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J Physiol (2003), 548.3, pp. 677-689
© Copyright 2003 The Physiological Society
DOI: 10.1113/jphysiol.2002.036426
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ABSTRACT |
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The cardiac Na+-Ca2+ exchanger (NCX1) is one of the major sarcolemmal Ca2+ transporters of cardiomyocytes. Structure-function studies suggest that-adrenergic inhibition of NCX1, as reported for frog, but not mammalian hearts, may be associated with a unique splice variant of frog cardiac NCX1 where insertion of an extra exon completes the coding of a nucleotide binding P-loop. To test the involvement of the P-loop in cAMP-mediated regulation of NCX1 we used four stably transfected human cell lines (a previously established line of baby hamster kidney (BHK) cells and three new lines of human embryonic kidney (HEK) cells) expressing: (1) wild-type dog NCX1 (dog NCX1); (2) wild-type frog NCX1 (frog NCX1); (3) chimeric frog-dog NCX1 incorporating the completed P-loop from the frog NCX1 into the dog NCX1 sequence (frog/dog NCX1); and (4) a mutated frog NCX1 where a putative protein kinase A (PKA) site was disrupted by substitution of a single serine residue with glycine (S374G frog NCX1). Structural expression of these NCX1 constructs was confirmed using Western blot analysis of extracted proteins and immunofluorescence imaging. The NCX1-generated current (INa-Ca) was reliably measured in cells expressing dog (2.0 ± 0.15 pA pF-1), frog (0.6 ± 0.1 pA pF-1) and frog/dog (0.6 ± 0.1 pA pF-1) NCX1, but less so in those expressing S374G frog NCX1 (0.3 ± 0.1 pA pF-1). Addition of 100 µM 8-bromoadenosine 3',5' cyclic monophosphate (8-Br-cAMP) suppressed INa-Ca of frog and frog/dog NCX1 by 60-80 %. The suppression of INa-Ca was smaller and transient in cells expressing S374G frog NCX1, and absent in cells expressing dog NCX1. Intracellular Ca2+ (Ca
)-transients, activated by rapid withdrawal of Na+, were also downregulated in the frog and frog/dog NCX1 and to a smaller and transient extent in S374G frog NCX1. Our findings suggest that the suppressive effect of
(Received 26 November 2002; accepted after revision 7 February 2003; first published online 7 March 2003)
Corresponding author L. Cleemann: Georgetown University, 4000 Reservoir Road NW, Washington, DC 20007, USA. Email: cleemanl{at}georgetown.edu
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INTRODUCTION |
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The sarcolemmal Na+-Ca2+ family of proteins (NCX) appear to have evolved throughout the biosphere, from cyanobacteria and yeasts to humans, with the function of removing Ca2+ from the cytosol in exchange for Na+ (Reuter & Seitz, 1968; Baker et al. 1969). This electrogenic transport process is driven exclusively by the electrochemical gradients of Na+ and Ca2+, which also appear to allosterically regulate intracellular Ca2+-sensing sites for activation (DiPolo, 1979), and Na+ sites for inactivation of the exchanger (Hilgemann, 1990). While this regulatory framework is generally conserved among species, other aspects of regulation of NCX appear to vary significantly from species to species or within an individual species, where functional diversity of NCX is produced by multiple genes (NCX1, NCX2, NCX3), each of which is expressed as tissue-specific splice variants (Quednau et al. 1997).
The molecular structure of NCX (~900-1000 amino acids) has clusters of membrane spanning domains at both ends (Fig. 1B) separated by a long regulatory cytoplasmic loop that comprises about half of the entire molecule and can be cleaved by chymotrypsin (Philipson et al. 1988; Hilgemann, 1990) or mostly deleted (Matsuoka et al. 1993; Ottolia et al. 2001) without significantly altering the coupled Na+-Ca2+ translocation. Some regulatory sites of the cytoplasmic loop, e.g. the XIP region associated with Na
-induced inactivation (Li et al. 1991) as well as acidic triads of aspartic acid associated with Ca-mediated activation (Matsuoka et al. 1995) are highly conserved in vertebrate and molluscan NCXs. In contrast, the alignment of vertebrate cardiac NCX1 primary amino acid sequences in Fig. 1A shows species-dependent variations in the occurrence of a putative protein kinase A (PKA) site and a P-loop. The putative PKA site (RKxxS) is present in both mammals and frog, but not in trout, as the phosphorylation reaction requires an essential serine residue. In addition, the presence of a P-loop (Saraste et al. 1990), or Walker A motif (GxxxxGKS), a potential nucleotide-binding site comprising nine amino acids found thus far only in the frog heart NCX1 ('exon X'; Fig. 1A), may be involved in mediating the cAMP-induced regulation of the frog NCX1.
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Figure 1. Structural properties and expression of cardiac Na+-Ca2+ exchanger (NCX1) constructs A, alignment of vertebrate amino acid sequences of cardiac NCX1 proteins in the vicinity of the protein kinase A (PKA) and P-loop motifs (numbering of residues corresponds to the dog NCX1 (Nicoll & Philipson, 1991); database protein accession numbers: dog AAA62766 (Nicoll et al. 1990), cat AAB41941 (Menick et al. 1996), human AAA35702 (Komuro et al. 1992), rat CAA48273 (Low et al. 1993), mouse AAB46708 (Kim & Lee, 1996), rabbit B53335/I46959 (Kofuji et al. 1993), trout AAF06363 (Xue et al. 1999) and frog CAA62345/CAA62344 (Iwata et al. 1996). B, illustration of the topology of NCX1 (top) and the variations in the main cytoplasmic loop of the isoforms we investigated expressed in different cell lines. The topology of the | ||
In this report we have examined if these sites are involved in cAMP-mediated regulation, based on the idea that the binding of ATP to the P-loop may be a required step preceding the PKA-dependent phosphorylation and on the finding that the cAMP-mediated suppression of INa-Ca in Xenopus oocytes is lost when exon X of the expressed recombinant frog NCX1 is deleted (Shuba et al. 1998).
