©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
An Alternative Splicing Site Modifies the Carboxyl-terminal Trans-membrane Domains of the Na/Ca Exchanger (*)

(Received for publication, December 15, 1994)

Nadia Gabellini (1)(§) Tomoko Iwata (2) Ernesto Carafoli (1) (2)

From the  (1)Department of Biological Chemistry, University of Padova, 35121 Padova, Italy and the (2)Laboratory for Biochemistry, Swiss Federal Institute of Technology (ETH), 8028 Zürich, Switzerland

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The 6-kilobase (kb) cDNA of pTB11 clone and its 5` fragment of 3.7 kb encoding the canine heart Na/Ca exchanger (Nicoll, D. A., Longoni, S., and Philipson, K. D.(1990) Science 250, 562-565) were transiently expressed in 293 cells to investigate the role of the 3`-``untranslated'' region. Both fragments yielded high levels of expressed protein that were well incorporated in the membranes. Cells expressing the 6-kb cDNA produced rearranged transcripts of smaller than expected size. A 120-kDa polypeptide was produced in cells expressing the modified exchanger, and Ca uptake was higher in this type of transfected cells. A constant stretch of nucleotides located at the 3` end of the 6 kb cDNA was found to be connected, by alternative RNA splicing, to four different upstream sequence positions. The deduced hydrophobic sequence of the spliced-in exon could replace the IX or the XI trans-membrane domain of the exchanger protein in two spliced isoforms. The new exon sequence was not completely included in the pTB11 insert, i.e. these two products were artificially truncated. The RNA processing of these two alternative 5`-splicing sites also occurred in tissues, as shown by RNase protection analysis. In a third type of isoform the splicing took place downstream of the originally proposed stop codon, whereas in a fourth type a stop codon was introduced after the V hydrophobic segment in the large intracellular loop.


INTRODUCTION

The Na/Ca exchanger (NCE) (^1)is an electrogenic transporter that couples the translocation of Na and Ca in opposite directions across the plasma membrane (see Reeves(1992)). Partially purified preparations of the heart sarcolemmal exchanger usually show a major component of about 120 kDa and two other species of 160 and 70 kDa, respectively (Philipson et al., 1988; Nicoll and Philipson, 1991). The cDNA sequence of the cloned NCE encoded a protein of 970 amino acids with a molecular mass of 108 kDa. The coding sequence was located in the 5`-proximal region (about 3 kb) of the 6-kb cDNA insert carried in clone pTB11. The full-length NCE transcript size was estimated at 7 kb (Nicoll et al., 1990). The deduced protein apparently corresponds to the 120-kDa species, the difference in size being attributed to glycosylation at Asn-9 (Hryshko et al., 1993). The deduced primary structure of the 108-kDa polypeptide indicates a cleavable NH(2)-terminal signal sequence and 11 trans-membrane domains (TM), interrupted by a large hydrophilic loop between TM segment V and VI. Cloning of the cardiac NCE cDNA from several mammalian species has revealed a high degree of sequence conservation (Nicoll et al., 1990; Aceto et al., 1992; Low et al., 1993; Kofuji et al., 1992; Komuro et al., 1992; Lee et al., 1994). However, tissue-specific isoforms with identical TM domains but with shortened hydrophilic segments are produced by alternative splicing of a single gene product (Furman et al. 1993; Kofuji et al. 1993, 1994). Recently a second NCE gene specifically expressed in brain was also identified (Li et al., 1994).

The exchange activity of the cardiac NCE expressed in oocytes by injection of the 2.9-kb cRNA coding sequence was found to be similar to that of heart sarcolemma, whereas an active NCE was not produced by injection of the 6-kb cRNA (Matsuoka et al., 1993), lacking the poly(A) tail (Huarte et al., 1992). The exchanger cDNA has been expressed in several heterologous systems, i.e. in the baculovirus Sf9 cell system, in COS-7 and 293 cells, and stably transformed CHO cells by transfection with plasmid DNA, and in vaccinia virus-infected HeLa cells (Li et al., 1992; Aceto et al., 1992; Kofuji et al., 1992; Pijuan et al., 1993; Low et al., 1993). In all these studies a shortened version of the 6-kb cDNA, i.e. a fragment of 3-3.7 kb, that included the proposed coding sequence was used. The large portion of cDNA downstream of the putative stop codon was omitted in the construction of all expression vectors. As a result, the function of this region, traditionally considered to be untranslated, has remained unknown. The study of its function has been the aim of the present contribution: specifically, its role in the expression of the NCE cDNA was explored. The approach used has been the transient transfection of 293 cells with plasmids carrying different lengths of the canine cardiac NCE cDNA, the analysis of the expression products, and the determination of NCE activity in the transfected cells. The results have led to the conclusion that the 6-kb cDNA is an incomplete copy of an immature transcript that could produce at least four differently spliced transcripts when expressed in 293 cells.


