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The Journal of Neuroscience, July 1, 2000, 20(13):4769-4775
Alternative Splicing in the Cytoplasmic II-III Loop of the
N-Type Ca Channel  1B Subunit: Functional Differences Are
 Subunit-Specific
Jennifer Qian
Pan and
Diane
Lipscombe
Department of Neuroscience, Brown University, Providence, Rhode
Island 02912
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ABSTRACT |
Structural diversity of voltage-gated Ca channels underlies much of
the functional diversity in Ca signaling in neurons. Alternative splicing is an important mechanism for generating structural variants within a single gene family. In this paper, we show the expression pattern of an alternatively spliced 21 amino acid encoding exon in the
II-III cytoplasmic loop region of the N-type Ca channel  1B subunit and assess its functional impact.
Exon-containing 1B mRNA dominated in sympathetic ganglia
and was present in ~50% of 1B mRNA in spinal cord and
caudal regions of the brain and in the minority of
1B mRNA in neocortex, hippocampus, and cerebellum (<20%). The II-III loop exon affected voltage-dependent inactivation of the N-type Ca channel. Steady-state inactivation curves were shifted
to more depolarized potentials without affects on either the rate or voltage dependence of channel opening.
Differences in voltage-dependent inactivation between 1B
splice variants were most clearly manifested in the presence of Ca
channel  1b or 4, rather than
2a or 3, subunits. Our results
suggest that exon-lacking 1B splice variants that
associate with 1b and 4 subunits will be
susceptible to voltage-dependent inactivation at voltages in the range
of neuronal resting membrane potentials ( 60 to 80 mV). In contrast,
1B splice variants that associate with either
2a or 3 subunits will be relatively
resistant to inactivation at these voltages. The potential to mix and
match multiple 1B splice variants and subunits
probably represents a mechanism for controlling the plasticity of
excitation-secretion coupling at different synapses.
Key words:
N-type calcium channel; 1 subunit; regulated alternative splicing; intracellular loop II-III; genomic
analysis; tissue distribution; subunit
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INTRODUCTION |
Structural diversity of
voltage-gated Ca channels is at the heart of the rich functional
diversity in Ca signaling in mammalian neurons. Neurons can select from
several distinct Ca channel 1 genes that
undergo extensive RNA processing, including alternative splicing
(Perez-Reyes et al., 1990 ; Soldatov, 1994; Kollmar et al., 1997;
Lin et al., 1997; Bourinet et al., 1999) and differential polyadenylation (Schorge et al., 1999). Each 1
subunit may also interact with multiple functionally distinct auxiliary
subunits (Scott et al., 1996; Walker and De Waard, 1998), expanding the capacity for diversity within each Ca channel family. The
1B subunit is the functional core of N-type Ca
channels that localize to synapses and control calcium-dependent
neurotransmitter release throughout the vertebrate nervous system
(Hirning et al., 1988; Takahashi and Momiyama, 1993; Dunlap et al.,
1995). Many neurotransmitters and neurohormones modulate
excitation-secretion coupling by regulating the gating of N-type Ca
channels via effects on the 1B subunit (Dunlap
et al., 1995). The 1B gene is also subject to
tissue-specific alternative splicing (Lin et al., 1997, 1999), which
probably represents an important mechanism for optimizing
neurotransmitter release in different regions of the nervous system.
The extent of splicing in the 1B gene is not
known, but its large size (>100 kb in human genome), together with
mismatches in 1B cDNAs isolated from different
tissues, supports the presence of multiple sites of alternative splicing.
In a previous study, we identified two short cassette exons in S3-S4
linkers of domains III and IV of the Ca channel
1B subunit gene (see Fig.
1A) (Lin et al., 1997, 1999). Alternative splicing of
these exons was tissue-specific and influenced both the
voltage-dependence and rate of Ca channel activation. A third variant
site in loop I-II of the of 1B gene involving
one amino acid (A415) originated from the
random use of alternative 3' acceptor-splice sites, but it is not
associated with any obvious change in channel gating (Lin et al.,
1997). In the present study, we characterize a larger alternatively
spliced sequence encoding 21 amino acids in the cytoplasmic II-III
loop of the 1B gene (see Fig.
