Cloning, expression, and characterization of the trout cardiac Na+/Ca2+ exchanger

Xiao-Hua Xue1, Larry V. Hryshko2, Debora A. Nicoll3, Kenneth D. Philipson3, and Glen F. Tibbits1

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


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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.


    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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 approx 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).


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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.


View larger version (89K):
[in this window]
[in a new window]
 
Fig. 1.   Nucleotide and deduced amino acid sequences of trout heart Na+/Ca2+ exchanger (NCX). Hydrophobic segments are underlined.

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, AATC<UNL>ATG</UNL>A (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).


View larger version (17K):
[in this window]
[in a new window]
 
Fig. 2.   Hydropathy plot of NCX-TR1. Hydrophobicity is indicated by positive numbers, and hydrophilicity is indicated by negative numbers. Hydrophobic regions are numbered 1-11. The first hydrophobic region is not numbered as it is designated a signal peptide (see text).

Like the hydropathy plot of mammalian NCX1, the NCX-TR1 hydropathy plot showed 12 hydrophobic segments and a long hydrophilic region forming an intracellular loop. The first hydrophobic segment is designated a signal peptide by analogy to NCX1. The cleavage site would be between amino acids 32 and 33 (30). Recent evidence (29) indicates that NCX1 has nine transmembrane segments and that two of the hydrophobic segments do not span the membrane. It is likely that NCX1 and NCX-TR1 have similar topological arrangements.

NCX-TR1 has six potential N-linked glycosylation sites. Glycosylation occurs at amino acid 40 (asparagine), or the ninth residue after the predicted site for cleavage of the leader peptide, of the mammalian NCX1 (10). Assuming NCX-TR1 undergoes glycosylation similar to that of NCX1, asparagine 42 (residue 10 postcleavage) would be glycosylated; however, this requires experimental confirmation. NCX-TR1 contains 10 potential protein kinase C (PKC) sites, two Ca2+/calmodulin-dependent kinase sites, and two tyrosine kinase sites, all shown in Fig. 3.


View larger version (69K):
[in this window]
[in a new window]
 
Fig. 3.   Amino acid sequence comparison of NCX-TR1 (upper sequence) and canine NCX1 (lower sequence). Signal sequence is italicized. Two dots indicate identity, and 1 dot indicates conservative amino acid substitution. The 11 hydrophobic segments of NCX1 are underlined. The alpha -repeats, exchanger inhibitory peptide (XIP) site, Ca2+ regulatory binding sites, and alternative splicing sites of NCX1 are also shown. * Potential N-linked glycosylation sites of the type NXS/T in NCX-TR1. Potential phosphorylation sites for protein kinase C (PKC), Ca2+/calmodulin-dependent kinase (CamK), and tyrosine kinase (TyrK) are indicated with bold letters and the residues to be phosphorylated are underlined. Exons determined from NCX1 are double underlined.

The sequence comparison of NCX-TR1 and dog NCX1 (28) shows ~75% identity at the amino acid level (Fig. 3) and 69% identity at the nucleotide level. The hydrophobic segments (especially the alpha -repeats) are highly conserved. The alpha -repeats (alpha 1 and alpha 2) are in hydrophobic segments 2-3 and 8-9, respectively, which are involved in the ion binding and translocation of the exchanger (27). The sequences in the vicinity of the two groups of three acidic amino acids are identical and appear to be directly involved in Ca2+ binding, thus regulating exchanger activity (19, 23). The exchanger inhibitory peptide (XIP) site associated with Na+-dependent inactivation of the exchanger (20) is also highly conserved. The high level of conservation of these regions suggests that the NCX-TR1 is also regulated by a similar modulation mechanism (7). The most divergent sequence is in the NH2 terminus of the protein. Other sequences with lower levels of homology are scattered in various regions of the intracellular loop besides the region where alternative splicing occurs (13, 17).

The frog cardiac NCX contains a novel exon of 27 bp inserted in the alternative splicing region, which completes a consensus ATP and GTP binding sequence, GXXXXGKS, and it is speculated that this sequence may influence cAMP regulation of exchange activity (12, 36). NCX-TR1, like the mammalian NCX1, does not have this nine-amino acid exon product in the corresponding region, although trout are evolutionarily closer to frogs. A phylogenetic tree is shown in Fig. 4, in which the position of NCX-TR1 based on percent amino acid identity can be seen relative to those of the various mammalian NCX1, NCX2, and NCX3 clones, as well as those of NCX from squid, Drosophila, and frogs.


