INTRODUCTION

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The sarcolemmal Na⁺-Ca²⁺ exchanger is one of the major Ca²⁺ extrusion pathways of the heart muscle. In nonmammalian species, the exchanger, in addition, may serve as a major Ca²⁺ influx pathway, as these hearts in general lack well developed intracellular Ca²⁺ release pools (1-3).

In mammalian species, most, if not all, tissues contain a transcript of the Na⁺-Ca²⁺ exchanger (NCX1) gene (4-13) which undergoes tissue-specific alternative splicing. Two additional genes are expressed in the brain (NCX2 and NCX3) and skeletal muscle (NCX2), but the primary structure of the exchangers remains highly conserved, especially within the 11 putative transmembrane domains. Relatively greater divergence has been found in the N-terminal regions and the large intracellular loop between transmembrane domains 5 and 6, where the high affinity Ca²⁺ regulatory site is located (14). Even though a putative protein kinase A phosphorylation site has been also identified in the mammalian isoform (4), no functional evidence for the cAMP/protein kinase A-dependent phosphorylation of the exchanger has as yet been found. Recently, however, it has been shown that the Na⁺-Ca²⁺ exchanger in frog but not in mammalian ventricular myocytes is regulated by isoproterenol via the activation of a -adrenoreceptor/adenylate-cyclase/cAMPdependent pathway (15). In this report we describe the functional expression of a recombinant cAMP-sensitive frog heart Na⁺-Ca²⁺ exchanger construct (fNCX1a) with a newly identified 9-amino acid exon which renders the molecule regulatable by cAMP.

	EXPERIMENTAL PROCEDURES

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Electrophysiology-- Procedures to inject and maintain the Xenopus oocytes were identical to those described previously (16). For the recording of Na⁺-Ca²⁺ exchange current the basic Cl-free extracellular (glass-funnel) solution contained (in mM): NaOH, 109; Ca(NO₃)₂, 5; MgSO₄, 1; HEPES, 20; niflumic acid, 0.1; flufenamic acid, 0.1; pH 7.3 (adjusted with methanesulfonic acid). Niflumic and flufenamic acids were used as Cl channel blockers (17). When necessary the extracellular solution was supplemented with either 1-5 mM Ni(NO₃)₂, 1 mM CdCl₂, 500 µM CPT-cAMP, or their combinations. The intracellular Cl-free solution contained (in mM): NaOH, 20; triethanolamine hydroxide, 100; HEPES, 10; Mg-ATP, 5; EGTA, 5; Ca(NO₃)₂, 3.2-4.99 (free Ca²⁺ 0.1-10 µM); pH 7.3 (adjusted with aspartic acid). All chemicals were purchased from Sigma, except for Ni(NO₃)₂ which was from Aldrich. Oocytes were perfused with intracellular solution through an intracellular glass cannula by applying discrete steps of positive pressure.

Photometry-- To measure Na⁺-dependent Ca²⁺ uptake both control and the frog clone (fNCX1a)-expressing oocytes were injected with (50 ng/oocyte) Ca²⁺-sensitive photoprotein, aequorin (dissolved in 1 mM EDTA buffer), 3 to 4 h before the measurements. Aequorin-injected oocytes were loaded with Na⁺ by incubating them for 30 min at room temperature in K⁺/Ca²⁺-free Barth's solution containing in mM: NaCl, 88; NaHCO₃, 2.4; MgSO₄, 0.82; HEPES, 15; pH 7.4 with NaOH, supplemented with 30 µM nystatin (Sigma, prepared as 10 mM stock in Me₂SO) and then transferred to K⁺/Ca²⁺-free Barth's without nystatin (18). Photon emission was measured from Na⁺-loaded oocytes placed in glass scintillation vials at 6-s intervals in a liquid scintillation counter (model LS250, Beckman). First, the basal photon emission was estimated in a scintillation vial filled with 1 ml of conditioning K⁺/Ca²⁺-free Barth's solution. The oocytes were then quickly transferred to a K⁺/Na⁺-free Barth's test solution containing in mM: triethanolamine chloride, 90; CaCl₂, 0.41; Ca(NO₃)₂, 0.33; MgSO₄, 0.82; HEPES, 15; pH 7.4 with triethanolamine hydroxide, and the measurements were repeated. To measure the Ni²⁺ sensitivity of the [Na⁺]_i-dependent Ca²⁺ uptake, photon emission was measured in the K⁺/Na⁺-free Barth's solution supplemented with 5 mM NiCl₂ prior to the use of test solutions.