1A) first identified in mouse neuroblastoma cells
(Coppola et al., 1994) and more recently shown by PCR analysis to be
present in rat brain (Ghasemzadeh et al., 1999). We report the genomic
structure of this site in the rat 1B gene,
detail the differential expression of this exon in the rat nervous
system, and assess its impact on channel function.
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MATERIALS AND METHODS |
Genomic analysis. A 6.3 kb region of the rat
1B gene was amplified from liver genomic DNA
using primers directed to 5' and 3' exons flanking the splice junction
of interest in the II-III loop region. Primer sequences were as
follows: Bup2170, (5'-GAG GAG ATG GAA GAG GCA GCC AAT-3'); and Bdw2361,
(5'-CTC CGG GTC CAT CTC ACT GTA CAG T-3'). The 50 µl PCR reaction mix
contained 250 ng of rat liver genomic DNA, 350 µM each deoxynucleotide and 0.4 µM each primer. After a 15 min preincubation at
92°C, 0.75 µl of enzyme mix (Expand Long Template; Boehringer
Mannheim, Indianapolis, IN) was added to "hot start" the
reaction. After 30 amplification cycles, a single 6.3 kb product was
generated and subsequently gel-purified, subcloned, and sequenced by
primer walking (Yale University Sequencing Facility, New Haven, CT).
The sequence is available under GenBank accession number AF222338.
Reverse transcription-PCR. Total RNA was isolated from
different regions of the adult rat nervous system using the
guanidium-thiocyanate method and used for first strand cDNA synthesis;
4-6 µg of RNA from CNS tissue or 1-2 µg of RNA from sensory and
sympathetic ganglia was used as template in each 20 µl reaction mix
containing 0.5 mM each deoxynucleotide, 50 ng/µl random hexamers, 10 mM DTT, and 200 U
Moloney murine leukemia virus reverse transcriptase (Life Technologies,
Grand Island, NY), incubated for 1 hr at 37°C. One microliter of cDNA
from each reverse transcription (RT) reaction (or 3 µl from pituitary
RT reaction) was used for PCR amplification in a 50 µl standard
reaction mix using the following protocol: 1 cycle at 94°C for 2 min,
30 cycles at 94°C for 10 sec, 59°C for 35 sec, 72°C for 50 sec,
and 1 cycle at 72°C for 8 min. Primers Bup2104 (5'-TTG AAC GTT TTC
TTG GCC ATT GCT GT) and Bdw2363 (5'- CTC CTC CGG GTC CAT CTC ACT GTA
CA) were located in 5' and 3' exons flanking the splice junction.
Negative controls that lacked template confirmed that reagents were not
contaminated by 1B cDNA clones. The primers
flanked a 6.1 kb intron so contamination by genomic DNA could be ruled
out, and reverse transcriptase-lacking controls were also performed.
Thirty rounds of amplification were used routinely in our analysis. To
control for the possibility of saturation, the number of cycles was
increased stepwise from 10, 15, 20, 25, to 30 in one experiment.
Products were first observed after 25 cycles, and relative band
intensities were identical to those observed after 30 cycles. Band
intensities were quantified and normalized to the size of the DNA
fragments using a gel documentation system (Alpha Inotech).
