(Received for publication, July 27, 1994; and in revised form, October 18, 1994)
From the
Calcium entry, via a dihydropyridine-sensitive pathway, is
required for differentiation in murine erythroleukemia cells (MELC).
Calcium channel currents have been identified physiologically in some
non-excitable cells, but little is known regarding the structure of
these channels. We show that a truncated form of the subunit of the cardiac voltage-gated calcium channel
(dihydropyridine receptor, DHPR) is expressed in MELC. This MELC
calcium channel lacks the first four transmembrane segments of the DHPR
(IS1 to IS4). A MELC calcium channel/cardiac DHPR chimera, co-expressed
with the
and
subunits of the DHPR, forms a
functional calcium channel in Xenopus oocytes.
Calcium (Ca) flux across cellular membranes is
a fundamental signal triggering pathways leading to numerous cellular
functions, including muscle contraction, neuronal signaling,
fertilization, and cell growth and differentiation. In excitable cells
voltage-dependent calcium channels (VDCC) (
)on the plasma
membrane are activated by depolarization and permit Ca
entry. Pathways for Ca
influx in non-excitable
cells, including T lymphocytes and hematopoetic precursors that lack
detectable VDCCs, have in most cases not yet been identified.
Biochemical characterization of Ca
channels present
in these non-excitable cells has been hindered by the lack of specific
high affinity ligands.
In non-excitable cells, including
hematopoetic cells, it has been proposed that Ca influx occurs via receptor- or second messenger- operated
channels(1, 2, 3, 4) . The existence
of a receptor-operated calcium channel was originally suggested on the
basis of agonist-induced dihydropyridine-insensitive calcium entry in
smooth muscle(5) . Second messenger-activated channels were
first detected with cholecystokinin activation of pancreatic cells (6) and gonadotrophin-releasing hormone stimulation of
gonadotrophs(7) . Receptor-operated calcium channels and second
messenger-activated channels have been proposed in numerous cell types
in which voltage-gated Ca
entry has not been
detected, including T lymphocytes, platelets, and endothelial
cells(8, 9, 10, 11, 12) .
Murine erythroleukemia cells (MELC) are a virally transformed cell line that has been studied extensively as a model for analyzing the molecular changes associated with differentiation in the erythroid lineage(13) . MELC are arrested in erythroid development at a stage comparable with the erythroid colony forming unit (CFU-e) and can grow indefinitely in appropriate culture conditions. They can be induced to terminal differentiation when cultured with various chemicals, including the polar compound hexamethylene bisacetamide (HMBA)(14) . Terminally differentiated MELC have characteristics of the differentiated erythroid phenotype, including globin gene expression and loss of proliferative capacity.
Changes
in [Ca]
have been
linked to several of the early events leading to terminal
differentiation(15, 16, 17) . As is the case
in many non-excitable cells, the mechanism controlling Ca
entry into MELC is not known. We previously reported that VDCCs
were not detected using whole cell patch clamp recordings of MELC and
that depolarization of the MELC membrane did not induce Ca
influx(17) . We found that HMBA stimulated Ca
influx within 3-6 min and that Ca
entry
was required but not sufficient for MELC growth and differentiation (17) . Nifedipine (1-10 µM) blocked
HMBA-induced Ca
influx and inhibited differentiation
by
60%.
The primary structure of the dihydropyridine receptor
(DHPR) class of VDCC has been determined from cDNA cloning in excitable
tissues including muscle and
brain(18, 19, 20, 21) . The
VDCC's described to date share a common predicted structure
consisting of four internally repeated motifs each containing six
putative transmembrane -helical segments (for a total of 24). In
addition there is a loop formed by antiparallel
sheets between
the fifth and sixth putative transmembrane segments in each motif that
is believed to form the channel lining(22) . In the fourth
transmembrane segment of each motif (S4 region) every third amino acid
residue is positively charged (21) forming the voltage-sensor.
A truncated form of the skeletal DHPR, comprised of only two motifs, is
expressed in newborn rabbit muscle (23) .