EXPERIMENTAL PROCEDURES

Materials

The expression vector pcDNAI and the TA-PCR vector were purchased from Invitrogen, San Diego, CA; the cell culture medium was purchased from Life Technologies, Inc., Eggenstein, Germany; fetal calf serum was from PAA, Linz, Austria; Hybond nylon membranes, [alpha-P]dCTP, [alpha-P]CTP, [-P]ATP, and Ca were from Amersham International, Little Chalfont, United Kingdom; filtration membranes were from Millipore, Bedford, MA; the random prime labeling kit, SP6/T7 transcription kit, and anti-rabbit IgG were from Boehringer, Mannheim, Germany; nitrocellulose membranes and electrophoresis reagents were from Bio-Rad; restriction enzymes, reverse transcriptase, exonuclease III, and the femtomole DNA sequencing system were from Promega, Madison, WI; and the ribonuclease protection assay kit was from Ambion, Austin, TX.

Construction of NCE Expression Vectors

The 3.7-kb region at the 5` end of the canine NCE cDNA was isolated by restriction with HindIII-XbaI from plasmid pTB11 (Nicoll et al., 1990) and inserted in the expression vector pcDNAI. The 2.3-kb fragment isolated from pTB11 by XbaI restriction was subsequently inserted in this construct. The expression vector carrying the whole 6-kb insert of clone pTB11 with the 2.3-kb XbaI fragment in the correct orientation was selected by restriction mapping with HindIII. Deletions in the polylinker region at the 3` end of the 6-kb insert have been performed by exonuclease III treatment from the unique BamHI site, and selected clones were analyzed by DNA sequencing.

Cell Culture and Transfection

Human embryonic kidney cells (293, ATCC, CRL 1573) were cultured in minimal essential medium supplemented with 10% fetal bovine serum. The cells were plated 24 h before transfection on plastic dishes (100 mm diameter) at a density of 2.5 times 10^6. Cell transfection was performed essentially as described (Chen and Okayama, 1987), using 15 µg of plasmid DNA/dish co-precipitated with Ca phosphate in 10 ml of growth medium and 15 h of incubation at 3% CO(2). Under these conditions 30-40% of cultured 293 cells became transiently transfected (Gabellini et al., 1993). After removal of the precipitate, the cells were cultured for additional 24-48 h to allow full expression of the plasmid DNA. The transient expression of heterologous polypeptides was maximal and constant during this period.

Northern Blotting

Total RNA was extracted from 293 cells with guanidinium isothiocyanate (Chirgwin et al., 1979) 30 h after transfection. Glyoxylated RNAs were separated on 1% agarose gels and blotted on nylon membranes. The probes were labeled with [alpha-P]dCTP by random priming.

Ribonuclease Protection

A 734-bp DNA fragment was amplified by PCR with oligonucleotides from base 2134 and the 3` sequence of pTB11 clone from the reverse-transcribed RNA of 293 cells expressing the 6-kb cDNA. This fragment was inserted in the TA-PCR vector, analyzed by DNA sequencing, cleaved with XbaI, and used as template for the SP6 RNA polymerase. The full-length antisense RNA fragment was gel-purified and hybridized with the RNA of interest at 42 °C for 18 h. The [alpha-P]CTP-labeled probe was used at 1.5 times 10^5 cpm/assay. RNase A + T(1) treatment was then performed at 37 °C for 30 min using 1:50, 1:100, and 1:200 dilutions. The protected RNA fragments were ethanolprecipitated and separated by electrophoresis on 5% acrylamide, 8 M urea gels.

Total Membrane Preparation

The transfected 293 cells were collected by scraping in 40 mM Tris-Cl, pH 7.4, 10 mM EDTA, 150 mM NaCl, and mechanically broken by 10 passages through a glass homogenizer in 10 mM Tris-Cl, pH 7.4, 5 mM EDTA, and 1 mM phenylmethylsulfonyl fluoride. Unbroken cells and organelles were discarded by a low speed centrifugation (1000 times g) for 10 min at 4 °C. The supernatant was centrifuged (250,000 times g) for 30 min at 4 °C. Cultured cells from three pooled dishes yielded about 100 µg of protein (Bradford, 1976) in the membrane fraction. Sarcolemmal membranes (microsomal fraction) were obtained from an explanted human heart that could not be used for transplantation, by ultracentrifugation of the post-mitochondrial supernatant. The same human tissue was also used for RNA extraction.

Western Blotting

Protein samples were separated on 15% acrylamide-SDS gels and electroblotted. The transferred proteins were transiently stained in 0.2% Ponceau red and 3% trichloroacetic acid to control the uniformity of gel loading. The immunoreaction was performed with a polyclonal antibody Ab P 8-10 (Philipson et al., 1988) used at 1:500 dilution. Immunostaining was obtained by a second reaction with an anti-rabbit IgG conjugated with alkaline phosphatase (1:2000).

