From the Department of Cellular and Molecular Medicine, University of Ottawa, Ottawa, Ontario K1H 8M5, Canada
Received for publication, July 31, 2000, and in revised form, September 27, 2000
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ABSTRACT |
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Novel splice variants of the
The voltage-gated ion channels determine membrane excitability and
regulate signal transduction (1-5). These ion channels consist of
multimeric complexes comprising a central The different pore-forming Changes in amino acid composition in the extramembrane regions of the
Because the Isolation of RNA and RT-PCR Analysis--
Total RNA was isolated
using the TriPure reagent (Roche Molecular Biochemicals) according to
method described by Chomczynski (48). Adult rabbit heart, brain, and
skeletal muscle were frozen in liquid nitrogen, and total RNA was
extracted in phenol thiocyanate. Total RNA (1.0 µg) was reverse
transcribed with either a gene-specific primer, based on the sequence
of the
Amplification of the loop II-III variant fragments encompassing the
predicted stop codon at nucleotide 2995 of the
Cav1.2 gene used upstream and downstream
primers anchored in transmembrane sequences encoding domains II and III
of cardiac Screening of Recombinant Clones and Cycle
Sequencing--
Recombinant clones of loop II-III RT-PCR amplicons
subcloned into either pCRII or pTZ18 were selected and inoculated
overnight at 37 °C in LB broth containing 75 µg/ml ampicillin.
Plasmid DNA was isolated using an alkaline miniprep (49). The
recombinant constructs were screened by restriction enzyme digestion
and agarose gel electrophoresis to identify loop II-III deletion
variants. The various inserts were then purified with a Qiagen miniprep plasmid preparation method to obtain high-purity templates suitable for
cycle sequencing with the Applied Biosystems Prism dye terminator cycle-sequencing method (PerkinElmer Life Sciences), and the sequences were analyzed using SeqAidII tools (University of Kansas).
Cloning of Full-length RNase Protection Assay--
The RNase protection assay used two
cRNA probes, RH-1 and RH-2, representing deletion variants of loop
II-III lacking exon 19 and exons 17 and 18. The
32P-radiolabeled deletion variant probes were generated by
in vitro transcription using MAXIscript reagents (Ambion)
with [ Antibodies, Western blot Analysis, and Subcellular Fractionation
of Cardiac Membranes--
Affinity-purified antibodies directed
against peptide sequences (KYTTKINMDDLQPSENEDKS) of the intracellular
loop II-II of the Cav1.2
Membrane fractions from rat myocardium were isolated by differential
and sucrose density gradient centrifugation and characterized for the
enrichment of markers for the sarcolemma (~20-fold enrichment in
Na+/K+-ATPase and adenylate cyclase activities)
and heavy sarcoplasmic reticulum (~10-fold enrichment in ryanodine
receptors) as described (67). Membrane proteins were subjected to
SDS-polyacrylamide gel electrophoresis followed by transfer to
nitrocellulose membranes, which were probed with affinity-purified
antipeptide antibodies.
Identification of Two Alternatively Spliced loop II-III Variants
of Cav1.2 Ca2+ Channels--
The second
intracellular loop connecting domains II and III of the
Evaluation of Genomic Organization of the Loop II-III
Variants--
The comparison of the deletion sites of the loop II-III
variants with the exon-exon boundaries of the human mRNA Expression of Cav1.2 Ca2+ Channel
Variants--
Confirmation of the expression of the Cav1.2
subunit mRNA variants detected by RT-PCR was sought by RNase
protection assays. Fig. 3 represents a
schematic of the predicated sizes of the protected fragments that would
be expected with the RH-1 and RH-2 probes. An antisense RNA probe of
RH-1 was constructed based on the 323-nt amplicon of loop II-III
(exons 16-18 and 20). The mRNA from cardiac and fast twitch
skeletal muscle was used to protect the RH-1 probe, and a
representative autoradiogram is shown. The mRNA from cardiac muscle
protected the 323-nt probe representing the RH-1 variant as well as
fragments of ~274, 144, and 46 nt (Fig. 3, lane 3). The
323-nt protected fragment represents the RH-1 type variant of loop
II-III and would correspond to a part of exon 16 through to the end of
exon 18 and the beginning of exon 20. Exon 19 of 133 nt is deleted in
the RH-1 splice variant. The 274-nt protected fragment denotes a part
the of the wild-type
An antisense RNA probe of RH-2 (320 nt) was constructed based on the
RT-PCR amplicon of loop II-III (lacking exons 17 and 18). Protection
of the RH-2 probe with cardiac and skeletal muscle mRNA was carried
out, and the respective autoradiogram is shown (Fig. 3, lanes
5-8). The mRNA from cardiac muscle protected fragments of
~320, 179, 144, and 46 nt (Fig. 3, lane 7). The 320-nt
protected fragment represents the RH-2 variant of loop II-III encoded
by part of exons 16 and 17 and exons 19 and 20. The 179-nt protected fragment would be contributed by the protection of the 3' end of the
wild-type loop II-III encoded by exons 19 and 20. Because the 3' 60 nt
of exon 17 and the entire exon 18 are deleted in the RH-2 type variant,
this region of loop II-III would not be protected and consequently
degraded by RNase. The theoretically expected fragments of 144 and 46 nt contributed by the 5' end of the wild type loop II-III (exon 16 and
the 5' end of exon 17) and the RH-1 deletion variant were also detected
but at low levels. Skeletal muscle mRNA did not protect the RH-2
probe (Fig. 3, lane 8). The protection of multiple fragments
by RNA from cardiac muscle is suggestive of the expression of the
RH-1and RH-2 deletion variant transcripts as well as the wild type loop
II-III. To estimate a relative level of expression of the three
transcripts, the ratio of RH-1 and RH-2 deletion variants to the
wild-type Cav1.2 loop II-III was quantified by using the
relative band intensities of the RH-1 (320-bp band) and the RH-2
(323-bp band) protected fragments and the appropriate protected
fragments contributed by the wild type loop II-III from the
autoradiogram, 274 and 174 bp, respectively. After normalization, the
analysis estimated a relative level of expression with a ratio
1:1.6:0.7 of wild-type loop II-III:RH-1:RH-2, respectively; however,
the estimate remains to be confirmed with protein expression levels.