Based on the
conservation of structure observed in the Ca channel
gene family, we hypothesized that non-excitable cells might contain a
Ca
channel that shared common structural features
with the VDCC of excitable tissues. To test this hypothesis we used
probes based on the sequence of the
subunit of the
DHPR to clone the complete cDNA encoding a putative voltage-insensitive
Ca
channel from MELC. We now report that this
putative novel Ca
channel expressed in MELC (MELC-CC)
is a truncated form of the
subunit of the cardiac
DHPR. The MELC-CC has a unique structure resulting from the use of an
internal transcription start site downstream from the cardiac DHPR
start site, and a unique pattern of alternative splicing. Elucidation
of the structure and function of this novel form of a calcium channel
should provide the basis for better understanding of mechanisms
controlling of Ca
entry in non-excitable cells.
Figure 1:
Schematic
representation of the locations of individual clones and the probes
used for cloning the MELC-CC cDNA. A, the rabbit cardiac DHPR
was aligned with MELC-CC. The solid bar represents the open
reading frame. Each of the predicted transmembrane repeat is indicated
by solid ovals, and four motifs are labeled I, II, III, and IV. B, the MELC-CC
cDNA sequence is represented. The solid bar represents the
protein coding region of MELC-CC cDNA. The 5`- and 3`-untranslated
regions are indicated by thin lines. The restriction sites
used in preparing probes and in making the full-length constructs are
indicated by letters. P, PstI; B, BamHI; H, HindIII; E, EcoRI; A, AccIII; S, SacII; F, FspI. 5MEL, 2MEL, and 3MEL were
cloned from an uninduced MELC cDNA library. 5M613, 5M623, and 5M713
were cloned from the HMBA-induced MELC cDNA library. The relative
positions of the important oligonucleotides and the cDNA fragment used
for later experiments (as probes and primers) are indicated. C, alignment of the deduced MELC-CC amino acid sequence
derived from 5MEL, 2MEL, and 3MEL with that of the rabbit cardiac DHPR
(C/DHPR). A 93% overall amino acid homology to the rabbit cardiac
subunit suggests that they are from the same gene.
Only 20 out of 24 putative transmembrane regions are conserved in
MELC-CC making this channel unique. In addition, deletions (indicated
by dots) and alternative splicing sites (double
underlined) were found within the MELC-CC sequence. The bold
letters located between IS6 and IIS1 represent a deduced amino
acid sequence inserted in the MELC-CC. Potential cAMP-dependent protein
kinase phosphorylation sites are underlined with dots.
Potential N-glycosylation sites are indicated with an asterisk below the amino acid residue. The putative cardiac
DHPR transmembrane regions are underlined and labeled over the
top of the fragment with Roman numerals I to IV. The numbers on the right side of the figure indicate the
position of the last amino acid in the row. An asterisk was
inserted in the sequence to maximize the
alignment.
Figure 2:
Upstream flanking sequence of the MELC-CC
revealed GATA and CACCC transcription elements. A, partial
sequence of MELC genomic clone, P2, is shown. This clone extends
3.5 kb upstream of the MELC-CC cDNA. The GATA repeats in the
distal region and GATA and CACCC elements in the proximal regions are boxed. These elements could represent promoter regions
regulating MELC-CC transcription. B, a diagram of the
relationship of the MELC genomic clone and the MELC-CC is shown. The
putative transcription initiation site is indicated by an arrow. C, proposed expression scheme for the MELC-CC
gene compared with the cardiac calcium channel gene and putative
channel topography. MELC-CC transcription initiates from an internal
intron (thin lines in between solid boxes) of the
calcium channel gene (possibly under the regulation of GATA and CACCC
regulatory elements and their corresponding transcription factors). A
truncated message is expressed in MELC (the thick lines represent open reading frames). The predicted channel topology of
the Ca
channel proteins within the cell membrane is
indicated below. Four H5 segments for both MELC and cardiac are
present, but MELC contains only 20 of the 24 transmembrane segments
present in the cardiac Ca
channel.