The strategy chosen in this study was to establish four stably transfected human kidney cell lines (HEK293 or BHK) expressing wild-type dog NCX1, wild-type frog NCX1, a dog-frog chimera incorporating the frog NCX1 exon X in the dog NCX1 sequence, and frog NCX1 with a single point mutation (S374G) deleting a potential PKA-phosphorylation site (Fig. 1B). We verified the expression of NCX1 using Western blot immunoassay, cell immunostaining, and measured Ni2+-sensitive INa-Ca (Fan et al. 1996; Hinde et al. 1999) and Ca2+ fluxes to determine the functional activity of the exchanger. We found that the P-loop from the frog NCX1 conveys the cAMP-mediated regulation on chimeric dog NCX1, and that disruption of the putative PKA site results in a somewhat smaller and transient regulatory response. These experiments were preceded by the observation that the chimeric dog NCX1 construct was inhibited by 50 µM 8-bromoadenosine 3',5' cyclic monophosphate (8-Br-cAMP) when expressed in Xenopus oocytes (Oz et al. 1998), which prompted the question whether such regulation was limited to amphibian cells or could it also be induced in mammalian cells.
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METHODS |
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NCX1 constructs
NCX1 constructs were based on the cloned frog (Iwata et al. 1996) and dog (Nicoll et al. 1990) NCX1, which share a high degree of identity, but differ in the main cytoplasmic loop (Fig. 1A) where the frog sequence has a nine-amino acid insertion (encoded by the 27 bp of exon X) that completes the consensus sequence for a nucleotide-binding domain (P-loop or Walker A motif; Iwata et al. 1996). To complete the otherwise disrupted P-loop in dog NCX1 we constructed a chimera by substituting its 528 bp PvuII/Xho I segment with the respective 555 bp Pvu II/Xho I segment from the frog NCX1 cDNA. This provides not only the insertion of the nine amino acids completing the P-loop but also 14 equivalent and five non-equivalent amino acid substitutions. The role of a putative PKA site was tested by introducing a single point S374G mutation in the frog NCX1 (corresponding to serine 357 in the dog NCX1). For this, the frog NCX1-coding Sal I, BamHI cassette was subcloned into pAlter-1 vector (Promega, Inc., Madison, WI, USA) using the Altered Sites II in vitro Mutagenesis System (Promega) according to the manufacturer's manual. The mutated primer was 5'-GCAAGGAAAGCTGTG gAGTATGCATGAAGT-3'.
Cell transfection and culture
BHK cells expressing dog NCX1 were generously provided by Dr Kenneth Philipson, UCLA (Nicoll et al. 1990, 1996; Linck et al. 1998). The three new NCX1 cDNAs were subcloned into a mammalian expression vector pcDNA3 and then transfected into HEK293 cells, which were chosen as hosts because they show no measurable Ni2+-sensitive NCX1 activity. Less auspiciously, it has been reported that protein kinase A (PKA)- and protein kinase C (PKC)-mediated regulation was reduced in HEK cells overexpressing a K+ channel (Martel et al. 1998). The frog NCX1 sequences were subcloned as Hin dIII, Mlu I (blunt) cassettes into pcDNA3 vector at its Hin dIII, Not I (blunt) sites. Transfection was carried out using the standard calcium phosphate method. To select the transfected cells, different concentrations of G418 (Geneticin, Gibco BRL 11811-031; Invitrogen, Inc., Carlsbad, CA, USA) were applied to the transfected and non-transfected cells. The effective concentration was identified by 100 % inhibition of non-transfected cells. Transfected cells were grown in MEM (Gibco-BRL) supplemented with G418, 1 % penicillin- streptomycin and 10 % fetal bovine serum at 37 °C in humidified air with 5-7 % CO2.
Clones of transfected cells were plated onto laminin-coated micro-coverslips at a density of ~400 cells mm-2 for patch clamp and immunofluorescent histochemical staining studies, or they were grown in 75 m1 culture flasks (2 
105 cells ml-1 suspension) for immunoblot assay.
Western blot analysis of expression of NCX1 protein
Cultured cells treated with 10 % trichloroacetic acid and acetone were homogenized in 50 volumes of buffer (10 mM Tris-HCl and 2 % SDS, pH 7.4) and centrifuged at 700 g for 10 min. Samples of homogenate (50 µg protein) from transfected or non-transfected HEK293 cells were separated on 8 % SDS-PAGE gels and transferred to a nitrocellulose membrane. The transferred blots were exposed to a blocking solution (20 mM Tris-HCl at pH 7.6, 137 mM NaCl, 5 % non-fat milk and 0.1 % Tween-20) overnight, and then exposed to primary antibody (1:1000) in the same solution for 4 h, and a secondary alkaline phosphatase-conjugated antibody to mouse IgG (1:5000) for 2 h. Tetrazolium was used as substrate for colour development. All processes were carried out at room temperature.
Immunofluorescent histochemical labelling of NXC1 protein in HEK293 cells
Fixation and indirect immunofluorescent staining of transfected HEK293 cells (Frank et al. 1992) was performed using a monoclonal antibody to mouse NCX1 diluted 1:200 in blocking solution (0.3 % Triton X-100, 5 % non-fat milk and 0.2 % sodium azide in PBS) followed by incubation with a FITC-conjugated secondary donkey anti-mouse IgG antibody (1:100). Control experiments were performed using secondary antibody alone. Coverslips were fastened with VectaShield mounting medium (Vector Laboratories, Burlingame, CA, USA), for the imaging on a Noran confocal fluorescence microscope.
Whole-cell voltage-clamp recording
For electrophysiological experiments, coverslips with attached transfected cells were placed in a chamber and perfused at a rate of 1-2 ml min-1 with basic external Tyrode solution (see below). Single cells were voltage-clamped in the whole-cell configuration (Hamill et al. 1981) using 2-5 M
patch pipettes (TWiSOF borosilicate glass capillaries; World Precision Instruments, Inc., Sarasota, FL, USA) and a model 9000 Dagan amplifier (Dagan Instruments, Minneapolis, MN, USA) controlled by a IBM-compatible computer equipped with hard- and software for data acquisition. After establishing the whole-cell patch, a 'ramp-pulse' protocol initiated by short-step depolarization to +40 mV (to activate NCX1-generated current (INa-Ca)), was applied to measure the voltage dependence of the INa-Ca (Fig. 2B). Collected current recordings were analysed using pCLAMP 5.5 software (Axon Instruments, Inc, Union City, CA, USA) and ORIGIN (OriginLab, Northampton, MA, USA).