Ca Uptake in Intact Cells

Pools of two dishes were used to determine the time course for each transfection experiment. Na loading of the cells was performed by incubation in 1 ml of: 160 mM NaCl, 20 mM Hepes, pH 7.4, 1 mM ouabain, 2 mM MgCl(2), and 25 µM nystatin, for 10 min at 37 °C on dishes. Cells were detached from the plastic surface by gentle pipetting and collected in 160 mM NaCl, 20 mM Hepes, pH 7.4, and 1 mM ouabain. For Ca uptake the cells were diluted 20-50-fold in uptake medium containing either NaCl or KCl (160 mM) and 20 mM Hepes, pH 7.4, 1 mM ouabain, 1 µM valinomycin, 25 µM Ca, 3 µCi/ml Ca in a stirred cuvette at 37 °C. The uptake reaction was started by the addition of cells; aliquots were taken at selected time points, placed on a Millipore membrane with 45-µm pore diameter, vacuum-filtered, washed immediately three times with 1 ml of ice-cold 160 mM KCl, 20 mM Hepes, pH 7.4, 1 mM EGTA, and then counted.

Reverse-transcribed PCR and DNA Sequencing

Reverse transcription of total RNA (4 µg) was performed by 1-h incubation at 42 °C using oligo(dT) (1 µg) and 16 units of avian myeloblastosis virus reverse transcriptase, in a 50 µl volume. The reaction was stopped by 10-min incubation at 70 °C, and cDNA samples were stored at -20 °C. PCR amplification of the cDNAs was performed using 10 pmol of primers, 58 °C annealing temperature, and 30 cycles of amplification by Taq polymerase (1.2 units). The amplified fragments of interest were directly ligated in the TA-PCR vector, and DNA sequencing of selected clones was determined using end-labeled [-P]ATP primers and cycle sequencing with Taq polymerase.


RESULTS

Analysis of NCE mRNA in Transfected Cells

Two expression vectors carrying different lengths of the canine NCE sequence were constructed. Cultured 293 cells were transfected under conditions that allow maximal DNA expression with either the construct comprising the total 6-kb insert of clone pTB11 (Nicoll et al., 1990) or its 3.7-kb HindIII-XbaI fragment (Fig. 1A).


Figure 1: Organization of the NCE expression vectors and of the amplified DNA fragments. Two constructs of pcDNAI including the whole 6-kb insert of clone pTB11 (Nicoll et al., 1990) or the 3.7-kb HindIII-XbaI fragment of canine NCE cDNA are shown in A, with the EcoRI, HindIII, and XbaI restriction sites as reference. The position of the eukaryotic cytomegalovirus promoter at the 5` end of the NCE coding sequence is indicated. The bacterial SP6 promoter at the opposite site of the polylinker is shown by a triangle, followed by the pcDNAI splice and poly(A) signal sequences. In B the position of the primers used for reverse-transcribed PCR amplification is indicated by arrows. The SP6 primer at the 3` end of cDNA is indicated by a triangle. In the amplified fragments of 1.3 kb, 1.1 kb, 280 bp, and 260 bp the dashed lines indicate the excised regions. Consensus sequences in the intron/exons junctions are indicated in lowercase and uppercase letters, respectively. The boxes represent the determined DNA sequence.



Northern blot analysis of total RNA extracted from the transfected cells showed that the 3.7-kb fragment yielded strong hybridization signals when used as a probe in both types of transfected cells (Fig. 2A, lanes 2 and 3). The hybridizing mRNA of cells expressing the 3.7-kb cDNA had the expected approximate size of 4 kb, when also considering the flanking sequence of pcDNAI included in the 3` end of the transcripts (Fig. 2A, lane 2). By contrast, the mRNA of cells transfected with the 6-kb cDNA failed to show a prominent band in the 6-kb region, but showed instead a smaller mRNA species (4 kb, lane 3). The similar intensity of the hybridization signals suggests that transfection with the two NCE constructs produced similar mRNA levels. This suggestion was supported by the control hybridization with beta-actin shown in lanes 1-3 of Fig. 3. No hybridizing band was observed with the RNA of 293 cells transfected with the vector pcDNAI (lane 1), confirming that natural NCE expression in this cell line is below detection level.


Figure 2: Northern blotting of NCE transcripts. Total RNA extracted from 293 cells transiently transfected with pcDNAI as control (lanes 1) and with the same vector either, including the 5` region of 3.7 kb (lanes 2) or the total 6 kb of the NCE cDNA (lanes 3), was loaded in duplicate (10 µg/lane) and analyzed by Northern blotting. The blots were hybridized separately with the following probes: the 3.7 HindIII-XbaI fragment (A), the 2.3-kb XbaI fragment (B) of the NCE cDNA, and subsequently with beta-actin (A and B) as control. The positions of the hybridizing bands of the expected 6-kb transcript and of the 28 and 18 S ribosomal RNA are indicated.




Figure 3: Immunostaining of NCE polypeptides. Total membranes purified from 293 cells expressing 3.7 (A) or 6 kb (B) of the NCE cDNA and membranes from cells transfected with pcDNAI (C) were analyzed by Western blotting with a specific anti-NCE antibody in parallel with human heart sarcolemmal membranes (D), using 50 µg of protein/lane. The position of the standard molecular mass markers is indicated in kilodaltons.