To determine whether the mRNA encoding
the complete murine cardiac DHPR subunit (including
the first four transmembrane segments) was also expressed in MELC, a
mouse cardiac DHPR
subunit cDNA (corresponding to the
5` portion of the cardiac DHPR mRNA encoding the first four putative
transmembrane regions lacking in the MELC-CC) was used to screen both
oligo(dT)- and random-primed MELC cDNA libraries. No clones were
isolated from 5
10
recombinants. Moreover, Northern
hybridizations (Fig. 3A), RNase protection (Fig. 3B) and PCR demonstrated that the 5` sequence
from the mouse cardiac DHPR
subunit (encoding the
first four putative transmembrane segments) was not expressed in
MELC-CC.
Figure 3:
Northern analysis of the expression of
MELC-CC and the cardiac calcium channel. A, total RNA from
mouse heart (15 µg) and MELC (30 µg) were run on formaldehyde
agarose gels. A 5` cDNA probe spanning IS1 to IS4 regions of the
cardiac DHPR and a 3` cDNA probe corresponding to 1-6,235 of the
MELC-CC were used. No detectable message was seen in MELC RNA probed
with the 5` cardiac DHPR probe, whereas three bands were detected in
mouse heart RNA, corresponding to mRNAs of 7.5, 9.5, and 22 kb. Thus
the 5` portion of the C-DHPR mRNA is not expressed in MELC. Using the
3` MELC-CC cDNA probe, the same three bands were also detected in mouse
heart RNA; however, three slightly smaller bands were now detected in
MELC RNA. This finding indicates that three DHPR mRNAs are expressed in
MELC. The difference in size between the mouse heart mRNAs and the MELC
mRNAs can be explained by deletion of the 5` sequences encoding the
first four transmembrane regions. In the lane labeled MELC-CC, in
vitro transcribed full-length MELC-CC RNA was loaded and
hybridized with the 3` MELC-CC probe as a size marker. Northern blots
were exposed 24 h, except for the lane labeled MELC which
represents a 7-day exposure. The position of the 28 S rRNA is also
indicated as a size marker. The lower panel represent the
ethidium bromide stain of the 28 S rRNA used to control for loading of
the RNA samples. B, tissue specific expression of the mouse
cardiac Ca channel. Using RNase protection with a
probe spanning IS1 to IS4 (corresponding to the amino acids
165-293). Expression of the mouse cardiac Ca
channel was detected in mouse heart and lung RNAs. These RNAs
protected a 384-bp fragment (indicated with arrows) using a
full-length probe (463 bp). No protected products were detected in
liver, MELC, and HL60. (MELC SC9 is a wild type cell line and MELC R1
is a vincristine-resistant cell line). Yeast RNA was used as a control
for RNase digestion. The schematic diagram shows the size of the
full-length probe as well as the size of the fully protected product.
The solid box represents the region that is homologous between
the cardiac and MELC-CC. The hatched box represents the IS1 to
IS4 transmembrane region of the cardiac CC that are not expressed in
MELC. The numbers in the open boxes represent the
plasmid nucleotides.
In the mouse heart, two major mRNA species migrating at
15.5 and
8.9 kb, and a third minor mRNA species migrating at
20 kb were identified by Northern hybridization (Fig. 3A). The hybridization pattern in MELC was
identical to that in mouse heart except that each mRNA species in MELC
was shifted to a smaller size. The difference in size between the
murine cardiac DHPR
subunit mRNAs and those from MELC
corresponds to the portion of the 5` sequence of the cardiac DHPR
subunit mRNA not expressed in the MELC-CC mRNA. No
signal was detected in MELC using a cDNA probe encoding the first four
transmembrane segments of the cardiac DHPR
subunit.
RNase protection was used to further analyze the structure of the
MELC-CC mRNA. Using a cRNA probe corresponding to the first four
transmembrane segments (IS1 to IS4) of the murine cardiac DHPR
subunit, a 384-bp protected fragment was seen in
mouse heart and lung (Fig. 3B). No protection was seen
in MELC, HL60, or liver. These results further indicated that the 5`
end of the cardiac DHPR
subunit mRNA was not
expressed in MELC, even at low amounts. Moreover, although the cardiac
DHPR
subunit mRNA was detected in heart and in lung,
there was no expression in liver or in HL60.
