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The Journal of Neuroscience, March 1, 2000, 20(5):1685-1693
Coexpression of Cloned  1B,  2a, and
2/ Subunits Produces
Non-Inactivating Calcium Currents Similar to Those
Found in Bovine Chromaffin Cells
Anne L.
Cahill,
Joyce H.
Hurley, and
Aaron P.
Fox
The Department of Neurobiology, Pharmacology, and Physiology, The
University of Chicago, Chicago, Illinois 60637
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ABSTRACT |
Chromaffin cells express N-type calcium channels identified
on the basis of their high sensitivity to block by  -conotoxin GVIA (-CgTx GVIA). In contrast to neuronal N-type calcium
currents that inactivate during long depolarizations and that require
negative holding potentials to remove inactivation, many chromaffin
cells exhibit N-type calcium channel currents that show little
inactivation during maintained depolarizations and that exhibit no
decrease in channel availability at depolarized holding potentials.
N-type calcium channels are thought to be produced by combination of the pore-forming  1B subunit and accessory  and
2/ subunits. To examine the molecular
composition of the non-inactivating N-type calcium channel, we cloned
the 1B and accessory (1b,
1c, 2a, 2b,
and 3a) subunits found in bovine chromaffin
cells. Expression of the subunits in either Xenopus
oocytes or human embryonic kidney 293 cells produced
high-threshold calcium currents that were blocked by -CgTx GVIA.
Coexpression of bovine 1B with 1b,
1c, 2b, or 3a
produced currents that were holding potential dependent. In contrast,
coexpression of bovine 1B with 2a
produced holding potential-independent calcium currents that closely
mimicked native non-inactivating currents, suggesting that
non-inactivating N-type channels consist of bovine
1B, 2/, and
2a.
Key words:
N-type calcium channel; 1B subunit;
subunits; non-inactivating calcium current; chromaffin cells; voltage-dependent calcium channel
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INTRODUCTION |
Adrenal chromaffin cells express L-,
N-, and P/Q-type calcium channels (Hans et al., 1990 ; Albillos
et al., 1993; Artalejo et al., 1994). In many chromaffin cells, N-type
calcium channels do not inactivate during long depolarizations, nor do
they exhibit decreased availability at depolarized holding potentials
(Artalejo et al., 1992). Similar non-inactivating N-type channels have
been described in presynaptic nerve terminals (Stanley and Goping, 1991). Interestingly, a significant fraction of N-type channels in
chromaffin cells exhibits robust inactivation, raising the question as
to why inactivation varies so much from cell to cell.
Voltage-dependent calcium channels are composed of at least three
different subunits designated 1,
2/, and and possibly a  subunit
(Dunlap et al., 1995; Jones, 1998; Letts et al., 1998). The
pore-forming 1 subunit alone encodes a
voltage-dependent calcium channel with kinetic properties different
from those of native channels (Lacerda et al., 1991; Varadi et
al., 1991; Stea et al., 1993). When and
2/ subunits are coexpressed with a variety
of cloned 1 subunits, current amplitude is
greatly augmented, and the kinetics of activation and inactivation more
closely resembles the kinetics of native channels (Birnbaumer et
al., 1998). Ten different calcium channel 1
subunits have been cloned
[1A-1I and
1S (Jones, 1998)].
1B subunits are required to make N-type calcium channels. Four -subunit genes have been identified
[1-4 (Birnbaumer et al., 1998)], each of
which has different effects on calcium channel inactivation.
Inactivation rates of channels produced by 1A
and 1E are slowest with
2a, fastest with 3a, and intermediate with 1b and
4 (Sather et al., 1993; Olcese et al., 1994;
Zhang et al., 1994; DeWaard and Campbell, 1995). Inactivation of
1B currents was greatest with
3a and least with 2a
(Patil et al., 1998). Thus expression of different accessory subunits
provides a potentially important regulatory mechanism for channel inactivation.
Certain 1-subunit regions appear to influence
channel inactivation. When the IS6 and I-II loop of
1A or 1B were
substituted for the analogous regions in 1E,
the chimeric channels had inactivation rates much closer to those of
1A and 1B than to
those of 1E (Zhang et al., 1994; Page et al.,
1997). It was reported recently that the splice variant of
1A with a valine inserted in the I-II loop
inactivated more slowly and less completely than did the variant
lacking this valine (Bourinet et al., 1999).
Our study was performed to determine whether the inactivation
properties of chromaffin cell N-type calcium channels result from a
unique 1B gene or whether specific accessory
subunit combinations were required to form these channels. We cloned
the 1B subunit and five subunits from
chromaffin cells and expressed the clones in Xenopus oocytes
and human embryonic kidney (HEK) 293 cells. Non-inactivating
currents that mimicked those found in many chromaffin cells were
observed when the bovine 1B clone was
coexpressed with 2a and
2/. Channels made by coexpressing the
1B clone with either
1b, 1c
2b, or 3a (and
2/) showed clear inactivation and resembled
the inactivating N-type calcium currents observed in some chromaffin cells.
