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Department of Physiology, University of Texas, Southwestern Medical Center at Dallas, Dallas, Texas 75235-9040
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ABSTRACT |
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S, in the presence of F– (0.2 mM) and vanadate (50 µM), and both effects reversed on application of a phosphatidylinositol-4',5'-bisphosphate antibody. NCX1 current was stimulated by ATP, but not by ATPS. Like NCX1 current, NCX-SQ1 current was strongly stimulated by phosphatidylinositol-4',5'-bisphosphate liposomes. In contrast to results in squid axon, NCX-SQ1 was not stimulated by phosphoarginine (5–10 mM). After chymotrypsin treatment, both the outward and inward NCX-SQ1 exchange currents were more strongly voltage dependent than NCX1 currents. Ion concentration jump experiments were performed to estimate the relative electrogenicity of Na+ and Ca2+ transport reactions. Outward current transients associated with Na+ extrusion were much smaller for NCX-SQ1 than NCX1, and inward current transients associated with Ca2+ extrusion were much larger. For NCX-SQ1, charge movements of Ca2+ transport could be defined in voltage jump experiments with a low cytoplasmic Ca2+ (2 µM) in the presence of high extracellular Ca2+ (4 mM). The rates of charge movements showed "U"-shaped dependence on voltage, and the slopes of both charge–voltage and rate–voltage relations (1,600 s–1 at 0 mV) indicated an apparent valency of –0.6 charges for the underlying reaction. Evidently, more negative charge moves into the membrane field in NCX-SQ1 than in NCX1 when ions are occluded into binding sites.
Key Words: sodium–calcium exchange • charge movements • Xenopus oocytes • patch clamp • phosphatidylinositols
Abbreviations: MES, 2-(morpholino)ethanesulfonic acid; NMG, N-methyl-D-glucamine; nts, nucleotides; PIP2, phosphatidylinositol-4',-5'-bisphosphate
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). Their major physiological role is to extrude Ca2+ in exchange for extracellular Na+, which is subsequently extruded by the ATP-dependent Na+-K+ pump.
Historically, two experimental models were used to characterize Na+–Ca2+ exchange function: cardiac sarcolemmal vesicles (for review, see Philipson and Reeves, 1989) and perfused squid giant axons (for review, see DiPolo, 1989; DiPolo and Beaugé, 1991). Molecular studies of the cardiac Na+–Ca2+ exchanger (NCX1) were made possible by its cloning (Nicoll et al., 1990). This was followed by the cloning and functional characterization of two other mammalian exchangers (NCX2 and NCX3; Li et al., 1994; Nicoll et al., 1996a, 1996b) and an NCX-type exchanger from Drosophila (Ruknudin et al., 1997; Schwarz and Benzer, 1997). Mutational studies of NCX1 have identified functional domains involved in both regulation and transport (Matsuoka et al., 1993, 1995, 1997; Levitsky et al., 1994; Nicoll et al., 1996a). Given the extensive nature of Na+–Ca2+ exchange studies in squid giant axons, cloning and molecular characterization of the squid exchanger could provide many unique insights.
Basic properties of the cardiac and squid exchangers are clearly similar. These include a 3 Na+ to 1 Ca2+ stoichiometry, regulatory activation of exchanger-mediated Ca2+ influx by cytoplasmic Ca2+ (DiPolo, 1979; Kimura et al., 1986), and stimulation by ATP-dependent mechanisms (DiPolo, 1974; Baker and McNaughton, 1976; Hilgemann, 1990). Nevertheless, recent results suggest that there are important differences in the function and regulation of the different exchangers. (a) The ATP dependence of the squid exchanger appears to reflect its phosphorylation by a protein kinase (DiPolo and Beaugé, 1994; DiPolo et al., 1997), while the ATP-dependent activation of the cardiac exchanger appears to reflect the generation of phosphatidylinositol 4'-,5'-bisphosphate (PIP2)1 from phosphatidylinositol (Hilgemann and Ball, 1997). ATPS activates the squid exchanger but not the cardiac exchanger. Cationic agents that bind anionic lipids inhibit the cardiac exchanger (Hilgemann and Collins, 1992), but agents such as pentalysine do not inhibit the squid exchanger (R. DiPolo and L. Beaugé, 1993). (b) The squid exchanger is regulated by a phosphoarginine-dependent process that may involve protein kinases unique to invertebrates (DiPolo and Beaugé, 1995); phosphoarginine is without effect on the cardiac exchanger (D.W. Hilgemann, unpublished observations). (c) The Ca2+–Ca2+ exchange operation of the squid exchanger (DiPolo et al., 1985; DiPolo and Beaugé, 1990) and of barnacle muscle (Rasgado-Flores et al., 1996) appears to be strongly voltage dependent, while Na+–Na+ exchange shows almost no voltage dependence in isotope flux studies. For the cardiac exchanger, on the other hand, Na+ transport has been shown to be strongly electrogenic (Hilgemann et al., 1991; Matsuoka and Hilgemann, 1992; Powell et al., 1993), while Ca2+–Ca2+ exchange is only weakly voltage dependent (Hilgemann et al., 1991; Matsuoka and Hilgemann, 1992; Powell et al., 1993; Niggli and Lederer, 1991; Kappl and Hartung, 1996).