To rule out the
possibility that a trace amount of the 5` end of the mouse cardiac DHPR
subunit mRNA was expressed in MELC, we used the 5`
rapid amplification of cDNA ends method (31) to amplify this
region of the sequence. The 5` region of the mouse cardiac DHPR
subunit mRNA was amplified from mouse heart as a
positive control. No product was obtained using MELC mRNA as the
template (data not shown).
Primer extension was used to identify the transcription start site of the MELC-CC mRNA. The antisense primer, 1440AS (a synthetic 20-base oligonucleotide complementary to the MELC-CC cDNA at nucleotide 45-64) was used to start the extension reaction. Fifty µg of MELC total RNA were extended using avian myeloblastosis virus reverse transcriptase, and two major extended products were generated (Fig. 4A). The two major bands were 75 and 105 bp in size, suggesting that there might be two transcription initiation sites for MELC-CC mRNA synthesis. The sites identified by primer extension are 11 and 41 nucleotides upstream from the end of the MELC-CC mRNA.
Figure 4:
A, primer extension analysis of the 5`
termini of MELC-CC RNA. 1440AS was used as an extension primer. MELC
total RNA was used in the extension assay and two major extended bands
at 75 and 105 bp were identified (indicated by arrows).
This finding is consistent with MELC-CC having two major transcription
initiation sites. An in vitro transcribed RNA derived from
clone 623 from the MELC-CC with a predicted extension product of 115 bp
(including 83 bp from pBluescript) was used as control. Size markers
were HaeIII digested
X174. B, the 5` terminus
of MELC mRNA was mapped using RNase protection analysis with MELC
genomic DNA (M/G DNA) probes. Two major bands at
100 and 130
nucleotides are protected with both probes as indicated by the vertical bar and arrow. An in vitro transcribed MELC-CC RNA was used as control and the predicted
66-bp band was generated as indicated by the arrowhead. The schematic
shows the probes derived from the MELC genomic DNA. Probe 1 was
generated with a BamI/SacI digestion, and probe 2 was
generated by a EcoRI/SacI digestion. The numbers below each enzyme indicate the distance from the end of that probe
relative to the first nucleotide of the MELC-CC cDNA. Size markers are HaeIII-digested
X174 DNA.
A MELC genomic library was screened using a 2MEL BamHI-HindIII cDNA fragment to isolate genomic clones suitable for use as probe for the region upstream from the 5` end of the MELC-CC mRNA. A positive clone (MELC13) was isolated and sequenced. A subclone of MELC13, pP2ApaI/SacI (located between -674 and +91 of the MELC-CC), overlapped with the 5` terminal region of the MELC-CC cDNA. pP2ApaI/SacI was cut into two probes of different lengths using BamI or EcoRI on the 5` end (which extend from +91 to -48 and -142, respectively) (Fig. 4B). When used in RNase protection assays these probes protected several fragments at 102-132 nucleotides in length (corresponding to -11 to -41 with respect to the first nucleotide of the MELC-CC cDNA) (Fig. 4B). Thus both the primer extension assay and the RNase protection identify the same two transcription start sites (Fig. 2A).
An 89-bp
insertion near the start site of transcription in one allele of the
murine cardiac subunit DHPR was identified by genomic
cloning. PCR amplification of MELC and DBA/2 (murine strain of origin
for MELC) genomic DNA using two synthetic oligonucleotide primers
complimentary to sequences surrounding this 89-bp insertion yielded two
bands (Fig. 5A), indicating that two alleles are
present in MELC while a single allele was detected in DBA/2 DNA.
Sequencing of the amplified genomic fragments confirmed the presence of
an 89-bp insertion in one allele at the junction between sequences
homologous and non-homologous to the
subunit of the
DHPR. This 89-bp insertion was not homologous to any sequence in
GenBank and was not present in the mature MELC-CC mRNA.