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MATERIALS AND METHODS |
Amplification of bovine chromaffin cell 1B
cDNA. Fragments of bovine chromaffin cell
1B cDNA were amplified from total chromaffin cell RNA by reverse transcriptase-PCR (RT-PCR) using the
Superscript Preamplification System (Life Technologies, Gaithersburg,
MD) and degenerate primers designed from amino acid sequences conserved in the N and C terminals of cloned human, rabbit, rat, and mouse 1B subunits. A 563 bp cDNA corresponding to
amino acids 65-252 of the N terminal of bovine
1B and a 683 bp cDNA corresponding to amino
acids 1751-1969 of the C terminal of 1B were obtained.
Cloning of 1B from a bovine chromaffin cell
library. The bovine 1B cDNAs obtained by
PCR were used to screen 0.9 × 106
plaques from a random- and polydT-primed bovine chromaffin cell library
in  ZapII (a gift from Dr. H. Pollard, National Institutes of
Health). The plaques were transferred to Hybond N+ membranes and
hybridized with 32P-labeled
1B cDNA using standard methods (Sambrook et
al., 1989). Positive plaques were picked from the library plates and
purified by two successive rounds of plating and hybridization. The
1B cDNA clones were excised in vivo
from ZapII using the ExAssist helper phage (Stratagene, La Jolla,
CA). The ends of each cDNA insert were manually sequenced using the T7
Sequenase version 2.0 plasmid sequencing kit. The largest cDNAs from
each screening were used to rescreen the library until clones
corresponding to the entire bovine 1B cDNA
were obtained. Ten screenings of the library yielded 44 independent
1B clones ranging in size from 150 to 3600 bp.
Construction of a full-length 1B cDNA. Eight
clones that spanned the entire coding region of
1B were used to construct a full-length
1B cDNA in the mammalian expression vector
pcDNA3.1(+) (Invitrogen, Carlsbad, CA). Fragments were cut from each
clone using selected restriction endonucleases and subcloned into
recipient plasmids using standard molecular biology techniques. The
full-length bovine 1B cDNA was sequenced in
its entirety by the University of Chicago Cancer Research Center
Sequencing Facility using an ABI 377 automated fluorescent
sequencer. Sequencing revealed a 2 bp deletion at the splice site for
the +SFMG splice variant described by Lin et al. (1997). This deletion
was repaired by subcloning the XbaI-Bsu36I
fragment (bp 3245-3852) from a different library clone into the
1B construct in pcDNA3.1(+). The
sequence of bovine 1B has been deposited in
GenBank with the accession number AF173882.
Cloning of 1b, 1c,
2a, 2b, and 3a from a
bovine chromaffin cell library. A 2690 bp fragment of human
1b was labeled with 32P and used to
screen the bovine chromaffin cell library by standard hybridization
techniques (Sambrook et al., 1989). Positive plaques were picked from
the library plates and purified by two more rounds of plating before
in vivo excision of the phagemid pBSII containing each cDNA
clone. This screening of the library yielded full-length clones for
bovine 1b, 1c, and
3a but no 2a or
2b clones. Therefore degenerate PCR primers
were designed to the 2 C-terminal amino acid
sequences EAYWKAT and EWNRDVYI and used in an RT-PCR protocol to
generate a 600 bp cDNA fragment of bovine 2.
This fragment was labeled with 32P and
used to screen the library as described above. This screening yielded
clones containing all but the first 48 bp of the
2a-coding region and the first 36 bp of the
coding region of 2b. The missing 5' end of
2a was cloned by RT-PCR from chromaffin cell
RNA and ligated to the longest library clone. The missing region of the bovine 2b was supplied by ligating the
appropriate fragment of rabbit 2b to the
bovine 2b. (The N-terminal amino acids 13-105 are identical between the bovine and rabbit clones. It appears likely
that the first 12 amino acids may be identical as well.) All
-subunit clones were sequenced and subcloned into pcDNA3.1(+) for
expression studies. The sequences of the five bovine subunits have
been deposited in GenBank with the accession numbers
AF174415-AF174419.
Other cloned calcium channel subunits. Other cDNA clones
used in this study were generous gifts from the following
investigators: human 2/ (GenBank accession
number M21948) and 1b (M92303) from Dr.
R. J. Miller (The University of Chicago), rat
2a (M80545) from Dr. E. Perez-Reyes (Loyola
University), and rabbit 2b (X64298) from Dr.
