pH 7.4. Lysates were stored at
 80°C until use.
Protein samples were loaded onto a 6% polyacrylamide gel and run at
120 V for ~2 hr. Thirty micrograms of protein were loaded for brain
homogenates, whereas 15 µg of 1E-transfected
and untransfected human embryonic kidney (HEK) cell membranes was used.
After transfer onto nitrocellulose, blots were blocked with 5% skim
milk in Tris-buffered saline for 2 hr at room temperature. Blots
were incubated with polyclonal 1E antibody at
1 µg/ml for 2 hr at room temperature.
Anti-human 1E antibody was generated by
collaborators at Eli Lilly and Company. Amino acid sequences specific
for 1E were prepared as glutathione
S-transferase (GST) fusion proteins in Escherichia
coli. Antigens were derived from the IIS-6/IIIS-1 cytoplasmic loop
region spanning amino acids 984-1099 of the human 1E subunit. The antibody was purified by
protein A chromatography and affinity chromatography [for a detailed
account of antibody preparation and purification, refer to Beattie et
al. (1997)]. Antibody specificity was confirmed by ELISA and
immunocytochemistry using human embryonic kidney cells transfected with
appropriate 1 subunits (Volsen et al., 1995;
Beattie et al., 1997). The specific use of the
1E antibody used in these studies has been
reported previously (Volsen et al., 1995; Day et al., 1996; Beattie et al., 1997; Grabsch et al., 1999). After washing, blots were incubated with HRP-conjugated secondary antibody (1:20,000; Promega, Madison, WI)
for 1 hr at room temperature. Bands were visualized by incubation in
ECL chemiluminescent reagent and development on chemiluminescent film.
Dorsal root ganglion neuron culture. Dorsal root ganglia
(DRGs) were rapidly removed from all spinal segments of 2- to
5-d-old neonatal knock-out (KO) and wild-type mice under aseptic
conditions and digested sequentially in collagenase (Sigma, St. Louis,
MO), collagenase and dispase (Boehringer Mannheim, Indianapolis, IN), and trypsin (Life Technologies, Gaithersburg, MD) in HBSS (Life Technologies) for 10 min, respectively, at 37°C. Ganglia were washed
in DMEM three times and resuspended in F12 media (Life Technologies),
supplemented with 10% FBS (Summit Biotechnology), N2 supplement (Life Technologies), 50 ng/ml nerve
growth factor (Collaborative Biomedical Products), and penicillin and
streptomycin (100 µg/ml and 100 U/ml, respectively). Single neuronal
cells were obtained by trituration through a flame-polished pipette ~10 times, and the cell suspension was centrifuged at 800 rpm for 5 min. The pellet was resuspended in F12 with the additives listed above,
except that serum was reduced to 0.5%. Isolated DRG neurons were
plated on polyornithine (Sigma)- and laminin (Collaborative Biomedical
Products)-coated glass coverslips (25 mm in diameter) and incubated in
the same medium that was replaced every 48 hr. Cultures were maintained
at 37°C in a water-saturated atmosphere with 5%
CO2 for up to 2 weeks.
Preparation of cultured cerebellar neurons. Cultured
cerebellar neurons were prepared in a coculture system as described
previously (Brorson et al., 1991). Briefly, 17- to 19-d-old embryos
were removed from pregnant mice anesthetized previously with ether and
killed by cervical dislocation. The cerebella were dissected out and
incubated in trypsin (Worthington, Freehold, NJ; 100 mg/10 ml; 0.5 ml
of trypsin in 4.5 ml of HBSS containing 25 mM HEPES) for 20 min at 37°C. After washing, tissue was resuspended in DNase and
triturated through a flame-narrowed pipette until no visible tissue
fragments remained. Cells were plated at a density of 3 × 105 cell/ml onto 15 mm round glass
poly-L-lysine-coated coverslips. Neurons were suspended
over a feeder layer of cortical astrocytes and maintained in serum-free
defined medium containing 15 mM HEPES. After 2 d in culture at 37°C, in a humidified 5% CO2
atmosphere, 5-fluoro-2-deoxyuridine (5 µM final
concentration) was added to maintain astrocyte numbers at near
confluency. The cultures were fed every 3-4 d by replacement of half
of the medium. This preparation generated both cerebellar Purkinje
cells and granule cells, which could be distinguished by their
differences in size. Granule cells were used for electrophysiological
analysis between 10 and 20 d in vitro.