Screening of the Library and the Construction of the Full-length Frog Na⁺-Ca²⁺ Exchanger-- The screening of Xenopus laevis heart cDNA library using a probe NcoI-MluI (nucleotides 269-2609) fragment of the dog cDNA (gift of K. D. Philipson, Los Angeles, CA) yielded two clones, H3 and H6. Further screening of the library did not produce clones which encode the N terminus. The remaining portion was retrieved from a X. laevis genomic library (Stratagene) using a PCR¹-derived fragment of H3 as a probe (primers: forward, 5'-gtggcagttactattgttcgtcgtgga-3', reverse, 5'-gctctttctgggtttcgcctggctt-3'). A positive clone X9 was found to contain a nucleotide sequence equivalent to the 1.8-kilobase pair mammalian exon 2. The full-length clone, fNCX1a (3.2 kilobase pairs) was then constructed from X9 and H6 DNAs in pGEM-T vector (Promega, Madison, WI), using a region encompassing the ATG start codon up to the BbsI (corresponding to amino acid residue F568) of X9, and the remainder from H6, including the entire coding sequence and a 400-base pair 3'-UTR (to the SpeI site).

Modifications of the Expression Construct-- In order to replace the whole 3'-untranslated region of fNCX1a with that of Na⁺-glucose co-transporter clone, pMJC424 (19), the coding region of fNCX1a was amplified by 10 PCR cycles using a sense primer that hybridizes at positions 1-20 of the coding region containing a SalI restriction site before the ATG initiation codon, and an antisense primer that hybridizes to the last 20 nucleotides of the coding region and contains additionally a MluI restriction site downstream to the stop codon. The amplification reaction was carried out in the presence of Pfu DNA polymerase (Stratagene) and 100 ng of fNCX1a plasmid. The PCR fragment was purified by agarose gel electrophoresis, digested with SalI and MluI, and ligated into pMJC424 (19) from which the coding region of the Na⁺-glucose co-transporter had been removed by digestion with SalI and MluI. The resultant plasmid, pF1a/MC, was sequenced.

In Vitro Transcription-- cRNA for the injection into the oocytes was synthesized in vitro from either plasmid pF1a/MC or pF1a/AGA using the mCAP^TM mRNA capping kit from Stratagene according to manufacture's instructions. Each oocyte was injected with 50 nl of cRNA solution in water at concentration 0.1 mg/ml.

Mutagenesis-- Deletion of the 27-nucleotide fragment (positions 1913-1939) of fNCX1a cDNA clone was made according to Kunkel et al. (21). CJ236 cells (Invitrogen) were transformed with the pF1a/MC plasmid. 2× YT medium supplemented with 0.25 µg/ml uridine was inoculated with a colony of recombinant clone, and the single-stranded form of the plasmid was isolated using R408 helper phage (Stratagene). A 34-base oligonucleotide complementary to 18 nucleotides upstream and 16 nucleotides downstream of the target sequence was phosphorylated with ATP and T4 polynucleotide kinase and annealed to the single-stranded plasmid. Conversion into closed circular double-stranded form was made with T4 DNA polymerase and T4 DNA ligase. The double-stranded plasmid was used for transformation of XL1-Blue cells (Stratagene). To confirm deletion, plasmid DNAs from several recombinant colonies were sequenced, and the clone containing the desired deletion was selected and designated pF1a/MC().

	RESULTS

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A Nucleotide-binding Motif in a Frog Na⁺-Ca²⁺ Exchanger-- The X. laevis heart cDNA library was screened with the cDNA pTB11, encoding dog heart Na⁺-Ca²⁺ exchanger as a probe. Two overlapping partial cDNA clones, H3 (corresponding to amino acid residues 388-656) and H6 (residue 544-3'-4TR), were isolated and sequenced (GenBank^TM accession no. X90838). Unlike H3, the H6 clone contained a 27-base pair insert located at a splicing junction between exons 7 and 8 (22) and representing a new exon (30).

View larger version (30K): [in this window] [in a new window]   Fig. 1.   Schematic representation of the structural differences between the mammalian and the frog NCX1 cDNAs. A, a model of the protein, showing the two clusters of transmembrane domains (filled bars) and the large cytoplasmic loop (open bar). B, arrangement of the mammalian exons, coding for the distal part of the cytoplasmic loop, and the pattern of their splicing in the dominant cardiac isoform. C, alignment of the two frog cDNA clones with the dog heart cDNA clone (2) and with the P-loop consensus sequence.