Functional assessment of the Ca channel
1B cDNA constructs. The N-type Ca
channel 1B-b splice variant (Lin et al., 1997) (GenBank accession number AF055477) referred to here as
 211B was used as template for constructing
the +211B splice variant (GenBank accession
number AF222337) by standard cloning methods. 211B and +211B
splice variants were cloned into the Xenopus -globin
vector pBSTA to facilitate functional expression (Goldin and Sumikawa,
1992). Functional properties of 211B and
+211B were assessed in the Xenopus
oocyte expression system using methods and procedures essentially as
described previously (Lin et al., 1997, 1999). Forty-six nanoliters of
1B (67-400 ng/µl) and (22-133 ng/µl)
cRNA mix (1B/ is 3:1 µg/µg) were
injected into defolliculated Xenopus oocytes, and currents
were recorded 3-5 d later. Before recording, oocytes were injected
with 46 nl of a 50 mM BAPTA solution to
reduce activation of the endogenous Ca-activated
Cl current (Lin et al., 1997). N-type
Ca2+ channel currents were recorded with
the two microelectrode voltage-clamp technique using electrodes of
0.8-1.5 and 0.3-0.5 M resistance (3 M KCl)
for voltage and current electrodes, respectively. Recording solutions
contained either 5 mM BaCl2 or 2 mM
CaCl2, 85 mM tetraethylammonium, 5 mM KCl, and 5 mM HEPES, pH adjusted
to 7.4 with methanesulfonic acid. The 1b subunit used in
this study was provided by K. P. Campbell (University of Iowa,
Iowa City, IA) (Pragnell et al., 1992); 2a and
4 was provided by E. Perez-Reyes (Loyola
University, Maywood, IL) (Perez-Reyes et al., 1992; Castellano et al.,
1993b), and 3 was cloned in our lab from rat
brain and is almost identical to the published rat brain sequence
(Castellano et al., 1993a). Both Ca channel 1B
splice variants expressed equally well in Xenopus oocytes
[e.g., in the presence of 3, the average N
channel current amplitude was 2.68 ± 0.26 µA (n = 8) compared with 2.46 ± 0.11 µA (n = 7) for
+211B and 211B,
respectively]. With the exception of 4, Ca
currents were twofold to threefold larger in oocytes expressing
1B together with a subunit.
4 did not increase current amplitude, but it
did modulate N channel gating, confirming that it was expressed (see
Results). Data were acquired on-line and leak subtracted using a P/4
protocol (pClamp V6.0; Axon Instruments, Foster City, CA). Voltage
steps were applied every 10-30 sec depending on the duration of the
step, from various holding potentials. Current-voltage and steady-state
inactivation relationships and activation and inactivation rates were measured.
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RESULTS |
We analyzed the 1B gene to determine
whether alternative splicing could explain the presence of
1B cDNA variants containing an extra 21 amino
acid encoding sequence in the II-III intracellular loop region. The
salient features of this region of the 1B gene are presented in Figure
1B. A single 6.3 kb
product was amplified from rat genomic DNA using PCR primers directed
to cDNA sequences flanking the putative alternatively spliced exon. We
sequenced this product and showed that codon A757 is interrupted by a
6125 bp intronic sequence that contains consensus gt and
ag splice junctions (Sharp and Burge, 1997). A 63 base
cassette exon was identified midway through the intronic sequence
flanked by ag-gt splice junction motifs (Fig.
1B) and a polypyrimidine track 5' to the exon (data
not shown). The sequence of the cassette exon corresponds to the 63 base insert, confirming that +211B and 211B variants that we (Fig.
2) (Pan et al., 1999) and others have
observed in the II-III loop region of 1B mRNA
(Coppola et al., 1994; Ghasemzadeh et al., 1999) arise from alternative
splicing. In their study, Coppola et al. (1994) showed that the
alternatively expressed sequence contained 66, rather than 63, bases.
This is because all 1B cDNAs isolated from
mouse neuroblastoma cells that lacked the 63 base exon sequence also
lacked three bases encoding R758. Figure 1B shows
that the R758 codon is not part of the isolated 63 base exon but rather
is contiguous with the 3' flanking exon, and its expression depends on
the use of alternative dinucleotide, ag, splice-acceptors
at this distal 3' intron-exon boundary (flanking nucleotide 2271)
(Fig. 1B). Use of the first splice-acceptor site
would result in 1B mRNA containing the R758 codon, whereas R758 would be skipped if the second splice-acceptor site is used. All 1B clones that we have so
far isolated from rat contain R758, but the presence of R758-lacking
1B clones in mouse neuroblastoma
cells (Coppola et al., 1994) and bovine chromaffin cells (Cahill et
al., 2000) confirms that both splice-acceptor sites are
used. We have not investigated the expression pattern or the functional
consequences of R758 but rather focused on the 63 base cassette
exon.
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Figure 1.