Whole-cell patch clamp. The tight-seal whole-cell
configuration of the patch-clamp technique (Hamill et al., 1981) was
used to record Ba currents. Recordings were made at room temperature (21-24°C). Currents were recorded using the Axopatch 1D (Axon Instruments, Foster City, CA) or EPC-7 (List Electronics, Darmstadt, Germany) amplifier, filtered at 2 kHz by the built-in filter of the
amplifier, and stored on the computer. Capacitative transients were
cancelled at 10 MHz, and their values were obtained directly, together
with the series resistance values from the settings of the Axopatch 1D
or EPC-7 amplifiers. Leak corrections were performed using a P/N
protocol. Command pulses were delivered at 30 sec intervals from 80
mV to +10 mV to elicit Ba current. Soft, soda-lime capillary glass was
used to make patch pipettes. Before seal formation, the resistances of
the recording electrodes were 4.5-6  M for granule cells and 2.5-4
M for DRG neurons. The extracellular buffer solution for whole-cell
voltage-clamp experiments was composed of (in mM): 151 tetraethylammonium chloride, 5 BaCl2, 1 MgCl2, 10 HEPES, and 10 glucose; pH was adjusted
to 7.4 with TEAOH. The standard internal solution consisted of (in
mM): 100 CsCl, 37 CsOH, 1 MgCl2, 10 BAPTA, 10 HEPES, 3.6 MgATP, 1 GTP, 14 Tris2CP, and 50 U/ml creatine
phosphokinase. The pH was adjusted to 7.3 with CsOH. The osmolarity of
the pipette solution was 300 mOsm/1, and the osmolarity of the
extracellular solution was between 325 and 340 mOsm/1. Toxins were
applied by bath perfusion (cerebellar granule cells) or locally by a
puff pipette (DRG neurons), always in the presence of 0.2 mg/ml chicken
egg ovalbumin (Sigma), to block the low-affinity binding sites.
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RESULTS |
Disruption of the Cacna1e gene does not
affect viability
A null allele of the Cacna1e gene was created using an
18 kb BamHI fragment that encodes all of domain II of the
1E protein. By the use of the homologous
recombination system of yeast, the S4-S6 regions of domain II were
replaced with a neomycin/URA3 selection cassette to generate a
targeting vector (Fig. 1). We believed
that by removing the pore-lining domain and its neighboring transmembrane domains that a null allele of Cacna1e would be
created. This construct was introduced into embryonic stem cells, and
the cells were screened for disruption of the Cacna1e locus.
Approximately 110 neomycin-resistant clones were recovered and analyzed
by Southern analysis. Of these 110 ES cells, 5 showed the desired
disruption at the Cacna1e locus. Chimeric animals produced
from blastocyst injection were then tested for the presence of mutant
alleles. Two lines were established from independent ES cell clones,
and intercross mating was used to generate homozygous mutant mice. Viable homozygous mutant animals were generated from both lines. Both
of the lines generated gave similar results. There were no observed
phenotypic differences between wild-type and homozygous knock-out
mice.
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Figure 1.
Generation of a mouse with a disrupted allele of
the Cacna1e gene. A, Targeting of the
Cacna1e gene is shown. Top, The genomic
structure of the wild-type allele of the Cacna1e gene is
shown with exons indicated by black boxes. Restriction
sites used for determination of the disrupted allele are indicated.
Bottom, A PGK neolURA3 expression
cassette replaced the fourth through eighth exons of this fragment. The
locations of probes used for genomic blot hybridization are shown
below the disrupted allele. B, Genomic
blot hybridization of DNA derived from neomycin-resistant ES cell
clones is shown. ES cell DNA was probed with a 3'-flanking probe
(left) and 5' internal probe (right).
Hybridization with the 3' probe shows a 6 kb wild-type band and 6.6 kb
mutant band, whereas the 5' internal probe revealed an 8.7 kb wild-type
band and 12 kb mutant band. C, Tail tip DNA from three
intercross littermates was probed with the 3'-flanking probe and showed
the expected size for wild-type, heterozygous, and homozygous mutant.
ko, Knock-out.