The RNA samples were also probed separately with the 2.3-kb XbaI fragment to clarify the origin of the 4-kb transcript produced by the transfection with the 6-kb cDNA, (Fig. 2B). This probe produced a clear signal in the region of 4 kb in cells transfected with the 6-kb cDNA template (lane 3). This mRNA species had the size of that revealed by the 3.7-kb probe, although its signal was less intense (Fig. 2A, lane 3). By contrast in cells transfected with the 3.7-kb cDNA, the probe produced a barely detectable signal (Fig. 2B, lane 2) that could have been due to a minimal cross-contamination of the probes. Only traces of the 6-kb NCE mRNA were detected under these conditions, supporting the suggestion that this transcript was unstable in 293 cells (Fig. 2B, lane 3). It could be mentioned at this point that a prominent mRNA species of about 7 kb was previously detected in heart, brain, kidney, and other tissues, although smaller hybridizing species were also revealed by NCE probes (Kofuji et al., 1992; Lee et al., 1994). The Northern blot analysis thus suggested that the 4-kb mRNA produced by transfection with the 6-kb cDNA included portions of the 2.3-kb XbaI fragment. This species therefore appeared to derive from the rearrangement of the 6-kb transcript.

Characterization of the Overexpressed NCE Protein

The NCE species produced by the expression of the NCE cDNA were analyzed by Western blotting of total membranes isolated from transfected 293 cells, using an antibody raised against the single components of a partially purified NCE preparation (Philipson et al., 1988). Strong immunoreactivity was detected in cells transfected with either the construct carrying the 3.7 kb or with that carrying the 6-kb cDNA (Fig. 3, lanes A and B). The staining intensity was very similar in the two cases and much stronger than in membranes of 293 cells transfected with pcDNAI as control (lane C). The amount of NCE protein in this cell line was evidently greater than in human cardiac membranes (lane D), especially when considering that only 30-40% of the cells were transfected under these conditions. A comparison of the antibody staining of equivalent aliquots of whole cells (Fig. 4B) and of their membrane fractions (Fig. 3), provided an approximate estimate of the proportion of the expressed NCE protein which had became incorporated in the membranes. The low speed centrifugation fraction, containing various organelles and unbroken cells, represented 25-30% of the total proteins, whereas the total membrane fraction contained 8-10% of them. The intensity of immunoreactivity became similar in the membrane fraction and in whole cells when a 3-fold excess of the latter was used ( Fig. 3and Fig. 4B). The largest portion of the overexpressed NCE protein had thus become inserted in the membranes.


Figure 4: Sodium-dependent Ca uptake in NCE-transfected 293 cells. The time course of Ca uptake (A) was measured in whole cells 24-48 h after transfection with the 3.7-kb HindIII-XbaI fragment (A, panel a), with the 6-kb NCE cDNA (A, panel b), and with the expression vector pcDNAI (A, panel c). Aliquots containing 100-200 µg of cell protein were taken for each time point. A typical immunostaining of corresponding samples of whole cells (150 µg of protein/lane) is also shown (B, lanes a-c). The values of Ca uptake are the average ± S.D. of three to four independent sets of transfection experiments. Standard molecular mass markers are indicated in kilodaltons.



The polypeptide pattern revealed by the antibody in transfected cells was similar to that of human heart sarcolemmal membranes (Fig. 3) and consistent with that reported in other expression systems. At least three species were resolved by SDS-polyacrylamide gel electrophoresis in the 120-kDa region (Fig. 3, lanes A, B, and D). The slowest migrating band could correspond to the most prominent species in human heart (lane D) the latter being only slightly larger. This band was very evident in 293 cells expressing the 6-kb cDNA (lane B), but was absent in cells expressing the 3.7-kb cDNA, the faint immunoreactivity sometimes observed being due to a small cross-contamination of the samples (lane A). The two other components of the 120-kDa triplet had approximately the same intensity in the two types of transfected cells (Fig. 3, lanes A and B) and were much more intense than the heaviest component of the triplet. In heart (lane D) the slowest moving component of the triplet was by contrast the most evident. It could thus be that the slowest migrating polypeptide of the triplet was a product of the 6-kb NCE cDNA. Differences in the mobility of the expressed 120-kDa species had been previously observed in COS and CHO cells and tentatively attributed to cell-specific post-transcriptional modification of the NCE proteins (Pijuan et al., 1993).

A second group of polypeptides stained by the antibody in both transfected cells and in heart membranes had lower molecular mass (70 kDa; Fig. 3, lanes A, B, and D). These products are generally assumed to be proteolytic products of the larger bands (Nicoll and Philipson, 1991). A difference with heart membranes was the presence in 293 cells and in other NCE-expressing cell lines of a 200-kDa band (Fig. 3, lanes A and B; Fig. 4B, lanes a and b) which could reflect the dimerization of misfolded overexpressed NCE proteins. Since the samples were treated with a reducing agent, it appears likely that the dimerization was due to hydrophobic interactions. At variance with these cells, heart membranes contained instead a 160-kDa species also assumed to be a native NCE (Nicoll and Philipson, 1991). The transfected cells showed additional polypeptides in the 80-kDa region (Fig. 4B, lanes a and b) which had not been seen in purified membranes (Fig. 3, lanes A and B). They could possibly arise from the degradation of misinserted overexpressed polypeptides.

