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 Previous Article | Next Article 
The Journal of Neuroscience, January 15, 1999, 19(2):684-691
Molecular Diversity of the Calcium Channel  2
Subunit
Norbert
Klugbauer,
Lubica
Lacinová,
Elsé
Marais,
Muriel
Hobom, and
Franz
Hofmann
Institut für Pharmakologie und Toxikologie der Technischen
Universität München, 80802 München, Germany
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ABSTRACT |
Sequence database searches with the  2 subunit as
probe led to the identification of two new genes encoding proteins with the essential properties of this calcium channel subunit. Primary structure comparisons revealed that the novel 2-2 and
2-3 subunits share 55.6 and 30.3% identity with the
2-1 subunit, respectively. The number of putative
glycosylation sites and cysteine residues, hydropathicity profiles, and
electrophysiological character of the 2-3 subunit
indicates that these proteins are functional calcium channel subunits.
Coexpression of 2-3 with 1C and
cardiac  2a or 1E and 3 subunits shifted the
voltage dependence of channel activation and inactivation in a
hyperpolarizing direction and accelerated the kinetics of current
inactivation. The kinetics of current activation were altered only when
2-1 or 2-3 was expressed with
1C. The effects of 2-3 on
1C but not 1E are indistinguishable from
the effects of 2-1. Using Northern blot analysis, it
was shown that 2-3 is expressed exclusively in brain,
whereas 2-2 is found in several tissues. In
situ hybridization of mouse brain sections showed mRNA
expression of 2-1 and 2-3 in the
hippocampus, cerebellum, and cortex, with 2-1
strongly detected in the olfactory bulb and 2-3 in
the caudate putamen.
Key words:
2 subunit; voltage-activated channel; calcium channel subunit; neuron; gene diversity; electrophysiology
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INTRODUCTION |
Voltage-gated calcium channels have
been purified and cloned from various tissues such as skeletal muscle,
heart, and brain. These channels are formed by heterooligomeric
complexes consisting of various combinations of an 1
protein with auxiliary 2, , and  subunits. To
date, seven genes encoding 1 subunits of the high
voltage-activated (for review, see Hofmann et al., 1994 ; Strom et al.,
1998; Bech-Hansen et al., 1998) and two genes of the low
voltage-activated calcium channels have been identified (Perez-Reyes et
al., 1998; Cribbs et al., 1998). This subunit accounts not only for the
ion channel pore but also contains the voltage sensor and the
determinants for binding of drugs and toxins. The current through the
1 subunit is modulated by interactions with the ,
2, and subunits. The molecular diversity of the subunit is not only caused by the expression of four genes, but
also by differential splicing. Until recently, only a single subunit in skeletal muscle had been described (Eberst et al., 1997),
but a novel neuronal form has since been identified in brain (Letts et
al., 1998).
Since the molecular cloning of the 2 subunit (Ellis
et al., 1988), several splice variants have been detected, but no
further 2 subunit genes have been identified. The
different splice variants arise from various combinations of three
alternatively spliced regions that result in five isoforms that are
expressed in a tissue-specific manner (Angelotti and Hofmann, 1996).
Structurally, the 2 subunit is a heavily glycosylated
175 kDa protein that is posttranslationally cleaved to yield a
disulfide-linked 2 and protein (DeJongh et al.,
1990; Jay et al., 1991). The part anchors the 2
protein to the membrane via a single transmembrane segment (Gurnett et al., 1996). The membrane topology of the 2 subunit
was further refined using anti-2 antibodies and
C-terminal deletion mutants (Brickley et al., 1995; Wiser at al.,
1996). Structural studies have shown that the extracellular
2 domain provides the structural elements required for
channel stimulation (Gurnett et al., 1996) and that the domain,
which contains the only transmembrane segment of 2
complex, harbors the regions important for the shift in voltage-dependent activation, steady-state inactivation, and the modulation of the inactivation kinetics (Felix et al., 1997). The
identification of new 2 subunits could present
further possibilities for differential and specific regulation of
calcium channels.
In studies designed to address the diversity and function of
2, cloning, hybridization, and patch-clamp techniques
were used to identify and characterize novel 2
subunits. Two new 2 genes were found by searching
databases with the 2 cDNA sequence (Ellis et al.,
1988). These genes encode proteins with essential features of
2 subunits, such as the high number of potential
glycosylation sites and cysteine residues, the hydrophobicity plots,
and electrophysiological characteristics. The novel
2-2 gene was found to be predominately expressed in
heart, pancreas, and skeletal muscle, with 2-3
expressed only in brain.