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Western blotting of 1E-deficient and
wild-type mice
Western blot analysis was used to confirm deletion of the
1E protein in
1E-deficient mice compared with wild-type mice
(Fig. 2). Cerebellum and whole brain
minus cerebellum extracts from both 129/SV and C57BL/6J wild-type mice
displayed a clear band at 230 kDa, the predicted size for the
1E subunit (Fig. 2). In contrast a
corresponding band was not observed in either the cerebellum or whole
brain minus cerebellum from 1E-deficient mice,
indicating the absence of the 1E subunit from
these mice (Fig. 2). Northern blot analysis revealed no detectable
1E transcript I in the homozygous knock-out mice using a probe located either within the deleted region
or downstream from the deletion (+5387-5966).
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Figure 2.
Western blot analysis of 1E protein
in wild-type and 1E-deficient mice. Brain lysates from
129/SV and C57BL/6J wild-type mice show the expression of the 230 kDa
1E protein, whereas the protein is clearly absent from
brain tissue of 1E KO mice. Specificity of the antibody
was confirmed by detection of a band in 1E-transfected
HEK cell membranes and the lack of staining in untransfected HEK cell
membranes.
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Electrophysiology of Ba currents
We examined the properties of the voltage-sensitive Ba currents in
cerebellar granule and DRG neurons cultured from
1E KO mice as well as their parental wild-type strains.
Ba currents in cerebellar granule and DRG neurons exhibited
differential sensitivity to a number of toxins and drugs that have been
shown previously to target different types of voltage-sensitive Ca
channels (Figs. 3,
4). In the case of cerebellar granule
neurons (Fig. 3), components of the Ba current were blocked by the
sequential addition of nimodipine (L-type blocker) -agatoxin VIA
(P/Q-type blocker), and -conotoxin GVIA (N-type blocker), leaving a
component of the current that we designated the R current on the basis
of previous criteria in the literature (Fig. 3). We examined the effect
of the 1E-selective toxin SNX-482 on the R
current in these neurons. In both of the parental lines SNX-482 at a
supramaximally effective concentration of 1 µM blocked
~30% of the granule cell R current. However, it was clear that the
majority of the R current was not blocked by this toxin. Although the
overall magnitude of the whole-cell Ca current in
1E KO mice was the same as that in wild-type
mice, the SNX-482-sensitive component of the Ba current was completely
absent from 1E mice. However, a large R
current still remained in these KO mice after sequential application of all of the drugs and toxins. We also noted that the size of the N-current was slightly larger in the granule neurons from
1E KO mice. The absolute magnitude of the
currents recorded in these experiments can be seen in Table
1.
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Figure 3.
Effects of drugs and toxins on the cerebellar
granule cell IBa. A, Plot of
the peak IBa versus time in an
1E KO mouse shows a resistant component of the high
voltage-activated IBa remaining after
consecutive bath application of specific drugs and toxins. SNX-482 had
no effect on the toxin-resistant current. Inset,
Representative traces at numbered points
are shown. B, Plot of the peak
IBa versus time from a wild-type C57BL/6J
mouse is shown. Inset, Representative
traces are shown. SNX-482 inhibited a component of the R
current remaining after nimodipine, -agatoxin IVA (-Aga
IVA), and -conotoxin GVIA (-CTx GVIA)
application. C, Plot of the peak
IBa versus time from a wild-type 129/SV
mouse is shown. SNX-482 also inhibited part of the R current in this
case. Inset, Representative traces are
shown. D, Comparison of inhibitory effects of specific
drugs and toxins on the granule cell IBa in
1E KO and control mice is shown. Drugs and toxins were
used in the following concentrations: nimodipine (Nimo),
500 nM; -conotoxin GVIA (CTx), 500 nM; -Aga IVA (Aga), 200 nM;
SNX-482 (SNX), 1 µM;
Cd2+, 100 µM. The data from the two
parental control mouse strains were pooled. SNX-482 was only effective
in inhibiting the IBa from control mice
(**p < 0.01). The numbers in
parentheses represent the number of experiments.
