©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Cloning and Expression of a Novel Truncated Calcium Channel from Non-excitable Cells (*)

(Received for publication, July 27, 1994; and in revised form, October 18, 1994)

Yongsheng Ma Evgeny Kobrinsky Andrew R. Marks (§)

From the Molecular Medicine Program, Department of Medicine, and Brookdale Center for Molecular Biology, Mount Sinai School of Medicine, New York, New York 10029

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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 alpha(1) 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 alpha(2) and beta subunits of the DHPR, forms a functional calcium channel in Xenopus oocytes.


INTRODUCTION

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) (^1)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 alpha-helical segments (for a total of 24). In addition there is a loop formed by antiparallel beta 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 alpha(1) 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 alpha(1) 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.


EXPERIMENTAL PROCEDURES

MELC Culture

MELC lines (a gift from Dr. Victoria Richon, Memorial Sloan-Kettering Cancer Center, New York) were maintained in a alpha-minimal essential medium containing 10% (v/v) fetal calf serum(24) . Cultures were initiated with an inoculum of 10^5 cells/ml; HMBA (Sigma) was added to cultures at a final concentration of 5 mM.

cDNA Cloning

MELC cDNA libraries (one random-primed and one oligo(dT)-primed) were made from mRNA isolated from MELC induced for 48 h with HMBA (5 mM). Each contained 90% inserts with an average size of 2.5 kb. These cDNA libraries were screened with a 570-bp PCR derived MELC-CC cDNA clone (17) randomly labeled to a specific activity of 1 times 10^9 cpm/µg. Three overlapping cDNAs isolated from two different MELC cDNA libraries were used to form the full-length MELC-CC cDNA.

Rapid Amplification of cDNA Ends to Clone the 5` End

Three specific antisense 20-oligonucleotide primers were designed denoted: 5MSacA, 5MBamA, and 5MASA (Fig. 1B). One µg of mRNA from MELC cultured in 5 mM HMBA for 56 h and from normal mouse heart were reverse-transcribed using the SuperScript Reverse Transcriptase (Life Technologies, Inc.) primed with 5MSacA. The specific first strand cDNA was then used as the template for PCR amplification. Synthetic oligonucleotide [-P]ATP-labeled probes (corresponding to sequences internal to the PCR primers) were hybridized to Southern blots of the PCR product to confirm its identity. PCR products hybridizing to these probes were subcloned and sequenced.




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 alpha(1) 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.



Genomic Cloning

MELC genomic DNA was digested with Sau3A and cloned into BamHI site of EMBL3. Two times 10^5 recombinants, grown in Le392 bacterial host, were screened using a probe corresponding to nucleotides 546-879 of the MELC CC cDNA. Positive clones were purified, restriction mapped and sequenced using standard procedures(25) .

RNA Preparation and Northern Blot Analysis

Total RNA was prepared from MELC using standard guanidinium thiocyanate lysis buffer and centrifugation over a cesium chloride cushion(26) . Northern blot analysis was performed as described previously (27) with hybridizations at 42 °C overnight and the final washing at 65 °C, with 0.2 times SSC. Autoradiography were carried out with a single intensifying screen at -80 °C for the time indicated. RNA quantity and quality in each lane on Northern blots were evaluated by ethidium bromide staining.

RNase Protection

RNase protection was performed using cRNA probes labeled with [P]CTP and T3 RNA polymerase. Hybridizations of total RNA (10-20 µg) with the radiolabeled cRNA (2 times 10^4 cpm) were performed in solution (80% deionized formamide, 40 mM PIPES, pH 6.4, 400 mM NaOAc, pH 6.4, 1 mM EDTA) overnight at 45 °C in a 20-µl reaction volume. Following incubation, reactions were digested with RNase A (0.33 unit/µl)/RNase T1 (100 units/µl) at 37 °C for 30 min with 300 µl of a solution containing 10 mM Tris, pH 7.5, 300 mM NaCl, 0.005 mM EDTA. Reactions were stopped with 10 µl of 10% SDS and 50 µl of proteinase K (1 mg/ml) followed by incubation for 15 min at 37 °C. Protected fragments were ethanol precipitated, resuspended in loading buffer, and size-fractionated on 6-8% polyacrylamide gels under denaturing conditions at 200 V for 4 h in Tris borate/EDTA buffer. Gels were dried and exposed to x-ray film at -80 °C with one intensifying screen.