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MATERIALS AND METHODS |
Isolation of RNA and cDNA library construction. Total
RNA from mouse brain was isolated by the guanidinium thiocyanate
method, and the poly(A) RNA was separated by oligo-dT cellulose
chromatography (Poly(A) Quik mRNA Isolation kit; Stratagene,
Heidelberg, Germany). Poly(A) RNA from mouse brain was
reverse-transcribed, and double-strand cDNA was synthesized using the
Superscript plasmid system (Life Technologies). The cDNA fragments were
ligated with BstXI/EcoRI adaptors (Invitrogen,
San Diego, CA) and size-fractionated in a low-melting agarose gel. Only
gel slices containing fragments >2000 bp were excised and digested
with Gelase (Biozym, Hessisch Oldendorf, Germany). Recovered
cDNA was ligated into the BstXI site of the pcDNAII vector
(Invitrogen) and transformed in the Escherichia coli
XL1-blue mrf' strain (Stratagene, Heidelberg, Germany). The cDNA
library was screened with a random-primed labeled PCR probe (see below,
nt 1501-1821). Sequencing of the clones was performed using the
dideoxy chain termination method on both strands. A full-length clone
was used for the preparation of the NotI-SphI
fragment with the entire open reading frame in pcDNA3 (Invitrogen)
yielding the expression plasmid pc32-3.
PCR amplification. The expressed sequence tag (EST)
with the accession number AA190607 was used to design the
primers NKAD1, GGC ACA GAT GTC CCA GTT AAA GA and NKAD2, TGT ATA GTA
GTA GTC ATT GGT CAT, with which the partial 2-3
subunit cDNA was amplified. The PCR fragment was cloned in pUC18 and
was sequenced on both strands.
Northern blot analysis. Human and mouse multiple tissue
Northern Blots were obtained from Clontech (Heidelberg, Germany) and hybridized according to the manufacturer's instructions. Random-primed labeled fragments (2-2, nt 2877-3249;
2-3, nt 2893-3377) were used as probes; these
regions share no significant homology with each other. A 3 hr
prehybridization step was followed by an overnight hybridization with
5 × 106 cpm/ml of probe at 42°C. The final
stringency wash performed was with 0.1× SSC, 0.1% SDS at 42°C.
In situ hybridization. Intact brains of adult female
mice were removed immediately after cervical dislocation and frozen in isopentane cooled to  40°C. Brain was sectioned into 16 µm slices in a cryostat at 20°C and thaw-mounted onto polylysine slides. The
tissue was vacuum-dried, fixed in 4% paraformaldehyde in PBS, and
washed in 0.5× SSC. The sections were acetylated in 0.25% acetanhydride in 0.1 M triethanolamine, pH 8.0, and washed
in 2× SSC. The tissue was dehydrated through a series of ethanol solutions, from 50 to 100%. The slides were vacuum-dried and stored at
80°C until used.
Murine 2-1 (nt 2760-3170), human
2-2 (nt 2877-3249), and murine 2-3
(nt 2744-3228) specific riboprobes were generated by in
vitro transcription as described previously (Ludwig et al., 1997).
Briefly, probe template DNA was amplified by PCR from murine 2-1 and -2 clones using primers specific for the
variable C-terminal region of 2. BamHI and
Asp718 restriction sites were added to the
2-1 forward and reverse primers, respectively, to
allow for sticky-end ligation. 2-3 was ligated to the
linearized vector by blunt end ligation. The integrity of each probe
was verified by dideoxy termination sequencing. 35S-UTP
(DuPont NEN, Wilmington, DE)-labeled sense and antisense riboprobes
were generated using a standard T3 and T7 polymerase in
vitro transcription procedure (T3 and T7 from New England Biolabs, Beverly, MA). Unincorporated nucleotides were removed using Sephadex G50 columns (Pharmacia, Freiburg, Germany).
Messenger RNA in situ hybridization was performed on
cryostat sections of mouse brain. A prehybridization step was performed at 45°C for 2 hr. Hybridization with 1 × 107
cpm/ml probe proceeded for 16 hr at 55°C. After a 20 µg/ml RNase A
(Boehringer Mannheim, Mannheim, Germany) digestion, a high-stringency wash of 0.1× SSC, 1 mM EDTA, and 1 mM
dithiothreitol was done for 2 hr at 60°C. The slides were
dehydrated in ethanol and analyzed by autoradiography.