Ca Uptake in NCE-expressing 293 Cells

Ca uptake was measured in whole 293 cells transfected in parallel with the two expression vectors (Fig. 4A). In the absence of Na, the Ca uptake of cells transfected with the vector carrying the 3.7-kb HindIII-XbaI fragment was in the range of 6 nmol/min/mg of protein (Fig. 4A, lane a), whereas the activity of cells transfected with the vector carrying the 6-kb cDNA was in the range of 18 nmol/mg/min (Fig. 4A, lane b). Maximal uptake was reached in both cases after 2 min of incubation, but was 2.5-fold higher in 293 cells expressing the 6-kb cDNA. When considering that only a portion of transfected cells produced NCE protein, these values were comparable to those in CHO cells stably expressing the NCE protein (Pijuan et al., 1993) and in line with the reported NCE activity of isolated heart sarcolemmal membranes (e.g. Reeves et al.(1976)). The presence of valinomycin stimulated the uptake activity of NCE-expressing 293 cells 2-3-fold, as previously reported (Caroni et al., 1980). The time course of the uptake in control cells transfected with the vector pcDNAI (Fig. 4A, lane c) was practically identical in the presence of Na or K, showing that, in agreement with previous observations (Kofuji et al., 1992), no NCE activity was detectable in untransfected 293 cells; the faint immunoreactivity observed in 293 membranes was probably due to cross-reactivity with other transporters. Samples of whole cells used for the measurements of Ca uptake were analyzed by Western blotting. The two types of transfected cells showed an equally intense immunoreaction except for the above mentioned difference in the slowest migrating component of the 120-kDa triplet (Fig. 4B, lanes a and b). It seems unlikely that this product, which represents only a minor part of the total immunoreactivity, would be responsible for the enhancement. However, the higher specific activity of NCE in 293 cells expressing the 6-kb cDNA suggested that the expression of this construct had produced a partial modification of the NCE coding sequence.

Alternative mRNA Splicing in the COOH-terminal Coding Sequence

The variability in the transcript length revealed by the Northern blotting analysis of 293 cells expressing the 6-kb cDNA was further investigated by PCR amplification of reverse-transcribed mRNA from transfected cells. When using primers complementary to the canine/human NCE coding sequence upstream of the XbaI site, at base 3700 (Fig. 1A), the size of the amplified DNA was as predicted from the nucleotide sequence of the NCE cDNA (Nicoll et al., 1990), irrespective of the cDNA length used for the transfection (Fig. 1B). Amplification of the NCE cDNA in the region encoding the proposed large intracellular loop also produced low amounts of irregularly small fragments with both types of cDNAs that were not further characterized. No specific amplification was obtained in pcDNAI-transfected 293 cells. Thus the composition of the five putative NH(2)-terminal trans-membrane segments and of the large intracellular loop were not substantially modified by the rearrangement of the 6-kb transcript.

When the amplification was performed with primers matching the sequence in the vicinity of the internal EcoRI sites, in combination with the SP6 oligonucleotide, adjacent to the 3` end of the NCE transcripts (Fig. 1A), an interesting discrepancy in the expected DNA length was detected. When using a primer from base 3043 in combination with SP6 (Fig. 1B), a 260-bp fragment was amplified only in cells expressing the 6-kb cDNA (Fig. 5A, lane 2). By contrast, this combination of primers amplified a DNA fragment of the expected size (580 bp) in 293 cells expressing the 3.7-kb cDNA (Fig. 5A, lane 1). Two DNA fragments of 1.3 and 1.1 kb were similarly amplified priming from base 1666 and SP6, whereas a third fragment of 280 bp was amplified only in low amount with this primer combination (Fig. 5B, lane 2). These products were much smaller than expected from the primer positions, located about 4 kb apart. However, in the system used the upper size limit of the amplification was 1.8 kb. No DNA fragments were amplified from the cDNA of 293 expressing the 3.7-kb cDNA with this primer combination (Fig. 5B, lane 1), the sequence being too large (2 kb).


Figure 5: Complementary DNA fragments at the 3` end of NCE transcript. Ethidium bromide staining of a 1.2% agarose gel showing the DNA fragments of the canine NCE sequence amplified from base 3043 with oligonucleotide 5`-CCTGCTGATGGAATCCAGCTTCA (A) and from base 1666 with oligonucleotide 5`-AGTGAGAGCATTGGCATCATGGAGG (B) in combination with primer SP6. The cDNAs used for the amplification were from cells expressing the 3.7-kb (lanes 1) or the 6-kb (lanes 2) NCE cDNAs. The size of standard DNA fragments (lanes 3) is indicated in base pairs.