Primer Extension

Primer extension assay was performed using 10 fmol of gel-purified 14- or 16-nucleotide synthetic primer 5` end-labeled with T4 polynucleotide kinase. Condition for hybridization of the primer to the target RNA species were as follows: (20-µl volume), 0.4 M NaCl, 40 mM PIPES, pH 6.5, 1 mM EDTA, and 80% formamide at 30 °C for 12 h. After precipitation with 0.5 volume of 7.5 M NH(4)OAc, reverse transcriptase was added at 37 °C for 1 h in the presence of 0.1 mM each of dTTP, dCTP, dGTP, and dATP to synthesize extended product. After RNase treatment, phenol/chloroform extraction and ethanol precipitation, the pellet was resuspended in 8 µl of loading buffer, denatured at 100 °C for 3 min, and size-fractionated on a 6-8% polyacrylamide gel electrophoresis. Gels were dried and exposed to x-ray film at -80 °C with one intensifying screen for 48 h.

Antibody Production

Synthetic peptides based on the deduced amino acid sequence of the cardiac DHPR (19) were made as follows: DHP-III (amino acids 1115-1129) and DHP-C (amino acids 2130-2143). These peptides were coupled to keyhole lympet hemocyanin and used to raise antibodies in rabbits for immunoblots as described (28) . Cell membrane protein samples were prepared from the MELC (SC9), Xenopus oocytes, and rabbit skeletal muscle and heart as described above.

Oocyte Expression and Electrophysiology

Xenopus laevis oocytes were injected with 5 ng of in vitro transcribed RNA and cultured for 3-7 days at 18 °C. Oocytes were incubated in ND96 solution, containing 2 or 10 mM calcium at holding potential = -60 or -80 mV and BAY K8644 (1-10 µM) or 5 mM HMBA was applied. Calcium channel expression was determined by monitoring the Ca-activated chloride current (endogenous to oocytes). To record barium currents, oocytes were perfused with a solution containing 50 mM sodium acetate, 40 mM barium acetate, 2 mM potassium acetate, and 5 mM HEPES, pH = 7.5. A voltage-ramp protocol, from -80 to +40 mV, was applied for 2 s. The same voltage-ramp protocol was applied in conditions permitting a challenge with Ca after preincubating oocytes with BAY K8644 (1-10 µM) or HMBA (5 mM). Cells were perfused with Ca-free/EGTA solution (86 mM NaCl, 2 mM KCl, 15 mM MgCl(2), 0.1 mM EGTA, 5 mM HEPES, pH = 7.5), containing either 5 µM BAY K8644 or 5 mM HMBA for 20-30 min. Subsequently a solution containing 5 mM CaCl(2) substituted for 5 mM MgCl(2) (no EGTA) was applied.