Functional Expression of the Frog Na⁺-Ca²⁺ Exchanger-- Initially we were unable to express fNCX1a in Xenopus oocytes. Two strategies were therefore employed to achieve functional expression. First strategy involved the replacement of the whole 3'-UTR of fNCX1a with that of Na⁺-glucose co-transporter clone pMJC424 (14), which includes a poly(A) tail (19) (gift of E. Wright, UCLA, CA). In addition, we replaced G for C in the 3 position to conform to a Kozak consensus sequence (27) and designated it pF1a/MC. In the second strategy, we replaced the 5'-UTR of fNCX1a with that of alfalfa mosaic virus RNA-4 and attached a 90-nucleotide poly(A) tail to the 3'-UTR of fNCX1a. This was done by subcloning the fNCX1a coding and 3'-UTR into the transcription-competent vector pAGA (20) (a gift of L. Birnbaumer, UCLA, CA). This plasmid was designated pF1a/AGA. We have chosen this strategy to verify: (a) if the whole 3'-UTR or only the poly(A) tail were important for translation, and (b) if the efficiency of the translation could be further increased by the presence of the 5'-UTR of alfalfa mosaic virus RNA-4. We found that the pF1a/AGA construct produced a 50% higher level of expression of the exchanger than did pF1a/MC (using I_Na-Ca in oocytes as a criterion). However, expression of pF1a/AGA was transient, reaching peak values within 24-48 h, and declining rapidly, while pF1a/MC produced a slow but steady level of expression for up to 6-7 days. Thus, the right Kozak consensus initiation site at the 5'-end and the presence of the poly(A) tail at the 3'-UTR are critical for the functional expression of the exchanger. Most of the experiments reported here were carried out using the pF1a/MC construct.

View larger version (27K): [in this window] [in a new window]   Fig. 2.   Expression of Na2+-Ca2+exchanger current (INa-Ca) in fNCX1a-injected Xenopus oocytes. A, superimposed membrane currents obtained from fNCX1a-injected Xenopus oocyte in response to the envelope voltage-clamp protocol shown in the upper row. These as well as all other currents illustrated were recorded using the glass funnel technique (16) following oocyte superfusion with Cl-free extra- and intracellular solutions containing 109 mM Na+, 5 mM Ca2+ and 20 mM Na+, and 10 µM Ca2+, respectively. B, membrane currents obtained under the same experimental conditions but after external application of 3 mM Ni2+. C, Ni2+-sensitive component of the current (INa-Ca) obtained as a result of the subtraction of current records shown in B from those in A. D, superimposed original current traces obtained in the absence and presence of 3 mM Ni2+(as indicated) in response to the ramp voltage-clamp protocol shown above the records. The Ni2+-sensitive INa-Ca was obtained after subtraction of the current recorded in presence of Ni2+ from those of control. E, current-voltage relation for Ni2+-sensitive INa-Ca constructed from the ramp portion of the difference current presented in D. F, the differences in Na+-dependent Ca2+ uptake in Na+-loaded control and fNCX1a-injected oocytes in the presence and absence of 5 mM Ni2+ as estimated by photon emission of aequorin. Oocytes from both groups were injected with aequorin and loaded with Na+ by incubation for 30 min at room temperature in conditioning K+/Ca2+-free Barth's solution supplemented with 30 µM nystatin. Photon emission was measured in both conditioning solution and following exposure of the oocytes to the test, K+/Na+-free Barth's solution. The number of oocytes tested is indicated above each column. The asterisk (*) denotes significantly different values from the value in test, K+/Na+-free Barth's solution at p < 0.05.

View larger version (21K): [in this window] [in a new window]   Fig. 3.   Voltage and Ca2+ dependence of INa-Cain fNCX1a-injected Xenopus oocytes. A, superimposed Ni2+-sensitive INa-Ca obtained from fNCX1a-injected Xenopus oocyte in response to the ramp envelope voltage-clamp protocol shown in the upper row; [Na+]o = 109 mM, [Ca2+]o = 5 mM, [Na+]i = 20 mM, [Ca2+]i = 1 µM. B, current-voltage relation for Ni2+-sensitive INa-Ca constructed from the ramp portions of the currents presented on A. Measured reversal potentials, ENa-Ca, as well as corresponding intracellular Ca2+ concentrations, [Ca2+]i, calculated according to the formula ENa-Ca = 3ENa  2ECa, are shown near the I-V values. The experimentally measured shifts of the reversal potential, ENa-Ca = ENa-Ca{1)  ENa-Ca(2)2, adequately corresponded to the change in the effective [Ca2+]i in accordance with the relation: ENa-Ca = ENa-Ca(1)  ENa-Ca(1) = 59 log([Ca2+]i(1)/[Ca2+]i(2)). The total amount of Ca2+ entering the oocyte by the time the exchanger reaches its reversal potential (determined by digital integration of the current traces presented on A) as well as the effective [Ca2+]i provide an estimate of the size of the confined space near the membrane for the accumulation of Ca2+ of about 27 nl (~2.7% of the average volume of the oocyte which is about 1 µl).