Transfection of HEK293 cells and electrophysiological
recordings. The full-length cDNAs of all subunits, i.e.,
1C, 1E, 2a, 3,
2a-1, and 2-3 were cloned into the
pcDNA 3 vector (Invitrogen). For more details, see Schuster et al.
(1996). HEK 293 cells were transfected with various combinations of an
1 subunit (1C,
1E), a subunit (2a, 3), and an
2 subunit (2-1,
2-3). This was achieved by lipofection with
Lipofectamine (Life Technologies) at a DNA mass ratio of 1:1 for
expression of two subunits or 1:1:1 for three subunits.
Electrophysiological recordings. Ionic currents from
transfected cells were recorded in whole-cell configuration of the
patch-clamp method. Ba2+ was used as the charge
carrier. The extracellular solution contained (in mM):
N-methyl-D-glucamine, 125;
BaCl2, 20; CsCl, 5; MgCl2, 1;
HEPES, 10; and glucose, 5, pH 7.4 (HCl). The intracellular solution
contained (in mM): CsCl, 60; CaCl2, 1;
EGTA, 11; MgCl2, 1; K2ATP, 5; HEPES, 10;
and aspartic acid, 50, pH 7.4 (CsOH). Currents were recorded using an
EPC-9 patch-clamp amplifier and corresponding Pulse software from Heka
Electronics (Lambrecht, Germany). Patch pipettes were pulled from
borosilicate glass. The pipette input resistance was typically between
1.8 and 2.2 M . The capacity of individual cells ranged between 25 and 90 pF, and series resistance ranged between 3.5 and 5.0 M.
Capacity transients were compensated using build-in procedure of the
Heka system. Curve fitting and statistical analysis were performed using the Origin 5.0 software package (Microcal, Northampton, MA). The
significance of observed differences was evaluated by nonpaired
Student's t test. Probability of 5% or less was considered to be significant.
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RESULTS |
Primary structures
A database search using the 2 subunit cDNA as
probe revealed three additional sequences similar to this calcium
channel subunit (Fig. 1), which were
derived from two genes named 2 and 3. Two of these sequences are human
full-length sequences of closely related isoforms of the
2 subunit (GenBank accession numbers: AF042792,
isoform I; AF042793, isoform II; M. H. Wei, F. Latif, F. M. Duh,
D. Andreazzoli-Angeloni, V. Kashuba, E. Zabarovsky, B. Johnson, and M. I. Lerman, unpublished observations). These sequences differ
only at the N terminus of the 2 protein, indicating that
both isoforms are derived from the same gene 2 and are generated by
differential splicing. The 5'-untranslated region upstream of the ATG
codon from isoform I is in good agreement with the Kozak sequence for
initiation of translation in eukaryotic cells, whereas the same region
of isoform II shows only a limited homology with this sequence.
Furthermore, only isoform I but not isoform II shows features of a
potential signal sequence. Both observations suggest that only isoform
I can form a functional calcium channel subunit. Isoform I, which we
describe here as the 2-2 subunit, (Wei, Latif, Duh,
Andreazzoli-Angeloni, Kashuba, Zabarovsky, Johnson, and Lerman,
unpublished observations) has 55.6% identity with the
2-1 subunit (Ellis et al., 1988). Eighteen potential
N-glycosylation sites can be identified in the primary
structure (Fig. 1), which is the same number of sites as in the
2-1 subunit (Ellis et al., 1988).
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Figure 1.
a, Amino acid alignment of the
2-1 (1), 2-2
(2), and 2-3
(3) subunits. The N-terminal region differing
between the 2-2 subunit isoform I and II is
underlined. Regions that are identical in all sequences
are boxed, and conserved cysteine residues are
additionally highlighted. The presumptive signal
peptides for classes 1 and 3 are shown in italics.
Potential N-glycosylation sites are printed in
bold. The arrow indicates the cleavage
site between the 2 and proteins of the
2-1 subunit. These sequence data are available from
the EMBL database under accession numbers M21948 for the
2-1 subunit, AF042792 for the
2-2(I) subunit, and AF042793 for the
2-2(II) subunit isoforms, respectively, and AJ010949
for the 2-3 subunit. b, Hydrophobicity
profile of the 2 subunits computed according to Kyte
and Doolittle (1982). The curve is the average of a
residue-specific hydrophobicity index over a window of nine
residues.