R. W. Tsien (Stanford University).
Xenopus oocyte expression and electrophysiology.
Calcium channel proteins were expressed in Xenopus laevis
oocytes after injection of in vitro-transcribed mRNA.
Full-length cDNAs of each of the cloned subunits were used to generate
mRNA from the T7 RNA polymerase promoter using an in vitro
transcription kit (mMessage Machine; Ambion, Austin, TX). Oocytes were
harvested from mature Xenopus laevis female frogs and
separated from follicle cells with 2 mg/ml collagenase type IA (Sigma,
St. Louis, MO). Oocytes were injected with 25 ng of both bovine
1B and human 2/
RNA and 10 ng of various RNAs in a total of 50 nl of DEPC-treated
H2O with a Drummond automatic microinjector.
Three days after injection, oocytes were voltage-clamped at various
potentials with two glass electrodes (filled with 3 M KCl and having a resistance of 0.5-2 M )
using an Axoclamp 2A (Axon Instruments, Foster City, CA). Oocytes were
initially superfused with 96 mM NaCl, 2 mM KCl, 1 mM
MgCl2, and 5 mM HEPES, pH
7.6, and N-type calcium channel currents were measured in a
Ba2+ recording solution designed to
minimize the oocyte's endogenous Ca2+-dependent
Cl current [40 mM
Ba(OH)2, 25 mM
tetraethylammonium (TEA)-OH, 25 mM NaOH, 2 mM CsOH, 5 mM HEPES, and 1 mM niflumic acid, pH adjusted to 7.5 with
methanesulfonic acid]. Membrane currents were recorded on a computer
using pCLAMP software (Axon Instruments). Leak currents were subtracted
on-line with a P/4 protocol.
Cell culture and transfection. HEK 293 (American Type
Culture Collection, Rockville, MD) cells were used for transient
transfection studies and were grown in MEM and 10% calf serum
supplemented with 2 mM glutamine, 100 U/ml penicillin, and
100 µg/ml streptomycin. Mammalian expression plasmids, containing the
complete cDNAs for each of the calcium channel subunits, and the marker
CD8 were purified on Qiagen (Hilden, Germany) columns and used
to transfect HEK 293 cells. Cells were transiently transfected with
cDNAs encoding the bovine 1B, human
2/, various subunits, and CD8 as a
reporter gene in a ratio of 15:12:5:3 using Lipofectin or Lipofectamine (both from Life Technologies). The human 2/
and various cDNAs were coexpressed with 1B
because they have been reported to increase the expression levels of
calcium channels and to normalize current kinetics (Lacerda et
al., 1991; Stea et al., 1993). Immediately after the
transfection, cells were replated onto polylysine-coated coverslips.
Calcium currents were recorded 2-3 d after transfection. Transfected
cells were detected visually by binding of anti-CD8 beads (Dynal, Great
Neck, NY).
Bovine chromaffin cells were prepared and cultured as described
previously (Artalejo et al., 1992). Briefly, bovine adrenal glands were
digested with collagenase, and cells were purified by density gradient
centrifugation. Cells were resuspended in bovine chromaffin cell media
and plated onto collagen-coated glass coverslips at a density of
0.15 × 106
cells/cm2. Cells were maintained at 37°C
in an atmosphere of 92.5% air and 7.5% CO2 and
90% humidity.
Electrophysiology on HEK 293 and chromaffin cells. Cells
were voltage-clamped in the whole-cell configuration of the patch-clamp technique (Hamill et al., 1981) with a List model L/M-EPC7 patch clamp.
Gigaohm seals were obtained in an extracellular Tyrode's solution (130 mM NaCl, 20 mM glucose, 10 mM
HEPES, 1 mM MgCl2, 2 mM
KCl, and 5 or 10 mM CaCl2, pH
adjusted to 7.3 with NaOH). Ionic currents were measured in an
extracellular TEA-Ba2+ solution
(140 mM TEA-Cl, 10 mM glucose, 10 mM HEPES, and 5 or 10 mM
BaCl2, pH adjusted to 7.3 with TEA-OH). For
chromaffin cell recordings, 1 µM tetrodotoxin and 1 µM nisoldipine were added to the
TEA-Ba2+ solution. Electrodes were pulled
from glass capillary tubes (Drummond, Broomall, PA), coated with
Sylgard (Dow Corning, Midland, MI), and fire-polished to a final
resistance of ~2.0 M when filled with a CsCl-based internal
solution (110 mM CsCl, 10 mM EGTA, 40 mM HEPES, 5 mM MgCl2, 0.3 mM GTP, and 2 mM ATP, pH adjusted to 7.3 with
CsOH, or 110 mM CsCl, 10 mM EGTA, 20 mM HEPES, 4 mM MgCl2, 0.3 mM GTP, 6 mM ATP, and 14 mM
creatine phosphate, pH adjusted to 7.3 with CsOH). -Conotoxin GVIA
(Alomone Labs, Jerusalem, Israel) was diluted in extracellular
recording solution and added to the bath at a final concentration of 1 µM.