Modulation of Exchanger Activity by cAMP-- In the frog ventricular myocytes the Na⁺-Ca²⁺ exchanger is suppressed via the -adrenoreceptor/adenylate-cyclase/cAMP-dependent pathway (15). Since the defolliculated and devitellinated oocytes lack -adrenoreceptor- and forskolin-stimulated adenylate cyclase (29), the membrane-permeable cAMP analog CPT-cAMP was used to determine the cAMP sensitivity of fNCX1a-injected oocytes. Fig. 4A shows that I_Na-Ca at 40 mV is slowly suppressed by 50-60% (n = 4) on rapid (<500 ms) application of CPT-cAMP. In the presence of CPT-cAMP, 5 mM Ni²⁺ failed to further inhibit the current, suggesting that virtually all the Ni²⁺-sensitive I_Na-Ca had been inhibited by CPT-cAMP. The CPT-cAMP-mediated suppression of the exchanger current was partially reversed by the wash out of extracellular CPT-cAMP, and the extensive perfusion of the oocyte's interior with cAMP-free internal solution (Fig. 4A). The recovery of the net current was primarily due to the recovery of I_Na-Ca, since all of the recovered current was blocked by 5 mM Ni²⁺ (Fig. 4A).

View larger version (26K): [in this window] [in a new window]   Fig. 4.   The effect of membrane-permeable cAMP analog, CPT-cAMP, on INa-Cain fNCX1a-injected Xenopus oocyte. A, the time course of the changes of the current amplitude elicited by depolarization to +40 mV in fNCX1a-injected Xenopus oocyte during interventions indicated by horizontal bars. The ramp voltage-clamp protocol used to elicit the currents as well as representative current traces acquired at the moments marked by corresponding numbers are shown in the inset. 5 mM Ni2+ was applied from the outside. B and C, superimposed membrane currents obtained from another fNCX1a-injected Xenopus oocyte in response to the envelope voltage-clamp protocol shown in the upper row of B before (B) and after 6 min of the oocyte exposure to 500 µM CPT-cAMP (C). For all currents presented [Na+]o = 109 mM, [Ca2+]o = 5 mM, [Na+]i = 20 mM, [Ca2+]i = 10 µM.

The Role of the 9-Amino Acid Motif in cAMP-dependent Modulation of Na⁺-Ca²⁺ Exchanger-- Since the presence of the 9-amino acid insertion comprising a putative nucleotide binding domain is the major distinguishing structural feature of the frog cardiac Na⁺-Ca²⁺ exchanger (fNCX1a), we constructed a deletion mutant, fNCX1a, without the 9-amino acid insertion. Fig. 5 compares the differences in Na⁺-dependent Ca²⁺ uptake measured by photoemission of aequorin in oocytes injected with the dog heart NCX1, the frog heart fNCX1a, and the mutated frog heart fNCX1a exchangers. Oocytes from each group were separated into two subgroups, one of which following the Na⁺ loading period was maintained in control conditioning solution and exposed to the control test solution, while the other was maintained in conditioning solution and subjected to the test solutions supplemented with 500 µM CPT-cAMP. The histograms of Fig. 5 show no significant differences in Na⁺-dependent Ca²⁺ uptake between the control and CPT-cAMP exposed oocytes expressing either the dog heart NCX1 or the mutated frog heart fNCX1a exchangers, both of which lack the 9-amino acid nucleotide binding domain. In sharp contrast, the Na⁺-dependent Ca²⁺ uptake was significantly smaller in the presence of CPT-cAMP in the oocytes expressing the frog heart fNCX1a isoform, consistent with the idea that the 9-amino acid domain, is critical for cAMP-mediated regulation of the exchanger.