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The third sequence identified by database searches was a partial human
EST sequence AC: AA190607. This sequence was used to design primers for
the amplification of cDNA from mouse tissues. The cDNA could be
amplified only from mouse brain mRNA and not from other tissues (Fig.
2). A cDNA library was constructed from mouse brain mRNA and screened with the PCR product as a probe. Several
independent clones could be identified that encode the novel
2 subunit, which we name 2-3 (Fig.
1). A detailed restriction analysis of the different clones showed that
there are no further isoforms in mouse brain. The sequence upstream of
the start ATG is in agreement with that for the initiation of
translation in eukaryotic cells (data not shown). The open reading
frame consists of 3273 bp encoding a protein with 1091 amino acid
residues. The 2-3 subunit has 30.3% identity with
the 2-1 subunit and 31.2% with the
2-2 subunit. The primary structure of
2-3 contains nine potential
N-glycosylation sites (Fig. 1a).
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Figure 2.
Northern blot analysis of the
2-2 and 2-3 subunits. For each
lane, ~2 µg of poly(A) RNA was run on a denaturing
formaldehyde-containing agarose gel, transferred to a nylon membrane,
and fixed by UV irradiation. a, Human multiple tissue
blot using a specific probe for the 2-2 subunit.
b, Mouse multiple tissue blot for the
2-3 subunit. Arrows indicate
predominant species of mRNA with sizes of 5.2 (2-2)
and 4.3 (2-3) kb.
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Hydrophobicity analysis of all three 2 subunits
indicates a similar membrane topology, including a hydrophobic
transmembrane segment at the C terminus of the domain (Fig.
1b). A sequence comparison reveals that as many as 14 cysteine residues are conserved in all of the three 2
genes, further strengthening the postulate that these subunits are
disulfide-linked proteins with similar higher order structures.
Tissue distribution
The expression of the novel 2 subunits was
examined by Northern and in situ hybridization. The
2-2 subunit is highly expressed in heart, pancreas,
and skeletal muscle tissue (Fig. 2), which can be seen even after 6 hr
of autoradiography of Northern blots. After 1 d of exposure, the
2-2 subunit can also be detected in kidney, liver,
placenta, and brain. This broad expression pattern was also observed
for the 2-1 subunit. An 8.0 kb transcript of the
2-1 subunit was detected in brain, heart, aorta,
skeletal muscle, and ileum (Ellis et al., 1988). In contrast to this
ubiquitous expression pattern, the 2-3 subunit is
only found in brain (Fig. 2). In each case, the Northern analysis
reveals one predominant species of mRNA with sizes of 5.2 (class 2) and
4.3 kb (class 3).
The mRNA expression of 2-1 and 2-3
calcium channel subunits in mouse brain was mapped by in
situ hybridization. Both 2 forms were detected
in several regions of the brain, with differential expression of the
mRNAs in some structures. Strong expression of 2-1
was seen in the pyramidal cell layer of Ammon's horn in the
hippocampus (CA1-3) and in the granular cell layer of the dentate
gyrus (Fig. 3). The olfactory bulb also
stained strongly, and expression in the mitral and glomerular cell
regions was seen by dark-field microscopy of slides coated with
photographic emulsion. Signals in the cerebellar cortex, and to a
lesser extent in thalamic nuclei, were also observed (Fig. 3).
Examination of emulsion-coated slides showed that the expression of
2-1 in the cerebellum was restricted to the granular
layer (data not shown).
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Figure 3.
Autoradiographs of 2-1 and
2-3 riboprobe hybridization to horizontal mouse brain
sections. Central (a, c) and more basal
(b, d) sections of the brain are shown.
Expression of 2-1 is seen in the
(a) hippocampus (H),
cerebral cortex (Co), cerebellum (Ce),
and (b) olfactory bulb (Ob).
2-3 mRNA was detected in the caudate putamen
(CPu), hippocampus (H),
entorhinal complex (E), cortex
(Co), and thalamic nuclei (T)
(c, d).
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The 2-3 mRNA was predominantly expressed in the
caudate putamen, entorhinal complex, hippocampus, and cortex (Fig. 3).