View larger version (16K):
[in this window]
[in a new window]
 
Fig. 4.   Phylogeny of NCX. Tree was constructed from percent amino acid sequence identities of all published full-length NCX clones in comparison with dog NCX1.1. Amino acid sequence derived from accession number was first analyzed with Signal P V1.1, which uses algorithms based on criteria of Nielsen et al. (30) to predict cleavage sites (CS) for leader peptide. Remaining sequence was compared with dog NCX1.1 with pairwise sequence alignment tool of Baylor College of Medicine search launcher. Data used in construction of tree are as follows (species/NCX isoform/accession no./cleavage site): dog/NCX1.1/M57523/32, human/NCX1.1/M91368/35, guinea pig/NCX1.1/U04955/32, cow/NCX1.1/L06438/32, rat/NCX1.1/Q01728/32, rabbit/NCX1.3/U52665/32, frog-A/NCX1/X90839/16, frog-B/NCX1/X90838, rat/NCX3/U53420/30, rat/NCX2/U081411/20, squid/NCX-SQ1/U93214/26, Drosophila/CalX1/AF009897/20. Frog-A is from genomic DNA, and frog-B is from cardiac cDNA. Full-length clone used in comparison was "constructed" by using genomic section coding for amino acids 1-403 and cDNA section coding for amino acids 404-963.

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.


View larger version (13K):
[in this window]
[in a new window]
 
Fig. 5.   Representative outward Na+/Ca2+ exchange currents obtained from excised oocyte membrane patch expressing NCX-TR1. Currents were activated by replacing 100 mM Li+ with 100 mM Na+ on cytoplasmic surface of patch. Pipette Ca2+ (transported) concentration was 8 mM, and currents were activated in presence or absence of regulatory Ca2+ (1 µM) on cytoplasmic surface. Na+i and Ca2+i, intracellular Na+ and Ca2+, respectively.

Figure 6 shows the current-voltage (I-V) relationship for NCX-TR1. Outward Na+/Ca2+ exchange currents were activated by applying 100 mM Na+i to the cytoplasmic surface of the patch in exchange for 8 mM pipette Ca2+. A series of 10-mV voltage steps from a holding potential of 0 mV were applied from -100 to 100 mV for 20 ms, with a return to the holding potential after each step. This voltage clamp protocol was applied in the presence or absence of Na+ to allow leak subtraction. The graph illustrates the I-V relationship obtained from an alpha -chymotrypsin-treated patch. After treatment of the cytoplasmic surface of the patch with alpha -chymotrypsin (1 mg/ml) for 1-2 min, the exchange currents were no longer regulated by cytoplasmic Ca2+ (data not shown), as has been demonstrated for the NCX1 (7). The I-V relationship obtained for NCX-TR1 is similar to that for NCX1.


View larger version (9K):
[in this window]
[in a new window]
 
Fig. 6.   Leak-subtracted current-voltage relationship for NCX1-TR1. In response to 10-mV voltage steps (-100 to 100 mV) from 0-mV holding potential, outward currents obtained in presence of 100 mM bath Li+ were subtracted from those obtained in presence of 100 mM bath Na+. Voltage was returned to 0 mV between each 20-ms voltage step. To avoid alterations associated with current inactivation, patch was treated with 1 mg/ml alpha -chymotrypsin for ~1 min. INaCa, Na+/Ca2+ exchange current.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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 alpha 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.


    ACKNOWLEDGEMENTS

The expert assistance of Chad Elias and Haruyo Kashihara is acknowledged.


    FOOTNOTES

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.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Bers, D. M. Species differences and the role of sodium-calcium exchange in cardiac muscle relaxation. Ann. NY Acad. Sci. 639: 375-385, 1991[Abstract].

2.   Bers, D. M., J. W. Bassani, and R. A. Bassani. Na-Ca exchange and Ca fluxes during contraction and relaxation in mammalian ventricular muscle. Ann. NY Acad. Sci. 779: 430-442, 1996[Abstract].

3.   Bers, D. M., C. W. Patton, and R. Nuccitelli. A practical guide to the preparation of Ca2+ buffers. Methods Cell Biol. 40: 3-29, 1994[Medline].

4.   Bridge, J. H., K. W. Spitzer, and P. R. Ershler. Relaxation of isolated ventricular cardiomyocytes by a voltage-dependent process. Science 241: 823-825, 1988[Medline].

5.   DiPolo, R., and L. Beaugé. Effects of vanadate on MgATP stimulation of Na-Ca exchange support kinase-phosphatase modulation in squid axons. Am. J. Physiol. 266 (Cell Physiol. 35): C1382-C1391, 1994[Abstract/Free Full Text].

6.   Dyck, C., K. Maxwell, J. Buchko, M. Trac, A. Omelchenko, M. Hnatowich, and L. V. Hryshko. Structure-function analysis of CALX1.1, a Na+-Ca2+ exchanger from Drosophila. Mutagenesis of ionic regulatory sites. J. Biol. Chem. 273: 12981-12987, 1998[Abstract/Free Full Text].