Sites of alternative splicing in the N-type
calcium channel 1B subunit. A, Putative
membrane topology of the 1B subunit and location of four
alternatively spliced sequences encoding Ala415 in
intracellular loop I-II (A), 21 amino acids in
intracellular loop II-III (FVKQTRGTVSRSSSVSSVNSP), four
amino acids in IIIS3-IIIS4 (SFMG), and two amino acids
in IVS3-IVS4 (ET). With the exception of
Ala415 whose expression depends on the use of
alternative 3' splice-acceptors, expression of the other three sites
is regulated by alternative splicing of isolated exon cassettes (Lin et
al., 1997, 1999). B, Genomic sequence derived from
analysis of the 1B gene in the region of the
intracellular II-III loop (middle) together with the
amino acid sequences of two cDNAs derived by RT-PCR from rat neurons
(top, bottom). The location of exons
(uppercase letters, shaded), introns
(lowercase), the 63 base exon cassette
(boxed), and splice junction consensus
ag-gt dinucleotide sequences (underlined)
are indicated. Nucleotides 2270 and 2271
and amino acids A757 (757) and
R758 (758) denote the splice junction
(numbering according to GenBank sequence M92905) (Dubel et al., 1992).
Dashed lines indicate the two patterns of alternative
splicing that give rise to +211B (top)
and 211B (bottom). The
genomic sequence of this region is available under GenBank accession
number AF222338.
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Figure 2.
Expression pattern of +211B and
211B mRNAs in various regions of the nervous system
of the adult rat. Top, Summary of RT-PCR analysis
showing the relative abundance of the +211B mRNA variant
expressed as a fraction of total 1B mRNA derived from
brain (Brain), superior cervical ganglia
(SCG), dorsal root ganglia (DRG), spinal
cord (SpC), pons and medulla (Pn/Med),
midbrain (MdBr), thalamus (Thal),
hypothalamus (Hypo), pituitary (Pituit),
hippocampus (Hippo), cerebellum (Cereb),
neocortex (NCtx), and template negative PCR controls
(). Data were averaged from analysis of at least three different RNA
samples for each tissue isolated from multiple rats (mean ± SE).
Bottom, Example of PCR-derived cDNAs separated by
electrophoresis in 2% agarose. Two cDNA products were amplified from
each sample (285 and 348 bp) corresponding to 211B
and +211B mRNA. The first lane shows 300 and 400 bp size markers. One microliter of RT reaction, except for
pituitary (3 µl), was used as template for PCR amplification for all
RNA samples. Relative band intensities were estimated using Alpha
Inotech gel documentation software and normalized for size
differences.
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To determine the expression pattern of the 63 base cassette exon, we
analyzed RNA isolated from different regions of the nervous system of
the adult rat by RT-PCR (Fig. 2). PCR primers that flanked the splice
junction were chosen to generate two readily separable size cDNA
products (348 and 285 bp) corresponding to
+211B and 211B
mRNAs, respectively. Contamination by genomic DNA could be excluded
because the primers flanked a 6.1 kb intron. By measuring relative band
intensities, we show that exon expression is differentially regulated
in different regions of the nervous system.
+211B mRNA dominates in sympathetic ganglia
(80.8 ± 0.2% of total 1B,
n = 3), whereas exon expression is suppressed in
1B mRNA isolated from whole brain (15.3 ± 0.8% of total 1B, n = 3).
Exon expression was not, however, suppressed uniformly throughout the
CNS. Although relatively low levels of
+211B mRNA (<20% total
1B) were present in neocortex, hippocampus,
and pituitary, reflecting the pattern in whole brain, significant
amounts of both splice variants (+211B and
211B) were detected in more caudal regions
of the CNS, including spinal cord and brainstem.
Having established that alternative splicing in the II-III
intracellular loop region of 1B is strongly
region-specific, we next determined whether exon expression affected
channel function. The kinetics and voltage dependence of N-type Ca
channel activation and inactivation were studied in oocytes expressing
211B and +211B
splice variants. At least three different subunits copurify with
1B in native membranes (Scott et al., 1996),
and heterologous expression studies have shown that multiple subunits modulate N-type Ca channel function (Walker and De Waard,
1998). We therefore thought it pertinent to assess the functional
impact of splicing in the presence of four different Ca channel subunits known to be expressed in rat brain
(1b, 2a,
3, and 4) (Pragnell et al., 1991; Perez-Reyes et al., 1992; Castellano et al.,
1993a,b).
Figure 3 compares N-type currents induced
by expressing 211B and
+211B splice variants in Xenopus
oocytes in the presence of each subunit.