Brain mRNA also protected multiple fragments of RH-1 and RH-2
similar in sizes to those observed with cardiac mRNA (data not shown). Thus data from the RT-PCR amplification of the loop II-III splice variants together with the data from the RNase protection assays
are indicative of the expression of these variants in cardiac and
neuronal tissue but not skeletal muscle and are therefore consistent
with the reported pattern of expression of the
Cav1.2 Domains I and II as Potential Structural-Functional Units of
Ca2+ Channels--
On the basis of the identification of
the loop II-III deletion variants, it was hypothesized that the
alternatively spliced forms of
The nucleotide sequence and the deduced amino acid sequence analysis of
the RH-1 and RH-2 variants are shown in Fig.
5, which indicates identical nucleotide
and amino acid sequences in domains I and II when aligned with the
wild-type Cav1.2
Confirmation of the in vivo expression of the two-domain
Cav1.2 Ca2+ channel polypeptides was carried
out by Western blot analysis of subcellular fractions from cardiac
muscle with antipeptide antibodies specific for loop II-III
(CNC-1) of the Cav1.2 calcium channel. The peptide
sequence used as antigen is also preserved in the RH-1 and RH-2
variants. Subcellular fractions enriched in SL and HSR were
immunoblotted with anti-Na+/K+-ATPase
monoclonal antibody to confirm the enrichment of SL versus the SR (Fig. 6). The
Na+/K+-ATPase staining is highly enriched in SL
compared with the original microsomes and fraction HSR, and
densitometery revealed that it was ~15-fold higher in SL compared
with the other fractions. Immunoblotting with the affinity-purified
CNC-1 antibody indicated the presence of an ~240-kDa polypeptide
representing the
The predicted topology of the two-domain
The intracellular loop of
Although the role of the
The generation of two distinct polypeptides by alternative splicing is
highly suggestive of functional specialization of the individual
truncated
In summary, the definition of the two new splice variants of the
Cav1.2 1 subunit of the Cav1.2 voltage-gated
Ca2+ channel were identified that predicted two truncated
forms of the
1 subunit comprising domains I and II
generated by alternative splicing in the intracellular loop region
linking domains II and III. In rabbit heart splice variant 1 (RH-1),
exon 19 was deleted, which resulted in a reading frameshift of exon 20 with a premature termination codon and a novel 19-amino acid
carboxyl-terminal tail. In the RH-2 variant, exons 17 and 18 were
deleted, leading to a reading frameshift of exons 19 and 20 with a
premature stop codon and a novel 62-amino acid carboxyl-terminal tail.
RNase protection assays with RH-1 and RH-2 cRNA probes confirmed the expression in cardiac and neuronal tissue but not skeletal muscle. The
deduced amino acid sequence from full-length cDNAs encoding the two
variants predicted polypeptides of 99.0 and 99.2 kDa, which constituted
domains I and II of the
1 subunit of the
Cav1.2 channel. Antipeptide antibodies directed to
sequences in the second intracellular loop between domains II and
III identified the 240-kDa Cav1.2 subunit in
sarcolemmal and heavy sarcoplasmic reticulum (HSR) membranes and a
99-kDa polypeptide in the HSR. An antipeptide antibody raised against
unique sequences in the RH-2 variant also identified a 99-kDa
polypeptide in the HSR. These data reveal the expression of additional
Ca2+ channel structural units generated by alternative
splicing of the Cav1.2 gene.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
subunit, which contains
the structural determinants for ion selectivity, conductance, and
voltage sensing, and several auxiliary subunits, which confer
regulation on the
subunits (6, 7). The
subunits of
K+, Na+, and Ca2+ channels show
overall structural similarity in that the
subunit of K+
channels consists of a single domain, which is predicted to contain six
membrane-spanning (S1-S6)1
regions, whereas the structural unit of Na+ and
Ca2+ channels consists of four such homologous domains (1).