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Figure 2. Measurement of the Ni2+-sensitive NCX1-generated current (INa-Ca) (A-C) and Ca2+ fluxes (D and E) in transfected HEK293 cells expressing frog/dog NCX1 A, suppression of INa-Ca by 5 mM Ni2+ (Ni2+). The whole-cell current was measured at +40 mV at 10 s intervals using the ramp-clamp protocol illustrated in B (top). The plotted value is the average current measured in a 100 ms interval at the end of the plateau phase of the ramp-clamp protocol. The arrow early in the record indicates the time when the basic external Tyrode solution was replaced by a modified Tyrode solution with 10 mM Ca2+ and zero K+. The current traces in B were recorded before (a), during (b) and after (c) exposure to 5 mM Ni2+ at the times indicated in A. The current-voltage relation in C is the Ni2+-sensitive component of the current ((Ia + Ic)/2 - Ib) measured at different potentials during the ramp clamp (cell capacitance = 37 pF). D, changes in Ca2+-dependent fluorescence of Fura-2 AM (F380) measured simultaneously in 7 individual cells exposed to Na+- and K+-free solution (Cs+ substitution) for ~30 s in the absence and presence of 5 mM Ni2+. Each trace represents [Ca2+]i in a single cell. Notice that [Ca2+]i was measured with Fura-2 AM using excitation at 380 nm, so that a decrease in fluorescence intensity corresponds to an increase in [Ca2+]i. E, average values of the change in fluorescence intensity evoked by Na+ withdrawal in the absence (0 Na+) and presence (0 Na+ + Ni2+) of 5 mM Ni2+. Changes in F380 were measured for individual cells in arbitrary units during two matched 30 s intervals (arrows in D) and the Ni2+-sensitive component was calculated as the difference (Diff.). | ||
Changes in the baseline current were often measured at +40 mV following Ni2+ exposure (Fig. 3D). Such changes appear to reflect a leak current that is insensitive to Ni2+, but sensitive to mechanical destabilization of the patch-electrode produced by the rapid exchange of 'puffing' solutions. A reduction in flow rate did not alleviate such problems since it resulted in currents that changed gradually from one voltage-clamp episode to the next, indicating also that application of Ni2+ during a single test pulse is not sufficient (Fig. 3A).
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Figure 3. Effect of 100 µM 8-Br-cAMP on isoforms of NCX1 expressed in different cell lines Each of the four panels illustrates the time course of the change in the whole-cell current during exposure to 100 µM 8-bromoadenosine 3',5' cyclic monophosphate (8-Br-cAMP) in one of the cell lines expressing variants of NCX1 as indicated by the labelling of the panels: A, dog NCX1; B, frog NCX1; C, frog/dog NCX1; D, S374G frog NCX1. The current was measured repeatedly every 20 s over a period of 400-700 s. Each data point represents the average current measured at +40 mV in a 100 ms interval at the end of the 400 ms plateau-phase, just prior to the onset of the descending ramp-clamp (cf. Fig. 4A and C). Repeated brief exposures to 5 mM Ni2+ (Ni2+) was used to ascertain magnitude of the Ni2+-sensitive component of the raw current. Typically Ni2+ was applied shortly before 8-Br-cAMP and 200-300 s later when the suppressive effect was often maximal, but the timing was sometimes varied, e.g. to test if the Ni2+ response was repeatable (Fig. 2C) or if the Ni2+-insensitive component remained constant while the Ni2+-sensitive component was changing (Fig. 2B). B, C, and D also show the average suppression of the Ni2+-sensitive component of current INa-Ca, measured at 40 mV after 40 and 200 s exposure to 8-Br-cAMP The number of voltage-clamped cells is indicated at the error bars. Since the Ni2+-insensitive background current (leak current, incomplete block of INa-Ca, etc.) was not always completely constant, we frequently used interpolation between two or more measured values when estimating INa-Ca e.g. to calculate INa-Ca, corresponding to 40 s exposure to 8-Br-cAMP. | ||
Intracellular Ca2+ measurements
For measurement of Ca2+-dependent fluorescence signals, cells were incubated for 20-30 min with 5 µM Fura-2 AM (Molecular Probes, Inc., Eugene, OR, USA) before experiments, and then transferred to a two-dimensional imaging system (intensified CCD camera, Attofluor; Atto Bioscience, Rockville, MD, USA) capable of simultaneous measurements of Ca from multiple cells at ~1 Hz. The fluorescence signals were obtained with excitation at 380 nm, where elevation of [Ca2+]i results in reduced fluorescence. One of the advantages of the imaging system is that it provides statistical significance by sampling many more cells than can be subjected to detailed voltage-clamp experiments.
Solutions and chemicals
The basic external Tyrode solution contained (mM): NaCl 137, KCl 5.4, CaCl2 2, Hepes 10, MgCl2 1, glucose 10 (pH 7.4). An electronically controlled multibarrelled puffing system (Cleemann & Morad, 1991) was used for rapid (< 50 ms) exchange of the external solution. For recording of INa-Ca, voltage-clamped cells were superfused with a modified K+-free Tyrode solution with 10 mM CaCl2 and 5.4 mM Cs+ instead of K+. The dialysing pipette solution contained (mM): KCl 40, potassium aspartate 60, TEA-Cl 10, NaCl 20, Mg-ATP 0.1, BAPTA 0.1 and Hepes 10 (pH 7.2). Tetraethylammonium (TEA) was included to block delayed outward rectifier K+ channels. Complete replacement of intracellular K+ with Cs+ or TEA was avoided to prevent possible unexpected effects of these cations. When measuring Ca in multiple cells, we activated NCX1 in the reverse mode using a Na+- and K+-free Tyrode solution containing 10 mM CaCl2 and Cs+ as the major monovalent cation. NiCl2 (5 mM) was used routinely as a blocker to assess the current or Ca2+ flux generated by NCX1. For this purpose Ni2+ was preferred to KB-R7943 (Iwamoto et al. 1996) due to its effectiveness (Hinde et al. 1999), fast kinetics and reversibility (Fan et al. 1996). Drugs were dissolved in the external Tyrode solution in the appropriate concentrations. All the experiments were performed at room temperature (22-23 °C).