As with 2-1, the pyramidal cell layer of the
hippocampus (CA1-3) and granular cell layer of the dentate gyrus
showed the highest degree of hybridization with the antisense probe
(Fig. 3). Hybridization of both probes was specific as judged by the
absence of signals when sense probes were applied (data not shown).
In situ hybridization for the 2-2 subunit
indicated a ubiquitous expression in cardiac tissues (data not shown).
Functional characterization
Because the Northern blot hybridization indicated a high
expression of the 2-3 subunit in brain, we performed
cotransfection studies with 1 subunits that have been
characterized in this tissue. Because in situ analysis did
not show an exact colocalization of 2-3 with known
1 subunits, coexpression studies were done with an
1 subunit of the dihydropyridine-sensitive class C and a
dihydropyridine-insensitive class E calcium channel. This approach allows for the identification of interactions with both evolutionary distant calcium channel 1 families.
Effects of the 2-3 subunit on barium current
through the 1C-type calcium channel
To study the effects of 2-3 subunit on channel
gating, we coexpressed this subunit with the 1C subunit
alone or in combination with both 1C and 2a subunits.
Voltage protocols are described in detail in the legend to Figure
4, and quantitative analyses are
summarized in Table 1. When coexpressed
with 1C alone, the effects of 2-3 on
IBa were less noticeable than when 2a was coexpressed.
2-3 did not affect the current density, time course of current inactivation, and voltage dependence of steady-state inactivation of 1C expressed singly (data not shown).
However, when expressed with 1C only,
2-3 shifted the activation curve by 4.4 mV in the
hyperpolarizing direction and slightly accelerated the time of current
activation during the depolarizing pulse (data not shown). The effects
on the channel kinetics became more prominent in the presence of the
2a subunit (Fig. 4, Table 1). In this combination,
2-3 significantly increased the current density, shifted the voltage dependence of current activation by 8.7 mV in a
hyperpolarizing direction, accelerated the time course of current
activation during the depolarizing pulse, accelerated the current
inactivation at positive membrane potentials, shifted the steady-state
inactivation curve in a hyperpolarizing direction, and significantly
changed its slope.
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Figure 4.
The 2 subunit affects
current through the 1C-type calcium channel. The
2-3 subunit was coexpressed with
1C and 2a. For comparison, the current through cells
coexpressing 2-1 subunit was coanalyzed.
a shows the voltage dependence of current activation
measured as the amplitude of current activated by a 40-msec-long
depolarizing pulse from a holding potential of 80 mV to voltages
marked on the ordinate and normalized to the maximal amplitude. Each
voltage dependence was fitted to the Boltzmann equation. Results of
these fits are summarized in Table 1. The inset in the
top left of a shows voltage dependence of
the kinetics of current activation. The ascending phase of the current
time course was fitted to single exponential. The resulting time
constants were averaged and plotted against corresponding membrane
potentials. In both graphs,  represents the 1C2a
channel,  the 1C2a2-1 channel,
and  the 1C2a2-3 channel. The
inset in the right of a
illustrates a typical family of currents measured during a series of
depolarizing pulses from the holding potential of 80 mV to membrane
potentials ranging from 20 to +70 mV with a step of +10 mV. ,
1C2a channel. The cell capacity was 83 pF, and the
resulting maximal current density was approximately 13 pA/pF; ,
1C2a2-3. The cell capacity was 31 pF, and the corresponding maximal current density was approximately
