1 Cardiac Membrane Research Laboratory, Simon Fraser University, Burnaby, British Columbia V5A 1S6; and 2 Institute of Cardiovascular Sciences, St. Boniface General Hospital Research Centre, The University of Manitoba, Winnipeg, Manitoba R2H 2A6, Canada; and 3 Cardiovascular Research Laboratories, School of Medicine, University of California, Los Angeles, Los Angeles, California 90095-1760
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ABSTRACT |
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Isoform 1 of the cardiac Na+/Ca2+ exchanger (NCX1) is an important regulator of cytosolic Ca2+ concentration in contraction and relaxation. Studies with trout heart sarcolemmal vesicles have shown NCX to have a high level of activity at 7°C, and this unique property is likely due to differences in protein structure. In this study, we describe the cloning of an NCX (NCX-TR1) from a Lambda ZAP II cDNA library constructed from rainbow trout (Oncorhynchus mykiss) heart RNA. The NCX-TR1 cDNA has an open reading frame that codes for a protein of 968 amino acids with a deduced molecular mass of 108 kDa. A hydropathy plot indicates the protein contains 12 hydrophobic segments (of which the first is predicted to be a cleaved leader peptide) and a large cytoplasmic loop. By analogy to NCX1, NCX-TR1 is predicted to have nine transmembrane segments. The sequences demonstrated to be the exchanger inhibitory peptide site and the regulatory Ca2+ binding site in the cytoplasmic loop of mammalian NCX1 are almost completely conserved in NCX-TR1. NCX-TR1 cRNA was injected into Xenopus oocytes, and after 3-4 days currents were measured by the giant excised patch technique. NCX-TR1 currents measured at ~23°C demonstrated Na+-dependent inactivation and Ca2+-dependent activation in a manner qualitatively similar to that for NCX1 currents.
teleosts; myocardium; contractility; sodium/calcium exchange
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INTRODUCTION |
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THE SODIUM/CALCIUM EXCHANGER (NCX) is an integral membrane protein that plays an important role in the regulation of cytosolic Ca2+ concentration in many cell types. NCX transports Ca2+ across the membrane with a stoichiometry of three Na+ to one Ca2+ and is therefore electrogenic (32). The role of the NCX in excitation-contraction (E-C) coupling in the mammalian heart has been studied extensively. Its function as the prime mechanism of Ca2+ extrusion from the cardiomyocyte and therefore an important component of relaxation is well documented (1, 2, 4). More controversial roles for NCX as a pathway for depolarization-induced Ca2+ influx (18) and/or Na+ current-induced Ca2+ influx (16) have also been proposed.
The NCX in the lower vertebrate heart has not been studied in great detail. Despite this, there is considerable circumstantial evidence from teleost and amphibian hearts that NCX plays a critical role in E-C coupling in these species. Electron micrographs of the teleost heart reveal both a scarcity of sarcoplasmic reticulum (SR) compared with mammal hearts and an absence of transverse tubules (34). Furthermore, the density of the SR Ca2+ release channel in the hearts of teleosts is substantially lower than that in the hearts of mammals (37), and ryanodine does not reduce the contractile force of the teleost ventricle under most physiological conditions (8). In addition, the surface-to-volume ratio is higher in teleost myocytes than in mammalian myocytes, implying that a greater rise in cytosolic intracellular Ca2+ concentration for a given Ca2+ influx occurs. Thus it has been suggested that, in the teleost heart, transsarcolemmal Ca2+ transport is sufficient to support contraction (38) and that reverse-mode NCX activity may contribute to the Ca2+ influx during E-C coupling. Furthermore, in the absence of a substantial SR in teleosts, it is likely that the NCX is the primary means of reducing cytosolic Ca2+ (38).
One prominent aspect of cardiac function in the rainbow trout (Oncorhynchus mykiss) is its ability to maintain adequate contractility under hypothermic conditions that are cardioplegic to mammals. It has been suggested that this capability requires at least some of the proteins involved in E-C coupling to have evolved differently in these species. Studies of Na+/Ca2+ exchange in trout heart sarcolemmal vesicles have demonstrated properties of this protein that are both unique and common to isoform 1 of the mammalian NCX (NCX1) (39). Immunoblots of trout sarcolemma (SL) probed with polyclonal antibodies raised against the canine NCX1 have a pattern of banding that is the same as that of immunoblots of mammalian SL, indicating similarities in antigenicity and molecular weight. Northern blots of trout mRNA, probed with a fragment of the canine NCX1 cDNA, demonstrate comparable transcript sizes (~7 kb), and this suggests substantial conservation of transcript sequence (39). Other similarities of the NCX1 from these divergent species include similarities of electrogenicity and stimulation by chymotrypsin treatment. However, despite these striking similarities of trout and mammalian NCX, the differences are also remarkable. Most notable is that reducing the temperature from 21 to 7°C results in a canine NCX1 activity <10% of the initial level, whereas in trout the activity remains >75% (39). This behavior is observed not only in the native membranes but also when both canine and trout NCX proteins are reconstituted into asolectin vesicles. These data strongly suggest that the differential temperature dependencies in the mammalian and teleost NCX are due to important differences in the primary structures of these isoforms. The cloning of the trout cardiac NCX (NCX-TR1) described here represents the first and crucial step in providing a mechanistic explanation of these important physiological differences.