Mouse monoclonal anti-NCX1 antibody IgM (MA3-926) to the common NCX amino acid sequence was purchased from Affinity Bioreagents, Inc. (Golden, CO, USA), FITC-conjugated secondary antibody (donkey anti-mouse IgG) was from Jackson ImmunoResearch Laboratories, Inc. (West Grove, PA, USA) and 8-Br-cAMP sodium salt was from Sigma Chemical Co. (St Louis, MO, USA).
Statistical analyses
All data are presented as means ± S.E.M. (n = number of observations). The statistical analysis was carried out using Student's t test. Differences were considered statistically significant when P < 0.05. All illustrations are representative of results found in three or more experiments.
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RESULTS |
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Structural expression of native and modified NCX1 in stably transfected cell lines
Four different cell lines stably expressing isoforms of NCX1 (Fig. 1B) were generated as described in the Methods section in order to compare cAMP-mediated regulation of dog and frog NCX1, and examine the roles of a nucleotide-binding P-loop and a putative PKA site. The P-loop, which is present only in the frog NCX1 sequence, was inserted into dog NCX1 in a chimeric construct (frog/dog NCX1). The involvement of the putative PKA site was tested by mutating its essential serine residue to glycine in the frog NCX1 (S374G frog NCX1). To evaluate the level of expression of frog, frog/dog and S374G frog NCX1 in our newly developed HEK293 cell lines, we used anti-NCX1 monoclonal antibody generated against the common amino acid sequence of NCX1.
Western blot analysis. This was performed to assess the expression of NCX1 proteins. Cells were homogenized to remove nuclei, and proteins were separated by SDS-PAGE, transferred to a nitrocellulose membrane, and probed with a monoclonal antibody to the mouse NCX1. The 120 and 160 kDa protein bands associated with the mature exchanger (Frank et al. 1992) were distinctly present in all three cell lines (Fig. 1C). Immunoreactivity was strong in the HEK293 cells expressing frog and frog/dog NCX1, but somewhat weaker in cells expressing the S374G frog NCX1, and no immunoreactivity was apparent in the non-transfected cells. These data suggest that Na+-Ca2+ exchanger protein is well expressed in the three new cell lines.
Immunolocalization. Exposure of transfected cells to monoclonal mouse NCX1 antibody followed by the FITC-labelled secondary antibody produced the fluorescence staining patterns shown in the confocal images of Fig. 1D. The transfected HEK293 cell lines produced bright fluorescence images confined mostly to the periphery of the cells. Non-transfected cells showed little or no fluorescence with identical exposures to the NCX1 antibody. The patterns of staining seen with confocal sectioning suggest that the expressed NCX1 proteins are targeted predominantly to the membrane.
Taken together the results from Western blot and immunofluorescent staining show that the NCX1 proteins are expressed in the three new cell lines and are translocated to the cell membrane.
Evaluation of functional expression of NCX1
To test for functional expression of NCX1, we measured INa-Ca in single voltage-clamped cells (Fig. 2A-C) and compared it to changes in Ca2+-induced fluorescence in response to Ca2+ influx recorded simultaneously in multiple cells (Fig. 2D and E). Figure 2 illustrates representative data from HEK293 cells expressing frog/dog NCX1. The activity of NCX1 in the Ca2+ influx mode (reverse mode, outward INa-Ca) was measured routinely at +40 mV in zero K+ external solutions containing high extracellular Ca2+ (10 mM). The current measured under these conditions was strongly, and reversibly suppressed by rapid application of 5 mM Ni2+ (Fig. 2A and B). Each depolarizing pulse to +40 mV was followed by a ramp clamp to -120 mV to check the voltage dependence of INa-Ca (Fig. 2B). The illustrated ramp-clamp protocol, preceded by an initial 200 (or 400) ms depolarization to +40 mV was intended in part to simulate the cardiac action potential and accompanying influx of Ca2+, prior to quantifying the activity of the exchanger at different potentials. The Ni2+-sensitive component of the current measured during the ramp clamp (Fig. 2C) had a reversal potential of around -20 mV, which was similar to that measured by others and by us in frog cardiomyocytes (Fan et al. 1996) and in Xenopus oocytes expressing the recombinant NCX1 (Shuba et al. 1998).
The magnitude of INa-Ca in different cell lines was quantified relative to the membrane capacitance, which is roughly proportional to the cell size. The densities of INa-Ca in the three HEK293 cell lines developed by us were compared (Table 1) with those measured in BHK cells expressing the dog NCX1. The measured current densities (0.3-2 pA pF-1) were also comparable to those measured in cardiomyocytes of different mammalian species (Sham et al. 1995; 1-4 pA pF-1). In cells expressing the S374G mutant frog NCX1 with a disrupted putative PKA site we consistently found relatively small and variable INa-Ca. Cells expressing the frog and frog/dog NCX1 had INa-Ca densities that were significantly smaller than in BHK cells expressing the dog NCX1. The voltage dependence of INa-Ca was similar in all three cell lines (data not shown). Non-transfected HEK293 cells produced no measurable Ni2+-sensitive INa-Ca.