Figure 5:
A, detection of MELC-CC gene rearrangement
using PCR amplification of MELC and DBA/2 mouse DNA. Primers used in
PCR were 1440S (sense) and 5MASA and 5MBamAS (antisense). PCR products
were electrophoresed on a 1% agarose TAE gel and stained with ethidium
bromide. Using the first pair of PCR primers, two amplified products,
446 and 357 bp, were obtained from MELC, whereas in DBA/2, only the
357-bp product was present. The extra band seen in MELC is due to an
89-base pair insertion in the MELC-CC gene. This was determined by
excising the bands and sequencing them. Using the second pair of
primers, a 510-bp product was seen both in MELC and DBA/2. The absence
of a second band in MELC suggests that a large insertion could have
prevented amplification from occurring using the primers of 5MASA and
5MBamAS. The positions of the primers, 1440S, 5MASA, and 5MBamAS are
indicated. HaeIII-digested X 174 was used as size marker. B, Southern analysis of the MELC (M) and DBA/2 (D) mouse Ca
channel genomic DNA structure.
Four pairs of DNA samples were digested with four restriction enzymes, BglII, NdeI, PstI, and XhoI,
respectively. DNAs were size fractionated on a 0.8% of agarose Tris
acetate-EDTA gel. The blot was probed with the 2MEL cDNA, BamHI-HindIII. The restriction fragment patterns in
the BglII, NdeI, and XhoI digests differ
between MELC and DBA/2. The band patterns from the PstI
digests are similar. This result suggests that gene rearrangement has
occurred within the MELC-CC gene. These results are also consistent
with a rearrangement, because only one allele is detected in the MELC
DNA, and it differs from the DBA/2 allele. HindIII-digested
phage DNA was used as size marker.
MELC is a virally transformed cell line and rearrangements have been documented frequently in other genes in MELC(32) . Genomic Southerns comparing MELC the parent mouse strain (DBA/2) were consistent with a rearrangement having occurred in the MELC-CC gene upstream from the exon encoding the putative transmembrane region IS5 in the MELC-CC. Fig. 5B shows the genomic Southern hybridization, in which a cDNA corresponding to bp 540-879 of the MELC-CC cDNA (corresponding to the IS5 region) was used as probe, demonstrated that a gene rearrangement had occurred in this region of the MELC-CC gene. Four restriction enzymes (NdeI, PstI, XhoI, and BglII) were used to digest MELC and DBA/2 genomic DNA. Hybridization patterns for the NdeI, XhoI, and BglII digestions differed between MELC and DBA/2. The band patterns from the PstI digestion were similar for both MELC and DBA/2. Both alleles in MELC contain rearrangements since the wild type (DBA/2) pattern was not seen in this region of the MELC genome. However, PCR data obtained by amplifying the region surrounding the junction between the homologous and non-homologous portions of the MELC-CC (Fig. 5A) revealed an 89-bp insertion found only in one allele (non-transcribed). This discrepancy indicates that although both alleles are the same in the IS5 region in MELC-CC, there must be differences in the junction region further upstream.
Alternative splicing in the MELC-CC mRNA defines a novel
splicing pattern that is regulated during induced cellular
differentiation (Fig. 6A). Mutually exclusive,
developmentally regulated splicing patterns have been reported for the
IVS3 region of the rat heart subunit(33) . In
MELC-CC the IVS3 region corresponds to nucleotides 1018-1045 (Fig. 1C). In MELC, exon S3A was expressed together
with exon D2 (Fig. 6A).
Figure 6:
A, comparison of the alternative splicing
pattern of MELC-CC subunits with that of the rat
cardiac
subunits. The genomic structure of exons for
the IVS3 transmembrane region and its adjacent regions are shown. The
amino acid positions corresponding to the rat cardiac
subunits are numbered. The S3A or S3B equivalent in the MELC-CC
amino acid sequence is 1018-1045. D1 equivalent in the MELC-CC
amino acid sequence is at amino acid 1046. A unique splicing pattern is
exhibited in MELC-CC. C, comparison of the COOH-terminal
sequences of the MELC-CC with that of the rabbit cardiac and rat aorta
VDCCs (VSM
). The non-homologous amino acids between
MELC-CC and rabbit cardiac VDCC are indicated by !. The non-homologous
amino acids between MELC and rat aorta VDCC are indicated by
&cjs1219;). Dots were inserted to maximize the sequence
alignment.