The amplified cDNA fragments, including the complementary sequences at the 3` end of the shortened transcripts, were cloned in the TA-PCR vector and further characterized by DNA sequencing (Fig. 1B). The DNA fragments of smaller than expected size included at their 3` end a constant stretch of 111 nucleotides that was also found at the 3` end of the 6-kb pTB11 insert. The 5` end of this sequence contained 47 bp of the NCE cDNA that were connected with base 3198 (6 bp downstream of the determined canine exchanger sequence; Nicoll and Philipson, 1990) in the 260-bp fragment mentioned above (Fig. 1B). This sequence was fused with a portion of the bluescript polylinker of pTB11 (45 bp, EcoRI-XbaI) followed by 19 bp of the SP6 promoter of pcDNAI. The approximate size of this modified mRNA was 3.9 kb, including the flanking vector sequence. It is unlikely that the predicted amino acid sequence was modified by this rearrangement, since it occurred downstream of the stop codon after base 2937 of NCE. Thus, the expression of both NCE inserts produced the canonical 108-kDa protein (Fig. 6A). Alternatively, the short NCE sequence adjacent to the splicing site could link up with G-2821 in the 1.3-kb fragment, with G-2620 in the amplified fragment of 1.1 kb or with G-1844 in the 280-bp fragment (Fig. 1B). The predicted total size of these three spliced mRNAs is about 3.5, 3.2, and 2.5 kb. The consensus sequences for RNA splicing in the junctions of the alternatively spliced intron shown in Fig. 1B include the invariant dinucleotides GU at 5` splice and AG at the 3` splice (Treisman et al., 1983; Krainer et al., 1984). The deduced amino acid sequence of the adjoining NCE exon found in frame with the NCE sequence in two spliced isoforms (Fig. 6, B and C) contained a hydrophobic stretch of 16 residues with only one positively charged Lys. The sequence continued artificially with 16 residues of hydrophilic character, specified by the polylinker of the vector and terminated with a stop codon in the SP6 promoter (Fig. 6C). The hydrophobic sequence was connected to the fifth putative extracellular loop at Gly-931 (Fig. 6B). When taking into account Ile-930 and 1-2 residues of the flanking polylinker sequence, its length would thus be adequate to replace the XI TM segment of the NCE protein. The deduced protein sequence could conserve the previously proposed membrane topology (Nicoll et al., 1990) assuming that the artificial hydrophilic segment had an intracellular location (Fig. 6B). This spliced isoform had a molecular mass of 103 kDa, including the 32 residues of the NH(2)-terminal signal peptide. In a third type of spliced transcript, the hydrophobic stretch was linked up with the fourth extracellular loop at Ile-864 and similarly could replace the IX TM domain when beginning with Ser-863 (Fig. 6C). This third NCE protein would have a predicted molecular mass of 96 kDa. Thus the sequence at the 3` end of the NCE transcripts represents the start of an additional exon only partially included in clone pTB11.


Figure 6: Schematic representation of the products of alternative splicing at the 3` end of the coding sequence. The predicted topology of the four NCE polypeptides derived by alternative splicing of the 6-kb NCE RNA is shown. Trans-membrane segments are represented by filled bars. The position of the amino (N) and of the carboxyl terminus (C) is indicated. The segment of 47 bp at the 3` end of the canine NCE cDNA is indicated by a filled box. This is connected in four different positions of the NCE sequence: base 3198 downstream of the stop codon of the deduced sequence (Nicoll et al., 1990) in A, base 2820 in B, and base 2621 in C. The deduced amino acid sequence of the adjoining exon is shown in single letter code. Residues encoded by the NCE sequence are indicated in bold (C). The remaining part of the sequence, also indicated by a triangle, is encoded by the polylinker of pTB11 (EcoRI-XbaI) and the SP6 promoter of pcDNAI, where a stop codon in frame with NCE sequence is marked by an asterisk. In D the spliced exon is connected after G-1844 with an in-frame stop codon following a Leu.



A deletion mutant in which the COOH-terminal amino acids encoded by the vector (Fig. 6C), found in the two NCE isoforms described above, were completely eliminated was also expressed in 293 cells to investigate the influence of the artificial sequence on the Ca uptake activity. In this construct the NCE sequence continued with a different and shorter sequence encoded by the vector: PYSIGVT, preceding a stop codon. The replacement of the artificial COOH-terminal sequence did not modify the uptake activity of the expressed protein that was in the same range of that measured with the original construct shown in Fig. 4A, panel b: 18 nmol/mg/min. This indicated that the increased activity of the expression products was probably due to the modification of the NCE sequence by the alternative splicing.

An additional type of splicing producing a complete NCE isoform was identified in the DNA fragment of 280 bp (Fig. 1B). This was not abundant in the amplification products shown in Fig. 5B, lane 2, but its small size made it well represented after cloning the total products in the TA-PCR vector. The predicted NCE isoform contained 607 amino acids, including only five TM and terminating in the large intracellular loop. The deduced sequence of this rearranged transcript that was modified after Lys-606 showed a stop codon in frame with the NCE coding sequence following one additional amino acid (Leu) encoded by the spliced exon (Fig. 6D).