Ca2+ movements mediated by different NCX1 clones were measured in multiple cells loaded with Fura-2 AM in response to withdrawal of extracellular Na+. The traces in Fig. 2D correspond to the Ca2+ signals measured simultaneously in a number of cells all showing a decrease in fluorescence (F380) reflecting an increase in Ca, when excited at 380 nm. To activate the reverse mode of NCX1, we exposed the transfected HEK293 cells for ~40 s to a Na+- and K+-free external solution containing 10 mM Ca2+. Figure 2D shows that this protocol produced a large decrease in fluorescence associated with an increase in Ca that was followed by nearly complete recovery when cells were returned to control solution and it was greatly reduced when 5 mM Ni2+ was added to the Na+-free solution to block NCX1. The Ni2+-sensitive component of the Ca signals was quantified as the average of the difference between the cellular responses recorded in the absence (0 Na+) and presence (0 Na+ + Ni2+) of Ni2+. The general slow decline of the fluorescence intensities during the recording period might appear as incomplete recovery following the exposures to Na+-free solution, but is probably due mostly to bleaching of Fura-2 AM, since it was found to depend on the intensity of light used for fluorescence excitation (data not shown).
The electrophysiological and fluorescence measurements produce consistent results for the activity of NCX1, and complement each other, since one set of data produces kinetic details on INa-Ca in single dialysed cells, while the other provides useful data on the properties of NCX1 in multiple intact cells.
cAMP-mediated regulation of NCX1
Suppression of INa-Ca. To examine the regulation of the various NCX1 clones via the -receptor/adenylate-cyclase/cAMP-dependent pathway (Fan et al. 1996), we exposed single voltage-clamped cells from each of the four cell lines to the cell-permeable non-hydrolysable cAMP analogue 8-Br-cAMP. The sampled records in Fig. 3 were selected to illustrate representative effects of cAMP and some difficulties associated with this approach.
To substantiate the validity of the data and relate them conclusively to the activity of the exchanger, we used repeated rapid applications of 5 mM Ni2+ to suppress the net current and recover it upon washout (Fig. 3A-D). Such records show that it is indeed the Ni2+-sensitive component of the total current that is suppressed by 8-Br-cAMP and that this suppression occurs gradually over a period of a few minutes (Fig. 3B and C). The major observation, derived from the current measured at +40 mV, is the extent of the inhibitory effects of cAMP on INa-Ca generated by different clones of NCX1. Figure 3A, for instance, shows little or no effect of cAMP on dog NCX1, while by contrast Fig. 3B shows that cAMP caused strong downregulation of frog NCX1. A similar suppression was observed in cells expressing frog/dog NCX1 (Fig. 3C), but in the S374G frog NCX1 with disrupted PKA site cAMP caused only a small and transient suppressive effect (Fig. 3D).
When evaluating the recordings of INa-Ca in all cell lines, we observed: (a) a small rapid shift in baseline current occasionally following brief exposure to Ni2+ or, in fact, following any solution change, and (b) a slow gradual change (typically a decline) both prior to application of 8-Br-cAMP (Fig. 2A) and in long-lasting control experiments in the absence of 8-Br-cAMP (data not shown). It is important to note, therefore, that in cells expressing dog NCX1 (> 30 cells examined) we never observed the fairly rapid onset of the 8-Br-cAMP effect illustrated in Fig. 3B-D, nor did we observe a significant long-lasting effect. In this sense, the absence of cAMP-mediated regulation in dog NCX1 as shown Fig. 3A was highly reproducible.
We also quantified the suppression of INa-Ca by 8-Br-cAMP in cells expressing frog and frog/dog NCX1 (Fig. 3B vs. C) and found no significant difference in the effectiveness of cAMP. In both cell lines, the response had a rapid onset, developed over a period of 2-4 min, and was sustained as long as 8-Br-cAMP was present. Recovery following washout was slow and often incomplete after 10-15 min. The Ni2+-sensitive INa-Ca of frog NCX1 was suppressed by 28 ± 7 % (n = 11) after 40 s of exposure to 8-Br-cAMP and by 66 ± 5 % (n = 11) after ~200 s (histograms in Fig. 3B). A similar suppressive effect was seen in chimeric frog/dog NCX1: 49 ± 6 % (n = 9) after 40 s and 72 ± 4 % (n = 8) after 200 s (Fig. 3C). Although the S374G frog NCX1 showed an initial suppressive effect of cAMP (27 ± 5 % (n = 6) after 40 s), the later suppressive effects after 200 s were virtually absent (Fig. 3D).
To examine the effect of 8-Br-cAMP on NCX1 operating in both forward and reverse mode, we measured the voltage dependence of the Ni2+-sensitive current using the ramp-clamp protocol (Fig. 4A and C). The current-voltage (I-V) relation measured in the cell line expressing frog/dog NCX1 was strongly suppressed both in the outward (> -20 mV, reverse mode) and inward (< -40 mV, forward mode) direction of INa-Ca after exposure of the cells to 8-Br-cAMP for 3 min. In fact, the results suggest that the suppression of the inward INa-Ca is as strong as that of the outward INa-Ca. It should be noted, however, that [Ca2+]i, and thereby the reversal potential of INa-Ca, may be expected to change substantially during the voltage-clamp protocol, as is apparent from the observed gradual decrease in the current measured during the 400 ms conditioning pulse to +40 mV and the shift in the holding current measured after returning to the holding potential (-80 mV) at the end of the ramp clamp (trace a, Fig. 4A). Therefore, the suppression of inward INa-Ca by 8-Br-cAMP, and the resulting shift of the reversal potential, in part reflects a decrease in the amount of Ca2+ brought into the cell during the conditioning pulse (cf. Woo & Morad, 2001). The results in Fig. 4A and B are typical of the chimeric frog/dog NCX1 and similar to results obtained with the cloned frog NCX1 or native frog cardiomyocytes. In contrast, only a small suppressive effect of 8-Br-cAMP was found in the S374G frog NCX1, suggesting that the putative phosphorylation site might be critical for a robust cAMP regulatory effect (Fig. 4C and D).