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MATERIALS AND METHODS |
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Screening of cDNA library. PCR was conducted with a pair of degenerate primers to amplify a fragment of NCX from a trout heart Lambda ZAP II cDNA library that we have constructed and described in detail previously (25). The primers were designed by using the highly conserved amino acid sequences of putative transmembrane segments 1 and 3 in NCX1 and NCX2. The sense primer was a 17-mer with 8-fold degeneracy (5'-GCIATGGTNTAYATGTT-3'), and the antisense primer was a 17-mer with 16-fold degeneracy (5'-AYRAACATRTTRAAIGC-3'). The PCR was performed under the following conditions: 94°C for 2 min, followed by 30 cycles of 94°C for 10 s, 50°C for 30 s, and 72°C for 30 s, and then 72°C for 10 min. The PCR product was subcloned into pCR-Script SK(+) (Stratagene, La Jolla, CA). After the sequence was identified as trout heart exchanger on the basis of sequence similarity with NCX1, this fragment was labeled and used as a probe to screen the trout heart cDNA library by using enhanced chemiluminescence random prime labeling and detection systems (Amersham Canada, Oakville, ON, Canada). A full-length clone (T6-1) was isolated, and both strands of the cDNA were sequenced by the dideoxynucleotide chain termination method using ABI's AmpliTaq dye terminator cycle sequencing.
Expression of trout NCX in Xenopus oocytes. T6-1 was subcloned from the original pBluescript into a modified vector, in which the multiple cloning site of pBluescript was removed and only BamH I and Hind III sites were left (27). The 3'-untranslated region of T6-1 was replaced with that of the Na+-glucose cotransporter clone, which contains a poly(A)+ tail (24). The Hind III site at nucleotide 2947 of T6-1 was removed by silent mutation with the Quickchange site-directed mutagenesis kit (Stratagene). The mutation was made in a 550-bp cassette generated by Aat II digestion. T6-1 was linearized with Hind III, and cRNA was synthesized with the T3 mMessage mMachine in vitro transcription kit (Ambion, Austin, TX). Oocytes were prepared as described by Longoni et al. (21). They were injected with 5 nl of cRNA, and exchange activity was measured 3-4 days after injection as Na+ gradient-dependent 45Ca2+ uptake (28) or as exchange current (see below).
Assay of
Na+/Ca2+
exchange activity.
Outward
Na+/Ca2+
exchange currents were measured by the giant excised patch technique,
as described previously (31). Borosilicate glass pipettes were pulled
and polished to a final inner diameter of 20-30
µm and coated with a Parafilm-mineral oil mixture. The vitellin layer was removed, and oocytes were placed in a solution containing (in mM) 100 KOH, 100 MES, 20 HEPES, 5 EGTA, and 5 MgCl2; the pH was maintained at
7.0 at room temperature with MES. Gigaohm seals were
formed via suction, and membrane patches (inside-out configuration)
were excised by movements of the pipette tip. A computer-controlled,
20-channel solution switcher was used for rapid solution changes. Axon
Instruments hardware and software were used for data acquisition and
analysis. The pipette solution contained (in mM) 100 N-methyl-D-glucamine-MES, 30 HEPES, 30 tetraethylammonium (TEA) hydroxide, 16 sulfamic acid, 8 CaCO3, 6 KOH, 0.25 ouabain, 0.1 niflumic acid, and 0.1 flufenamic acid; the pH was maintained at 7.0 with MES. Outward
Na+/Ca2+
exchange currents were activated by switching from intracellular Li+
(Li+i)- to intracellular
Na+
(Na+i)-based bath solutions
containing (in mM) 100 Na+ or
Li+ aspartate, 20 MOPS, 20 TEA
hydroxide, 20 CsOH, 10 EGTA, 0-7.3 CaCO3, and 1.0-1.13
Mg(OH)2; the pH was maintained at
7.0 with MES or LiOH. Mg2+ and
Ca2+ were adjusted to yield free
concentrations of 1.0 mM and 0 or 1 µM, respectively, with MAXC
software (3). All experiments were conducted at room temperature
(22-23°C).