The COOH-terminal sequence
portion of the MELC-CC cDNA coding region (amino acids 1552-1619)
differs from that of the rabbit cardiac DHPR (19) (Fig. 6B). This region of the MELC-CC
sequence exhibits 81% identity with that of the rat aorta DHPR
VSM(20) , in contrast the MELC-CC has only 56%
identity with the rabbit cardiac DHPR in this region. Thus, in this
region of the MELC-CC a smooth muscle-specific exon appears to have
been introduced as opposed to the cardiac form.
Figure 7:
A, tissue-specific expression of MELC-CC
RNA in MELC and mouse heart was analyzed using RNase protection.
MELC-CC mRNA was detected in mouse heart and MELC. Partial and fully
(possibly due to the unprocessed RNA contamination) protected products
were detected in the heart while the partially protected product
accounts for the majority of the signal. Only the fully protected
product was seen in MELC, suggesting that this is the only form of the
mRNA expressed in these cells. The sizes of the full-length of the
probe and the fully and partially protected products are indicated with arrows. The schematic shows that a MELC-CC probe spanning
nucleotides 1-550 was used. The solid box represents the
homologous region between the cardiac and MELC CC 1 subunits. The hatched box represents the MELC-CC-specific region where the
sequence diverges from the cardiac DHPR. The numbers in the open boxes represent the plasmid. Size markers are HaeIII-digested
X174 DNA. Tissue-specific expression was
also examined in differentiated (D) and undifferentiated
cells, including the following: BC3H1 (murine muscle cells), NIH3T3
(murine fibroblasts), and MELC. Full protection was seen in heart,
NIH3T3, and MELC. Partial protection was seen in heart and NIH3T3
cells. B, Northern analysis of MELC-CC mRNA expression during
HMBA-induced MELC differentiation. Total RNA was obtained from MELC
after 3, 12, 24, 36, 72, and 100 h of HMBA treatment. The MELC-CC
expression peaked after 36 h of HMBA treatment. 20 µg of total RNA
from each sample was size fractionated on a 1% formaldehyde agarose
gel. The blot was hybridized with the entire 2MEL cDNA probe. The
position of the 28 S rRNA is indicated. Levels of actin mRNA expression
were determined to normalize differences in RNA loading in each
lane.
MELC-CC mRNA levels were also regulated during HMBA-induced differentiation. Northern hybridization analysis was performed on MELC cultured with HMBA (5 mM) under conditions that we had previously shown to induce 80% of the cells to commit to terminal differentiation(17) . Compared with control, uninduced cells, there was an increase in MELC-CC mRNA levels beginning at 12 h and reaching a peak at 36 h of HMBA exposure (Fig. 7B).
Immunoblots were performed on MELC membranes,
membranes from oocytes injected with MELC-CC mRNA, murine cardiac
tissues membranes, and membranes from insect cells overexpressing the
subunit of the cardiac DHPR. Two different anti-DHPR
antibodies were used. One of these, DHP-III, recognizes an epitope in
motif III and the other, DHP-C, recognizes the carboxyl terminus of the
subunit of the cardiac DHPR. Both identified an
200-kDa protein on immunoblots containing
subunit of the cardiac DHPR overexpressed in insect cells
(provided by Dr. M. Hosey). This result indicated that both antibodies
were specific for the
subunit of the cardiac DHPR.
However, neither antibody identified a protein in any of the other
preparations, including murine cardiac membranes, oocytes expressing
the
subunit of the cardiac DHPR, or the MELC-CC or
MELC membranes.