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Figure 4. Changes in the voltage dependence of INa-Ca measured during inhibition of chimeric frog/dog (A and B) and S374G frog (C and D) NCX1 by 100 µM 8-Br-cAMP A and C, currents measured during the ramp-clamp protocol (top) under control conditions (a), during brief exposure to 5 mM Ni2+ (b) and after exposure to 100 µM 8-Br-cAMP (c). The current-voltage relations of the Ni2+-sensitive components (a - b and c - b) of the currents measured at different potentials during the ramp clamp are shown in B and D. The current-voltage relations in B show the steady-state suppression of frog/dog NCX1 measured ~300 s after addition of 8-Br-cAMP (cell capacitance = 28 pF), while D shows the smaller, typically transient, suppression of the S374G frog NCX1 measured ~40 s after addition of 8-Br-cAMP (cell capacitance = 26 pF). | ||
Suppression of Ca2+ fluxes. The effects of 8-Br-cAMP on different isoforms of NCX1 were also evaluated by simultaneously measuring [Ca2+]i in multiple cells. Figure 5 illustrates how the Ni2+-sensitive component of the Ca2+ influx activated on exposure to Na+-free solution (Fig. 5A), in cells expressing the frog/dog NCX1, was strongly suppressed following 5 min incubation with 8-Br-cAMP (Fig.5B), but recovered partially after 5 min washout (Fig. 5C). Each of the histograms to the right of the traces show the average amplitude of the decline in the Ca2+-dependent fluorescence measured in the absence and presence of 5 mM Ni2+ in Na+-free solution as well as the difference between them. Measured in arbitrary units of fluorescence intensity this difference was 16 ± 2 under control conditions, was reduced to 3 ± 1 after preincubation with 8-Br-cAMP and recovered to 12 ± 1 after washout. These results indicate that the strong inhibition of Ca2+ influx in the presence of cAMP in frog/dog NCX1 chimera is reversible following 5 min of washout. Similar effects were observed in cells transfected with frog NCX1 (data not shown).
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Figure 5. Suppression of the Ni2+-sensitive Ca2+ influx by 8-Br-cAMP in 12 cells expressing frog/dog NCX1 Ca2+ signals (F380) were measured with Fura-2 AM loaded cells during three periods of 200 s: before (A, 0-200 s), during (B, 500-700 s) and after (C, 1000-1200 s) exposure to 100 µM 8-Br-cAMP. Intervening periods of 300 s were used for preincubation with 8-Br-cAMP and washout, and were passed without fluorescence excitation, and therefore without measurements, to limit exposure to UV light (380 nm). Na+-free solution was applied twice during each 200 s interval of Ca2+ measurement, first in the absence (0 Na+) and then in the presence (0 Na+ + Ni2+) of 5 mM Ni2+. The histograms at the right side of each panel show average changes in the Ca2+ signals corresponding to the Ca2+ influx during 40 s in Na+-free solution in the absence (0 Na+) and presence (0 Na+ + Ni2+) of Ni2+ and the difference (Diff.) between these two values. | ||
Figure 6 shows the response of the [Ca2+]i, in cells expressing the S374G frog NCX1, to repeated exposures to 0 Na+ solution in the absence and presence of Ni2+. The conditions and protocol are basically the same as in Fig. 5 except that the time dependence the 8-Br-cAMP effect was probed by repeating the measurements of Ca2+ influx before and after evaluating the Ni2+ response. Due, in part, to bleaching, such repeated measurements generally produced Ca2+-dependent fluorescence transients of declining amplitude (Fig. 6A). It is significant therefore that following incubation with 8-Br-cAMP, the 0 Na+-induced fluorescence transients that were strongly suppressed (Fig. 6B, *), partially recovered in the continued presence of 8-Br-cAMP (Fig. 6B, **) consistent with the electrophysiological measurements in this cell line (Fig. 3D). Note also that this was the case even though less time was allowed for re-equilibration between the exposures to Na+-free solution. Thus it appears that Ca2+ influx is only transiently suppressed by 8-Br-cAMP in cells expressing the S374G frog NCX1 (cf. Fig. 3D). Furthermore, it should be noted that on Na+-withdrawal, S374G frog NCX1 (Fig. 6) did not produce the type of saturation ([Ca2+]i >> Kd 
140 nM; Cleemann & Morad, 1991) seen with frog/dog NCX1 3374G, where the unprocessed Fura-2 AM signals tended to settled near the dark-current level of the image intensifier tube (F380 15; Fig. 2D and Fig. 5). Although more quantitative measurements would be preferable, the Ca2+ data are consistent with reduced INa-Ca in cells expressing S374G frog NCX1 (Table 1).
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Figure 6. Transient suppression of Ca2+ influx by 8-Br-cAMP in 5 cells expressing the S374G frog fNCX1 The measurements were performed during three periods of 300 s before (A), during (B) and after (C) exposure to 100 µM 8-Br-cAMP with intervening periods of 300 s (not shown) used for preincubation with or washout of 8-Br-cAMP. The cells were exposed to Na+-free solution three times during each 300 s measurement interval to bracket an exposure where Ni2+ was also applied. The histograms at the right side of each panel have columns corresponding to the average Ca2+ influx observed during each of these three episodes of exposure to Na+-free solution. | ||
Suppression of INa-Ca by forskolin. We also used the adenylyl cyclase activator forskolin as a means to increase the concentration of intracellular cAMP (Manolopoulos & Lelkes, 1993; Goaillard et al. 2001). In voltage-clamped cells expressing the chimeric frog/dog NCX1, forskolin slowly but strongly suppressed the Ni2+-sensitive INa-Ca at +40 mV (Fig. 7C) as it suppressed the currents generated by the ramp-clamp protocol (Fig. 7A), and produced a negative shift in the reversal potential in the I-V relation (Fig. 7B). The suppression of INa-Ca at +40 mV ranged ~75 % (n = 4) and the changes in the I-V relations were similar to those observed with 8-Br-cAMP (cf. Figs 3C, 4A and 4B), but the effects of forskolin were not equally reversible as it has been also observed in intact cardiomyocytes (Woo & Morad, 2001). We cannot entirely exclude the possibility that suppression of INa-Ca also includes an element of run-down.
