Correspondence to: Larry V. Hryshko, Institute of Cardiovascular Sciences, St. Boniface General Hospital Research Centre, 351 Tache Avenue, Winnipeg, Manitoba, Canada, R2H 2A6. Fax:204-233-6723 E-mail:lhryshko{at}sbrc.umanitoba.ca.
Released online: 25 October 1999
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Abstract |
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Ion transport and regulation of Na+Ca2+ exchange were examined for two alternatively spliced isoforms of the canine cardiac Na+Ca2+ exchanger, NCX1.1, to assess the role(s) of the mutually exclusive A and B exons. The exchangers examined, NCX1.3 and NCX1.4, are commonly referred to as the kidney and brain splice variants and differ only in the expression of the BD or AD exons, respectively. Outward Na+Ca2+ exchange activity was assessed in giant, excised membrane patches from Xenopus laevis oocytes expressing the cloned exchangers, and the characteristics of Na+i- (i.e., I1) and Ca2+i- (i.e., I2) dependent regulation of exchange currents were examined using a variety of experimental protocols. No remarkable differences were observed in the currentvoltage relationships of NCX1.3 and NCX1.4, whereas these isoforms differed appreciably in terms of their I1 and I2 regulatory properties. Sodium-dependent inactivation of NCX1.3 was considerably more pronounced than that of NCX1.4 and resulted in nearly complete inhibition of steady state currents. This novel feature could be abolished by proteolysis with -chymotrypsin. It appears that expression of the B exon in NCX1.3 imparts a substantially more stable I1 inactive state of the exchanger than does the A exon of NCX1.4. With respect to I2 regulation, significant differences were also found between NCX1.3 and NCX1.4. While both exchangers were stimulated by low concentrations of regulatory Ca2+i, NCX1.3 showed a prominent decrease at higher concentrations (>1 µM). This does not appear to be due solely to competition between Ca2+i and Na+i at the transport site, as the Ca2+i affinities of inward currents were nearly identical between the two exchangers. Furthermore, regulatory Ca2+i had only modest effects on Na+i-dependent inactivation of NCX1.3, whereas I1 inactivation of NCX1.4 could be completely eliminated by Ca2+i. Our results establish an important role for the mutually exclusive A and B exons of NCX1 in modulating the characteristics of ionic regulation and provide insight into how alternative splicing tailors the regulatory properties of Na+Ca2+ exchange to fulfill tissue-specific requirements of Ca2+ homeostasis.
Key Words: sodiumcalcium exchange, regulation, alternative splicing
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INTRODUCTION |
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Sodiumcalcium exchangers constitute a family of ion counter transporters that play a prominent role in cellular Ca2+ homeostasis (
The consequences of alternative splicing of Na+Ca2+ exchangers are largely unknown. One possibility is that expression of different exon combinations could provide a mechanism for directing Na+Ca2+ exchangers to appropriate cellular locations. For example, in kidney, the exchanger may reside exclusively in the basolateral membrane of renal cells (
In an effort to gain a better understanding of the transport and regulatory consequences of alternative splicing among Na+Ca2+ exchangers, we selected two versions of NCX1 that differ only in terms of which of its two, mutually exclusive exons (i.e., A and B) is expressed: NCX1.4 (exons AD) and NCX1.3 (exons BD). Since exon A splice variants appear to be expressed predominantly in excitable tissues (e.g., neurons, heart, skeletal muscle), whereas exon B isoforms are more widely distributed (e.g., kidney, liver, lung, astrocytes) (
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METHODS |
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Construction of NCX1.3 and NCX1.4
Kidney and brain samples were collected from adult dogs, rapidly frozen in liquid N2 and stored at -70°C. Total RNA was subsequently isolated by the method of
Synthesis of NCX1.3 and NCX1.4 cRNA
Complementary DNAs encoding NCX1.3 and NCX1.4 were linearized with HindIII (New England Biolabs Inc.) and cRNA synthesized using T3 mMessage mMachine in vitro transcription kits (Ambion Inc.) according to the manufacturer's instructions. After injection with 5 ng of cRNA encoding NCX1.3 or NCX1.4, oocytes were maintained at 16°C in solution B minus BSA (see below). Electrophysiological measurements were typically obtained from days 46 after injection.