Occurrence of the Spliced Transcripts in Tissue

Samples of RNAs from tissues known to express the NCE were analyzed by ribonuclease protection to establish the presence of the spliced transcripts found in 293 cells. A PCR-amplified fragment cloned in the TA-PCR vector was used as template to generate a riboprobe. This included 724 bases of the canine NCE sequence and 131 bases of the flanking polylinker region (Sp6 promoter-XbaI). The NCE probe shown in the scheme of Fig. 7A could detect two of the spliced isoforms. It could be partially protected (486 bases) from RNase digestion when hybridizing with the mRNA modified after G-2620. The expected RNA fragment of 486 bases was detected in 293 cells expressing NCE (Fig. 7B, lane 5) that were used as a positive control. This fragment was clearly protected by human heart RNA (Fig. 7B, lanes 3 and 4) and became evident when the RNase concentration was increased (lane 4 versus 4a). Interestingly, the relative content of the mRNA spliced after base 2620 was higher in heart than in transfected 293 cells.


Figure 7: RNase protection. A, the full-length RNA probe (855 bases) consisted of 724 bases of NCE sequence and 113 bases of the flanking vector sequence. The NCE fragment of 724 bp was amplified by PCR with the oligonucleotides indicated by the arrows from base 2134 (5`-CTGGGAGAACACACCAAGCTGGAAG) and from base 5863 (5`-AGGAACAGCACAATTAGTGCACTTC) of the canine NCE cDNA. The NCE probe was complementary to the mRNA modified after base 2821, which could protect it completely. It included 687 bases of the alternatively spliced 5` exon, indicated by the shaded rectangles, that could be protected by the unspliced transcript and by that spliced at base 3198. The black box represents 37 bases of the 3` exon and the dashed line the excised intron (not on a linear scale). This probe could be partially protected by the NCE mRNA modified after base 2620 (486 bases). B, total RNA (10 µg) isolated from rat spleen (lanes 1 and 1a) rat brain (lane 2), normal human heart (lane 3), a hypertrophic human heart (lanes 4 and 4a), and 0.5 µg of total RNA of 293 cells expressing NCE (lane 5) were hybridized with the riboprobe. No RNase treatment was performed on rat spleen RNA (lane 1), whereas a RNase dilution 1:100 was used in lanes 2, 3, 4, and 5. A higher RNase dilution (1:200) was used in lane 4a. The protected RNA fragments of 724, 687, and 486 bases are indicated.



A fully protected NCE RNA hybrid of 724 bp was expected when the splicing occurred at base 2821. If this alternative 5`-splicing site would not be processed, a 37-bp shortened hybrid could be expected (Fig. 7A). The heart RNA samples examined (Fig. 7B, lanes 3, 4, and 4a) revealed two equally intense RNA fragments of the correct size (724 and 687 bases). These are most clearly seen in Fig. 7B, lane 4a, in which a lower RNase concentration and a shorter exposure time have been used to improve the resolution. Consistent with previous reports (Holtz, 1993), the human RNA samples from a hypertrophic heart (Fig. 7B, lanes 4 and 4a) showed higher NCE mRNA levels than normal heart tissue (lane 3). The proportion of the protected species was however similar in the two cases, the most abundant RNA fragments being the largest. Also the RNA of 293 cells expressing the 6-kb cDNA (lane 5) protected more efficiently the largest RNA fragments than that of 486 bases. However, these fragments were much more abundant in 293 cells expressing the NCE than in tissues, although much less RNA was used for hybridization in the former. In particular, the 687-base fragment could be the most prominent in 293 cells. This fragment was expected to be protected by the 6-kb mRNA and by the transcript spliced at base 3198. In agreement with the results of Northern blotting that indicated very high levels of a NCE transcripts at 4 kb, the protected 687-base fragment probably represented the mRNA that is spliced at base 3198, with a total size of about 3.9 kb.

The lower content of NCE mRNA in brain (Fig. 7B, lane 2) as compared with heart (lanes 3, 4, and 4a) and possibly also the expression of a second NCE gene in brain (Li et al., 1994) made it impossible to resolve the pattern of heaviest bands. However, traces of the protected species of 486 bases were also visible in brain (Fig. 7B, lane 2). No protection was obtained when rat spleen RNA was used as a negative control (lane 1a), whereas the full-length RNA probe (855 bases) was recovered after incubation with rat spleen RNA in the absence of RNase (lane 1). The results of RNase protection thus indicated that post-transcriptional modifications of the NCE mRNA involving regions coding for the COOH-terminal TM domains also occurred in heart and probably also in brain.