7.   Hilgemann, D. W. Regulation and deregulation of cardiac Na+-Ca2+ exchange in giant excised sarcolemmal membrane patches. Nature 344: 242-245, 1990[Medline].

8.   Hove-Madsen, L. The influence of temperature on ryanodine sensitivity and the force-frequency relationship in the myocardium of rainbow trout. J. Exp. Biol. 167: 47-60, 1992[Abstract].

9.   Hryshko, L. V., S. Matsuoka, D. A. Nicoll, J. N. Weiss, E. M. Schwarz, S. Benzer, and K. D. Philipson. Anomalous regulation of the Drosophila Na+-Ca2+ exchanger by Ca2+. J. Gen. Physiol. 108: 67-74, 1996[Abstract].

10.   Hryshko, L. V., D. A. Nicoll, J. N. Weiss, and K. D. Philipson. Biosynthesis and initial processing of the cardiac sarcolemmal Na+-Ca2+ exchanger. Biochim. Biophys. Acta 1151: 35-42, 1993[Medline].

11.   Iwamoto, T., Y. Pan, T. Y. Nakamura, S. Wakabayashi, and M. Shigekawa. Protein kinase C-dependent regulation of Na+/Ca2+ exchanger isoforms NCX1 and NCX3 does not require their direct phosphorylation. Biochemistry 37: 17230-17238, 1998[Medline].

12.   Iwata, T., A. Kraev, D. Guerini, and E. Carafoli. A new splicing variant in the frog heart sarcolemmal Na-Ca exchanger creates a putative ATP-binding site. Ann. NY Acad. Sci. 779: 37-45, 1996[Medline].

13.   Kofuji, P., W. J. Lederer, and D. H. Schulze. Mutually exclusive and cassette exons underlie alternatively spliced isoforms of the Na/Ca exchanger. J. Biol. Chem. 269: 5145-5149, 1994[Abstract/Free Full Text].

14.   Kozak, M. The scanning model for translation: an update. J. Cell Biol. 108: 229-241, 1989[Abstract].

15.   Kyte, J., and R. F. Doolittle. A simple method for displaying the hydropathic character of a protein. J. Mol. Biol. 157: 105-132, 1982[Medline].

16.   Leblanc, N., and J. R. Hume. Sodium current-induced release of calcium from cardiac sarcoplasmic reticulum. Science 248: 372-376, 1990[Medline].

17.   Lee, S. L., A. S. Yu, and J. Lytton. Tissue-specific expression of Na+-Ca2+ exchanger isoforms. J. Biol. Chem. 269: 14849-14852, 1994[Abstract/Free Full Text].

18.   Levi, A. J., K. W. Spitzer, O. Kohmoto, and J. H. Bridge. Depolarization-induced Ca entry via Na-Ca exchange triggers SR release in guinea pig cardiac myocytes. Am. J. Physiol. 266 (Heart Circ. Physiol. 35): H1422-H1433, 1994[Abstract/Free Full Text].

19.   Levitsky, D. O., D. A. Nicoll, and K. D. Philipson. Identification of the high affinity Ca2+-binding domain of the cardiac Na+-Ca2+ exchanger. J. Biol. Chem. 269: 22847-22852, 1994[Abstract/Free Full Text].

20.   Li, Z., D. A. Nicoll, A. Collins, D. W. Hilgemann, A. G. Filoteo, J. T. Penniston, J. N. Weiss, J. M. Tomich, and K. D. Philipson. Identification of a peptide inhibitor of the cardiac sarcolemmal Na+-Ca2+ exchanger. J. Biol. Chem. 266: 1014-1020, 1991[Abstract/Free Full Text].

21.   Longoni, S., M. J. Coady, T. Ikeda, and K. D. Philipson. Expression of cardiac sarcolemmal Na+-Ca2+ exchange activity in Xenopus laevis oocytes. Am. J. Physiol. 255 (Cell Physiol. 24): C870-C873, 1988[Abstract/Free Full Text].

22.   Matsuoka, S., D. A. Nicoll, Z. He, and K. D. Philipson. Regulation of cardiac Na+-Ca2+ exchanger by the endogenous XIP region. J. Gen. Physiol. 109: 273-286, 1997[Abstract/Free Full Text].

23.   Matsuoka, S., D. A. Nicoll, L. V. Hryshko, D. O. Levitsky, J. N. Weiss, and K. D. Philipson. Regulation of the cardiac Na+-Ca2+ exchanger by Ca2+. Mutational analysis of the Ca2+-binding domain. J. Gen. Physiol. 105: 403-420, 1995[Abstract].