E, The effects of the specific drugs and toxins on the
IBa from the two control mouse strains did
not differ significantly from each other. The numbers in
parentheses represent the number of experiments.
Resist, Resistant; wt, wild type.
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Figure 4.
Effects of drugs and toxins on the
IBa in DRG neurons from wild-type and
1E KO mice. A, Plot of the peak
IBa versus time in a DRG neuron from an
1E KO mouse. An R current still remained after
sequential application of the different drugs and toxins. The
IBa from 1E KO mice was
insensitive to SNX-482. Inset, Representative currents.
B, Time course of the peak
IBa from a wild-type DRG neuron exhibiting
sensitivity to SNX-482. C, Time course of the peak
IBa from a wild-type DRG neuron that was
insensitive to SNX-482. D, Average inhibition (mean ± SEM) of the peak IBa by specific drugs
and toxins. Drugs and toxins were used in the following concentrations:
nimodipine, 2 µM; -conotoxin GVIA, 1 µM;
-Aga IVA, 200 nM; SNX-482, 1 µM. DRG
neurons from 1E KO mice (n = 7) and
a population of DRG neurons from wild-type mice (n = 8) were insensitive to SNX-482 application. The
IBa inhibition by SNX-482 in sensitive DRG
neurons (n = 10) was significant
(p < 0.01). E,
Interrelationship between sensitivity to -Aga IVA and SNX-482 in
wild-type DRG neurons (n = 8). The correlation was
significant (p = 0.0265) using the Spearman
rank correlation test.
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In DRG neurons sequential addition of drugs and toxins also inhibited
different components of the Ba current, leaving a substantial R current
(Fig. 4). Interestingly, we observed two populations of DRG neurons
from wild-type mice. In the first type, addition of SNX-482 was
ineffective in blocking the R current. In a second population of
neurons, addition of SNX-482 produced a clear inhibition of the R
current although, as in the case of cerebellar granule neurons, the
majority of the R current was not blocked by SNX-482. Both types of DRG
neurons (i.e., SNX-482 sensitive and insensitive) were observed in both
wild-type parental strains. Interestingly, in neurons that exhibited
SNX-482-sensitive R currents, we found that -agatoxin IVA was
relatively ineffective (Fig. 4E). DRG neurons from
1E KO mice exhibited Ba currents with the same
magnitude as those from wild-type mice. The Ba currents in these
neurons exhibited components that were blocked by nimodipine,
-agatoxin IVA, and -conotoxin GVIA, leaving a substantial R
current, but this was not blocked by SNX-482.
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DISCUSSION |
Voltage-sensitive Ca channels are a diverse family of molecules
(Ertel et al., 2000). Of the nine genes whose expression produces channels of this type, the properties and functions of the
1E channel are the least well understood.
Although 1E subunits appear to be widely
distributed throughout the CNS and peripheral nervous system (Volsen et
al., 1995; Day et al., 1996), it is not clear which types of Ca
currents result from their expression. One of the problems has been the
lack of a drug or toxin that specifically targets these channels,
although the recent identification of the
1E-selective toxin SNX-482 has helped to
alleviate this problem (Newcombe et al., 1998). Furthermore, because
1E subunits are structurally diverse
(Pereverzev et al., 1998; Vajna et al., 1998; Spaetgens and Zamponi,
1999) and because their combination with different ancillary subunits
results in a variety of Ca currents with differing biophysical
characteristics (Parent et al., 1997; Nakashima et al., 1998), there is
still considerable uncertainty about the types of Ca currents
1E subunits produce in neurons and other cells.
To overcome this problem some investigators have resorted to antisense
approaches to examine the relationship between Ca currents and the
expression of 1E subunits. For example
Piedras-Renteria and Tsien (1998) observed that antisense
oligonucleotides for 1E partially reduced the
size of the R current in rat cerebellar granule cells. They suggested
either that more than one 1 subunit was
responsible for the R current in these cells or that the apparent heterogeneity was caused by the expression of multiple forms of the
1E subunit that were differentially sensitive
to antisense treatment. Furthermore, Newcombe et al. (1998) reported
that the R current in cerebellar granule neurons was insensitive to
SNX-482, suggesting a dissociation between this current and
1E expression. On the other hand, Tottene et
al. (2000) reported that the R current in cerebellar granule neurons
consisted of multiple components, some of which were sensitive to
SNX-482 and some of which were resistant. However, these authors also
demonstrated that antisense treatment was nearly completely effective
in reducing both the toxin-sensitive and -insensitive components of the
current. Thus, it is still unclear whether the R current, at least in
cerebellar granule neurons, is a homogeneous entity or not and what its
relationship to 1E expression might be.