To better compare the function and structure of the exchangers, we have now cloned the squid neuronal Na+–Ca2+ exchanger, successfully expressed it in Xenopus oocytes, and studied its function in giant membrane patches. We describe here functional similarities and differences of the two exchangers, which should facilitate the development of structure/function models and the elucidation of exchanger regulatory mechanisms.
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Cloning Procedures
PCR with degenerate primers was used to amplify a fragment of NCX-SQ1 from squid optic lobe cDNA. Primers were synthesized and reverse-phase purified by Retrogen (San Diego, CA). The primer pairs were designed from conserved amino acid sequences of putative transmembrane segments 6 and 9 of NCX1 and NCX2. The forward primer was a 29-mer with 32-fold degeneracy (5'-TTAAGAATTCTGGAA(A/G)GT(C/T) CT(C/G) TT (C/T) GC(A/C)T-3') and the reverse primer was a 33-mer with 64-fold degeneracy (5'-TTAAGAATTCCCAG(A/G)AA(C/G)AC(A/G)TT (C/G)AC(A/C)GC(A/G) TTG-3'). The PCR reaction was carried out for 30 cycles (94°C, 30 s; 42°C, 60 s; 72°C, 120 s). The PCR product was cloned into the pCRII vector using the TA Cloning Kit (Invitrogen Corp., San Diego, CA). Two identical clones were identified as squid exchanger based on their sequence similarity to NCX1 and NCX2 and were used as probes to screen a squid optic lobe 
ZAPII cDNA library. A partial clone of 1.5 kb was isolated with a nucleotide sequence 
60% identical to the 3' end of NCX1, but no clones extending further into the missing 5' end sequence were isolated. An EcoRI fragment (0.5 kb) from the 5' end of the 1.5-kb clone was then used to screen a squid stellate ganglion ZAPII cDNA library. Two clones (SG12 and SG14), each containing the complete coding sequence of the squid exchanger, were isolated.
Expression of NCX-SQ1 in Xenopus Oocytes
Initially, expression level of SG12 was relatively low in Xenopus oocytes. Therefore, we used the same strategy that improved expression of NCX1 by replacing the 3' untranslated region of SG12 with that of the Na+-glucose cotransporter clone, which possesses a poly(A)+ tail (Matsuoka et al., 1993). Expression of NCX-SQ1 was improved only moderately after modification. Expression was optimized when SG12 was subcloned from pBluescript SK+ into the pBST4 vector (provided by Dr. Bezanilla's laboratory) at the BglII site. The vector contains the 5' and 3' untranslated regions of Xenopus β globin, which flank the NCX-SQ1 coding region in the final construct. The pBluescript SK+ or pBST4 vector containing the NCX-SQ1 full-length cDNA was linearized with XhoI or SacII, and cRNA was synthesized using the T3 or T7 mMessage mMachine Kit (Ambion Inc., Austin, TX), respectively. Unincorporated nucleotides were removed with Chromaspin-100 columns (Clontech, Palo Alto, CA). Oocytes were prepared as described by Longoni et al. (1988). Oocytes were injected with 46 nl of cRNA or water, and exchange activity was measured 4 d after injection as Na+ gradient-dependent 45Ca2+ uptake (Longoni et al., 1988; Nicoll et al., 1990) or as exchange current across giant excised patches (see below).
Northern Blot Analysis
Total RNA from squid optic lobe and stellate ganglion were prepared using the protocol of Chomczynski and Sacchi (1987) as modified by Quednau et al. (1997). Poly (A)+ RNA was isolated from 100 µg of total RNA using the Poly (A)Tract mRNA Isolation System (Promega Corp., Madison, WI). 1 µg poly (A)+ RNA from each tissue was fractionated on a 1% agarose/6% formaldehyde gel, transferred to Hybond-N membrane (Amersham Corp., Arlington Heights, IL), hybridized with an antisense probe (see below), and washed as described previously (Nicoll et al., 1996). Final washing was at 0.2x SSC at 42°C.