24.   Matsuoka, S., D. A. Nicoll, R. F. Reilly, D. W. Hilgemann, and K. D. Philipson. Initial localization of regulatory regions of the cardiac sarcolemmal Na+-Ca2+ exchanger. Proc. Natl. Acad. Sci. USA 90: 3870-3874, 1993[Abstract].

25.   Moyes, C. D., T. Borgford, L. LeBlanc, and G. F. Tibbits. Cloning and expression of salmon cardiac troponin C: titration of the low-affinity Ca2+-binding site using a tryptophan mutant. Biochemistry 35: 11756-11762, 1996[Medline].

26.   Nicoll, D. A., L. V. Hryshko, S. Matsuoka, J. S. Frank, and K. D. Philipson. Mutagenesis studies of the cardiac Na+-Ca2+ exchanger. Ann. NY Acad. Sci. 779: 86-92, 1996[Medline].

27.   Nicoll, D. A., L. V. Hryshko, S. Matsuoka, J. S. Frank, and K. D. Philipson. Mutation of amino acid residues in the putative transmembrane segments of the cardiac sarcolemmal Na+-Ca2+ exchanger. J. Biol. Chem. 271: 13385-13391, 1996[Abstract/Free Full Text].

28.   Nicoll, D. A., S. Longoni, and K. D. Philipson. Molecular cloning and functional expression of the cardiac sarcolemmal Na+-Ca2+ exchanger. Science 250: 562-565, 1990[Medline].

29.   Nicoll, D. A., M. Ottolia, L. Lu, Y. Lu, and K. D. Philipson. A new topological model of the cardiac sarcolemmal Na+-Ca2+ exchanger. J. Biol. Chem. 274: 910-917, 1999[Abstract/Free Full Text].

30.   Nielsen, H., J. Engelbrecht, S. Brunak, and G. von Heijne. Identification of prokaryotic and eukaryotic signal peptides and prediction of their cleavage sites. Protein Eng. 10: 1-6, 1997[Abstract].

31.   Omelchenko, A., C. Dyck, M. Hnatowich, J. Buchko, D. A. Nicoll, K. D. Philipson, and L. V. Hryshko. Functional differences in ionic regulation between alternatively spliced isoforms of the Na+-Ca2+ exchanger from Drosophila melanogaster. J. Gen. Physiol. 111: 691-702, 1998[Abstract/Free Full Text].

32.   Philipson, K. D., D. A. Nicoll, and Z. Li. The cardiac sodium-calcium exchanger. Soc. Gen. Physiol. Ser. 48: 187-191, 1993[Medline].

33.   Quednau, B. D., D. A. Nicoll, and K. D. Philipson. Tissue specificity and alternative splicing of the Na+/Ca2+ exchanger isoforms NCX1, NCX2, and NCX3 in rat. Am. J. Physiol. 272 (Cell Physiol. 41): C1250-C1261, 1997[Abstract/Free Full Text].

34.   Santer, R. M. Morphology and innervation of the fish heart. Adv. Anat. Embryol. Cell Biol. 89: 1-102, 1985[Medline].

35.   Shigekawa, M., T. Iwamoto, and S. Wakabayashi. Phosphorylation and modulation of the Na+-Ca2+ exchanger in vascular smooth muscle cells. Ann. NY Acad. Sci. 779: 249-257, 1996[Medline].

36.   Shuba, Y. M., T. Iwata, V. G. Naidenov, M. Oz, K. Sandberg, A. Kraev, E. Carafoli, and M. Morad. A novel molecular determinant for cAMP-dependent regulation of the frog heart Na+-Ca2+ exchanger. J. Biol. Chem. 273: 18819-18825, 1998[Abstract/Free Full Text].

37.   Thomas, M. J., B. N. Hamman, and G. F. Tibbits. Dihydropyridine and ryanodine binding in ventricles from rat, trout, dogfish and hagfish. J. Exp. Biol. 199: 1999-2009, 1996[Abstract/Free Full Text].

38.   Tibbits, G., C. D. Moyes, and L. Hove-Madsen. Excitation-contraction coupling in the teleost heart. In: Fish Physiology, edited by D. Randall, and A. P. Farrell. New York: Academic, 1992, p. 267-304.

39.   Tibbits, G. F., K. D. Philipson, and H. Kashihara. Characterization of myocardial Na+-Ca2+ exchange in rainbow trout. Am. J. Physiol. 262 (Cell Physiol. 31): C411-C417, 1992[Abstract/Free Full Text].

40.   Tsuruya, Y., M. M. Bersohn, Z. Li, D. A. Nicoll, and K. D. Philipson. Molecular cloning and functional expression of the guinea pig cardiac Na+-Ca2+ exchanger. Biochim. Biophys. Acta 1196: 97-99, 1994[Medline].


Am J Physiol Cell Physiol 277(4):C693-C700
0002-9513/99 $5.00 Copyright © 1999 the American Physiological Society