RESULTS

Primary Structure of the MELC Calcium Channel

The MELC-CC cDNA sequence 3` to nucleotide 369 demonstrated 95% deduced amino acid identity with the rabbit cardiac DHPR alpha(1) subunit(19) . The sequence 5` to nucleotide 369 diverged completely from the cardiac DHPR, suggesting an RNA splice site at this junction. An AG was present at this junction consistent with a donor splice site(29, 30) . Five overlapping cDNA clones with identical sequences corresponding to this junction region were isolated and sequenced. Subsequent cloning of portions of the murine cardiac DHPR alpha(1) subunit cDNA revealed identity with the corresponding regions of the MELC-CC cDNA, indicating that the two transcripts were derived from a single gene. Thus the MELC-CC cDNA (7,554 bp) encodes an mRNA that is transcribed from the murine cardiac DHPR alpha(1) subunit gene using a start site downstream from the one used in heart (Fig. 2). The resulting mRNA encodes a molecule that lacks the first four putative transmembrane regions of motif I of the cardiac alpha(1) DHPR. A 1,864 amino acid open reading frame with the first in frame ATG located at the nucleotide 405-407 was present (Fig. 1C). This open reading frame encodes a protein with a predicted molecular mass of 210,769 daltons. Comparison of the MELC-CC sequence with consensus sequences for protein phosphorylation sites exhibited a threonine residue and 17 serine residues in the intracellular domains of the MELC-CC, suggesting these may be potential cyclic AMP dependent phosphorylation sites. In addition to 18 putative phosphorylation sites found at probable intracellular locations, three N-linked glycosylation sites were identified at amino acid residues 64, 1118, and 1169 in the putative extracellular domain. With the exception of the first 4, 20 out of the 24 putative transmembrane regions were highly conserved, including three of the four S4 (voltage sensor) transmembrane regions. The 3`-untranslated region comprises 1,618 nucleotides with a conventional polyadenylation signal (AAUAAA) at nucleotide 7524. The four missing putative transmembrane regions (designated IS1 to IS4 in the DHPR) correspond to the most NH(2)-terminal regions, including the first S4 region (Fig. 2C).


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 alpha(1) subunit (including the first four transmembrane segments) was also expressed in MELC, a mouse cardiac DHPR alpha(1) 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 times 10^6 recombinants. Moreover, Northern hybridizations (Fig. 3A), RNase protection (Fig. 3B) and PCR demonstrated that the 5` sequence from the mouse cardiac DHPR alpha(1) 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 alpha(1) subunit mRNAs and those from MELC corresponds to the portion of the 5` sequence of the cardiac DHPR alpha(1) 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 alpha(1) 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 alpha(1) 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 alpha(1) subunit mRNA was not expressed in MELC, even at low amounts. Moreover, although the cardiac DHPR alpha(1) 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 alpha(1) 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 alpha(1) 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).

Structure of the Calcium Channel Gene in MELC

To further characterize the structure of the 5`-flanking region of the MELC-CC gene, the genomic clone MELC13 (15 kb) was analyzed and restriction fragments selected by Southern hybridization using two synthetic oligonucleotides 5MASA and 1440AS located near the 5` end of the MELC-CC cDNA as probes. A genomic fragment, pP2, extended 3.6 kb upstream from the 5` cap site of the MELC-CC cDNA sequence. Two types of transcription factor recognition elements were identified in this region: 1) three GATA boxes with the consensus sequence (A/T)GATA(G/A) at -81, -350, and -434 bp upstream from the putative cap site of MELC-CC and 2) two CACCC boxes at -58 and -127 bp (Fig. 2A). No TATA or CAAT motifs were present. Moreover, there was a stretch of 15 GATA repeats located 2.8 kb upstream of the putative transcription start site.

An 89-bp insertion near the start site of transcription in one allele of the murine cardiac alpha(1) 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 alpha(1) 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 DeltaX 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.

Regulation of MELC-CC Alternative Splicing during HMBA-induced Differentiation

A 25-amino acid insertion, located intracellularly between motif I and II in MELC-CC at amino acid 200, was identified in two overlapping cDNA clones, 2MEL and 5MEL, isolated from a cDNA library made from undifferentiated MELC (Fig. 1). This 25-amino acid insertion was not present in three cDNA clones isolated from a library made from MELC cultured in the presence of 5 mM HMBA for 48 h (to induce differentiation). This insertion displayed no homology to any protein sequence in GenBank and was hydrophilic, containing four positive charges (1 arginine and 3 lysines). There is a potential phosphorylation site (STE) in the insertion and no glycosylation sites. No conventional splicing signal (29, 30) is present at either end of this 75-bp insertion. A 20-amino acid deletion from amino acid 692 to 711 in the MELC-CC corresponding to most of the IIIS2 transmembrane region was also only detected in cDNA clones isolated from undifferentiated MELC libraries.

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 alpha(1) 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 alpha(1) subunits with that of the rat cardiac alpha(1) 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 alpha(1) 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 (VSMalpha(1)). 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 VSMalpha(1)(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.