A 32P-labeled antisense probe was generated by asymmetric PCR as described by Sturzl and Roth (1990). A plasmid containing the ApaI to SacI cDNA fragment of NCX-SQ1 (nucleotides [nts] 1987–2592) was linearized by ApaI restriction enzyme digestion downstream from the primer annealing site. Phenol-purified template (200 ng) was used together with 200 pmol primer in a 100-µl reaction mix containing 50 µCi of [
-32P]dCTP, 5 U Taq DNA Polymerase (GIBCO BRL, Gaithersburg, MD). The primer for PCR was a 20-mer (5'-ATACGCACTT CCACTTCACC-3'; nts 2574–2554). PCR was carried out for 35 cycles (94°C, 45 s; 55°C, 1 min; 72°C, 2 min). Unincorporated nucleotides were removed by two consecutive steps of ammonium acetate precipitation.
Preparation of NCX-SQ1 Fusion Protein and Antibody Production
An expression construct containing the cDNA coding for the large intracellular loop of NCX-SQ1 was constructed by PCR. The 5' end of the forward primer (5'-AAGCATGCGGTGTGATTGTCCAATGT-3'; nts 1767–1785) included an introduced SphI restriction site and the reverse primer (5'-TTCTGCAGAATGGCCTCAATAAACTG-3'; nts 3007–2989) contained an introduced PstI restriction site at the 3' end. pBluescript SK+ vector containing the NCX-SQ1 full-length cDNA (10 ng) was amplified using 0.2 mM each of dATP, dCTP, dGTP, and dTTP, 1 mM MgCl2, 5% DMSO, 50 pmol of forward and reverse primer, and 2.5 U of Taq DNA polymerase to produce a 1,240-bp fragment (nts 1767– 3007; amino acids 259–665). PCR was carried out over 35 cycles (94°C, 30 s; 55°C, 1 min; 72°C, 2 min). The PCR product was cloned into the pCR II vector using the TA Cloning Kit, and the identity of the clone was confirmed by sequencing. The PCR construct was digested with SphI and PstI and the resulting fragment cloned into the expression vector pQE (QIAGEN Inc., Chatsworth, CA). The loop fusion protein was expressed and purified as described by He et al. (1997) and used as antigen for generation of polyclonal antisera in rabbits (HRP Inc.; Denver, PA).
Western Blot
Samples from oocytes expressing NCX-SQ1 protein were prepared as follows: 10 oocytes were sonicated in 100 µl of homogenization buffer (0.1 M NaCl, 1% Triton X-100, 20 mM Tris-HCl, pH 7.6) and centrifuged at 12,000 rpm for 10 min. The supernatant was filtered twice through Spin-X filters to remove lipids and yolk protein. Supernatant (5 µl) or squid optic lobe vesicles (20 µg total protein) were electrophoresed on a 7% SDS-polyacrylamide gel. Proteins were transferred onto nitrocellulose membrane and the filter was blocked with 5% (wt/vol) milk in MTBST buffer (140 mM NaCl, 20 mM MOPS/Tris, pH 7.4, 0.05% TWEEN 20). The membrane was incubated with the antiserum raised against the loop fusion protein of NCX-SQ1 or preimmune serum at a dilution of 1:5,000, followed by incubation with goat anti–rabbit IgG conjugated with horseradish peroxidase (1:3,000; Bio-Rad Laboratories, Richmond, CA) in MTBST. The antigen–antibody complexes on the membrane were visualized using 3,3'-diaminobenzidine.
Electrophysiological Analyses: Endogenous Oocyte Conductances
Na+–Ca2+ exchange currents were isolated and studied in giant excised membrane patches from Xenopus oocytes, as described previously, using solutions that minimize endogenous currents in the oocyte membrane (Matsuoka et al., 1993, 1995). The extracellular solution contained 4 mM Ca-sulfamic acid, 1 mM Mg- sulfamic acid, 40 mM Na-2-(morpholino)ethanesulfonic acid (MES), 20 mM Cs-MES, 20 mM tetraethyl ammonium (TEA)- MES, 40 mM N-methyl-D-glucamine (NMG)-MES, and 20 mM HEPES, adjusted to pH 7.0 with NMG. The cytoplasmic solution contained (mM): 10 EGTA, 6 Ca-sulfamic acid, 0.5 Mg-sulfamic acid, 60 Cs-MES, 20 TEA-MES, and either 40 additional Cs-MES or 40 Na-MES to activate outward exchange current, at pH 7.0 with NMG (pCa 6.5). Gigaohm seals were made in a solution containing (mM): 80 K+-aspartate, 40 KCl, 4 MgCl2, 5 EGTA, and 10 HEPES, at pH 7.0 with NMG. Experiments were performed at 32°C. Concentrated stock solutions of nucleotides were prepared as Mg2+/TRIS salts, with the Mg2+ concentration adjusted to 75% of the total nucleotide concentration. In this way, the free Mg2+ concentration (0.3 mM) is not changed on addition of nucleotide. Free Ca2+ and Mg2+ concentrations were calculated with the binding constants given by Fabiato (1988), and for ANP-PNP (adenosine 5'-(β,-imido)triphosphate) we assumed the same Mg binding constant as for ATP. Unless indicated otherwise, the membrane potential was 0 mV. PIP2 liposomes were prepared by sonicating 1 mM PIP2 (Boehringer Mannheim, Mannheim, Germany) in distilled water. Reconstituted monoclonal PIP2 antibody (PerSeptive Biosystems, Framingham, MA) was diluted 40-fold into the experimental solution.