Preparation of Xenopus laevis Oocytes
Xenopus laevis were anaesthetized in 250 mg/liter ethyl p-aminobenzoate (Sigma Chemical Co.) in deionized ice-water for 30 min. Oocytes were then removed and washed in solution A containing (mM): 88 NaCl, 15 HEPES, 2.4 NaHCO3, 1.0 KCl, 0.82 MgSO4; pH 7.6 at room temperature (rt). The follicles were teased apart and the oocytes transferred to 5 ml of solution A containing 80 mg collagenase (Type II; Worthington Biochemical Corp.) and incubated for 4560 min at rt with gentle agitation. The oocytes were washed several times in solution B containing (mM): 88 NaCl, 15 HEPES, 2.4 NaHCO3, 1.0 KCl, 0.82 MgSO4, 0.41 mM CaCl2, 0.3 mM Ca(NO3)2, 1 mg/ml BSA (Fraction V; Sigma Chemical Co.); pH 7.6 at rt, and then transferred to 5 ml of 100 mM K2HPO4, pH 6.5 at rt, containing 1 mg/ml BSA. After incubation at rt for 1112 min with gentle agitation, the oocytes were washed several times in solution B at rt. Defolliculated stage VVI oocytes were selected and incubated at 16°C in solution B minus BSA until injection the following day.
Measurement of Na+Ca2+ Exchange Activity
Na+Ca2+ exchange current measurements were obtained using the giant excised patch clamp technique, as described previously (2025 µm and coated with a ParafilmTM:mineral oil mixture to enhance patch stability and reduce electrical noise. To expedite removal of the vitelline layer before membrane patching, oocytes were shrunk slightly by incubation in a solution containing (mM): 100 K-aspartate, 100 KOH, 100 MES, 20 HEPES, 5 EGTA, 5 MgCl2, pH 7.0 at rt, for
15 min at rt. After removal of the vitelline layer by dissection, oocytes were placed in a solution containing (mM): 100 KOH, 100 MES, 20 HEPES, 5 EGTA, 510 MgCl2, pH 7.0 at rt (with MES), and G
seals formed via gentle suction. Membrane patches (inside-out configuration) were excised by progressive movements of the pipette tip. Rapid solution changes (i.e.,
200 ms) were accomplished using a custom-built, computer-controlled, 20-channel solution switcher. Hardware (Axopatch 200a; Axon Instruments) and software (Axotape) were used for data acquisition and analysis. For outward current measurements, pipette (i.e., extracellular) solutions contained (mM): 100 N-methyl-glucamineMES, 30 HEPES, 30 tetraethylammonium (TEA)-OH, 16 sulfamic acid, 8.0 CaCO3, 6 KOH, 2.0 Mg(OH)2, 0.25 ouabain, 0.1 niflumic acid, 0.1 flufenamic acid; pH 7.0 at 30°C (with MES). Outward Na+Ca2+ exchange currents were elicited by switching from Li+i- to Na+i-based bath solutions containing (mM): 100 [Na + Li]-aspartate, 20 MOPS, 20 TEA-OH, 20 CsOH, 10 EGTA, 09.91 CaCO3, 1.01.5 Mg(OH)2, pH 7.0 at 30°C (with MES or LiOH). Magnesium and Ca2+ were adjusted to yield free concentrations of 1.0 mM and 030 µM, respectively, using MAXC software (
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RESULTS |
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Our aim was to determine which aspects of the ionic regulatory profile of NCX1 can be directly attributed to expression of its mutually exclusive exons, A and B. We chose the splice variant pair NCX1.4 (exons AD) and NCX1.3 (exons BD) and characterized their ionic regulatory phenotypes under a variety of conditions.
Inspection of the aligned sequences of exons A (encoding 35 amino acids) and B (encoding 34 amino acids) in Figure 1 reveals substantial similarity between NCX1.3 and NCX1.4. 13 identities are found and the exons are 63% similar with respect to conservative substitutions, with the greatest similarity occurring towards their NH2 termini. Charge reversal occurs at two positions, substitution of charged for neutral residues is observed at six, polar residues coincide with hydrophobics at three, and the overall electric charge of exon A is -2, whereas exon B is +1. Exon B encodes a cysteine residue at position 585 (
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Na+i-dependent Regulation of NCX1.3 and NCX1.4
We examined the [Na+]i dependence of peak and steady state outward Na+Ca2+ exchange currents mediated by NCX1.4 and NCX1.3 to obtain estimates of Na+ transport affinities, as well as the rate and extent of I1 inactivation. Figure 2 shows representative current traces obtained in response to the rapid (i.e., <200 ms) application of 10100 mM Na+i to the cytoplasmic surface of the patches in the continuous presence of 1 µM regulatory Ca2+i. Transport Ca2+o in the pipette was constant at 8 mM. After each current activation event, patches were allowed to recover for 3248 s in Li+i-containing solution plus 1 µM Ca2+i before delivery of the next Na+i pulse. With increasing [Na+]i, the isoforms exhibited similar increases in peak current and in the extent of current inactivation, characteristic of Na+i-dependent, or I1, inactivation (-chymotrypsin (Figure 2, bottom), a procedure known to deregulate Na+Ca2+ exchangers (
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Figure 3 summarizes the Na+i dependence of peak and steady state outward Na+Ca2+ exchange currents derived from pooled data obtained with NCX1.3 and NCX1.4 in the presence of 1 µM regulatory Ca2+i, as above. Currents were normalized to the values obtained at 100 mM Nai+. For both isoforms, peak currents progressively increased with increasing [Na+]i. Estimates of exchanger affinity for Na+i based on peak current measurements provided nearly identical values of Kd (33 ± 5 mM, n = 4 vs. 31 ± 4 mM, n = 6, for NCX1.3 and NCX1.4, respectively). For comparison, the peak current-derived Na+i affinity of the cardiac exchanger, NCX1.1, is 27 mM (
-chymotrypsin, a Kd value of 26 ± 1 mM (three determinations from two patches) was estimated for NCX1.3, similar to that of peak currents for both exchangers.