View larger version (31K): [in this window] [in a new window]   Fig. 5.   The differences in Na+-dependent Ca2+ uptake in Na+-loaded oocytes injected with dog heart NCX1, intact frog heart fNCX1a, and mutated frog heart fNCX1a() Na+-Ca2+ exchangers in the presence and absence of 500 µM CPT-cAMP. Oocytes from all groups were injected with aequorin and loaded with Na+ by incubation for 30 min at room temperature in conditioning K+/Ca2+-free Barth's solution supplemented with 30 µM nystatin. After Na+ loading control subgroups of the oocytes were maintained in regular conditioning solution, whereas test subgroups of the oocytes were maintained in conditioning solution supplemented with 500 µM of CPT-cAMP. Photon emission was measured following exposure of the oocytes from the control subgroups to the regular test, K+/Na+-free Barth's solution and oocytes from the test subgroups to the test, K+/Na+-free Barth's solution supplemented with 500 µM CPT-cAMP. The number of oocytes tested is indicated above each column. The asterisk (*) denotes a significantly different value from the corresponding control value at p < 0.05.

	DISCUSSION

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We have successfully expressed a functional cAMP-regulated frog Na⁺-Ca²⁺ exchanger (fNCX1a) in Xenopus oocytes. The most distinguishing structural feature of the construct is the presence of a consensus ATP/GTP binding 9-amino acid motif (P-loop), located between residues 636 and 646 of the main cytoplasmic linker. Deletion of this motif from the clone (fNCX1a) abolished the cAMP-dependent regulation of the exchanger (Fig. 5). fNCX1a, constructed from H3/H6 and a genomic clone X9, contained a small (7.9%) divergence in the overlapping 178-amino acid sequence and only 6.4% divergence at the nucleotide level at the same region. This divergence is in the order of interspecies polymorphism for orthologous genes, since it is substantially lower than that observed between the genes of the same family in mammals (e.g. 35-40% divergence in rat) (23). Whether this divergence arises from polymorphic variation of the frog subspecies (where the structure of genus Xenopus is not well defined) or from the unlikely presence of a novel member of NCX family is not as yet clear. Irrespective of whether the clone represents a chimera of orthologous genes from related species, the presence of the putative protein kinase A phosphorylation site and Ca²⁺ binding domain, the similarity in cAMP-mediated regulation between the expressed protein and the exchanger in native frog myocytes (15), as well as the abundance of fNCX1a sequence in mRNA of frog heart compared with other tissues,² suggests that the clone represents a legitimate molecular model to study the functional implication of the novel motif.

Voltage clamp studies in early 1970s have shown that developed tension in frog heart has a dominant tonic and a small phasic component (1, 2, 31). In the presence of catecholamines, however, the phasic (I_Ca-dependent) component of tension is strongly enhanced, while the sigmoid Na⁺-dependent tonic component is strongly suppressed (32). This dual effect of catecholamines was thought to result from both increased Ca²⁺ influx via the Ca²⁺ channels and enhanced uptake of Ca²⁺ by the sarcoplasmic reticulum. In light of recent studies showing the virtual absence of SERCA II gene of Ca-ATPase and phospholamban proteins in the frog heart (33-35), the exchanger may be the molecular site that mediates the relaxant effects of catecholamines. At first the suppressive effect of cAMP on the exchanger appears contraintuitive, but considering that the Ca²⁺ channel and the plateau of the action potential are significantly enhanced in the presence of catecholamines (32, 36), necessarily increasing Ca²⁺ influx via the exchanger, the cAMP-dependent suppression of the exchanger may be the appropriate evolutionary solution to stem the tide of large Ca²⁺ influx that would result. Thus, the suppression of the tonic Ca²⁺ influx pathway may contribute to the early fall in tension observed during depolarizing pulses in the presence of isoproternol (32).

The -agonist/protein kinase A-induced temporal shift of the fraction of contractile Ca²⁺, transported into the cell during the action potential from the exchanger to the Ca²⁺ channel, may be related to the evolutionary requirement of the fight and flight reflex in almost all nonmammalian vertebrates (and possibly prenatal mammals) lacking significant SERCA II and intracellular Ca²⁺ release pools. Thus, the heart of these animals may utilize the same -adrenergic regulatory mechanism for both the control of phasic (Ca²⁺ channel) and tonic (exchanger) transport of Ca²⁺ and development of tension. Under sedentary conditions the exchanger would primarily deliver and extrude the contractile Ca²⁺ into and out of the cell. Upon sympathetic stimulation, as the heart shifts to the faster Ca²⁺ delivery pathway via the phosphorylated Ca²⁺ channel, the influx of Ca²⁺ via the exchanger would be suppressed, providing the heart with faster but shorter contractions to accommodate the faster heart rate. Whether such a protein kinase A-dependent-regulatory mechanism can be made to operate by genetic manipulation of the mammalian Na⁺-Ca²⁺ exchanger when the exchanger is overexpressed (37, 38) in heart failure remains to be tested.