From the Departments of Pharmacology and Medicine, Georgetown
University Medical Center, Washington, DC 20007 and
Laboratory of Biochemistry, Swiss Federal Institute of
Technology (ETH), Universitatsstrasse 16, CH-8092
Zürich, Switzerland
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ABSTRACT |
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Na+-Ca2+ exchanger
is one of the major sarcolemmal Ca2+ transporters of
cardiac myocytes. In frog ventricular myocytes the exchanger is
regulated by isoproterenol via a
-adrenoreceptor/adenylate-cyclase/cAMPdependent signaling
pathway providing a molecular mechanism for the relaxant effect of the
hormone. Here, we report on the presence of a novel exon of 27-base
pair insertion, which generates a nucleotide binding motif (P-loop) in
the frog cardiac Na+-Ca2+ exchanger. To examine
the functional role of this motif, we constructed a
full-length frog heart Na+-Ca2+ exchanger
cDNA (fNCX1a) containing this exon. The functional expression
of fNCX1a in oocytes showed characteristic voltage dependence, divalent
(Ni2+, Cd2+) inhibition, and sensitivity to
cAMP in a manner similar to that of native exchanger in frog myocytes.
In oocytes expressing the dog heart NCX1 or the
frog mutant (
fNCX1a) lacking the 9-amino acid exon, cAMP failed to
regulate Na+-dependent Ca2+ uptake.
We suggest that this motif is responsible for the observed cAMP-dependent functional differences between the frog and
the mammalian hearts.
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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.
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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.
During subcloning of the fNCX1a coding and 3'-UTRs into transcription-competent vector pAGA (20) (a gift of L. Birnbaumer, UCLA, CA) in order to reduce the chances of point mutations due to PCR, only the 5'-terminal part (positionIn Vitro Transcription-- cRNA for the injection into the oocytes was synthesized in vitro from either plasmid pF1a/MC or pF1a/AGA using the mCAPTM 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().
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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 (GenBankTM 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).
The deduced amino acid sequence of H6 and H3 showed 87.8% identity to the corresponding part of the dog sequence. However, unlike mammalian clones, the 27-base pair insertion generates an ATP/GTP binding motif (P-loop) by adding the GKS sequence to GKILY (essential amino acids are underlined), thus forming the consensus P-loop structure (A/G)-X4-G-K-(S/T) (25). In the sequence encoded by H3 and H6, the putative protein kinase A phosphorylation site (4), and the Ca2+-binding domain (26) seen in the mammalian NCX1 proteins are conserved (Fig. 1). To examine the functional role of this motif, we constructed the full-length frog Na+-Ca2+ exchanger. Since the frog heart cDNA library lacked coding sequences for the N terminus of the Na+-Ca2+ exchanger, the X. laevis genomic library was screened using a fragment (nucleotides 1626-1773) produced by PCR at the N terminus of the H3 clone. The stretch coding for the remainder of the region was found in X9 (GenBankTM accession no. X90839). A small diversity was found in the region of the overlap between H3/H6 and X9 (534 nucleotides, 178 amino acids). Alignment of the amino acid sequences showed the identity of 92.1%, and the similarity of 94.5% (1-amino acid insertion, 13-amino acid changes, and 17-nucleotide substitutions which do not cause changes in amino acid residues). It should be noted that the portion that was supplemented by the genomic sequence does not contain any known regulatory motifs (4). We therefore constructed the full-length "composite" frog clone (fNCX1a) for expression studies in Xenopus oocytes.
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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 INa-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.
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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
INa-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 INa-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 INa-Ca,
since all of the recovered current was blocked by 5 mM
Ni2+ (Fig. 4A).
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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.
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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 (ICa-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.
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FOOTNOTES |
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* This work was supported in part by National Institutes of Health Grant R01-HL16152-22 (to M. M.) and Swiss National Science Foundation Grant 31-30859.91 (to E. C.).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. Section 1734 solely to indicate this fact.
§ To whom all correspondence should be addressed: Depts. of Pharmacology and Medicine, Georgetown University Medical Center, 3900 Reservoir Road, NW, Washington, DC 20007. Tel.: 202-687-8464; Fax: 202-687-8458; E-mail: moradm{at}gunet.georgetown.edu.
1 The abbreviations used are: PCR, polymerase chain reaction; UTR, untranslated region; CPT-cAMP, 8-(4-chlorophenylthio)-cAMP.
2 A. Kraev and E. Carafoli, unpublished data.
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REFERENCES |
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