41 pA/pF. b shows averaged steady-state inactivation
curves measured from a holding potential of 80 mV. Current was
inactivated by a 5-sec-long prepulse to the potentials marked on the
ordinate. This was followed by a 5-msec-long return to the holding
potential and a 40-msec-long test pulse to the maximum of the
current-voltage relationship. Solid lines are fitted to
the Boltzmann equation. The inset shows the time course
of the current during a 5-sec-long depolarizing pulse to +20 mV with
zero level indicated by a horizontal line. Currents
shown are averaged time courses from 9 to 12 experiments scaled to the
same amplitude. Individual measurements were fitted to the sum of two
exponentials. Results of all fitting procedures are summarized in Table
1. Symbols are as in a.
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We further compared the effects of 2-3 with those of
the previously described 2-1 subunit (Fig. 4, Table
1). The changes in gating-related channel characteristics elicited by
both subunits were qualitatively similar, and in most cases the
measurements (current density, voltage dependence of current activation
and inactivation, and time course of current activation) were not significantly different. Both 2-1 and -3 subunits
accelerated the time course of current inactivation at a membrane
potential of +20 mV by enhancing the proportion of the fast time
constant,  1. 2-3 changed this constant from
273 ± 24 msec to 156 ± 10 msec. Surprisingly,
2-1 significantly increased the value of the slow
time constant 2 from 1.16 ± 0.08 sec to 3.54 ± 0.46 sec,
but this effect was apparently overwhelmed by the effect of the
increased proportion of the current inactivating with fast time
constant 1, with the result that the overall time course of
inactivation was accelerated (Fig. 4b, inset).
The effects of both 2-3 and 2-1
subunits on whole-cell current parameters, which reflect the gating of
the 1C channel, are virtually identical and they require
the presence of (in this case 2a) to become prominent.
Effects of the 2-3 subunit on barium current
through 1E-type calcium channel
Although both 1C and 2-1 subunits
are fairly abundant in mammalian tissues, 1E and
2-3 are predominantly expressed in neuronal tissue.
We therefore selected 1E for studying the effects of
2-3. For all experiments, 3 subunit was
coexpressed with 1E. This subunit was suggested to
modulate the current through the 1E channel (Ludwig et
al., 1997). As with the 1C channel, both
2-1 and 2-3 affected most of the
gating-related parameters except for the time constant of current
activation during membrane depolarization (Fig.
5, Table 1). In contrast to
1C2a channel, the effects of 2-3
and 2-1 on the voltage dependence of current activation (Fig. 5a) and inactivation (Fig. 5b)
of 1E3 channel were significantly different. The
2-1 subunit shifted both activation and steady-state
inactivation curves in a hyperpolarizing direction, but the change in
current activation was not statistically significant. In both curves,
the shift evoked by 2-3 was significantly larger (Table 1). In contrast, the effects of both 2s on the
time course of current inactivation at a membrane potential of +20 mV
were identical and restricted to diminution of the noninactivating part
of the current (Table 1).
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Figure 5.
The 2 subunit affects current
through the 1E-type calcium channel. Unless otherwise
indicated, the voltage protocols used were the same as those described
in the legend to Figure 4. represents the 1E3
channel; the 1E32-1 channel,
and the 1E32-3 channel.
Boltzmann fits of voltage dependencies of current activation are
summarized in Table 1. The inset in the
right of a shows a typical family of
currents measured during a series of depolarizing pulses from a holding
potential of 100 mV to membrane potentials ranging from 30 mV to
+60 mV with step of +10 mV. , 1E3 channel, with a
cell capacity of 19 pF and a maximal current density of approximately
34 pA/pF; , 1E32-3 channel,
with a capacity of 43 pF and a maximal current density of approximately
74 pA/pF. b shows the steady-state inactivation curve
measured from a holding potential of 100 mV using a 5-sec- long
conditioning pulse to membrane potentials marked on the ordinate.
Solid lines represent Boltzmann fits. The
insert illustrates the inactivation of
IBa during a 300-msec-long depolarizing
pulse from a holding potential of 100 mV to +20 mV scaled to the same
amplitude. Eight to 10 measurements were averaged for each channel
type. The 1E32-1 and
1E32-3 current traces are
indistinguishable from each other. Individual time courses of current
inactivation were fitted to a single exponential with a small
proportion of noninactivating current. Results of all fits are
summarized in Table 1. Symbols are as in
a.
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DISCUSSION |
We present here the first account of the existence of multiple
2 calcium channel subunits. The previously known
2 we have named 2-1, and the new
forms have been designated 2-2 and 2-3 based on their similarity to the original
subunit. An amino acid alignment reveals that there is only a
significant degree of homology in the core region of the
2 protein, whereas the proteins show identities only
with respect to the cysteine residues. Despite the low degree of
homology at the primary structure level between these forms, other
features such as the number of glycosylation sites, cysteine residues,
and hydrophobicity profiles are very similar. For these reasons we
conclude that all three 2 subunits are
disulfide-linked proteins with similar higher order structures. The
2-1 and -2 subunits are more ubiquitously expressed
than 2-3, which has only been identified in brain.
Comparisons of the expression patterns of the 1 and 3 class
2 subunits with 1 subunits in brain
slices (Ludwig et al., 1997) do not indicate specific
1-2 combinations. We cannot exclude
the possibility that 2-3 also interacts with other
ion channels or even other membrane proteins.