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Figure 7. Suppression of INa-Ca by forskolin in a voltage-clamped HEK293 cell expressing frog/dog NCX1 A, currents measured during the repeated application of the ramp-clamp protocol at the times indicated (a, b, c and d) in C as the cell was exposed briefly to Ni2+ twice (a and d) and for 5 min to 10 µM forskolin (c). B, the current-voltage relation of INa-Ca in the absence (b - a) and presence (c - d) of forskolin (cell capacitance 20 pF). | ||
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DISCUSSION |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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The major finding of this report is that the insertion of the P-loop from the amino acid sequence of the frog NCX1 protein into the mammalian NCX1 confers cAMP-mediated regulation to the recombinant wild-type mammalian exchanger expressed in HEK293 cells. This finding is consistent with the hypothesis that a nucleotide binding P-loop, or Walker A motif, that is present in the frog heart NCX1 (Iwata et al. 1996), but not in the dog NCX1 (Nicoll et al. 1990), is essential to -adrenergic regulation of the exchanger. Since -adrenergic regulation is generally thought to involve phosphorylation, we also probed the involvement of a potential PKA site located in the main cytoplasmic loop (Fig. 1A) and found that disruption of this site reduced and shortened the cAMP-mediated effect on the cell lines expressing the S374G mutated frog NCX1. We have not as yet used PKA inhibitors to investigate whether the activity of S374G frog NCX1, or the other NCX1 constructs, might be modulated by basal levels of cAMP in HEK 293 cells.
These results were obtained by stably expressing variant clones of NCX1 in human cell lines (HEK293 and BHK cells) where they produced Ni2+-sensitive INa-Ca in control solutions (Figs 2A-C, 3, 4 and 7) and a rise in [Ca2+]i upon rapid withdrawal of extracellular Na+ (Figs 2D and E, 5 and 6). The level of expression of various clones was of some concern, since the measured currents in some cells, especially those expressing the S374G frog NCX1, were of relatively modest amplitude (Table 1). We therefore used monoclonal antibodies to the mouse exchanger (MA3-926) to verify expression and found that the profile of protein expression in different cell lines (Fig. 1B) matched that of INa-Ca (Table 1), and that S374G frog NCX1 was clearly concentrated at the cell membrane (Fig. 1C), so that impaired translocation to the membrane was not likely to be responsible for the relatively low INa-Ca amplitude of S374G frog NCX1 (Table 1).
Measurements of Ca2+ signals were used as an independent means of assessing functional expression. Such measurements could be performed on multiple non-dialysed cells at the same time (increasing the reliability of Ca2+ measurement) and for longer periods of time than possible for voltage-clamp experiments. Nevertheless, the fluorescence measurements did not provide the same time resolution as the voltage-clamp experiments. The measurements of Ca2+ fluxes were generally consistent with the measurements of INa-Ca, but did not lend themselves to detailed numerical analysis, since they showed considerable variability from cell to cell. Further, the exact level of the membrane potential in unclamped cells was uncertain. Nevertheless, the combined evidence from structural and functional voltage-clamp and optical experiments indicate that different variants of NCX1 could be stably expressed in transfected mammalian cell lines at levels that allow detailed examination of their properties, including cAMP-mediated regulation.
Role of the P-loop
The cell lines expressing frog and frog/dog NCX1 produced indistinguishable results with respect to the level of expression using Western blots, immunofluorescence imaging, or the degree of cAMP-mediated suppression of INa-Ca and Ca signals (Fig. 1C and D; Table 1; Fig. 3B and C; Fig. 4). Since dog NCX1 showed no such regulatory properties, the finding may imply that the frog NCX1 P-loop is essential in conferring cAMP-mediated regulation onto the dog NCX1. The expression of the chimeric frog/dog NCX1 in HEK293 cells demonstrates strong cAMP-mediated suppression of NCX1 for the first time in a human cell line indicating that the effect is not exclusive to the Xenopus expression system. Since a DNA sequence that may complete the coding of the P-loop is found in the human NCX1 gene in 5' region of intron 8, immediately downstream from exon 7 (Kraev et al. 1996), our findings also raise the possibility that alternative splicing may conceivably produce a cAMP-sensitive phenotype of NCX1 in mammalian cardiomyocytes, e.g. during embryonic and postnatal development, when t-tubules are absent, allowing most of the centrally located sarcoplasmic reticulum (SR) Ca2+-release sites to remain dormant. The SR in neonatal mammalian heart is poorly developed and excitation-contraction coupling signalling indeed appears to be similar to that of the amphibian heart (Morad & Goldman, 1973; Fabiato & Fabiato, 1978; Maylie, 1982). Similarly, -adrenergic agonists enhance the twitch and suppress KCl-induced contractures (Morad, 1969; Morad & Rolett, 1972), consistent with their enhancement of ICa and suppression of INa-Ca as described above.
Role of the PKA site
Our study showed that point mutation S374G of the putative PKA site (RKxxS) decreased the sensitivity of frog NCX1 to cAMP and made the response transient (Fig. 3D and Fig. 6B). This suggests that multiple sites with different rates of phosphorylation and dephosphorylation are involved, and may provide the explanation as to why somewhat divergent results have been obtained for cAMP-mediated regulation in mammalian cardiac NCX1, both in myocytes and heterologous expression systems (Han & Ferrier, 1995; Ballard & Schaffer, 1996; Main et al. 1997; Iwamoto et al. 1998; Linck et al. 1998; Perchenet et al. 2000; Ruknudin et al. 2000). In vitro experiments indicate that mammalian cardiac NCX1 is subject to PKA-mediated phosphorylation (Ruknudin et al. 2000), but the stoichiometry and exact site(s) involved are not yet known. Short tryptic peptide fragments (16 kDa) from scallop show PKA-mediated phosphorylation mapping to the long cytoplasmic loop of squid NCX (Chen et al. 2000). The location, however, is different from that examined here, which conversely appears to have no equivalent site in squid NCX. While there is as yet no conclusive evidence that the putative PKA site is indeed functionally phosphorylated by activation of the adenylate cyclase pathway, its serine residue (S357) appears to be weakly phosphorylated as a secondary effect when the PKC pathway is activated (Iwamoto et al. 1998).