Because the K+ channel genes encode only one domain, it is
proposed that homo- and heterotetrameric co-assembly of single-domain
K+ channel subunits is required to constitute the
four-domain channel complexes (8-10).
subunits are encoded by distinct gene
families (68). Ten genes encoding the voltage-gated Ca2+
channels have been identified; seven (denoted
Cav1.1-1.4 and
Cav2.1-2.3) encode the high
voltage-activated channels (5, 11-17), and three
(Cav3.1-3.3) encode the low
voltage-activated channels (18-21). Genes encoding different
1 subunits of the voltage-gated Ca2+
channels exhibit distinct channel properties, which are determined by
subtle changes in amino acid composition and appear to be expressed in
a tissue- and cell-specific manner (22-28). Alternative splicing, posttranslational modifications, and modulation by auxiliary subunits can generate further diversity in Ca2+ channel
heterogeneity, although the four-domain structure is maintained
(29-36). For example, alternative splicing of mutually exclusive exons
encoding the S3 segment of domain IV of the
1 subunit of
Cav1.2 channels serves as a developmentally regulated switch in cardiac tissue coinciding with major changes in excitation (29). Variability in the carboxyl-terminal region of the
1 subunit of Cav1.2 channels generated by
alternative splicing influences the kinetics as well as
Ca2+ and voltage dependence of the L-type Ca2+
channels (32, 33). Moreover, dihydropyridine sensitivity of cardiac and
vascular
1 subunits of Cav1.2
Ca2+channels may be attributed to tissue-specific
expression of an alternatively spliced S6 segment of domain I of the
Cav1.2 gene (36).
1 subunit appear to have given rise to specialized physiological roles for the different classes of
1
subunits of the Ca2+ channel family (37-41). For example,
the structure of the intracellular loop connecting domains II and III
appears to be a critical determinant of the mode of signal transmission
in the Cav1.1 and Cav1.2
1 subunits (42-44), because this structure of the skeletal
(Cav1.1) but not cardiac (Cav1.2)
1 subunits can directly interact with the
Ca2+ release channel of the sarcoplasmic reticulum to
determine the contractile characteristics of skeletal muscle (45-47).
Furthermore, loop II-III of Cav2.1 (P/Q-type) and
Cav2.2 (N-type) Ca2+ channels associates with
syntaxin and synaptosomal associated protein 25 to regulate
excitation-secretion coupling (50-56).
1 subunit of Cav1.2
Ca2+ channels is expressed in tissues such as the
myocardium and brain, which are composed of a heterogeneous population
of cells, it has been postulated that alternatively spliced variants
may be expressed to serve functions in a cell type-specific manner. In
view of the functional importance of the loop II-III structure, we
examined whether alternative splicing will result in the generation and
expression of structural variants in this critical region. We report
here the identification of two splice variants of Cav1.2
Ca2+ channels that generate truncated forms of the
1 subunits, which were predicted to constitute domains I
and II with unique carboxyl-terminal tails. The expression of these
variants in cardiac and neuronal tissue suggests that further diversity
in Ca2+ channel function and regulation may be generated
through alternative structural units.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
1 subunit of the
Cav1.2 gene from rabbit myocardium
described by Mikami et al. (Ref. 17; primer JW6,
GGTGAAGATCGTGTCGTTGAC, nt 2990-2970) or random hexamers and
SuperScript reverse transcriptase. The first-strand cDNA synthesis with the gene-specific primer results in exclusive reverse
transcription of the Cav1.2 transcripts
from rabbit myocardium, thus yielding increased sensitivity of the PCR
amplification by increasing the representation of the rare messages. To
amplify the Cav1.2 subunit loop II-III, the specific
primers JW5 (GTGGACAACCTGGCTGATGCTGAG, nt 2538-2561) and JW6 were
used. The PCR was performed under the following conditions: 94 °C
for 45 s, 50 °C for 30 s, and 72 °C for 30 s,
repeated 30 times. There were two negative controls used, one with
water to control for contamination of the master mix and a second
control reaction that contained all reaction components except reverse
transcriptase (i.e. RNA but no first-strand cDNA) to
control for contamination from genomic DNA and partially processed
mRNA. The PCR reaction was run in an MJ Research thermocycler. The
products were analyzed by 1% gel electrophoresis, gel purified, and
cloned into the pCRII vector (Invitrogen). Twenty bacterial colonies were selected and screened by restriction mapping.
1 subunit. The sense primer loop5'
(TCCTACTGAATGTGTTGG, nt 2509-2529) and antisense primer loop3'
(CAGGATG TTGAAGTAGTTC, nt 3199-3179) also incorporated BamHI and EcoRI restriction sites, respectively,
to facilitate directional cloning of the PCR products into pTZ18
(United States Biochemical, Cleveland, OH). Similar to RT-PCR
performed with JW5 and JW6 primers, synthesis of the first-strand
cDNA was primed with a gene-specific antisense loop3'
oligonucleotide. The following cycling condition were used: 94 °C
for 45 s, 52 °C for 35 s, and 72 °C for 35 s,
repeated 32 times. The final elongation was performed at 72 °C for
10 min. The RT-PCR products were analyzed on a 1% agarose gel, gel
purified, and subcloned into the pTZ18 vector. Sixty bacterial colonies
were selected and screened by restriction mapping and direct sequencing.
1 Subunit Variant cDNAs--
Total
RNA (1.0 µg) prepared as described above was reverse transcribed with
antisense primer 5'-CAGGATGTTGAAGTAGTTC-3' (nt 3199-3179) and
SuperScript at 50 °C for 45 min. PCR amplification was carried out
at standard conditions with sense primer 5'-TGGAAACTGACAATGCTTCGAGCC-3' (nt 180-203) and 3' antisense splice-specific primers
5'-ATCCTCTTCTCCTTGGCCTCCTC-3' and 5'-GAGGCGGAACCTGTGGTTTCC-3',
with first denaturation at 94 °C for 2 min followed by
94 °C for 45 s, 70 °C for 35 s, 72 °C 2 min 30 s, which was repeated 32 times, and the final elongating at 72 °C
for 10 min. The PCR amplicons were electrophoresed on a 0.9% agarose
gel, TA cloned into pCRII vector (Invitrogen), and cycle sequenced
(PerkinElmer Life Sciences).