211B and +211B
splice variants expressed equally well in oocytes, and their
normalized, peak current-voltage curves and channel activation
kinetics were indistinguishable. Peak current-voltage curves for Ca
channels recorded from oocytes expressing 3
were shifted by ~10 mV in the depolarizing direction relative to
1b, 2a, and
4 similarly for both
1B splice variants (see also Fig.
5A). We therefore conclude that the presence of an
additional 21 amino acids in the cytoplasmic II-III loop of the
1B subunit does not affect the kinetics or
voltage dependence of N-type Ca channel opening.
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Figure 3.
+211B and 211B
splice variants have similar voltage- and time-dependent activation
properties. Normalized, averaged peak current-voltage plots were
calculated from Xenopus oocytes expressing
211B ( ) and +211B ( ) subunits
together with Ca channel 1b (A),
2a (B), 3
(C), and 4
(D) subunits. Currents were activated by brief
depolarizations to various test potentials from a holding potential of
80 mV. Barium (5 mM) was the charge carrier. Normalized,
averaged current traces for 211B (thick
line) and +211B (thin line) are
shown superimposed as insets in A-D.
Currents shown were activated by depolarization to 10 mV for
1b, 2a, and
4 and, to compensate for its different voltage-dependent
activation, to 0 mV for 3. Activation midpoints for
currents induced by the expression were as follows:
+211B/1b, 12.7 ± 0.8 mV (n = 5);
+211B/2a, 12.1 ± 0.7 mV (n = 6);
+211B/3, 5.5 ± 0.7 mV (n = 5); and
+211B/4, 13.2 ± 0.5 mV (n = 7). These values were not significantly
different from 211B (see legend to Fig. 5 for
211B values).
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N-type Ca channels recorded from native cells vary considerably in
their voltage dependence and kinetics of inactivation (Bean, 1989). We
therefore compared both the time course of inactivation and
steady-state inactivation curves of 211B
and +211B splice variants in the presence of
different subunits. Normalized, averaged currents recorded from
oocytes expressing each 1B/ combination are
shown as insets in Figure
4A-D. Inactivation
kinetics of the splice variants are indistinguishable in the presence
of a given subunit, and superimposed averaged currents overlap almost perfectly. The kinetics of N-type Ca channel inactivation did,
however, vary depending on the type of subunit expressed. For
example, N-type Ca channel currents recorded in
1b- and 3-expressing oocytes inactivate with relatively fast time courses compared with
2a similarly for both
1B splice variants (Fig. 4). These findings
are consistent with other studies that associate the presence of
2a with relatively slowly inactivating Ca
channels (Olcese et al., 1994; Walker and De Waard, 1998). Alternative splicing in the II-III loop of the 1B subunit
does not, therefore, affect channel inactivation kinetics during step
depolarizations to relatively positive voltages. However, a significant
difference in the N-type Ca channel availability curve was observed
between 211B and
+211B when the holding potential was varied.
N-type Ca channels induced by expressing +211B
splice variant with 1b and
4 inactivated at membrane potentials that were
~10 mV more depolarized relative to 211B
(Fig. 4A,D; see also Fig.
6B). These differences were only revealed in the
presence of specific 1B/ subunit
combinations because steady-state inactivation curves were similar
between 1B splice variants when coexpressed
with 2a and only slightly different in the
presence of 3 (Fig.
4B,C; see also Fig.
6B).
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Figure 4.
+211B and 211B
splice variants have different steady-state inactivation curves
depending on which subunit is coexpressed. Normalized, averaged
steady-state inactivation curves were calculated from currents
activated by brief depolarizations to 0 mV from various holding
potentials in oocytes expressing 211B () and
+211B () subunits together with Ca channel
1b (A), 2a
(B), 3 (C),
and 4 (D) subunits. Normalized,
averaged currents activated by long depolarizations to +20 mV are also
shown for 211B (thick line) and
+211B (thin line) to compare inactivation
kinetics (insets, A-D). Barium (5 mM) was
the charge carrier. Midpoints of steady-state inactivation curves for
currents induced by the expression were as follows:
+211B/1b, 58.1 ± 0.8 mV (n = 6);
+211B/2a, 33.0 ± 0.8 mV (n = 5);
+211B/3,=41.5 ± 0.9 mV (n = 5); and
+211B/4, 63.3 ± 0.8 mV (n = 6). Values for 211B
currents are in the legend to Figure 5.