Tissue Distribution and Regulation of the MELC Calcium Channel mRNA Expression

To determine the tissue distribution of the MELC-CC mRNA RNase protection was performed using probes overlapping the 5` region of the MELC-CC cDNA corresponding to the junction between cardiac alpha(1) subunit homologous and non-homologous sequences. MELC-CC specific mRNA was detected in MELC mouse lung, heart, and 3T3 cells (Fig. 7A). Cardiac alpha(1) subunit specific mRNA was detected in heart and 3T3 cells but not in MELC (Fig. 7A). The possibility that the MELC-CC is a channel common to non-excitable cells was tested by investigating the expression of its message in a variety of tissues and cells. Northern analysis was performed on total RNA from T lymphocytes, liver, kidney, and NIH3T3 fibroblasts probed with 2MEL cDNA. No signals were detected (data not shown). When the same RNA samples were analyzed using RNase protection with probes corresponding to nucleotide 1-550 of the MELC-CC cDNA, two protected bands of 550 and 178 bp each were seen in mouse cardiac muscle and in NIH3T3, whereas only a fully protected fragment at 550 bp was seen in MELC. The 178-bp fragment is specific for the intact cardiac DHPR alpha(1) subunit mRNA and the 550 bp represents the MELC-CC specific mRNA. The low levels of MELC-CC mRNA in NIH3T3 cells explains the lack of signal in Northern hybridizations. Interestingly, the novel MELC-CC 5` sequence was detected using RNase protection in murine cardiac muscle where it accounted for 1% of the total DHPR message (Fig. 7A).


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 alpha1 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).

Dihydropyridine Binding in MELC Membranes and in Xenopus Oocytes Expressing the MELC-CC and Immunoblot Analyses of Expressed MELC-CC Protein

MELC membrane preparations were used to determine [^3H](+)-PN200-110 binding (DuPont NEN, 87.0 Ci/mmol]. PN200-110 is high affinity ligand for the L-type Ca channel. The MELC-CC structure possesses all the domains necessary for DHP binding (S6 helices of the third and fourth repeating domains as well as the loop linking S5 and S6 of the third domain(34, 35) . In addition, physiological data indicated that a DHP-sensitive molecule resided on the MELC plasma membrane(17) . MELC membranes (50 µg) showed only very weak binding activity compared with mouse heart membrane (30 µg) (data not shown). This lack of DHP binding is not surprising in light of a recent report that DHP binds only when the alpha(1) is expressed with other subunits (including alpha(2) and beta)(36) .

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 alpha(1) 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 alpha(1) subunit of the cardiac DHPR. Both identified an 200-kDa protein on immunoblots containing alpha(1) subunit of the cardiac DHPR overexpressed in insect cells (provided by Dr. M. Hosey). This result indicated that both antibodies were specific for the alpha(1) subunit of the cardiac DHPR. However, neither antibody identified a protein in any of the other preparations, including murine cardiac membranes, oocytes expressing the alpha(1) 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 alpha(1) subunit plus the entire MELC-CC was expressed using a plasmid created by ligating the 5` end of the murine cardiac DHPR alpha(1) 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 alpha(1) subunit protein. This result also demonstrates the requirement for the first four transmembrane segments of the cardiac DHPR alpha(1) 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 alpha(1) subunit (A) and chimeric alpha(1) (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 alpha(2) and beta 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).




DISCUSSION

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 alpha(1) 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 alpha(1) 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](i) 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 alpha(1) 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 alpha(1) 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 alpha(1) 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.


FOOTNOTES

*
This work was supported by National Institutes of Health Grant RO1-NS29814 and by a grant-in-aid from the American Heart Association (to A. R. M.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Bristol-Meyers/Squibb Established Investigator of the American Heart Association. To whom correspondence should be addressed: Molecular Medicine Program, Brookdale Center for Molecular Biology, Box 1269, Mount Sinai School of Medicine, One Gustave L. Levy Place, New York, NY 10029. Tel.: 212-241-0309; Fax: 212-996-4498.