Species-specific differences in cAMP-mediated NCX1 regulation
The role and regulation of NCX1 in the heart has been associated with the development of a functional SR with a rapidly releasable pool of Ca2+. In response to activation of the adenylate cyclase/-adrenergic pathway, the activity of mammalian NCX1 measured in adult cardiomyocytes with well-developed SR or in expression systems has been reported to be either unchanged (Ballard & Schaffer, 1996; Main et al. 1997) or somewhat enhanced (Han & Ferrier, 1995; Perchenet et al. 2000; Ruknudin et al. 2000). The observed differences in the mammalian response may be related to tissue-specific splice isoforms (Quednau et al. 1997; Ruknudin et al. 2000), temperature (Perchenet et al. 2000), long-term upregulation of NCX1 expression (Smith et al. 1996), condition of the cell culture (Pabbathi et al. 2002), PKC activation (Iwamoto et al. 1998), difficulties in dissociating the direct response of NCX1 from those of other established or potential targets of cAMP-mediated regulation (SR Ca2+-ATPase, Ca2+ channel, ryanodine receptor, ADP/ATP ratio, metabolism, [Na+]i etc.) and finally species-specific variability. Nevertheless, we have not found any reports indicating that mammalian NCX1 is strongly suppressed by cAMP as has been found in frog (Fan et al. 1996) and shark (Woo & Morad, 2001) cardiomyocytes both with rudimentary SR (Page & Niedergerke, 1972; Morad & Goldman, 1973) and with expression of the recombinant frog NCX1 in the Xenopus oocytes (Shuba et al. 1998). It appears as if the cAMP-mediated regulation is qualitatively different in frog and mammalian heart and that this may be related to the differences in the amino acid sequence of the respective NCX1s.
Physiological role of cAMP-mediated regulation
The role of -adrenergic regulation of cardiac NCX1 is likely to be different in amphibians and in mammals since relaxation of the heart beat in amphibians depends almost exclusively on NCX1, while in mammals most activator Ca2+ is recirculated into the SR by a Ca2+-ATPase (SERCAII) that serves as one of the major targets of adrenergic stimulation.
The effects of adrenaline in the frog ventricular strips were examined in early voltage-clamp studies showing diametrically opposite effects on twitch tension and tonic tensions. The dominant phasic (ICa-dependent) component was strongly enhanced in the presence of catecholamines, while the smaller Na+-dependent tonic component was suppressed (Kavaler & Morad, 1966; Vassort & Rougier, 1972; Morad & Goldman, 1973; Morad et al. 1981). Such dual effects of catecholamines were originally thought to result from both increased Ca2+ influx via the Ca2+ channels and enhanced uptake of Ca2+ by the SR. In light of more recent studies showing the virtual absence of the SERCAII gene, Ca2+-ATPase (Vilsen & Andersen, 1992), and phospholamban proteins in the frog heart (Kurebayashi & Ogawa, 1995), it would appear, however, that NCX1 is the molecular site that mediates the relaxant effects of catecholamines. The suppressive effect of cAMP on NCX1 may be counterintuitive, but considering that the Ca2+ channel current and the plateau of the action potential are significantly enhanced by catecholamines (Morad et al. 1981; Fischmeister & Hartzell, 1986), 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. 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 isoproterenol (Morad et al. 1981).
In contrast, NCX1 in mammalian heart is neither downregulated by catecholamines nor can it be specifically targeted with presently limited pharmacological agents. Millimolar concentrations of Ni2+ effectively block NCX1 (Hinde et al. 1999), but in addition to being toxic, they also block Ca2+ channels. KB-R7943 is potentially a more useful agent (Iwamoto et al. 1996; Woo & Morad, 2001), which has been reported to primarily block NCX1 in the reverse mode (outward INa-Ca) (Watano et al. 1996; Woo & Morad, 2001), but appears to also block Ca2+ channels (Watano et al. 1996). It has long been thought that digitalis modulates NCX1, secondary to inhibition of Na+ pump and elevation of [Na+]i,. This is supported by the recent finding that cardiac glycosides have no effect in heart tubes from embryonic (embryonic day (E) 9.5) homozygous NCX1 knockout mice (Reuter et al. 2002).
Nevertheless, clinical NCX1 drugs, should they be found, might be useful to treat conditions of Ca2+ overload with impaired relaxation (e.g. dilated cardiomyopathy) and to block large inward INa-Ca that may trigger arrhythmias. Such agents might be particularly useful if the forward and reverse modes could be targeted independently as observed for KB-R7943 in mammals (Watano et al. 1996) and for isoproterenol in elasmobranches (Woo & Morad, 2001). Other strategies would be to interfere with the expression of NCX1, either via inherent regulatory pathways or via gene therapy. The results presented here suggest that this might be done by expressing an NCX1 with a complete P-loop, or even by inducing the expression of a yet-to-be-discovered 'frog-like', perhaps embryonic, splice variant of the mammalian NCX1 gene.
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Acknowledgements
We thank Dr K. D. Philipson for providing BHK cells expressing the wild-type dog NCX1, K. O'Brien for help with molecular biology, Dr Steven N. Ebert for help with immunofluorescence cytochemistry and Dr Toshio Kitazawa for help with Western blot analysis. This work was supported by grants from NIH (RO1 16152) and the American Heart Association (4230751).
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-helical transmembrane segments is based on Iwata et al. (1996), but modified according to Nicholl et al. (1999). The isoforms of NCX1 expressed in the four cell lines are based on the wild-type dog and frog NCX1 and differ with respect to the absence or presence of a complete P-loop and a putative PKA site. C, Western blots testing the expression of the NCX1 protein in different transfected and non-transfected HEK293 cell lines using anti-NCX mouse monoclonal antibody (MA3-926). The positions of the 120 and 160 kDa protein bands associated with the NCX1 expression are marked on the left side. D, confocal fluorescence images of different HEK293 cell lines (newly developed and non-transfected) immunostained with anti-NCX antibody and FITC-labelled secondary antibody. All images were obtained under identical conditions.