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RESULTS |
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Cloning of trout heart NCX.
To obtain a probe for library screening, we conducted PCR amplification
on trout heart cDNA. Degenerate primers were designed on the basis of
conserved amino acid sequences of putative transmembrane segments 1 and
3 of the mammalian exchangers NCX1 and NCX2. A PCR product of the
appropriate size (~290 bp) was isolated, subcloned into pCR-Script
SK(+) and sequenced. Sequence analysis suggested that this cDNA
fragment coded for a protein with a high degree of conservation
compared with NCX1 and NCX2. The PCR product was used to screen a trout
heart cDNA library. After secondary screening, we obtained several
positive clones. Partial sequencing indicated one of the clones,
T6-1, contained full-length cDNA, and the cDNA was subsequently
sequenced in both directions. The complete nucleotide and deduced amino
acid sequences of T6-1 are shown in Fig.
1.
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The structure of trout heart NCX.
The T6-1 clone has a nucleotide sequence of 3302 bp with an open
reading frame of 2904 bp from base 72 to 2975 encoding a protein of 968 amino acids with a deduced molecular size of 108 kDa, designated
NCX-TR1. The nucleotide sequence around the start site,
AAA (start codon underlined), is
similar to the sequence of the Kozak consensus initiation site
(14). No poly(A)+
tail is shown in the 3'-untranslated region. The result of a hydropathy analysis of the deduced amino acids of NCX-TR1 is shown in
Fig. 2, which was determined by a
modification of the method of Kyte and Doolittle (15).
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Functional expression of NCX-TR1 in Xenopus oocytes. To confirm that a functional trout exchanger had been cloned, NCX-TR1 cRNA was synthesized in vitro and injected into Xenopus oocytes. After 3 days of incubation, expression was assessed by measuring 45Ca2+ uptake. The uptake of Ca2+ in the presence of an outwardly directed Na+ gradient was on the average more than an order of magnitude greater than that in the absence of a Na+ gradient (data not shown).
Exchange current of the NCX-TR1.
Figure 5 shows outward
Na+/Ca2+
exchange currents for the NCX-TR1 exchanger expressed in
Xenopus oocytes. Currents were
activated by the application of 100 mM
Na+ to the cytoplasmic surface of
an excised patch of oocyte membrane. The pipette contained 8 mM
Ca2+ (extracellular surface). As
indicated on the overlapping current traces, records were obtained in
the presence or absence of regulatory Ca2+ (1 µM) at the cytoplasmic
surface. Outward
Na+/Ca2+
exchange currents show characteristics similar to those of other mammalian exchangers. That is, both peak and steady-state outward currents were larger in the presence of regulatory
Ca2+, demonstrating positive
regulation of exchange current by intracellular Ca2+
(Ca2+i). Also, in response to
Na+i application, the current peaks and
then slowly decays, indicative of
Na+i-dependent inactivation (7). The
results shown are representative of those obtained with three
additional patches.
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DISCUSSION |
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We have cloned and expressed the trout heart NCX, NCX-TR1. The hydropathy plot is similar to that for mammalian NCX1 and predicts 12 hydrophobic segments and a large hydrophilic domain. The mammalian NCX1 has a cleavable signal peptide that is removed from the protein during biosynthesis in the endoplasmic reticulum (10, 28). A potential cleavage site of NCX-TR1 is predicted to exist between amino acids 32 and 33 on the basis of the sequence homology between mammalian NCX1 and NCX-TR1 and the criteria of Nielsen et al. (30). Thus the topology of NCX-TR1 is not different from that proposed for NCX1, which is now modeled to have nine transmembrane segments (29).
A sequence comparison, including the cleaved leader peptide, showed ~75% identity at the amino acid level to the dog NCX1 and 61 and 66% identity to rat NCX2 and NCX3, respectively. Like all NCXs, NCX-TR1 has the most divergent sequence at the NH2 terminus (40). Sequence identity becomes very high (85%) within the putative transmembrane segments, consistent with their functional significance for ion translocation. The last putative transmembrane segment is the least well conserved transmembrane domain (26). Overall, the amino acid sequence of the intracellular loop is 73% identical to that of NCX1 and, as expected, is less conserved than those of the transmembrane segments. However, those regions within the loop with known functional importance are well conserved. The endogenous XIP site, consisting of 20 amino acids at the NH2 terminus of the loop, exhibits a high degree of conservation. There is strong evidence that the XIP site of NCX1 is involved in Na+-dependent inactivation (20). The regulatory Ca2+ binding domains characterized by three consecutive aspartic acid residues have been found to be highly conserved in the NCX family (6) and are completely conserved in NCX-TR1.