-32P]UTP (PerkinElmer Life Sciences);
104 cpm of the respective RNA probe was hybridized with 15 µg of mRNA in a Hybspeed hybridization at 68 °C for 10 min
followed by RNase digestion (RNase A/T1, 1:100) at 37 °C for 45 min.
Reactions were inactivated by addition of inactivation-precipitation
mix (Ambion) and ethanol precipitated. Pellets were resuspended in 10 µl of gel loading buffer and analyzed on 5% polyacrylamide-8 M urea-1× Tris borate-EDTA vertical gel and subjected to
autoradiography. The protected fragments appearing on the
autoradiograms were quantified on a Kodak Science image analysis
station. To assess relative expression of
Cav1.2 loop II-III variants, the
intensities of protected fragments were size normalized and expressed
as a ratio. Century RNA markers (Ambion) were used as molecular size standards.
1 subunit were generously provided by Dr. William
Catterall. Polyclonal antibodies directed against the carboxyl-terminal
sequences of RH-1 and RH-2 variants were raised in New Zealand White
rabbits. The only unique amino acid sequence (TTGSASSVTVSSTTRSSPT) of
the common 19-amino acid carboxyl terminus of RH-1 and RH-2 was found
to be nonantigenic and gave no immune response. On the other hand, two
peptide sequences, KRMRRSLRCLSAPALGHS and GHSPSCTLRRRPC, were selected
to raise antibody 1929 selective for the RH-2 variant. The peptides
were conjugated to keyhole limpet hemocyanin carrier protein via
the carboxyl-terminal carboxyl group and used in six consecutive
immunizations per each of the New Zealand White rabbits (Genosys). The
antisera was collected in four bleeds per animal. The
Na+/K+-ATPase antibodies were provided by Dr
Kathleen Sweadner.
RESULTS AND DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
1 subunit of voltage-gated Ca2+ channels is
believed to reside at the cytoplasmic phase of the membrane and
determines the mode of signal transduction in excitation-contraction coupling in muscle cells (42-47) and excitation-secretion coupling in
nerve cells (50-56). Given the structural and functional importance of
this region, we determined whether diversity in Ca2+
channel structure can be created through alternative splicing in this
region of the
1 subunit gene. The intracellular loop connecting domains II and III of Cav1.2
1
subunits was amplified from rabbit myocardium by RT-PCR using primers
based on the cDNA sequence reported by Mikami et al.
(17). The first-strand cDNA synthesis was primed either with the
Cav1.2 subunit-specific oligonucleotide JW6 (nt 2990-2970)
or random hexamers. The purpose of priming with the gene-specific
oligonucleotide was to increase representation of the rare messages of
the Cav1.2 transcript family. The PCR amplification of loop
II-III was then carried out with JW5 (nt 2538-2561) and JW6 (nt
2990-2970) primers and analyzed by 1% agarose electrophoresis (Fig.
1A). The PCR with the JW6
primer yielded a major 452-nt product as well as smaller diffuse
fragments (Fig. 1A, lane 1), whereas PCR of the
first-strand cDNA primed with random hexamers yielded a single
fragment of 452 nt (Fig. 1A, lane 3). Fig.
1A, lane 2, represents a PCR-negative control.
Subcloning and direct sequencing of the PCR products shown in
lane 1 revealed that the 452-nt product matched perfectly
the sequence of the intracellular loop linking domains II and III of
the rabbit heart
1 Cav1.2
subunit gene, whereas that of the smaller diffuse fragment matched the
452-nt loop II-III sequence except for a deletion of 133 nt (from 2812 to 2945). Open reading frame analysis of the 320-nt amplicon containing
the 133-nt deletion indicated that the deletion was predicted to shift
the translational reading frame of full-length
1
Cav1.2 and to introduce a premature chain
termination at nt 2995 (data not shown). To obtain longer cDNAs
encompassing the entire loop II-III and the predicted stop codon,
RT-PCR was performed with another set of primers nested in the
transmembrane segment S6 of domain II and S3 segment of domain III
(loop 5', nt 2509-2529; and loop 3', nt 3199-3179, respectively).
Primers were designed to contain BamHI and EcoRI restriction enzyme sites to facilitate subcloning and analysis of the
PCR amplicons. Fig. 1B depicts RT-PCR of rabbit heart, where
the first-strand cDNA was performed with the gene-specific antisense oligonucleotide loop 3', and PCR amplification was carried out with loop 5' and loop 3' primers. Similar to RT-PCR analysis carried out with the JW5 and JW6 primers, amplification with the loop
5' and loop 3' primers produced two products, an amplicon of ~700 nt
and a lower band of ~560 bp (Fig. 1B, lane 1).
Moreover, the size difference between the top and bottom products
was ~ 140 nt, analogous to the RT-PCR performed with the JW5 and
JW6 primers. After subcloning of the amplified products, three
representative amplicons were characterized by restriction mapping and
direct sequencing (Fig. 1C). Lane 1 shows the
presence of a 690-nt product, representing the expected amplification
product of
1 Cav1.2 with
the loop 5' and loop 3' primers, and lanes 2 and
3 represent two clones isolated and characterized from the
lower RT-PCR amplification band. The nucleotide sequence analysis and
alignment shown in Fig. 2A
indicated that the 690-nt product represented the nucleotide sequence
of the expected amplified fragment encompassing the wild-type intracellular loop II-III of Cav1.2 (Rab-H), whereas the
smaller products denoted two distinct variants of this sequence.