Ionic currents were activated by step depolarizations of 20-200 msec
duration from various holding potentials. For chromaffin cell
recording, the cells were continuously perfused with recording solution
at a rate of 2-3 ml/min by gravity flow. Leak currents were generated
by averaging hyperpolarizing steps. All data presented here are leak
and capacitance subtracted. Series resistance was partially compensated
(>60%) using the series resistance compensation circuit of the patch clamp.
Data analysis. For I-V curve calculations, the
peak current from each cell was recorded, and groups were pooled to
calculate the average and SEM. For steady-state inactivation
curves, currents from each cell at each holding potential were
normalized to the peak current at the most hyperpolarized holding
potential. The normalized data were averaged across cells and fit to
single Boltzmann functions.
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RESULTS |
We have described previously a biophysically distinct N-type
calcium channel found in bovine chromaffin cells (Artalejo et al.,
1992). This "atypical" N-type calcium channel does not exhibit voltage-dependent inactivation while retaining other defining characteristics of N-type channels such as -conotoxin GVIA (-CgTx GVIA) sensitivity. In addition, a large subset of chromaffin cells exhibits N-type calcium channel currents that show robust inactivation and thus resemble neuronal N-type channels. Examples of both varieties of chromaffin cell N-type calcium channels are shown in Figure 1. The method used to isolate N-type
calcium currents from whole-cell calcium currents is illustrated in
Figure 1A. The whole-cell calcium currents in Figure
1A labeled Total consist only of N- and
P/Q-type currents because the specific L-type calcium channel blocker
nisoldipine was included in the extracellular medium. Whole-cell
currents were elicited by test depolarizations to +10 mV from holding
potentials in the range of  90 to 50 mV. Note that current
traces from five holding potentials are superimposed in each
part of Figure 1A, indicating that the calcium
currents in this cell are not sensitive to changes in the holding
potential. Addition of 1 µM -CgTx GVIA to
the extracellular medium blocked approximately two-thirds of the
current at each of the holding potentials. Five superimposed traces
illustrating the P/Q-type calcium currents remaining after -CgTx
GVIA application are plotted in Figure 1A,
middle (labeled -CTx GVIA insensitive).
Subtraction of the -CgTx GVIA-insensitive current from the total
current yields the -CgTx GVIA-sensitive N-type calcium current in
isolation (Fig. 1A, labeled N-type). The
currents from this cell were stable for >30 min and exhibited little
rundown. Although many chromaffin cells exhibit calcium currents that
are holding potential insensitive, other cells exhibit strong
inactivation of currents. Figure 1B shows N-type
current from a different cell, isolated in a similar manner, that
showed a strong holding potential dependence; over one-half of the
current was inactivated by a change in holding potential in the range of 90 to 60 mV. Figure 1C shows a plot of peak N-type
current elicited at each holding potential, using the data from the
non-inactivating cell shown in Figure 1A,
right. Each current has been normalized to the current
observed at the 90 mV holding potential. The data demonstrate that
the N-type channels in this cell are not sensitive to changes in
holding potential. Channel availability was not tested at very
depolarized potentials (more than 40 mV) because these potentials
activate Ca2+ currents in chromaffin
cells. N-type Ca2+ current inactivation
appears to involve a component of
Ca2+-dependent inactivation (Cox and
Dunlap, 1994), which would complicate our studies of voltage-dependent
inactivation.
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Figure 1.
Some chromaffin cell N-type calcium channel
currents observed in isolation are insensitive to changes in the
holding potential. A, The strategy used to isolate
N-type calcium channel currents in chromaffin cells is shown.
Leak-subtracted calcium current traces were obtained by depolarizing a
chromaffin cell to +10 mV for 24 msec from holding potentials in the
range of 90 to 50 mV. Each holding potential was maintained for 60 sec before eliciting the test depolarization. Left,
Total represents the total current obtained in the
absence of any calcium channel antagonist. Middle,
-CTx GVIA-insensitive shows calcium currents elicited
after application of 1 µM -CgTx GVIA.
Right, N-type shows currents after
subtraction of -CgTx GVIA-insensitive currents from the total
currents. For each condition there are five superimposed current
records, one from each holding potential in the range of 90 to 50
mV (10 mV steps). B, Isolated N-type current from a
different cell that exhibited strong holding potential dependence is
shown. More than one-half of the current was inactivated when the
holding potential was changed from 90 to 60 mV. C,
Peak N-type current amplitude as a function of holding potential
(HP) is shown. The peak current at each
HP was normalized by dividing by the current observed at
an HP of 90 mV. Inset, The
voltage-clamp protocol.