Functional Expression of the MELC-CC Xenopus
Oocytes-In vitro transcribed MELC-CC mRNA was
injected into X. laevis oocytes for functional expression. No
L-type Ca channel currents were detected using
standard voltage-clamp techniques and protocols designed to activate
the channel using either HMBA (5 mM), BAY K8644 (see
``Experimental Procedures'' for details). A fusion protein
comprised of the first four transmembrane segments of the murine
cardiac DHPR
subunit plus the entire MELC-CC was
expressed using a plasmid created by ligating the 5` end of the murine
cardiac DHPR
subunit cDNA to the complete MELC-CC
cDNA. In vitro transcribed RNA from this chimeric cDNA
(Card/MELC-CC) expressed a typical L-type current in oocytes (Fig. 8). This calcium channel current was compared with that of
the wild type rabbit cardiac DHPR also expressed in oocytes. The
maximum of I-V curve for the wild type channel was 5.7 ± 2.9 mV
and 15.0 ± 2.2 mV for chimeric calcium channel (n = 7, p < 0.05). Both the wild type and the
chimeric channels showed similar sensitivities to BAY K8644 (5
µM); calcium channel current was increased 4.1 ±
0.6 times for the wild type and 3.6 ± 0.5 times for the chimera (n = 6). The shift in the peak of the I-V curve after
application of BAY K8644 was -15 mV for both channels. At the
same voltage the chimeric channel exhibited more rapid activation and
slower inactivation compared to the wild type rabbit cardiac DHPR (Fig. 8). These findings indicate that the MELC-CC cDNA was
capable of being translated into a functional Ca
channel
subunit protein. This result also
demonstrates the requirement for the first four transmembrane segments
of the cardiac DHPR
subunit in terms of making a
functional voltage-gated Ca
channel.
Figure 8:
Normal
(wild type) and chimeric calcium channels expressed in Xenopus oocytes. Whole cell currents recorded from Xenopus oocytes injected with cRNA from the wild type rabbit heart
subunit (A) and chimeric
(B) subunit with 40 mM barium
solution(41) : 40 mM Ba(OH)2, 50 mM NaOH, 2
mM KOH, 5 mM HEPES, pH adjusted to 7.4 with
methanesulfonic acid. Chloride channels were inhibited by 300
µM niflumic acid. Data were filtered at 1 kHz and sampled
at 2.5 kHz. Net calcium channel current was obtained using the cadmium
(0.4 mM) subtraction procedure. Currents were recorded with
standard two-microelectrode voltage clamp, using depolarization pulses
200 ms long from a holding potential of -80 mV. All cells were
co-injected with skeletal muscle
and
calcium
channel subunits. Currents were measured 3-5 days after injection
of cRNA. On the top, calcium channel currents in control
conditions and after application of the dihydropyridine agonist BAY K
(5 µM). Holding potential -80 mV, test potential 0
mV. Scale bars, 1 µA and 50 ms. On the bottom,
current-voltage relationships before (open symbols) and after (filled symbols) application of BAY K (5
µM).
In the present study we have characterized the structure and
regulation of a novel calcium channel expressed in transformed
erythroleukemia cells that require Ca influx for
induced differentiation. To study Ca
channels in
non-excitable cells we took advantage of the evolutionary conservation
among ion channels. We hypothesized that a Ca
channel
in non-excitable cells would have homology to the structures of the
known calcium channels in excitable tissues. Indeed, we found that a
truncated form of the cardiac DHPR
subunit is
expressed in MELC. This truncated molecule is unlike other
Ca
channels that have been described as it lacks the
first four putative transmembrane segments.
Although the predicted
topography of the MELC-CC based on its primary structure makes it
unique among Ca channels, the inward rectifying
potassium channel, IRK1, exhibits the same truncated
structure(37) . IRK1 is predicted to have only two
transmembrane segments, equivalent to S5 and S6, and an H5 pore region.
This structure would be the same as that of the first motif of the
MELC-CC. The functional role of the S1, S2, and S3 regions has been
debated. One possibility is that these transmembrane segments provide
electrostatic shields between the charged S4 voltage sensor regions and
the hydrophobic membrane(38) .
The ability of the chimeric
molecule combining the 5` portion of the murine cardiac DHPR
subunit with the complete MELC-CC to express a
functional Ca
channel in oocytes also demonstrates
that the MELC-CC cDNA encodes a portion of a functional channel. That
is, none of the structural alterations introduced by alternative
splicing prevent this molecule from supporting Ca
channel function. The activation and inactivation kinetics of the
chimeric channel, although similar to those of the wild type, exhibited
some differences; activation was faster and inactivation was slower.