Variant 1, denoted RH-1, and variant 2, denoted RH-2, appear to be
derived from the full-length loop II-III sequence spanned by the loop 5' and loop 3' primers, as a consequence of internal deletions of 133 and 130 nt, respectively. The RH-1 variant contained deletion of nt
2812-2945, and it represents a deletion variant already characterized
in the RT-PCR analysis carried out with the JW5 and JW6 primers. The
RH-2 variant displayed deletions of nt 2681-2811, and it represents a
novel variant cloned from the smaller amplification product of loop
II-III. Analysis of the open reading frame of the deletion variants
RH-1 and RH-2 in relation to the wild-type sequence confirmed that both
deletions shifted the open reading frame, thus introducing a common
termination codon (TGA) at nucleotide 2995. The amino acid sequence
alignment of the variants indicated that both variants introduce novel
amino acid sequences past their respective deletions depicting novel
carboxyl terminal sequences of the truncated polypeptides (Fig.
2B). Because both variants use the same stop codon, the
terminal 19 amino acids were identical in the two variants. An
independent isolation of the RH-1 and RH-2 cDNAs was obtained with
RT-PCR of RNA from the whole heart and cardiac left ventricle but not
skeletal muscle.
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Fig. 1.
RT-PCR of loop II-III of the cardiac
1 subunit. A, loop
II-III of the rabbit cardiac
1 Cav1.2
subunit of the L-type Ca2+ channel was amplified by RT-PCR
with JW5 and JW6 primers. The RT reaction was primed with the antisense
JW6 oligonucleotide (lane 1) or with random hexamers
(lane 3). A control reaction containing no first-strand
cDNA template (i.e. no reverse transcriptase) is
represented in lane 2. The amplification of the predicted
452-nt product is seen in lanes 1 and 3. In
addition, lane 1 also indicates the presence of smaller
products of ~300 bp. B, results of the RT-PCR analysis of
the rabbit cardiac
1 Cav1.2 with loop 5' and
loop 3' primers, encompassing loop II-III and the predicted stop codon
at nt 2995. The analysis reveals the presence of two amplification
products, similar to the RT-PCR shown in A, thus confirming
the expression of deletion variants of loop II-III. C,
EcoRI-BamHI digests of representative recombinant
plasmids derived by cloning DNA fragments eluted from a gel in which
the RT-PCR products from cardiac RNA were amplified with primers
anchored in the membrane-spanning segments S6 of domain II (loop 5', nt
2509-2529) and S3 of domain III (loop 3', nt 3199-3179). The products
from the RT-PCR reaction were separated by gel electrophoresis, and the
amplification products were subcloned as described under
"Experimental Procedures." Three representative clones were
characterized by the restriction mapping and direct sequencing
(lanes 1-3).
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Fig. 2.
Characterization of PCR products.
A, DNA sequence analysis and alignment of the loop II-III
variants. Shown are the nucleotide sequence and alignment of the
various RT-PCR products of the 1 subunit loop II-III
amplified with loop 5' and loop 3' primers from Fig. 1B.
RH-1 and RH-2 denote the two smaller fragments;
Rab-H represents the larger product in Fig. 1B.
The sequence and alignment reveal that RH-1 and RH-2 contain deletions
of 133 and 130 nt, respectively, when compared with the sequence of the
full-length (wild-type) loop II-III (Rab-H). The sequence
gaps are denoted by dashes, and the deduced in-frame stop
codons are shaded. The splice junction primers are
single or double underlined. All
sequence positions are relative to the rabbit cardiac
1 subunit
(X15539) identified by Mikami et al. (43). B,
amino acid alignment of the loop II-III variants. Shown is alignment
of the deduced amino acid sequence of the loop II-III splice variants,
where boldface denotes the novel carboxyl terminus common to
RH-1 and RH-2 splice variants, and the underlined
sequence represents the unique RH-2 sequence. C,
genomic organization of the loop II-III variants. A schematic
representation of the loop II-III splice variants based on the exon
organization of intact loop II-III of the cardiac
1
subunit gene is indicated. Hum-H represents the full-length
(wild-type) loop II
III; RH-1 and RH-2 represent
the two deletion variants. It is depicted that the RH-1 variant would
arise because of deletion of exon 19, and as a result there would be a
reading frameshift of exons 20. The RH-2 variant, on the other hand,
would be missing a fragment of exon 17 and the entire exon 18; this
would also result in a reading frameshift of exons 19 and 20. The
reading frameshifts in both variants would introduce a common premature
termination codon in exon 20.
1
Cav1.2 subunit gene is summarized (Fig.
2C). Depicted are the exons encoding loop II-III of the
Cav1.2
1 subunit gene as
determined by Soldatov (57) and how the variants would potentially
arise as a result of internal deletions. The RH-1 variant lacks exon 19, which results in a reading frameshift of exon 20, thus generating a
novel 19-amino acid carboxyl-terminal peptide with an in-frame premature termination codon. The RH-2 variant, on the other hand, could
arise because of to the deletion of a 3' 60-nt fragment of exon 17 and
the entire exon 18. The putative alternative 5' donor site of the
variant would be in exon 17, whereas the 3' splice site of exon 19 would be used as a 3' acceptor. The deletion of the 3' part of exon 17 and the entire exon 18 predicts a reading frameshift, such that exons
19 and 20 are translated with respect to a new reading frame.