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In Figure 5 we compare normalized
current-voltage and steady-state inactivation curves of the four
different subunits expressed with one 1B
splice variant (211B). With the exception
of 3, which is associated with Ca channel
currents activating at more depolarized voltages
(V1/2 of approximately 5 mV),
current-voltage relationships were similar between subunits (Fig.
5A). However, a comparison of steady-state inactivation
curves highlights how similar inactivation profiles of N-type Ca
channels recorded from 1b- and
4-expressing oocytes are to each other on the
one hand and different from 2a and
3 on the other. This correlation could be
significant in light of the fact that differences between
1B splice variants are only significant when
coexpressed with 1b and
4 but not 2a and
3. Figure 5B shows that N-type Ca
channels in 1b- and
4-expressing oocytes inactivate at more
hyperpolarized voltages (V1/2 of 60 to
80 mV) relative to N-type Ca channels associated with
2a and 3, which
inactivate at significantly more depolarized membrane potentials
(V1/2 of 35 to 45 mV) (Fig. 5B). Similar results were also obtained using 2 mM extracellular calcium rather than 5 mM barium as the permeant ion (Fig.
6).
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Figure 5.
Functional differences in 1B
coexpressed with different Ca channel subunits. N-type Ca channel
currents induced by coexpressing 211B with
1b(), 2a (), 3( ),
and 4 ( ) subunits in Xenopus oocytes.
Barium (5 mM) was the charge carrier. A,
Currents activated by step depolarizations to various test potentials
from a holding potential of 80 mV. Normalized, averaged peak
current-voltage plots for each subunit are shown. Each point is
mean ± SE. Activation midpoints were as follows:
211B/1b, 13.6 ± 0.6 mV (n = 6);
211B/2a, 11.7 ± 1.1 mV (n = 6);
211B/3, 4.6 ± 1.0 mV (n = 4); and
211B/4, 13.2 ± 0.5 mV (n = 5). In the presence of
3, currents activated at voltages ~10 mV more
depolarized compared with 1b,
2a, and 4. B,
Average, steady-state inactivation curves of 211B
channels expressed with different subunits. Currents were activated
by depolarizations to 0 mV from different holding potentials. Peak
currents were measured and expressed relative to maximum current
(holding potential, 120 to 100 mV). Inactivation midpoints
were as follows:
211B/1b, 68.5 ± 0.8 mV (n = 6);
211B/2a, 34.1 ± 0.7 mV (n = 5);
211B/3, 43.8 ± 1.0 mV (n = 5); and
211B/4, 71.2 ± 0.7 mV (n = 6).
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Figure 6.
Functional differences between 1B
splice variants are subunit-specific. Average shifts in channel
activation (A) and steady-state inactivation
(B) midpoints between 211B and
+211B splice variants in the presence of different Ca
channel subunits. Currents were measured using 2 mM Ca
(dark shading) and 5 mM Ba (light
shading) as the permeant ions. Midpoints of activation
calculated from current-voltage plots using 2 mM Ca were
as follows: 211B/1b,
13.9 ± 1.2 mV (n = 7);
211B/2a, 7.9 ± 0.8 mV (n = 6);
211B/3, 5.3 ± 0.6 mV (n = 4); and
211B/4, 14.9 ± 1.1 mV (n = 5); and compared with
+211B/1b, 16.1 ± 1.2 mV (n = 5);
+211B/2a, 8.6 ± 0.6 mV (n = 6);
+211B/3, 7.0 ± 0.6 mV (n = 5); and
+211B/4, 15.0 ± 0.5 mV (n = 5). Midpoints from steady-state
inactivation curves using 2 mM Ca as charge carrier were as
follows: 211B/1b,
72.1 ± 0.6 mV (n = 6);
211B/2a, 37.0 ± 1.6 mV (n = 5);
211B/3, 41.9 ± 0.6 mV (n = 5); and
211B/4, 72.7 ± 1.3 mV (n = 7); and compared with
+211B/1b, 64.4 ± 0.5 mV (n = 6);
+211B/2a, 38.0 ± 1.0 mV (n = 5);
+211B/3, 40.4 ± 0.7 mV (n = 5); and
+211B/4, 64.1 ± 0.7 mV (n = 5). Values are mean ± SE. See legends
to Figures 3-5 for values with 5 mM Ba as charge
carrier.