(^1)
The abbreviations used are: VDCC, voltage-dependent calcium channel; MELC, murine erythroleukemia cells; HMBA, hexamethylene bisacetamide; DHP(R), dihydropyridine (receptor); MELC-CC, murine erythroleukemia cell calcium channel; kb, kilobase pair(s); bp, base pair(s).


ACKNOWLEDGEMENTS

We thank Dr. Richard Axel for helpful discussions and Dr. Steven Siegelbaum for helpful discussions and for reviewing parts of this manuscript. We thank Dr. Nathan Dascal for providing cardiac DHPR alpha(1) subunit RNA for oocyte injection as a positive control, Dr. Marlene Hosey for providing immunoblots containing membranes from insect cells expressing the cardiac DHPR alpha(1) subunit, and Dr. Victoria Richon for providing MELC. We are most grateful to Dane Worley for excellent technical assistance in earlier phases of this project.


REFERENCES

  1. Meldolesi, J., and Pozzan, T. (1987) Exp. Cell Res. 171, 271-283 [Medline] [Order article via Infotrieve]
  2. Penner, R., and Neher, E. (1988) J. Exp. Biol. 139, 329-345 [Abstract]
  3. Hallam, T. J., and Rink, T. J. (1989) Trends Pharmacol. Sci. 10, 8-10 [Medline] [Order article via Infotrieve]
  4. Hockberger, P. E., and Swandulla, D. (1987) Cell. Mol. Neurobiol. 7, 229-236 [Medline] [Order article via Infotrieve]
  5. Benham, C. D., and Tsien, R. W. (1987) Nature 328, 275-278 [CrossRef][Medline] [Order article via Infotrieve]
  6. Maruyama, Y., and Petersen, O. H. (1982) Nature 300, 61-63 [Medline] [Order article via Infotrieve]
  7. Mason, W. T., and Waring, D. W. (1986) Neuroendocrinology 43, 205-219 [Medline] [Order article via Infotrieve]
  8. Rink, T. J. (1988) Nature 334, 649-650 [Medline] [Order article via Infotrieve]
  9. Zschauer, A., van Breeman, C., Buhler, F. R., and Nelson, M. T. (1988) Nature 334, 703-705 [CrossRef][Medline] [Order article via Infotrieve]
  10. Merrit, J. E., and Rink, T. J. (1987) J. Biol. Chem. 262, 4958-4960 [Abstract/Free Full Text]
  11. Jacob, R. (1990) Biochim. Biophys. Acta 1052, 427-438 [Medline] [Order article via Infotrieve]
  12. Pecht, I., Corcia, A., Liuzzi, M. P. T., Alcover, A., and Reinherz, E. L. (1987) EMBO J. 6, 1935-1939 [Abstract]
  13. Friend, C. (1977) Harvey Lect. 253-281
  14. Reuben, R., Wife, R., Breslow, R., Rifkind, R. A., and Marks, P. A. (1976) Proc. Natl. Acad. Sci. U. S. A. 73, 862-866 [Abstract]
  15. Bridges, K., Levenson, R., Housman, D., and Cantley, L. (1981) J. Cell Biol. 90, 542-544 [Abstract]
  16. Hensold, J. O., Dubyak, G., and Housman, D. E. (1991) Blood 77, 1362-1370 [Abstract]
  17. Gillo, B., Ma, Y. S., and Marks, A. R. (1993) Blood 81, 783-792 [Abstract]
  18. Mori, Y., Friedrich, T., Kim, M.-S., Mikami, A., Nakai, J., Ruth, P., Bosse, E., Hofmann, F., Flockerzi, V., Furuichi, T., Mikoshiba, K., Imoto, K., Tanabe, T., and Numa, S. (1991) Nature 350, 398-402 [CrossRef][Medline] [Order article via Infotrieve]
  19. Mikami, A., Imoto, K., Tanabe, T., Niidome, T., Mori, Y., Takeshima, H., Narumiya, S., and Numa, S. (1989) Nature 340, 230-233 [CrossRef][Medline] [Order article via Infotrieve]