Consequently, exons 19 and 20 are predicted to translate a novel
62-amino acid carboxyl-terminal polypeptide.
1 subunit loop II-III, from exons
16 to 18. Because exon 19 is spliced out in the RH-1 type variant, this
exon is unprotected and consequently degraded by RNase. The
theoretically expected fragments of 144 and 46 nt, which would be
contributed by the RH-2 variant and the 3' end of the wild-type loop
II
III, respectively, were detectable, although at low levels, perhaps
reflecting low specific activity of the probe and the sensitivity of
the assay as well as the expression levels. A fragment of 454 nt
probably represents trace amounts of undigested cDNA template that
co-precipitated with RH-1 cRNA probe as it migrates at the same level
as the free undigested probe (Fig. 3, lane1). RNA from
skeletal muscle failed to protect any fragments (Fig. 3, lane
4). The protection of multiple fragments by RNA from cardiac
muscle is suggestive of the expression of the RH-1-type alternatively
spliced transcript and the wild type loop II-III of the
Cav1.2 gene.
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Fig. 3.
RNase protection analysis of loop
II-III. Expression analysis of loop II-III variant mRNAs was
performed with an RNase protection assay using RH-1 and RH-2 cRNA
probes. A schematic depicts the expected products of the RNase
protection assay of the 1 subunit of Cav1.2
Ca2+ channels with the deletion variant probes isolated by
RT-PCR. The line diagram reveals the expected sizes of the
protected fragments contributed by wild type (wt) and the
deletion variants of the
1 subunit loop II-III
mRNA. Solid bars denote protected fragments;
dotted lines represent deleted exons in RH-1 and RH-2
variants. Expected sizes of the protected fragments are indicated above
the respective products. Bottom panel, results of the RNase
protection of cardiac and skeletal muscles with RH-1 and RH-2 cRNA
probes. The protection of the mRNA from rabbit heart (lane
3) and skeletal muscle (lane 4) with the RH-1 cRNA
probe, lacking exon 19, is shown. Lane 1, migration of the
free probe (open arrowhead); lane 2, its
digestion with RNase. The RH-1 probe protected fragments of ~320,
274, 160, 144, and 46 nt in heart (solid arrows, lane 3)
that closely corresponded to the sizes of the expected products.
Interestingly, however, protection of an ~160-nt fragment
(solid arrowhead) may perhaps be indicative of an additional
deletion variant of loop II-III that has not yet been characterized.
The RNase protection of mRNA from heart (lane 7) and
skeletal muscle (lane 8) with the RH-2 type cRNA probe,
lacking exons 17 and 18, is presented. Lane 5, migration of
the free probe; lane 6, its digestion with RNase. The RH-2
probe protects fragments of ~320, 179, 144, and 46 nt, which closely
correspond to the sizes of the expected fragments in the top
panel.
1 subunit gene.
1 subunit of RH-1 and
RH-2 would generate two-domain truncated forms of the
Cav1.2 Ca2+ channels. To verify the hypothesis,
the full-length cDNAs encoding two-domain polypeptides needed to be
selectively isolated. RT-PCR of cardiac RNA was performed with a common
5' sense primer (nt 180-203, TGGAAACTGACAAT GCTTCGAGCC) and the
respective splice junction primers selective for the RH-1 and RH-2
variants (ATCCTCTTCTCCTTGGCCTCCTC-3' and 5'-GAGGCGGAACCTGTG GTTTCC,
respectively). RT-PCR yielded two products of ~2.6 (Fig.
4, lane 1) and 2.4 (Fig. 4, lane 2) kb, representing RH-1 and RH-2 variants, respectively, which were subsequently TA cloned and then cycle sequenced. Lane 3 represents a PCR-negative (water) control. A control with RNA but
without RT (for contamination with genomic and partially processed
mRNA) was also run, and it was negative.
View larger version (94K):
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Fig. 4.
RT-PCR of RH-1 and RH-2 deletion
variants. To amplify the full-length two-domain channels RH-1 and
RH-2, RT-PCR was performed using a common 5' primer
(TGGAAACTGACAATGCTTCGAGCC) as well as splice junction-specific primers
for RH-1 (ATCCTCTTCTCCTTGGCCTCCTC) and RH-2 variants
(GAGGCGGAACCTGTGGTTTCC). Products of the RT-PCR analysis of rabbit
cardiac RNA electrophoresed on a 0.9% agarose gel stained with
ethidium bromide are shown. Lane 1, RH-1 amplicon of ~2.6
kb generated with the 5' primer and an RH-1-specific primer; lane
2, RH-2 variant of ~2.4 kb generated with the 5' primer and an
RH-2-specific primer; lane 3, negative PCR (water) control.
Products were gel extracted, TA subcloned, and sequenced.
1 subunit. The salient
sequence differences were present in loop II-III as indicated The RH-1
variant would generate a polypeptide of predicted molecular mass
of 99.0 kDa, whereas the RH-2 variant would encode a polypeptide of
99.2 kDa.
View larger version (65K):
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Fig. 5.