Endogenous conductances of the oocyte membrane were found to be activated by ATP and anionic lipids, so more extensive control experiments were needed to test for the influence of contaminating currents. Most importantly, we established conditions such that current changes in patches from uninjected (or water-injected) oocytes amounted to at most a few picoamperes using the same conditions and protocols employed in RESULTS (i.e., at 0 mV).
Since our efforts to understand and minimize endogenous conductances are relevant to many expression studies with Xenopus oocytes, we describe here the two major oocyte currents activated by ATP and PIP2; Ca2+-activated Cl– and voltage-activated Na+ currents. Activation of Ca2+-activated Cl– current by ATP (Hilgemann, 1997) and anionic lipids in oocyte patches (Hilgemann, 1995) has been described previously. As shown in Fig. 1, Ca2+-activated Cl– current is stimulated by many polyvalent anions (0.1–2 mM) and by F– when cytoplasmic free Ca2+ is submaximal. Fig. 1 A shows results for phosphate. Activation by polyvalent anions typically takes 1 min, but the effect decays on removal of anions in only a few seconds. Possibly, these effects reflect chelation of trace polyvalent cations in solutions (or from the pipette) by these anions (Hilgemann, 1997). Since the pipette tip can become contaminated with Cl– during seal formation, there is a danger that outward Cl– current can occur during exchange current measurements. Inclusion of Cl– current blockers in the pipette solution (0.3 mM niflumic acid + 0.3 mM flufenamic acid) effectively blocked the residual Cl– currents, while results with PIP2 and nucleotides in exchanger-expressing patches were not changed.
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), this current activates and deactivates rapidly in response to voltage steps after it has first been primed by a long depolarization. Fig. 2 B shows the steady state current–voltage relation of an oocyte patch with symmetrical Na+-containing solutions (40 mM). We stress that the Na+ conductance was always negligible at potentials more negative than –20 mV. In the absence of ATP and PIP2, the magnitude of the Na+ conductance was variable and depended on the oocyte batch. Na+ conductance could be induced in the majority of patches by ATP or anionic lipids. Fig. 2 C shows the activation by ATP, reversal after ATP removal, and suppression by 40 mM aluminum in the presence of 10 mM EGTA (50 ms cumulative voltage pulses). To avoid contamination of exchange currents with Na+ current, we made our recordings with 40 mM Na+ on both sides at 0 mV (i.e., at the reversal potential of the Na+ current). Also, we monitored current–voltage relations using 2-ms voltage steps and verified that the current activated by ATP and PIP2 remained outward in direction at potentials as negative as –70 mV with 40 mM extracellular and cytoplasmic Na+ concentrations. Thus, the underlying mechanism could not involve Na+ channels.
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320 bp) was subcloned. Sequence analysis indicated that the DNA coded for a protein homologous to NCX1. The PCR product was used to screen a squid optic lobe library and a partial clone of 1.5 kb was isolated with 60% identity to NCX1 at the amino acid level. Longer clones were not found in the optic lobe library. A fragment of the partial 1.5-kb clone was then used to screen a squid stellate ganglion cDNA library. Two clones containing the complete coding sequence of the squid exchanger were isolated. These clones were identical at the 3' end. The complete nucleotide and amino acid sequences of the longest clone, SG14, are shown in Fig. 3. SG14 is 4,096 bp long with an open reading frame of 2,676 nucleotides encoding for a protein of 892 amino acids, which we refer to as NCX-SQ1. The most 5' ATG, in the proper reading frame, begins at nucleotide 994 with some features of a Kozak (1989) consensus initiation site. The 3' end of the cDNA terminates with 16 adenosines preceded by multiple consensus polyadenylation sites.