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Figure 4 illustrates the Na+i dependence of the NCX1 isoforms in terms of their ratios of steady state to peak currents, or Fss values ( 25 mM, the decrease in Fss is more pronounced for NCX1.3 than for NCX1.4. For example, Fss values calculated from currents acquired in response to the application of 100 mM Na+i were 0.24 ± 0.03 and 0.07 ± 0.01 (n = 20 and 25 determinations from 14 patches) for NCX1.4 and NCX1.3, respectively. Note that for NCX1.3, the extent of I1 inactivation is
90% at high [Na+]i, whereas at 10 mM Na+i it is
20%. This substantial inactivation of NCX1.3-mediated transport is likely to be responsible for the peculiar nature of the Na+i dependence of its steady state currents, as shown in Figure 3. That is, the increase in steady state current in response to Na+i appears to be largely offset by the extensive inactivation that occurs as [Na+]i is raised. Consequently, steady state Na+Ca2+ exchange currents mediated by NCX1.3 do not exhibit a hyperbolic response to rising [Na+]i unless this regulatory mechanism is eliminated by proteolysis with
-chymotrypsin.
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Figure 5 illustrates the currentvoltage (IV)1 relationships for NCX1.3 and NCX1.4. Outward currents were activated by switching from 100 mM Li+i-containing perfusing solution to 100 mM Na+i, and 1 µM regulatory Ca2+i was present throughout. The IV relationship was determined before (a) and during (b) exchange current activation, and the former values were subtracted from the latter. From a holding potential of 0 mV, 40-ms voltage steps, in 10-mV increments, were applied from -100 to +100 mV, with a return to the holding potential between each step. Pooled data shown in Figure 5 (bottom) are from three NCX1.3 and four NCX1.4 patches, with currents normalized to the values obtained at 0 mV. Note that a reversal potential is not observed under these conditions as the pipette solution does not contain Na+o. The exchanger isoforms exhibited similar IV relationships to that observed for the cardiac exchanger, NCX1.1 (
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Ca2+i-dependent Regulation of NCX1.3 and NCX1.4
Figure 6 illustrates the effects of removal and reapplication of regulatory Ca2+i on outward Na+Ca2+ exchange currents mediated by NCX1.3 and NCX1.4. Currents were elicited by applying 100 mM Na+i, with 1 µM Ca2+i present before activation. Regulatory Ca2+i was then removed and reapplied in the middle of the current trace in the continuous presence of 100 mM Na+i. For NCX1.4, Ca2+i removal led to rapid and nearly complete inhibition of outward exchange current, with a half-time for current decay of 0.52 ± 0.05 s (n = 5). Similarly, steady state levels were rapidly restored when Ca2+i was reapplied, with a half-time of 0.49 ± 0.14 s (n = 5). For comparison with NCX1.1, the equivalent protocols yield half-times for loss and reacquisition of steady state current levels of 10.8 and 7.5 s, respectively (
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Figure 7 shows representative traces that illustrate the dependence of outward Na+Ca2+ exchange currents mediated by NCX1.3 and NCX1.4 upon [Ca2+]i. Regulatory Ca2+i, at the indicated concentrations, was present for 3248 s before, during, and after current activation with 100 mM Na+i. For both isoforms, exchange currents are augmented by Ca2+i. In particular, NCX1.4 behaves similar to the cardiac exchanger, NCX1.1, in that regulatory Ca2+i not only stimulates exchange activity, but also alleviates I1 inactivation. At 10 µM Ca2+i, Na+i-dependent inactivation is nearly eliminated and the current recording adopts a square appearance. With NCX1.3, however, I1 inactivation is still prominent at 10 µM Ca2+i, in sharp distinction to both NCX1.4 and NCX1.1. That is, regulatory Ca2+i is not only incapable of alleviating I1 inactivation for NCX1.3, it appears to inhibit outward current generation. These relationships are illustrated graphically in Figure 8.