RH-1 and RH-2 splice variants encode
two-domain Cav1.2 Ca2+ channels. Sequence
comparison of the full-length deletion variants of RH-1 and RH-2 type
with 1 subunit of the Cav1.2
Ca2+ channel are shown. Truncated channel variants contain
identical sequences in domains I and II similar to those in
1 Cav1.2, whereas the loop II-III sequences
are divergent because of alternative splicing. The unique
carboxyl-terminal sequences of truncated channels are enclosed in a
black box. RH-1 and RH-2 variants encode two-domain
1 Cav1.2 truncated channels of 99.0 and 99.2 kDa, respectively. Shaded sequences represent transmembrane
segments based on predicted channel topology. The membrane-spanning
regions (S1-S6) are shaded. ·, sequence identities;
, termination codons. The putative PKC phosphorylation
sites in the carboxyl-terminal sequences of the deletion variants are
denoted in bold and underlined. The RH-1 variant
contains one putative PKC phosphorylation site, whereas RH-2 predicts
three. The putative CK 2 phosphorylation site is represented in
bold italics. The CK 2 putative site is not present in the
deletion variants.
1 Cav1.2 subunit in
microsomes (Fig. 6, lane R) that was found to enrich in SL, although a substantial amount (~40%) of this immunoreactive
polypeptide was also present in HSR. Interestingly, the CNC-1 antibody
also recognized an ~99-kDa polypeptide in the microsomal fractions that was found to enrich in HSR but was completely absent from the
sarcolemmal fraction. The immunoreactivity of CNC-1 with the ~240-
and ~99-kDa polypeptides was completely negated by preincubation of
the antibody with the antigen (data not shown). The 99-kDa peptide
detected with the CNC-1 antibodies may represent the two-domain polypeptides generated by alternative splicing in loop II-III. To
further confirm this, antibodies were raised against unique sequences
in the RH-1 and RH-2 channel variants. The carboxyl-terminal 19 amino
acids in RH-1 and RH-2 were used as the antigen; however, this peptide
was found to be nonantigenic. Two peptide sequences unique to RH-2
(KRMRRSLRCLSAPALGHS and GHSPSCTLRRRPC) were found to be antigenic and
used to obtain antiserum 1929. Fig. 6 demonstrates that antiserum 1929, selective for the RH-2 variant, immunoreacted with a 99-kDa polypeptide
in the microsomal fractions as well as the HSR fractions but not the SL
fraction. No reactivity was seen in any of the fractions with the
preimmune serum. Thus the antibodies raised against the RH-2 variant
identified a 99-kDa polypeptide that was absent from the SL fractions
but was enriched in the heavy SR fraction as observed with the
anti-Cav1.2 (CNC-1) antibody. These data suggest the
presence of the two-domain Cav1.2 Ca2+ channel
polypeptides generated by alternative splicing in loop II-III as
components of the heavy SR membrane in cardiac muscle but not the
SL.
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Fig. 6.
Subcellular distribution of Ca2+
channel polypeptides in myocardium. Crude microsomes from rat
hearts were separated by differential and sucrose density gradient
centrifugation to enrich for SL membranes and HSR and immunoblotted
with specific antibodies to the Na+/K+-ATPase,
anti-loop II-III sequence of the Cav1.2 Ca2+
channel (CNC-1) and Anti-RH-2 (1929). Immunoblotting with
anti-Na+/K+-ATPase and the reactivity at ~110
kDa enriched in SL fractions compared with the crude microsomes
(R). Immunoblotting with the anti-Cav1.2
Ca2+ channel (CNC-1) revealed reactivity with ~240- and
~99-kDa polypeptides in the various fractions. Immunoblotting with
antisera 1929 raised against unique peptide sequences
(KRMRRSLRCLSAPALGHS and GHSPSCTLRRRPC) of RH-2 indicated reactivity
with a polypeptide of ~99 kDa in the various fractions with immune
serum but not preimmune serum.
1
Cav1.2 subunit variants generated by alternative splicing
from the proposed four-domain Ca2+ channel structure is
shown (Fig. 7). The full-length RH-1
variant renders a truncated, two-domain polypeptide composed of normal domains I and II, as well as 78 amino acids of the wild-type loop and a
novel 19-amino acid carboxyl-terminal tail. The RH-2 variant, on the
other hand, contains normal domains I and II and 35 amino acids of the
wild-type loop II-III and a unique 62-amino acid carboxyl-terminal
peptide. The terminal 19 amino acids in both variants would be
identical.
View larger version (37K):
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Fig. 7.
Topology of full-length
1 Cav1.2 and the deletion
variants (RH-1 and RH-2). The topology of the full-length cardiac
Cav1.2 Ca2+ channel and the predicted topology
of the two-domain channels are shown. The RH-1 two-domain channel
arises as a result of alternative splicing of exon 19, which results in
reading frameshift of exon 20 and a premature termination codon. The
RH-2-type channel, on the other hand, is generated by deletion of a
fragment of exon 17 and entire exon 18, which results in a reading
frameshift of exons 19 and 20. RH-1 and RH-2 both share a common
19-amino acid carboxyl terminus; however, RH-2 in addition contains a
unique 43-amino acid sequence. The two-domain polypeptides retain the
critical determinants of channel function, including activation,
inactivation, voltage sensor, ion selectivity, and pore structure,
which are contained within domains I and II. The
-subunit binding
sites will also be retained in the two variants, whereas the ligand
binding sites for phenylalkylamines (PAA), the
benzothiazepines (BTZ), and the dihydropyridines
(DHP) will not be retained. The putative carboxyl-terminal
PKC phosphorylation sites are indicated.