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We have attempted to determine the molecular basis of the inactivating
or non-inactivating N-type calcium channels found in bovine chromaffin
cells by cloning the 1B subunit and a variety of accessory subunits from a bovine chromaffin cell cDNA library and then characterizing the calcium currents resulting from the expression of the cloned subunits. The cDNA library was screened as
described in Materials and Methods, and a full-length bovine 1B cDNA was assembled from eight overlapping
library clones. The full-length clone was sequenced, and the deduced
amino acid sequence for the bovine chromaffin cell
1B subunit was aligned with reported sequences
for human 1B (Williams et al., 1992a), rabbit
1B (Fujita et al., 1993), rat
1B (Dubel et al., 1992), and mouse
1B (Coppola et al., 1994), and a consensus
sequence was generated (Fig.
2). The bovine
1B was most similar to the human
1B with 93% of the deduced amino acid
residues being identical. Identities with the
1B subunits cloned from mouse, rat, and rabbit ranged from 89 to 91%. The N terminal and the four putative
transmembrane domains of 1B were most highly
conserved with 98-99% of the deduced amino acids being identical to
the consensus. There was significantly more variation among species in
the C terminal and the putative intracellular loop between
transmembrane domains II and III with 89-93% of the C-terminal
residues for each species being identical to the consensus sequence and
83-84% of the II-III loop residues agreeing with the consensus.
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Figure 2.
Alignment of the deduced amino acid sequence of
bovine chromaffin cell 1B with those from human, rabbit,
rat, and mouse 1B. The consensus sequence
(Con) calculated from this alignment is shown on the
top line, and only differences from the
consensus are shown for the other sequences. The putative transmembrane
domains and the 1 interaction domain
(AID) are underlined and
labeled in bold above the
consensus sequence. The splice variants identified here and by
Lin et al. (1997) are indicated by *. The GenBank accession numbers and
the sources of the sequences are as follows: Bov, bovine
chromaffin cell (AF173882) (this study); Hum, human
neuroblastoma (M94172) (Williams et al., 1992a); Mus,
mouse neuroblastoma (U04999) (Coppola et al., 1994);
Rab, rabbit brain (D14157) (Fujita et al.,
1993); and Rat, rat brain (M92905) (Dubel et al., 1992).
(Figure 2 continues.)
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We also compared the bovine 1B sequence with
the reported splice variants for cloned 1B.
All of the three library clones containing the C terminal of bovine
1B were similar to the longer splice form
described by Williams et al. (1992a) for human
1B. None of the four clones covering the
II-III loop region of bovine 1B contained the
22 amino acid insert found in mouse 1B
(Coppola et al., 1994), but interestingly a different splice variant
was observed at this same splice site. Two of the four library clones and the full-length bovine 1B construct
contained an arginine residue at this splice site (Fig. 2, R757), and
the other two library clones did not. Three sites for alternative
splicing of rat 1B have been described as
follows: ±A(415), ±SFMG (1242-1245), and ±ET (1574-1575) (Lin et
al., 1997). None of three library clones coded for A, and none of three
library clones coded for SFMG, but we found one library clone that was
+ET and one that was ET. The full-length expressed bovine
1B was A, +R, SFMG, and ET. The IS6
region, which has been suggested to be important in N-type channel
inactivation (Zhang et al., 1994), contained a single amino acid
substitution, leucine in bovine 1B rather than isoleucine.
Five accessory subunits (1b,
1c, 2a,
2b, and 3a) were
cloned from the chromaffin cell cDNA library. The bovine
1b, 1c, and
3a subunits were recovered from the library as
full-length clones and were sequenced in their entirety. The predicted
amino acid sequences of these three subunits were 97-98%
identical with those of human clones (Powers et al., 1992; Williams et
al., 1992b; Collin et al., 1994). The initial screen of the chromaffin cell cDNA library yielded several clones corresponding to the region
common to 2a and 2b,
but no clones contained the complete and unique N terminals of
2a or 2b. Therefore
the N terminal of 2a was cloned from
chromaffin cell RNA by RT-PCR and ligated to a library clone to produce
full-length 2a. Overall the bovine 2a subunit was 94% identical to human
2a, but almost all of the differences were
confined to the common C terminal shared by 2a
and 2b. This region was only 82% identical to
human 2a. The full-length
2b was obtained by ligating the incomplete
2b library clone with a cDNA fragment from
rabbit 2b that coded for the first 12 missing
amino acids. No human 2b clone has yet been reported.