These subtle differences could be due to charge differences in the
carboxyl terminus between the wild type and chimeric channels. Indeed,
the carboxyl-terminal region of the channel exhibited only limited
(56%) homology with the rabbit cardiac channel (Fig. 6B) making this region one of the most divergent
between the murine and rabbit forms of the calcium channel. There are
several possible explanations for the failure of the MELC-CC to form a
detectable VDCC in oocytes. One possibility is that a VDCC is expressed
after injection of oocytes with MELC-CC RNA, but that the conductance
through the channel is substantially reduced, preventing detection with
the protocols that we used. This type of channel, with extremely low
conductance, could still pass enough Ca
to raise the
[Ca
]
from basal (
100
nM) to
1 µM, as we observed after HMBA and
Bay K8644 stimulation(17) . Another possibility is that a
co-factor or subunit normally present in MELC required for MELC-CC
functional expression is missing from Xenopus oocytes. We were
also unable to detect MELC-CC protein either in MELC or in oocytes
injected with MELC-CC mRNA. However, despite the fact that we showed
that our antibodies were specific for the
subunit of
the DHPR on immunoblots, our inability to detect the protein in MELC
was not surprising. Using the same antibodies we were unable to detect
the
subunit of the DHPR in cardiac membranes, where
we can easily detect a voltage-gated Ca
current and
measure DHP binding sites. Therefore, the MELC-CC may be expressed at
too low a level to detect in MELC.
Structural diversity among the
VDCC has been created by alternative splicing of RNA transcripts.
Alternative usage of two exons at the IVS3 transmembrane region had
been reported during rat heart development (33) . Three
mutually exclusive, developmentally regulated splicing patterns have
been previously identified in this region of the rat heart subunit(33) . The MELC-CC pattern defines a novel fourth
splicing pattern that is regulated during induced cellular
differentiation (Fig. 6A). In MELC-CC the IVS3 region
corresponds to nucleotides 1018-1045 (Fig. 1C).
Exon S3A was expressed with exon D2, a pattern that has not been
observed previously. Interestingly, exon S3A corresponds to the fetal
form of this putative transmembrane region that has previously been
reported to be expressed with exon D1 which is not expressed in the
MELC-CC cDNA (33) . Thus the MELC-CC exhibits a unique pattern
of alternative splicing that is regulated during cellular
differentiation. This alternative splicing is most striking in the IVS3
region where we found a novel combinatorial pattern of exons that was
regulated during induced differentiation in MELC (Fig. 6A). This is the first example of alternative
splicing regulated during cellular differentiation in calcium channels.
We demonstrated that a gene rearrangement had occurred in the MELC-CC gene. It is possible that this gene rearrangement resulted in the placement of two GATA boxes just upstream of the start site of transcription of the MELC-CC gene. GATA sequences, (A/T)GATA(G/A), with enhancer activity have been associated with transcriptional regulation during hematopoetic differentiation(39, 40) . GATA-1 is an erythroid transcription factor expressed in all stages of vertebrate erythroid development, and it plays a important regulatory role in erythropoiesis (39) . Expression of the MELC-CC mRNA may be regulated by the transcription factors that bind to the GATA boxes located upstream of the start site of transcription (Fig. 2A). Regulation by GATA-1 or a similar transcription factor could explain the induction of MELC-CC mRNA during HMBA-induced differentiation.
The MELC-CC mRNA was expressed in MELC, but also in cardiac tissue and in NIH 3T3 cells. One possibility is that this RNA could represent unprocessed RNA or nuclear RNA. The MELC-CC message represented approximately 1-5% of the total cardiac DHPR mRNA in cardiac total RNA. If this message were translated it is possible that it could encode a molecule that binds DHP under the appropriate circumstances (e.g. in the presence of other subunits), but does not form a VDCC. Although the MELC-CC message might account for only 1 to 5% of the total cardiac calcium channel mRNA, the ratio for protein levels could be different (i.e. more MELC-CC protein than message). This could in part explain the observed discrepancy between DHP binding sites and functional calcium channels in cardiac tissue.