  20. Koch, W. J., Ellinor, P. T., and Schwartz, A. (1990) J. Biol. Chem. 265, 17786-17791 [Abstract/Free Full Text]
  21. Tanabe, T., Takeshima, H., Mikami, A., Flockerzi, V., Takahashi, H., Kangawa, K., Kojima, M., Matsuo, H., Hirose, T., and Numa, S. (1987) Nature 328, 313-328 [CrossRef][Medline] [Order article via Infotrieve]
  22. Heinemann, S., Terlau, H., Stuhmer, W., Imoto, K., and Numa, S. (1992) Nature 356, 441-443 [CrossRef][Medline] [Order article via Infotrieve]
  23. Malouf, N. N., McMahan, D. K., Hainsworth, C. N., and Kay, B. K. (1992) Neuron 8, 899-906 [Medline] [Order article via Infotrieve]
  24. Melloni, E., Pontremoli, S., Damiani, G., Viotti, P., Weich, N., Rifkind, R. A., and Marks, P. A. (1988) Proc. Natl. Acad. Sci. U. S. A. 85, 3835-3839 [Abstract]
  25. Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual , Cold Spring Harbor Laboratory, Cold Spring Harbor, NY
  26. Maniatis, T., Fritsch, E. F., and Sambrook, J. (1982) Molecular Cloning: A Laboratory Manual , Cold Spring Harbor Laboratory, Cold Spring Harbor, NY
  27. Marks, A. R., Tempst, P., Chadwick, C. C., Riviere, L., Fleischer, S., and Nadal-Ginard, B. (1990) J. Biol. Chem. 265, 20719-20722 [Abstract/Free Full Text]
  28. Harlow, E., and Lane, D. (1988) Antibodies, A Laboratory Manual , Cold Spring Harbor Laboratory, Cold Spring Harbor, NY
  29. Sharp, P. (1985) Cell 42, 397-400 [Medline] [Order article via Infotrieve]
  30. Reed, R., and Maniatis, T. (1986) Cell 46, 681-690 [Medline] [Order article via Infotrieve]
  31. Frohman, M. A., Dush, M. K., and Martin, G. R. (1988) Proc. Natl. Acad. Sci. U. S. A. 85, 8998-9002 [Abstract]
  32. Peters, G. (1990) Cell Growth & Differ. 1, 503-510
  33. Diebold, R., Koch, W., Ellinor, P., Wang, J., Muthuchamy, M., Wieczorek, D., and Schwartz, A. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 1497-1501 [Abstract]
  34. Nakayama, H., Taki, M., Striessnig, J., Glossman, H., Catterall, W., and Kanaoka, Y. (1991) Proc. Natl. Acad. Sci. U. S. A. 88, 9203-9207 [Abstract]
  35. Striessnig, J., Glossman, H., and Catterall, W. A. (1990) Proc. Natl. Acad. Sci. U. S. A. 87, 9108-9112 [Abstract]
  36. Puri, T. S., Zhao, X. L., Ladner, M. B., and Hosey, M. M. (1994) Biophys. J. 66, A319
  37. Kubo, Y., Baldwin, T., Jan, Y., and Jan, L. (1993) Nature 362, 127-133 [CrossRef][Medline] [Order article via Infotrieve]
  38. Hille, B. (1992) Ionic Channels of Excitable Membranes , Sinauer Associates Inc., Sunderland, MA
  39. Orkin, S. (1990) Cell 63, 665-672 [Medline] [Order article via Infotrieve]
  40. Miller, I. J., and Bieker, J. J. (1993) Mol. Cell. Biol. 13,, 2776-2786 [Abstract]
  41. Zhang, J., Randall, A., Ellinor, P., Horne, W., Sather, W., Tanabe, T., Schwarz, T., and Tsien, R. (1993) Neuropharmacology 32, 1075-1088 [CrossRef][Medline] [Order article via Infotrieve]

©1995 by The American Society for Biochemistry and Molecular Biology, Inc.