DISCUSSION

This study has shown that the large 3` end portion of the NCE cDNAs contains an intron sequence that can be alternatively spliced when expressed in 293 cells. Thus the 3` region of the cDNA was not completely untranslated as previously thought. Expression of the 6-kb NCE cDNA yielded spliced mRNAs encoding polypeptides whose COOH-terminal domains differed from those of the canonical NCE. The deduced sequence of the spliced-in domain had predominant hydrophobic character and was connected to the extracellular loops following TM segment VIII or X in the accepted NCE topological model (Nicoll et al., 1990). The newly identified exon, with few adjacent residues, could replace TM IX or XI. The adjoining NCE sequence was incomplete, terminating with an hydrophilic stretch of amino acids encoded by the vector that could represent an artificial cytoplasmic COOH-terminal domain. Therefore, two of the isoforms produced by transfecting cells with the 6-kb cDNA represented truncated versions of larger homologues: they could for example correspond to the 160-kDa NCE protein normally seen in gels of heart sarcolemmal membranes. On the contrary the shorter isoform identified in this study was complete, since a termination signal following one Leu, encoded in an other frame of the same exon, was introduced by the alternative 5` splicing. This small NCE protein, including only the five NH(2)-terminal TM domains and a shortened intracellular loop, could correspond to the 70-kDa polypeptide revealed by the NCE antibody in sarcolemmal membrane preparations. The complex immunoreaction pattern in heart and transfected cells made it difficult to identify each polypeptide. However, the slowest moving component of the 120-kDa triplet was produced only in cells expressing the 6-kb cDNA. As discussed under ``Results,'' it is unlikely that the appearance of a 120-kDa polypeptide was linked to the glycosylation of the expressed protein as proposed for the heterologous expression in CHO cells (Pijuan et al., 1993). It could also be added that the splicing of the 6-kb transcripts would reduce the number of the extracellular loops, thus eliminating three potential glycosylation sites (Asn-866, Asn-871, and Asn-796). The individual expression of each isoform will eventually correlate the deduced polypeptide sequence with the bands detected in gels.

The mRNA processing producing the two isoforms modified after the VIII or the X TM domains also occurs in heart, as shown by the results of RNase protection. This alternative 5` splicing involving G-2821 or G-2620, is likely to provide an important source of variability for the cardiac NCE protein. The relative amount of the NCE mRNA modified at G-2620 was greater when naturally expressed in heart than in transfected 293 cells. This mRNA encoding the NCE protein modified after the VIII TM domain could also be produced in brain. In addition to the 7-kb transcript, smaller mRNAs are also recognized by NCE probes in heart and other tissues (Kofuji et al., 1992; Lee et al., 1994): particularly abundant is a transcript of about 2 kb that could encode the smaller spliced isoform produced only in low quantity in 293 cells. The full-length 7-kb NCE transcript is evidently more stable in tissues than its shorter homologue (6 kb) was in 293 cells, in which the most abundant species revealed by Northern blotting was of 4 kb. This mRNA was probably spliced at base 3198, as also suggested by RNase protection. A tissue-specific alternative 5`-splicing pattern could be produced by different concentrations of alternative splicing factors (ASF or SF2). The high concentration of SF2 in 293 cells was found to promote the use of the 5` splice site nearest the 3` site in the SV40 tumor antigens expression (Green, 1991). A similar control of alternative 5` splice site selection could also be occurring in the expression of NCE.

The splicing phenomenon described here had an additional point of interest, since it apparently led to the elimination of one, three, or six COOH-terminal TM domains. It is normally assumed that the hydrophobic domains of membrane proteins are essential for their correct insertion in the membrane and naturally, for their proper function. The overexpressed protein described here, and thus presumably its truncated species as well, became nevertheless associated with the membrane. The finding that the COOH-terminal TM composition of the NCE may vary as a result of alternative splicing and that this is accompanied by an increase of the transport function of the protein is interesting and may help to identify TM domains involved in transport regulation. Whether the increased transport activity was due to the elimination of inhibitory domains or whether the spliced-in hydrophobic sequence functioned as an enhancer is not known. However, the possibility that the vector sequence at the COOH terminus of two of the spliced isoforms contributed to the enhanced activity has been ruled out, since deletion of the artificial sequence did not alter the transport function. The variations in the trans-membrane hydrophobic domains brought about by the alternative splicing could influence transport by altering the structural interactions with phospholipid. The NCE activity has been claimed to be regulated by the phospholipid environment of the protein (Hilgemann and Collins, 1992; Collins et al., 1992).


FOOTNOTES

*
The work was made possible by the financial contribution of the National Research Council of Italy (Progetto finalizzato Biotecnologie). 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.

§
To whom correspondence should be addressed: Dipartimento di Chimica Biologica, Universita' degli studi di Padova, Via Trieste, 75, 35121 PADOVA, Italy. Tel.: 39-49-8286470; Fax: 39-49-8073310.

(^1)
The abbreviations used are: NCE, the Na/Ca exchanger; TM, trans-membrane domain(s); PCR, polymerase chain reaction; bp, base pair(s); kb, kilobase(s); CHO, Chinese hamster ovary.


ACKNOWLEDGEMENTS

We are grateful to Dr. K. D. Philipson, Los Angeles, for the kind gift of the antibody and of plasmid pTB11 and to Dr. A. Kraev, Zürich, for the gift of several primers. The authors also thank Dr. G. Salviati and Dr. C. Presotto, Padova, for providing samples of human heart sarcolemmal membranes. They also thank Dr. C. Ballarin, Padova, for her help in the preparation of the illustrations.


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