To settle this issue, we have generated mice that completely lack the
1E transcript and protein. It is therefore
clear that any Ca currents recorded from these animals cannot result
from the expression of 1E. It is important to
note therefore that there was a substantial R current in all of the
neurons from 1E KO mice from which we made
recordings. Therefore, we can conclude that the majority of R currents,
at least in cerebellar granule and DRG neurons, do not result from the
expression of 1E subunits. Having said this,
it is also true that there appears to be a component of the R current
in some cell types that does result from the expression of
1E, although this component is relatively
small. The situation in mouse cerebellar granule cells therefore seems to be similar to that reported by Piedras-Renteria and Tsien (1998). Although the magnitude of 1E-dependent R
current in their studies was larger than that in ours, their conclusion
that the R current in granule neurons did not completely result from
the expression of 1E seems similar to ours.
Furthermore, our results are also consistent with the work of Newcombe
et al. (1998), who observed SNX-482-resistant R currents in several
types of neurons.
The identity of R currents in sensory neurons also seems to be complex.
It is interesting to note that two different patterns of Ca current
sensitivity were observed in these neurons. We only identified an
SNX-482-sensitive component in those wild-type neurons with a
relatively small -agatoxin IVA-sensitive current. We do not yet know
what this phenotype means in terms of the overall classification of DRG
neurons. However, as in the case of granule neurons, the magnitude of
the SNX-482-sensitive current, in those DRG cells in which it is
observed, was relatively small.
If the expression of 1E is not responsible for
the majority of the R current in most neurons, then what is? It is
interesting to note that Jun et al. (2000) have shown recently that
there was a large decline in the magnitude of the cerebellar granule neuron R current in 1A KO mice [although,
interestingly, Piedras-Renteria and Tsien (1998) found that
1A antisense did not reduce the R current in
these neurons]; the decline in R current magnitude in these mice was
much greater than that in the 1E KO mice
reported here. Jun et al. (2000) demonstrated that ~80% of the R
current was absent in their 1A KO mice. Thus,
it is conceivable that this portion, together with the
SNX-482-sensitive portion described here, accounts for virtually all of
the granule neuron R current, although exactly what the relationship is
between the non-1E-dependent R current and
1A is not clear at this time. Nevertheless, it is also interesting to note that in our studies on DRG neurons there
was a negative correlation between the magnitude of the -agatoxin
IVA-sensitive and the SNX-482-sensitive currents. It is also of
interest that the SNX-482-sensitive current was restricted to a
particular population of DRG neurons. Exactly what this population represents from the functional point of view awaits further
identification but could indicate a role for
1E-based currents in the processing of a
particular type of sensory information, such as pain (Saegusa et al.,
2000).
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FOOTNOTES |
Received June 5, 2000; revised Aug. 28, 2000; accepted Sept. 1, 2000.
This study was supported by Public Health Service Grants
DA-02121, MH-40165, NS-33826, DK-44840, and NS-21442 to R.J.M. and grants from the National Cancer Institute Department of Health and
Human Services to N.A.J. and N.G.C. We are grateful to Dr. G. Miljanich
of Elan Pharmaceuticals for the gift of SNX-482.
Correspondence should be addressed to Dr. Richard J. Miller, Department
of Neurobiology, Pharmacology, and Physiology, The University of
Chicago, 947 East 58th Street (MC 0926), Chicago, IL 60637. E-mail:
rjmx{at}midway.uchicago.edu.
| |
REFERENCES |
-
Beattie RE,
Volsen SG,
Smith D,
McCormack AL,
Gillard SE,
Burnett JP,
Ellis SB,
Gillespie A,
Harpold MM,
Smith W
(1997)
Preparation and purification of antibodies specific to human neuronal voltage-dependent calcium channels.