The high degree of conservation of the known regulatory components of the intracellular loop is reflected in the NCX-TR1 giant excised patch current records shown in Fig. 5. The current decay or inactivation seen after peak current requires the presence of the XIP site in mammalian NCX1 (22). The modulation of the NCX current by regulatory Ca2+ seen in both NCX1 and NCX-TR1 is dependent on the presence of the Ca2+ binding sites of the cytoplasmic loop (23). The regulation of the NCX-TR1 current by Ca2+ is positive, making it similar to NCX1 and the opposite of the cloned Drosophila melanogaster NCX, CalX, which is characterized by reduced exchange current magnitude in response to increasing cytoplasmic Ca2+ (9).
A regulatory role for phosphorylation of the exchanger has been
described for squid axons (5) and vascular smooth muscle cells (35).
The frog NCX has also been reported to be modulated by cAMP (36).
Recently, Iwamoto et al. (11) examined the phosphorylation regulation
of mammalian NCX and found that the activities of both NCX1 and NCX3
but not NCX2, when expressed in CCL-39 fibroblasts, are stimulated by a
pathway involving PKC. Their results also suggested that the large
cytoplasmic loop is required for stimulation, although it may not
directly involve phosphorylation. Each of the exchangers contains
several consensus sites for phosphorylation by different protein
kinases. Potential sites for phosphorylation by PKC,
Ca2+/calmodulin-dependent kinase,
and tyrosine kinases are shown in Fig. 3. NCX-TR1 has one potential
tyrosine kinase site, KVISEGTGY (Tyr-509), which is unique in the
exchanger family. This site is located next to one of the regulatory
Ca2+ binding domains, suggesting
that phosphorylation may play a role in the
Ca2+ regulation of exchange
activity. Residue Thr-119 in NCX-TR1 could be a potential
phosphorylation site of PKC or calmodulin-dependent kinase and is also
present in NCX3. Because this site is located near the 1 repeat,
phosphorylation of this residue may be involved in modulating ion
transport (26).
The gene coding for NCX is characterized by a cluster of six exons (A-F) coding for a variable region in the COOH terminus of the large intracellular loop (13). Alternative splicing of these exons generates multiple tissue-specific variants of NCX (17). Exons A and B are mutually exclusive and are used in conjunction with the other four exons (C-F) to produce all NCX isoforms. On the basis of these findings, NCX-TR1 uses exons A and C and D and F (Fig. 3). Compared with NCX1.1 cDNA, which is composed of exons A and C-F (33), NCX-TR1 cDNA apparently lacks exon E, and this accounts for the five-amino acid deletion in the alternate-splicing region.
This initial characterization of NCX-TR1 is an important first step in understanding how the teleost heart functions normally over a range of temperatures (4-15°C) that is debilitating to the mammalian heart. Furthermore, this represents the first nonmammalian heart-specific full-length NCX clone to be expressed and characterized. Although the full-length clones NCX-SQ1 and CalX for squid and Drosophila NCXs, respectively, have been expressed, neither is heart specific. The published NCX clone of the amphibian Xenopus laevis (12, 36) represents the splicing of frog heart cDNA clones with a genomic clone, with only 93% identity in the 178-amino acid overlap region in the cytoplasmic loop.
Thus knowledge of the sequence and biophysical properties of NCX-TR1 can contribute to our understanding of the evolution of the NCX (as the teleosts are ~400 million years removed from mammalian species) and how proteins in general can evolve to function under hypothermic conditions.
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ACKNOWLEDGEMENTS |
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The expert assistance of Chad Elias and Haruyo Kashihara is acknowledged.
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FOOTNOTES |
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The support of the Natural Sciences and Engineering Research Council of Canada (Grant OGP0002321) to G. F. Tibbits is greatly appreciated. L. V. Hryshko and K. D. Philipson were funded by the Medical Research Council of Canada (Grant GEC3) and the National Heart, Lung, and Blood Institute (Grants HL-48509 and HL-49101), respectively.
The NCX-TR1 cDNA sequence described in this paper has been granted accession number AF175313.
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: G. F. Tibbits, Cardiac Membrane Research Laboratory, Simon Fraser Univ., Burnaby, BC V5A 1S6, Canada (E-mail: tibbits{at}sfu.ca).
Received 26 April 1999; accepted in final form 10 June 1999.
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