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The pooled data shown in Figure 8 illustrate the regulatory Ca2+i dependence of peak and steady state outward currents mediated by NCX1.3 and NCX1.4 in response to the application of 100 mM Na+i. Currents were normalized to the values obtained at 1 µM regulatory Ca2+i. Both exchangers initially exhibit an increase in peak outward current as regulatory Ca2+i is raised (Figure 8, top). For NCX1.4, data were fit to the Hill equation over the [Ca2+]i range 03 µM, providing a Kd value of 0.2 ± 0.06 µM. Beyond 3 µM Ca2+i, however, deviation from simple hyperbolic behavior became evident, and further increases in [Ca2+]i often led to decreased peak outward currents (Figure 7). This is presumably due to competition between Ca2+i and Na+i at the intracellular transport site, a phenomenon previously documented for NCX1.1 and two alternatively spliced isoforms of the Drosophila Na+Ca2+ exchanger, CALX1 (
The effects of regulatory Ca2+i on Fss, the ratio of steady state to peak currents are shown in Figure 8 (bottom). For NCX1.4, a U-shaped Ca2+i dependence was obtained. The value of Fss initially declines from 00.3 µM Ca2+i, and then rises as Ca2+i is increased beyond 0.3 µM. This behavior reflects the observation that, in the absence of Ca2+i, outward currents are small and show relatively little inactivation. As Ca2+i increases, however, peak current progressively rises, causing Fss to decrease at intermediate [Ca2+]i. Finally, as [Ca2+i] is further increased, the extent of I1 inactivation is reduced to near-zero levels and Fss returns to higher values. These characteristics of Fss are typical of the cardiac exchanger, NCX1.1 (
The observation that NCX1.3 appears to be inhibited at higher concentrations of regulatory Ca2+ could occur if this exchanger had a higher affinity for Ca2+ at the intracellular transport site. Consequently, lower concentrations of Ca2+ could compete for Na+i and reduce current magnitude. To test this possibility, we examined inward Na+Ca2+ exchange currents for both NCX1.3 and NCX1.4. Pipettes contained 100 mM Na+ and inward currents were activated by applying different Ca2+ concentrations (0.1100 µM) to the cytoplasmic surface of the patch. Typical inward current recordings are shown in Figure 9. We did not observe any major differences for inward Na+Ca2+ exchange currents produced by NCX1.3 and NCX1.4. The apparent affinities calculated for Ca2+ activation of inward Na+Ca2+ exchange currents were 9.0 ± 1.9 µM (mean ± SD, n = 5 patches) for NCX1.3 and 8.1 ± 1.4 µM (mean ± SD, n = 3 patches) for NCX1.4. These values are similar to that reported for NCX1 (e.g., 7 µM;
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Figure 10 illustrates representative current recordings obtained for paired-pulse experiments conducted at two different regulatory Ca2+ concentrations (0.3 and 10 µM Ca2+i). In each case, currents were activated by 100 mM Na+i and transported Ca2+o in the pipette was constant at 8 mM. Regulatory Ca2+i, at the indicated concentration, was present throughout the entire paired-pulse trials. For NCX1.4, the second test pulse is substantially reduced after a 4-s interval at 0.3 µM Ca2+i, whereas the two pulses are nearly identical in magnitude at 10 µM Ca2+i. This behavior illustrates the ability of Ca2+i to accelerate exit from, and/or reduce entry into, the I1 inactive state, and is typical of NCX1.1 (
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DISCUSSION |
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The NCX1 gene encodes a variety of alternatively spliced Na+Ca2+ exchangers, several with unique tissue distributions. Although the physiological significance of this diversity is unknown, it may reflect different requirements for the maintenance of Ca2+ homeostasis in various cell types. Thus, we examined the ionic regulatory properties of two splice variants of NCX1: NCX1.3 and NCX1.4. These particular exchangers were selected for two reasons. First, NCX1.3 is a prominent splice variant in kidney, whereas NCX1.4 is abundant in brain. Second, these isoforms differ only in terms of expression of the mutually exclusive exons A (NCX1.4) and B (NCX1.3), with expression of the D cassette exon common to both. Therefore, our results provide direct insight into the functional role(s) of the mutually exclusive exons of NCX1. From structurefunction considerations, our results point to prominent interactions between the alternative splicing region and other domains subserving the Na+i- (i.e., I1) and Ca2+i- (i.e., I2) dependent regulatory processes. These observations also provide a foundation towards understanding the physiological behavior of these transporters in their native environments.