The cloned 1B and subunits were expressed
both in HEK 293 cells and in Xenopus oocytes (Fig.
3). Although the experiments could be
done more efficiently in oocytes where virtually every injected cell
exhibited large currents, HEK 293 cells were also used in certain
experiments to exclude possible effects of the endogenous
3xo subunit in oocytes (Tareilus et al.,
1997). HEK 293 cells transiently transfected with mammalian expression
plasmids containing bovine 1B and human
2/ and 1b subunits
expressed a high-threshold calcium current (Fig. 3A) that
was blocked by 1 µM -CgTx GVIA (Fig.
3B). Sham-transfected HEK 293 cells did not have any
observable calcium currents (data not shown). Oocytes were injected
with in vitro-transcribed mRNAs for each subunit, and current recordings were done 2-3 d later. Oocytes expressing all
three calcium channel subunits exhibited a large inward
Ba2+ current that was not present in
uninjected oocytes. Small outward currents (<50 nA) were occasionally
seen in oocytes injected with only the 2/
and 1B subunits (data not shown). Figure
3C shows data from an oocyte injected with mRNA for bovine
1B and human 2/
and 1b. The high-threshold currents observed
were blocked by -CgTx GVIA. Thus in both oocytes and HEK 293 cells
the bovine 1B subunit when coexpressed with a
and an 2/ subunit appears to make
N-type calcium channels.
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Figure 3.
Coexpression of bovine 1B, human
1b, and 2/ in HEK 293 cells and Xenopus oocytes. A, Peak
currents in HEK 293 cells plotted as a function of voltage. Transfected
cells were depolarized for 200 msec to a variety of test potentials
from an HP of 80 mV; peak current was measured and then plotted as a
function of test potential. B, Peak currents plotted as
a function of time. The HEK 293 cell was depolarized to +10 mV every 15 sec from an HP of 80 mV. -CgTx GVIA was added to the bath as
indicated by the horizontal bar. Toxin
application produced >95% inhibition of the calcium current.
C, Peak currents in oocytes expressing
1B, 1b, and
2/ plotted as a function of voltage. Oocytes
were depolarized for 200 msec to a variety of test potentials from an
HP of 120 mV. The experiment was repeated after the addition of 1 µM -CgTx GVIA.
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We next investigated the inactivation properties of the cloned
1B subunit expressed with
2/ and a variety of subunits. Figure
4A shows data from
three different oocytes expressing bovine 1B
and human 2/ in addition to the subunit
indicated in the figure. The families of current records were obtained
by depolarizing cells for 200 msec to +10 mV from a variety of holding
potentials in the range of 90 to 30 mV. Each holding potential was
maintained for 60 sec before the test depolarization. Expression of
either 1b, 1c, or
3a produced currents that were strongly
dependent on holding potential. Figure 4B plots
current-voltage relationships for each of the cells shown in Figure
4A. The cells were depolarized to a variety of test
potentials from an HP of 100 mV; peak current was measured and then
plotted as a function of test potential. Figure 4C graphs
voltage-dependent inactivation curves made by averaging data from
groups of cells expressing different subunits. The curves were
constructed by normalizing the currents; the currents observed at each
holding potential were divided by the current recorded at 90 mV. Note
that the currents generated by the channels containing any of the three
subunits used in this experiment produced strong inactivation.
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Figure 4.
Coexpression of bovine 1B with
bovine 1b, 1c, or
3a and 2/ in
Xenopus oocytes produced inactivating calcium currents.
A, Families of current traces obtained by
depolarizing oocytes for 200 msec to +10 mV from holding potentials in
the range of 90 to 30 mV. Each holding potential was maintained for
60 sec before the test depolarization. B,
Current-voltage relationship for each of the cells in
A. The oocytes were depolarized for 200 msec to a
variety of test potentials from an HP of 100 mV. C,
Mean normalized peak current as a function of holding potential for
groups of cells expressing the subunit indicated. The
number of cells in each group is indicated in
parentheses.
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In contrast, expression of rat 2a with
1B produced currents that were virtually
holding potential independent. Figure
5A shows a family of current
records obtained by depolarizing an oocyte for 200 msec to +10 mV from
a variety of holding potentials in the range of 90 to 40 mV. Each
holding potential was maintained for 60 sec before the test
depolarization. Figure 5B plots the current-voltage
relationship for this cell. The cell was depolarized to a variety of
test potentials from an HP of 90 mV; peak current was measured and
then plotted as a function of test potential. Figure 5C
plots the voltage-dependent inactivation curve obtained by averaging
data from five oocytes expressing bovine 1B
and rat 2a and illustrates the holding
potential independence of N-type calcium channels constructed with
2a subunits. Similar results were obtained
when the human 2/ subunit was expressed along with 1B and rat
2a (data not shown).