Functional coexpression of the 2-1 subunit with
various combinations of 1 and subunits results in an
increase in the current densities or dihydropyridine
(DHP)-binding sites (Singer et al., 1991; Welling et al., 1993;
De Waard et al., 1995; Shistik et al., 1995; Bangalore et al., 1996;
Gurnett et al., 1996; Felix et al., 1997; Parent et al., 1997;
Jones et al., 1998), acceleration of current activation and
inactivation (Singer et al., 1991; De Waard et al., 1995; Bangalore et
al., 1996; Felix et al., 1997; Qin et al., 1998; Shirokov et al.,
1998), and a shift of the current-voltage curve in a hyperpolarizing
direction (Singer et al., 1991; Felix et al., 1997). Regardless of the
1 subunit used in this study, the 2-3
subunit was found to increase the current density, which is in
agreement with the results of these groups. In addition to this
effect, coexpression of 2-3 caused a shift of the
voltage dependence of channel activation and inactivation in a
hyperpolarizing direction and an acceleration in the kinetics of
current inactivation.
When the results shown in Figures 4 and 5 are compared, it can be seen
that 2-1 caused a smaller shift in the voltage
dependence of activation and inactivation of 1E3 as
compared with the 1C2a channel. The effects of
2-3 on the electrophysiological properties of
1C coexpressed with 2a was found to be similar to
those of 2-1. In contrast, coexpression of
2-3 with 1E and 3 produced more
pronounced differences in the current characteristics associated with
channel gating than the coexpression of 2-1.
The mechanism whereby 2 modulates the conductances of
1 is not clearly understood. The increase in density of
current and DHP-binding sites can be explained by an improved targeting
of expressed 1 subunit to the cell membrane and
maturation of the channel complex (Shistik et al., 1995), which leads
to an increased amount of charge moved during channel activation
(Bangalore et al., 1996; Qin et al., 1998). It was suggested that the
increase in current requires the presence of an intact 2
protein, whereas the shift of voltage-dependent activation and steady
state inactivation as well as the acceleration of the inactivation
kinetics are caused by the transmembrane protein (Felix et al.,
1997). However, based on the amino acid similarity of the three subunit
forms, it seems more likely that 2 harbors the relevant
residues responsible for the observed effects on 1 and
that the domain functions only as an membrane anchor for
2. This interpretation is further strengthened by the
sequences of the proteins, which are not conserved.
These results, together with the brain-specific expression of
2-3, suggest that the 2-3 subunit
may have a distinct physiological role in neuronal tissue. The
2 protein has been implicated as the in
vivo target for the antiepileptic drug gabapentin (Gee et al.,
1996), which apparently inhibits calcium currents in isolated rat brain
neurons (Stefani et al., 1998). It was proposed that gabapentin binds
preferentially to the 2-1 subunit. This is supported
by evidence that the partial N-terminal amino acid sequence of the
gabapentin-binding protein obtained from porcine brain membranes is
identical with an aminoterminal peptide of 2-1. This
sequence is not present in classes 2 or 3. Further support for this
postulate is that our mRNA in situ analysis of
2-1 in brain showed the same distribution as that of
gabapentin-binding sites (Taylor et al., 1998), which differs from that
of 2-3. For these reasons it is more likely that
gabapentin targets the 2-1 subunit. However, another
study using a different purification procedure showed that there may be
an additional gabapentin-binding protein in brain, which was detected
with a polyclonal 2 antibody (Brown et al., 1998) and
which could be another 2 form. Further investigations
need to be undertaken to elucidate the binding of gabapentin to the
2 subunit.
| |
FOOTNOTES |
Received Sept. 14, 1998; revised Nov. 2, 1998; accepted Nov. 3, 1998.
This work was supported by the Deutsche Forschungsgemeinschaft and Fond
der Chemie.
Correspondence should be addressed to Dr. Norbert Klugbauer, Institut
für Pharmakologie und Toxikologie der Technischen
Universität München, Biedersteiner Strasse 29, 80802 München, Germany.
Dr. Lacinová contributed to this paper while on leave from
Institute of Molecular Physiology and Genetics, Slovak Academy of
Sciences, Vlarska 5, 833 04 Bratislava, Slovakia.
| |
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Abstract
Introduction