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DISCUSSION |
Alternative splicing is region-specific
Alternative splicing in intracellular loop II-III of the Ca
channel 1B subunit mRNA is differentially
regulated in distinct regions of the nervous system. Sympathetic
ganglia express the highest levels of exon-containing
1B mRNA (>80% of total), and in the CNS,
+211B mRNA levels decrease in an approximately
caudal to rostral pattern, from ~50% in brainstem to <20% in
neocortex. Overall, exon skipping at this splice site prevails in whole
brain, consistent with the absence of the exon in
1B cDNAs isolated previously from rat
brain-derived cDNA libraries (Dubel et al., 1992; Fujita et al., 1993).
It has been suggested recently that the II-III intracellular loop exon
of 1B is preferentially expressed in brain
regions of the rat enriched in monoaminergic neurons, based on in
situ hybridization and RT-PCR analysis (Ghasemzadeh et al., 1999).
Our results do not test this hypothesis directly but nonetheless do
favor more widespread distribution. For example, there is a relatively
high representation of +211B mRNA in regions of the CNS not particularly rich in monoaminergic neurons, such as
spinal cord and hypothalamus (Fig. 2). The expression pattern of the
II-III intracellular loop exon differs from other splice sites that we
have studied previously in the IIIS3-IIIS4 and IVS3-IVS4 extracellular linkers of the 1B subunit,
suggesting that the 1B gene contains multiple,
independently regulated sites of alternative splicing.
Functional consequences of alternative splicing
The functional effect of lengthening the II-III intracellular
loop of the 1B subunit by 21 amino acids is
summarized in Figure 6. Exon inclusion did not affect the voltage
dependence of channel activation but did induce a shift in the channel
availability curve to more depolarized membrane voltages similarly with
calcium or barium as permeant ion. These results contrast nicely with our analysis of alternative splicing in the IVS3-IVS4 extracellular linker of the 1B subunit, a domain close to
the putative voltage sensor (S4). Splicing in IVS3-IVS4 affected both
the voltage dependence and kinetics of channel activation but not
inactivation (Lin et al., 1997, 1999).
Functional differences between +211B and
211B splice variants were only observed in
the presence of Ca channel 1b or
4 subunits that also shift channel
steady-state inactivation curves into the physiologically interesting
range of potentials between 60 and 90 mV. In contrast, small or no
shifts were observed between the splice variants coexpressed with
either 2a or 3 that
form N channels that inactivate at significantly more depolarized voltages (50 to 20 mV). Xenopus oocytes express an
endogenous Ca channel subunit that is highly homologous to
mammalian 3 (3XO)
(Tareilus et al., 1997). If present at high enough levels, endogenous
3XO could partially mask the actions of
exogenously expressed subunits; consequently, hyperpolarizing
shifts in N-type Ca channel steady-state inactivation curves associated with 1b and 4 might
be slightly underestimated. Nonetheless, our results demonstrate that
functional differences between 1B splice
variants depend on their interactions with specific subunits.
How does this subunit specificity arise? One possibility is that
1b and 4 subunits,
but not 2a or 3,
specifically interact with the 21 amino acid insert in the II-III
intracellular loop of 1B. Although the primary
subunit binding site on 1 is in the
intracellular loop between domains I and II (Pragnell et al., 1993; De
Waard et al., 1994), secondary -interaction sites in the
C-terminal region of 1 have been
identified recently (Walker et al., 1998, 1999), leaving open the
possibility that additional -interaction sites in
1, such as in the II-III loop, might exist. Alternatively, subunit specificity might not depend on direct interactions between subunits and the II-III intracellular loop of
1B. For example, modification of N-type Ca
channel inactivation might be most permissible at membrane potentials
between 60 and 90 mV; consequently, differences between splice
variants would surface in the presence of 1b
or 4 but be less obvious with 2a or 3 subunits. In
this case, functional differences between 1B
splice variants in native cells might also depend on interactions with
other Ca channel subunits (e.g., 2 ) and
modulators (e.g., G-proteins) that, like subunits, affect channel inactivation.