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Figure 5.
Coexpression of bovine 1B with rat
2a in Xenopus oocytes produced
non-inactivating calcium currents. A, Family of current
traces obtained by depolarizing an oocyte for 200 msec
to +10 mV from holding potentials in the range of 90 to 40 mV. Each
holding potential was maintained for 60 sec before the test
depolarization. B, Mean normalized peak current as a
function of holding potential for eight cells expressing the bovine
1B and rat 2a subunits. C,
Current-voltage relationship for the cell in A. The
oocyte was depolarized for 200 msec to a variety of test potentials
from an HP of 100 mV.
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Bovine 2a differs significantly from rat
2a primarily in the C terminal where only 81%
of the predicted amino acids are identical. Coexpression of bovine
2a along with 1B and
2/ produced non-inactivating N-type calcium
channel current (Fig. 6A). In contrast,
coexpression of bovine 2b along with
1B and 2/ produced
a robustly inactivating N-type calcium channel current (Fig.
6B). These results suggest that the C terminal is not
likely to contribute to the unique properties of
2a because similar results were obtained with
both rat and bovine 2a. The N terminal of
2a is, however, a good candidate for mediating
the non-inactivating calcium currents seen with
2a, because 2a
differs from 2b only in its short N
terminal.
View larger version (12K):
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|
Figure 6.
Coexpression of bovine 1B and human
2/ with bovine 2a but not
2b produced a non-inactivating calcium channel current
in HEK 293 cells. A, Family of current
traces obtained by depolarizing an HEK 293 cell
expressing bovine 1B and 2a along with
human 2/ for 140 msec to +10 mV from holding
potentials in the range of 100 to 60 mV. Each holding potential was
maintained for 60 sec before the test depolarization. B,
Family of current traces obtained by depolarizing an HEK
293 cell expressing bovine 1B and 2b
along with human 2/. The voltage-clamp
protocol is described in A.
|
|
| |
DISCUSSION |
In an attempt to identify the molecular basis for the
non-inactivating N-type calcium channels observed in bovine chromaffin cells (Fig. 1) (Artalejo et al., 1992), we cloned the
1B, 1b, 1c, 2a,
2b, and 3a calcium
channel subunits from a chromaffin cell library and expressed these
clones in both HEK 293 cells and Xenopus oocytes. In both
systems expression of the cloned 1B and
2a along with human
2/ yielded N-type calcium currents that
resembled the non-inactivating N-type currents in bovine chromaffin
cells. However, expression of the 1B and
2/ with 1b,
1c, 2b, or
3a yielded calcium currents that showed
distinct inactivation during prolonged depolarizations as well as
decreased availability from depolarized holding potentials. These
results suggest that the 2a subunit is a key
determinant of the non-inactivating properties of the N-type calcium
channel in chromaffin cells. Previous studies with cloned
1A, 1B, and
1E have shown that inactivation rates of these
calcium channels depended on which subunit was coexpressed;
inactivation was slowest with 2, fastest with
3, and intermediate with
1 and 4 (Ellinor et
al., 1993; Sather et al., 1993; Olcese et al., 1994; Zhang et al.,
1994; DeWaard and Campbell, 1995; Parent et al., 1997; Patil et al., 1998). In addition coexpression of 2a has been
found to shift the voltage-dependent inactivation curves obtained with
1E to more depolarized potentials and to
result in incomplete steady-state inactivation (Sather et al., 1993;
Olcese et al., 1994; Parent et al., 1997). However, steady-state
voltage-dependent inactivation of 1A and
1E was never totally abolished even with the
2a subunit as was seen in this study that used
bovine 1B and either the bovine or rat
2a. A recent study using single-cell RT-PCR
linked 2a expression to a slowly inactivating
Q-type current in neostriatal neurons, whereas
1b expression was linked to fast-inactivating Q-type currents in cortical neurons (Mermelstein et al., 1999).
Several previous studies have suggested that the N terminal of the subunit is an important region regulating channel inactivation (Olcese
et al., 1994; Cens et al., 1999). The different inactivation rates that
we observed with bovine 2a and
2b (Fig. 6) support this hypothesis because
2a and 2b differ from
each other only in their short N terminal. Palmitoylation of the two
cysteines in the N terminal of 2a has been
shown to be essential for certain modulatory effects of
2a on 1C and
1E (Chien et al., 1996; Qin et al., 1998). The
C terminals of bovine 2a and
2b were markedly more different from this
region of the human, rabbit, rat, and mouse 2a
or 2b than these were from each other. To determine whether these C-terminal differences were functionally important, we also expressed the rat 2a (in
which 19% of the amino acids in the C terminal differ from those in
bovine 2a) with the bovine
1B and found that this combination of subunits also yielded non-inactivating N-type calcium channels. Thus it appears
that the differences in the C terminal of 2a
are not functionally important in terms of regulating calcium channel inactivation.