1 subunits connecting domains
II and III of the Ca2+ channel family of proteins is a
critical determinant of the mode of signal transduction through
protein-protein interactions (45-47, 50-57), which can be further
modulated by post-translational mechanisms such as phosphorylation of
amino acid residues in this loop (51, 58-60). Although the consensus
sequence prediction program PROSITE predicts putative PKC (TTK) and
casin kinase 2 (TTGE) phosphorylation sites in the wild-type
loop II-III, the demonstration of their phosphorylation and any
functional consequences remains to be determined. Interestingly,
however, the putative PKC site is deleted in the RH-2 variant, and the
casin kinase 2 site is deleted on both RH-1 and RH-2, whereas the novel
carboxyl termini introduce additional consensus PKC sites, one in RH-1
and three in RH-2. In view of the significant changes in the amino acid
composition and perhaps tertiary structure of loop II-III in the two
variants, it is speculated that these polypeptides (if functional)
could markedly influence signal transduction via these structural units.
1 subunit variants producing
truncated, two-domain Ca2+ channel polypeptides remains to
be elucidated, existence of other two-domain channel polypeptides in
the voltage-dependent superfamily has also been reported. A
two-domain, truncated form of Cav1.1
1
subunits comprising domain I and chimeric domain IV generated by
splicing of the S2 segment of domain II to the S2 segment of domain IV
has been observed predominantly in muscle from neonates (61). In this
regard, we observed that the RH-1 and RH-2 variants were expressed in
fetal and neonatal hearts (data not shown). Genetic mutations of the
neuronal Cav2.1 (P/Q-type Ca2+ channel,
CACNL1A4), associated with inherited episodic ataxia, predict synthesis
of a truncated, two-domain channel polypeptide composed of domains I
and II and a part of domain III, suggesting that these molecules have
functional consequences (62-64). In addition, a
voltage-dependent Na+ channel
subunit,
SCN8A, generates a predicted polypeptide comprising domains I and II as
a result of alternative splicing of exon 18N, which introduces an
in-frame termination codon (65). The predicted topography of truncated
CACNL1A4 is similar to that proposed for the SCN8A Na+
channel polypeptide. Recent studies revealed that the
-conotoxin MVIIC receptor associated with Cav2.1 (P/Q-type)
Ca2+ channels consisted of a 95-kDa polypeptide that
comprised domains I and II and a part of loop II-III (66). The 95-kDa
polypeptide was able to bind the auxiliary
subunits, and it remains
to be determined whether the polypeptide can form functional channels. Furthermore, it would be intriguing to elucidate whether the 95-kDa polypeptide is also generated by the alternative splicing mechanisms of
the Cav2.1
1 subunit gene,
similar to the Cav1.2 gene. It is also
notable that a unique cDNA has been isolated from kidney that
encodes a two-domain polypeptide that exhibits ~20% identity with
the voltage gated Na+ and Ca2+ channels
(69).
1 subunit isoforms described here. The
presence of the critical determinants of channel function, including
activation and inactivation domains, the voltage sensor, ion
selectivity, and pore structure within domains I and II, suggests that
the truncated channel polypeptides could perhaps homodimerize and heterodimerize to form functional channels. The RH-1 and RH-2 variants
may also play roles as dominant negative regulators of the wild type
Cav1.2 Ca2+ channels. It is notable that
subcellular distribution of the 240-kDa
1 subunit
indicated the presence of a substantial pool of this isoform in the
heavy SR membranes, probably representing the dyad structure known to
be enriched in this fraction. The exclusive presence of the 99-kDa RH-1
and RH-2 variants in this fraction suggests that these polypeptides may
play a role in excitation-contraction coupling at the dyad
junctions. In this regard, purified dyads were found to be highly
enriched in both the 240- and 99-kDa polypeptides (data not shown). The
RH-1 and RH-2 polypeptides may also serve regulatory roles by
sequestering auxiliary channel subunits, such as the
subunit,
because the binding site for this modulator would be retained by the
two variants. In view of heterogeneity of the tissues in which the
Cav1.2
1 subunits are expressed, it is
conceivable that the splice variants may also be present in a subset of
cells geared for specialized regulation of calcium entry. The level of
expression of the two variants compared with the wild type is
indicative of a stoichiometric relationship between the various
isoforms. Studies indicate that there may be a
voltage-dependent component to the excitation-contraction
coupling mechanism in cardiac tissue (70), and whether the two domain
polypeptides serve a role in this process remains to be defined.
1 subunit gene,
which form two-domain polypeptides, implies a novel mechanisms for
Ca2+ channel structure and function and/or regulation under
normal physiological conditions. Moreover, these results lend further support for an evolutionary model of a four-domain channel structure created by two subsequent gene duplications of an ancestral one-domain gene.
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FOOTNOTES |
---|
* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Recipient of a grant from the Medical Research Council of Canada
and Career Investigator of the Heart and Stroke Foundation of Ontario.
To whom correspondence should be addressed: Dept. of Cellular and
Molecular Medicine, University of Ottawa, 451 Smyth Rd., Ottawa,
Ontario, Canada K1H 8H5. Phone: 613-562-5800 (ext. 8355); Fax:
613-562-5434.
Published, JBC Papers in Press, September 28, 2000, DOI 10.1074.jbc.M006868200
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ABBREVIATIONS |
---|
The abbreviations used are: S1-S6, membrane-spanning regions 1-6; loop II-III, second intracellular loop between domains II and III; RT-PCR, reverse transcription-polymerase chain reaction; RH, rabbit heart splice variant; nt, nucleotide; bp, base pair; SL, sarcolemma; SR, sarcoplasmic reticulum; HSR, heavy sarcoplasmic reticulum; PKC, protein kinase C; CNC-1, antipeptide antibody to loop II-III.
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