Our studies suggest that all 1B/
combinations are permissible and form functional channels in the
Xenopus oocyte expression system, but which combinations
occur in native cells? There is significant overlap in the distribution
of 1B and 3 mRNA and protein in mammalian brain but the correlation is not perfect (Ludwig
et al., 1997), and biochemical studies have demonstrated significant
levels of 1B/3 and
1B/4 complexes in
native N-type Ca channel proteins isolated from brain (Scott et al.,
1996). The study by Scott et al. (1996) does not, however, distinguish between different 1B splice variants. Although
1B, 3, and
4 mRNAs are broadly distributed in brain, in
brainstem 1b and 4 mRNAs are present to the exclusion of 2a and
3 (Ludwig et al., 1997). Because significant
levels of 211B and
+211B mRNAs (Fig. 2) are also present in
brainstem, we suggest that both 1B splice variants probably form native N-type Ca channels with subunits other than 3 (i.e.,
1b or 4). In light of
our results, it will now be important to establish which subunits
associate with which specific 1B splice
variant, 211B and
+211B, in different regions of the nervous system.
The potential to mix and match multiple 1B
splice variants and subunits may represent a mechanism for
fine-tuning excitation-secretion coupling at different synapses. We
speculate that the availability of N-type Ca channels at synapses
dominated by
211B/1b or
211B/4 complexes
may be more sensitive to modulation by agents or stimuli that induce
relatively prolonged changes in the resting membrane potential between
60 and 80 mV compared with 2a- or
3-dominant synapses containing either
211B or +211B.
Heterogeneity in 1B/ complexes (Scott et
al., 1996) probably account for native N-type Ca channel currents in
neurons that differ in their sensitivity to inactivation as the
membrane potential is depolarized (Bean, 1989; Kongsamut et al., 1989;
Plummer et al., 1989). Our results also highlight the II-III
intracellular loop of the Ca channel 1B
subunit as a domain important for regulating N-type Ca
channel availability at voltages close to the resting membrane
potential. The demonstration that syntaxin, a SNARE [SNAP (soluble
N-ethylmaleimide-sensitive factor attachment protein)
receptor] that binds to a region in the II-III intracellular loop
overlapping the splice junction, also affects the position of the
steady-state inactivation curve is consistent with this hypothesis
(Bezprozvanny et al., 1995).
Future directions
Our findings provide the framework to begin to address other
questions related to the functional significance of splicing in the
II-III intracellular loop of the Ca channel
1B subunit. For example, the alternatively
spliced exon is unusually enriched in serine and threonine residues (9 of 21), suggesting that 211B and
+211B splice variants might be differentially
modulated by protein kinase. Furthermore, because the exon overlaps the
synaptic protein interaction site on 1B
(synprint) (Catterall, 1999), it would be interesting to determine how
its presence influences SNARE binding (Sheng et al., 1994; Charvin et
al., 1997; Catterall, 1999). Along these lines, there is evidence for
isoform-specific interactions of SNAREs with II-III intracellular loop
variants of the closely related 1A subunit
(Rettig et al., 1996; Catterall, 1999), although the isoforms of
1A reported by Catterall and colleagues do not
correspond to the 211B and
+211B splice variants described here. Finally,
we know very little about the mechanisms that regulate expression of
this exon and other alternative spliced exons of
1B in different regions of the nervous system.
The identification of regulatory elements presumably in introns
upstream of the exon might provide clues as to the nature of the
proteins that direct these functionally significant splicing events
(Grabowski, 1998).
| |
FOOTNOTES |
Received Feb. 11, 2000; revised April 4, 2000; accepted April 11, 2000.
This work was supported by National Institutes of Health Grants NS29967
and NS01927 (D.L.). We are grateful to Kevin Campbell for
1b cDNA and Edward Perez-Reyes for 2a and
4 cDNAs. Julie Nam assisted in some of the PCR
experiments. We thank members of the Lipscombe laboratory and Dr. Yael
Amitai for comments on this manuscript.
Correspondence should be addressed to Diane Lipscombe, Department of
Neuroscience, 192 Thayer Street, Brown University, Providence, RI
02912. E-mail: diane_lipscombe{at}brown.edu.
| |
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ABSTRACT
INTRODUCTION