Brain Res Brain Res Protoc
1:307-319[Medline].
-
Bourinet E,
Zamponi GW,
Stea A,
Soong TW,
Lewis BA,
Jones LP,
Yue DT,
Snutch TP
(1996)
The 1E calcium channel exhibits permeation properties similar to low voltage activated calcium channels.
J Neurosci
16:4983-4993[Abstract/Free Full Text].
-
Brorson JR,
Bleakman D,
Gibbons SJ,
Miller RJ
(1991)
The properties of intracellular calcium stores in cultured rat cerebellar neurons.
J Neurosci
11:4024-4043[Abstract].
-
Catterall WA
(1998)
Structure and function of neuronal Ca channels and their role in neurotransmitter release.
Cell Calcium
24:307-323[Web of Science][Medline].
-
Cribbs LL,
Lee JH,
Yang J,
Satin J,
Zhang Y,
Daud A,
Barclay J,
Williamson MP,
Fox M,
Rees M,
Perez-Reyes E
(1998)
Cloning and characterization of 1H from human heart, a member of the T-type Ca2+ channel gene family.
Circ Res
83:103-109[Abstract/Free Full Text].
-
Day NC,
Shaw PJ,
McCormack AL,
Craig PJ,
Smith W,
Beattie R,
Williams TL,
Ellis SB,
Ince PG,
Harpold MM,
Lodge D,
Volsen SG
(1996)
Distribution of 1A, 1B and 1E voltage-dependent calcium subunits in the human hippocampus and parahippocampus gyrus.
Neuroscience
71:1013-1024[Web of Science][Medline].
-
Ertel EA,
Campbell KP,
Harpold MM,
Hofmann F,
Mori Y,
Perez-Reyes E,
Schwartz A,
Snutch TP,
Tanabe T,
Birnbaumer L,
Tsien RW,
Catterall WA
(2000)
Nomenclature of voltage gated calcium channels.
Neuron
25:533-535[Web of Science][Medline].
-
Grabsch H,
Pereverzev A,
Weiergraber M,
Schramm M,
Henry M,
Vajna R,
Beattie RE,
Volsen SG,
Klockner U,
Hescheler J,
Schneider T
(1999)
Immunohistochemical detection of 1E voltage-gated Ca2+ channel isoforms in cerebellum, INS-1 cells and neuroendocrine cells of the digestive system.
J Histochem Cytochem
47:981-994[Abstract/Free Full Text].
-
Hamill OP,
Marty A,
Neher E,
Sakmann B,
Sigworth FJ
(1981)
Improved patch-clamp techniques for high-resolution current recording from cells and cell-free membrane patches.
Pflügers Arch
391:85-100[Web of Science][Medline].
-
Hilaire C,
Diochot S,
Desmadryl G,
Richard S,
Valmier J
(1997)
Toxin resistant calcium currents in embryonic mouse sensory neurons.
Neuroscience
80:267-276[Web of Science][Medline].
-
Hoffman F,
Lacinova L,
Klugbauer N
(1999)
Voltage-dependent calcium channels: from structure to function.
Rev Physiol Biochem Pharmacol
139:33-87[Web of Science][Medline].
-
Jun K,
Piedras-Rentera E,
Smith SM,
Wheeler DB,
Lee SB,
Lee TG,
Chin H,
Adams ME,
Scheller RH,
Tsien RW,
Shin HS
(2000)
Ablation of P/Q type Ca channel currents, altered synaptic transmission, and progressive ataxia in mice lacking the 1A subunit.
Proc Natl Acad Sci USA
96:15245-15250[Abstract/Free Full Text].
-
Lee JH,
Daud AN,
Cribbs LL,
Lacerda AE,
Pereverzev A,
Klockner U,
Schneider T,
Perez-Reyes E
(1999)
Cloning and expression of a novel member of the low voltage-activated T-type calcium channel family.
J Neurosci
19:1912-1921[Abstract/Free Full Text].
-
Meir A,
Dolphin AC
(1998)
Known calcium channel 1 subunits can form low threshold small conductance channels with similarities to native T-type channel.