Ionic Regulation of Na+Ca2+ Exchangers
Sodium-dependent, or I1, inactivation of Na+Ca2+ exchange current describes the ionic regulatory process that occurs in response to the cytoplasmic application of Na+i. The phenomenon manifests as a rapid (i.e., 200 ms) rise in outward exchange current to a peak value, followed by a relatively slow (i.e.,
30 s) decay to steady state levels of activity. This mechanism has been characterized extensively in giant excised patch experiments (
Calcium-dependent, or I2, regulation describes the stimulatory (e.g., NCX1.1) or inhibitory (e.g., CALX1.1) effect of micromolar [Ca2+]i on both inward and outward currents associated with the forward (i.e., Ca2+ efflux) and reverse (i.e., Ca2+ influx) modes, respectively, of Na+Ca2+ exchange. Like I1, this process has also been well documented in giant excised patch clamp studies using both cardiomyocytes and Xenopus oocytes expressing NCX1.1 (
Functional Role of the Alternative Splicing Region
Before recognition of the existence of alternatively spliced isoforms of Na+Ca2+ exchangers, structurefunction studies aimed at delineating the bases of ionic regulation of NCX1 examined the consequences of deleting substantial portions of its large cytoplasmic loop (240-679 and
562-685) eliminated the alternative splicing region, as well as appreciable flanking sequences. With
562-685, I1 regulation was ablated, whereas
240-679 was associated with loss of both I1 and I2 regulation (
Qualitatively, NCX1.4 behaves in a similar fashion to NCX1.1 in terms of its Na+i and Ca2+i dependencies and I1 and I2 regulatory profiles. The main difference we observed between NCX1.1 and NCX1.4 lies in the rapidity with which removal and reapplication of regulatory Ca2+i influences steady state Na+Ca2+ exchange currents (Figure 6). The response of NCX1.4 to this maneuver is at least 10x faster than that of NCX1.1 (
The ionic regulatory behavior of NCX1.3 is considerably different from both NCX1.1 and NCX1.4. Specifically, I1 inactivation appears to be much more prominent for this exchanger. This inactivation process resulted in steady state currents that were inhibited 90% compared with peak current values. Furthermore, regulatory Ca2+i was incapable of alleviating this inhibition, in contrast to the behavior of NCX1.1 and NCX1.4. Finally, we observed a unique behavior of NCX1.3, which suggested that regulatory Ca2+i exhibits both stimulatory and inhibitory effects (Figure 7). This is unlikely to reflect differences in competition between Ca2+ and Na+ at the intracellular transport site, as both NCX1.3 and NCX1.4 show similar Ca2+ affinities for inward currents. Consequently, the expression of the B exon adds a novel aspect to the ionic regulatory phenotype of NCX1.3.
Characterization of regulatory phenotypes has only been undertaken for a few members of the Na+Ca2+ exchanger family (i.e., NCX1.1, NCX1.3, NCX1.4, CALX1.1, and CALX1.2). For example, we have shown that alternatively spliced CALX1 exchangers, which differ by five amino acids, show marked differences in their I1 and I2 regulatory properties (0.53 Hz. Frequency-encoded signaling within neuronal tissue, however, occurs considerably faster (e.g., 300 Hz), whereas Ca2+ reabsorption in the kidney is likely to be a less dynamic process. As a Ca2+ efflux (and possibly influx) mechanism, the activity of the Na+Ca2+ exchange system must be able to keep pace with this wide range of Ca2+ flux requirements. If ionic regulation contributes to the ability of Na+Ca2+ exchangers to accommodate these various cellular Ca2+ flux rates, then the activity of exchangers in a physiological setting may be governed, in part, by the time-averaged consequences of ionic regulation, in addition to the thermodynamic influences of the Na+ and Ca2+ electrochemical gradients.
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Acknowledgements |
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This work was supported by a Medical Research Council of Canada grant (GEC-3) and Heart and Stroke Foundation of Canada grant to L.V. Hryshko, and by the National Institutes of Health grants HL-48509 and HL-49101 (K.D. Philipson). L.V. Hryshko is supported by a Scholarship from the Medical Research Council of Canada.
Submitted: 14 June 1999
Revised: 7 September 1999
Accepted: 27 September 1999
1used in this paper: IV, currentvoltage
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