Despite the apparent importance of the 2a
subunit in regulating the rate of inactivation of N-type calcium
channels, a role for the 1B subunit itself
cannot be excluded. Both the IS6 transmembrane domain and the adjacent
I-II cytoplasmic loop have been implicated previously in the rate of
inactivation exhibited by cloned 1A and
1B subunits (Zhang et al., 1994; Page et al.,
1997). Bovine 1B differs from the consensus in
only one position within the IS6 region and in two positions in the
I-II loop region, but one of these is the absence of alanine 415. The
presence or absence of alanine 415 is a known splice variant in
1B (Lin et al., 1997) that corresponds to the
recently described splice variant ±valine 421 in
1A (Bourinet et al., 1999). The presence of
valine 421 confers much slower inactivation on
1A. None of the three library clones
corresponding to the alanine 415 region that were isolated from the
bovine chromaffin cell library contained alanine 415. Another splice
variant of 1B [+A, SFMG, and +ET (Lin et
al., 1997)] was originally suggested to have slower inactivation
kinetics than +A, +SFMG, and ET, but more recent work has suggested
that the differences between these splice variants are primarily in the
rates of activation and depend primarily on +ET (Lin et al., 1999). Our
expressed 1B was A, SFMG, and ET and
thus did not correspond exactly to either of these forms. However, two
of the four library clones covering the ±ET region did have +ET. Work is in progress to incorporate this splice variant into full-length 1B. In discussing the various splice variants
of 1B, it should also be noted that we do not
know the exact splice variant composition of the native
1B in chromaffin cells. The clones we used to
construct the full-length 1B may not be the
ones normally spliced together, a limitation that is true of all
full-length 1 subunits that have been
constructed from more than one cDNA library clone. RT-PCR of smaller
portions of 1B could easily determine which of
the individual known splice variants of 1B
(e.g., ±A415 or ±ET1575) are expressed in chromaffin cells, but it
still would not answer the question of which combinations of splice
variants make up a single mRNA.
In addition to modifications to inactivation made by different subunits or by changes to the 1 subunits
described above, inactivation of N-type
Ca2+ channels can be altered by
interactions with synaptic vesicle docking and fusion machinery.
Coexpression of syntaxin 1A with N-type channels in Xenopus
oocytes dramatically decreased channel availability because of the
stabilization of channel inactivation (Bezprozvanny et al., 1995).
Interestingly, we observed no such changes in inactivation when we
coexpressed syntaxin 1A with 1B, 2a, and 2/
(our unpublished observations), which suggests that syntaxin 1A
alters inactivation exclusively in N-type channels that exhibit inactivation.
N-type calcium channels, which are widely distributed throughout the
nervous system, have been unambiguously linked to a variety of
important physiological processes including synaptic transmission (Takahashi and Momiyama, 1993; Turner et al., 1993; Wheeler et al.,
1994). In secretory cells, like chromaffin cells, activation of N-type
calcium channels by themselves can trigger catecholamine secretion
(Artalejo et al., 1994). It seems likely that non-inactivating N-type
calcium channels would operate more effectively during periods of
intense stimulation, which would lessen the availability of
inactivating channels thereby diminishing
Ca2+ influx. In addition to expressing
inactivating or non-inactivating N-type calcium channels, chromaffin
cells also expressed inactivating or non-inactivating P/Q-type calcium
channels (our unpublished observation), which should also have
dramatic effects on Ca2+ influx. Thus, the
changes in channel inactivation properties because of alterations in
-subunit channel composition that are described in this study may
have important functional consequences for a wide variety of
Ca2+-dependent processes including
catecholamine secretion.
| |
FOOTNOTES |
Received Aug. 23, 1999; revised Dec. 10, 1999; accepted Dec. 13, 1999.
This work was supported by National Institutes of Health (NIH) grants
to A.P.F. and by an NIH training grant to J.H.H. We would like to thank
Drs. H. Pollard (NIH) for the bovine chromaffin cell cDNA library,
R. W. Tsien (Stanford University) for the rabbit 2b
cDNA clone, E. Perez-Reyes (Loyola University) for the rat 2a, and R. J. Miller (The University of
Chicago) for the human 1b and
2/ cDNA clones.
Correspondence should be addressed to Dr. Anne L. Cahill, The
Department of Neurobiology, Pharmacology, and Physiology, The University of Chicago, 947 East 58th Street, Chicago, Illinois 60637. E-mail: acahill{at}Drugs.bsd.uchicago.edu.
| |
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ABSTRACT
INTRODUCTION