Neuron
20:341-351[Web of Science][Medline].
-
Nakashima YM,
Todorovic SM,
Pereverzev A,
Heschler J,
Schneider T,
Lingle CJ
(1998)
Properties of Ba2+ currents arising from human a1E and 1E
 3 constructs expressed in HEK293 cells: physiology, pharmacology, and comparison to native T-type Ba2+ currents.
Neuropharmacology
37:957-972[Web of Science][Medline]. -
Newcombe R,
Szoke B,
Palma A,
Wang G,
Chen XH,
Hopkins W,
Cong R,
Miller J,
Urge L,
Tarczy-Hornoch K,
Loo JA,
Dooley DJ,
Nadasdi L,
Tsien RW,
Lemos J,
Miljanich G
(1998)
Selective peptide antagonist of the class E calcium channel from the venom of the tarantula Hysterocrates gigas.
Biochemistry
37:15353-15362[Medline].
-
Parent L,
Schneider T,
Moore CP,
Talwar D
(1997)
Subunit regulation of the human brain 1E calcium channel.
J Membr Biol
160:127-140[Web of Science][Medline].
-
Pereverzev A,
Klockner U,
Henry M,
Grabsch H,
Vajna R,
Olyschlager S,
Viatchenko-Karpinski S,
Schroder R,
Heschler J,
Schneider T
(1998)
Structural diversity of the voltage dependent Ca channel 1E subunit.
Eur J Neurosci
10:916-925[Web of Science][Medline].
-
Perez-Reyes E,
Cribbs LL,
Daud A,
Lacerda AE,
Barclay J,
Williamson MP,
Fox M,
Rees M,
Lee JH
(1998)
Molecular characterization of a neuronal low-voltage-activated T-type calcium channel.
Nature
391:896-900[Medline].
-
Piedras-Renteria E,
Tsien RW
(1998)
Antisense oligonucleotides against 1E reduce R-type calcium currents in cerebellar granule cells.
Proc Natl Acad Sci USA
95:7760-7765[Abstract/Free Full Text].
-
Randall A,
Tsien RW
(1995)
Pharmacological dissection of multiple types of Ca channel currents in rat cerebellar granule neurons.
J Neurosci
15:2995-3012[Abstract].
-
Saegusa H,
Kurihara T,
Zang S,
Minowa O,
Kazuno A,
Han W,
Matsuda Y,
Yamanaka H,
Osanai M,
Noda T,
Tanabe T
(2000)
Altered pain responses in mice lacking 1E subunit of the voltage dependent Ca channel.
Proc Natl Acad Sci USA
97:6133-6137.
-
Spaetgens RL,
Zamponi GW
(1999)
Multiple structural domains contribute to voltage dependent inactivation of rat brain 1E calcium channels.
J Biol Chem
274:22428-22436[Abstract/Free Full Text].
-
Tottene A,
Moretti A,
Pietrobon D
(1996)
Functional diversity of P-type and R-type calcium channels in rat cerebellar neurons.
J Neurosci
16:6353-6363[Abstract/Free Full Text].
-
Tottene A,
Volsen S,
Pietrobon D
(2000)
1E subunits form the pore of three cerebellar R-type calcium channels with different pharmacological and permeation properties.
J Neurosci
20:171-178[Abstract/Free Full Text].
-
Vajna R,
Schramm M,
Pereverzev A,
Arnhold S,
Grabsch H,
Klockner U,
Perez-Reyes E,
Heschler J,
Schneider T
(1998)
New isoform of the neuronal Ca channel 1E subunit in islets of Langerhans and kidney.
Eur J Biochem
257:274-285[Web of Science][Medline].
-
Volsen SG,
Day NC,
McCormack AL,
Smith W,
Craig PJ,
Beattie R,
Ince PG,
Shaw PJ,
Ellis SB,
Gillespie A,
Harpold MM,
Lodge D
(1995)
The expression of neuronal voltage-dependent calcium channels in human cerebellum.
Brain Res Mol Brain Res
34:271-282[Medline].
Copyright © 2000 Society for Neuroscience 0270-6474/00/20238566-06$05.00/0
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|
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|
|
TOP
ABSTRACT
INTRODUCTION