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) is shown in Fig. 4. Like NCX1, NCX-SQ1 has 12 hydrophobic segments that could form transmembrane segments. In NCX1, the first hydrophobic segment at the NH2 terminus is a cleaved leader peptide and is removed from the protein during biosynthesis in the endoplasmic reticulum (Durkin et al., 1991; Hryshko et al., 1993). NCX-SQ1 has a potential signal peptidase-recognition site (Von Heijne, 1983) after alanine 26, and we predict that a signal peptide is removed from this protein as well. Thus, the mature protein is modeled to have 11 transmembrane segments with a large intracellular loop separating transmembrane segments 5 and 6. The NH2 terminus would be extracellular and the COOH terminus would be intracellular. NCX-SQ1 has nine potential sites for N-linked glycosylation. If the glycosylation pattern is similar to that of NCX1 (Hryshko et al., 1993), only the first site (asparagine 31) would be glycosylated. Overall, the mature NCX-SQ1 and NCX1 proteins are 58% identical, ignoring the gap in NCX-SQ1 in the intracellular loop region. More sequence analysis will be presented below.
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120 kD and two bands at lower molecular weights. NCX1 produces a similar pattern on immunoblots (Nicoll et al., 1990). Preimmune serum from the same rabbit failed to produce any signal (not shown). Expression of exchanger protein in Xenopus oocytes injected with NCX-SQ1 cRNA was also examined. A protein band of 100 kD was detected in a membrane fraction from cRNA- injected oocytes, whereas no signal was detected in control water-injected oocytes (Fig. 5 B, lanes 2 and 3). The small difference in apparent molecular weight between the native squid exchanger and NCX-SQ1 expressed in oocytes is possibly due to a difference in glycosylation.
Functional Expression of NCX-SQ1 in Xenopus Oocytes
cRNA encoding NCX-SQ1 was synthesized from linearized plasmids and injected into Xenopus oocytes. Expression was optimized when the 5'- and 3'-untranslated regions of Xenopus β globin flanked the NCX-SQ1 coding region. Expression of Na+–Ca2+ exchange activity was assessed by measuring 45Ca2+ fluxes into intact oocytes and by measuring exchanger currents using the giant excised patch technique. An example of Na+ gradient-dependent 45Ca2+ uptake into Na+-loaded Xenopus oocytes is shown in Fig. 6. The first columns show that NCX-SQ1 RNA induces a substantial uptake of 45Ca2+ in the presence of an outwardly directed Na+ gradient (K+0). This uptake is abolished in the absence of the Na+ gradient (Na+0). We have validated the use of this approach to measure Na+–Ca2+ exchange activity in previous studies (Longoni et al., 1988; Nicoll et al., 1990). No Ca2+ uptake was observed in control water- injected oocytes.
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; DiPolo and Beaugé, 1986). Oocytes expressing NCX1-SQ1 were injected with EGTA before the Ca2+ uptake assay. Chelation of internal Ca2+ blocked most Ca2+ uptake (Fig. 6), which is consistent with previous studies of squid axons and NCX1 (Baker and McNaughton, 1976; DiPolo and Beaugé, 1986; Kimura et al., 1986; Hilgemann et al., 1992a, 1992b).
Outward Exchange Current of the Squid (NCX-SQ1) Na+–Ca2+ Exchanger
Fig. 7 shows basic properties of outward Na+–Ca2+ exchange current in patches from oocytes expressing the NCX-SQ1 exchanger. In brief, no obvious property of the current is different from NCX1 exchange current. On application of cytoplasmic Na+, the current activates in the solution switch time and then shows partial inactivation over several seconds (see Fig. 7, first record, in the presence of 1 µM cytoplasmic free Ca2+). When cytoplasmic Ca2+ is removed, the current magnitude decreases, and inactivation on application of Na+ is subsequently faster. Exchange current remains substantial (second record). With 5 µM free cytoplasmic Ca2+ (third record), or higher concentrations (not shown), the current did not increase further and ran down with time. Current run-down prevented us from determining Ca2+–current relations in more detail. Application of -chymotrypsin (1 mg/ml) activated the exchange current over 1 min. The final current magnitude was typically more than twice the peak current magnitude obtained on applying Na+. After chymotrypsin, the exchange current was insensitive to changes of cytoplasmic free Ca2+ from 0 to 5 µM.
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). However, in our initial studies of the cardiac exchanger (NCX1) in oocyte patches, no effects of ATP were observed (Matsuoka et al., 1993). We now report that ATP indeed can be effective when the phosphatase inhibitors, F– and vanadate, are included in the cytoplasmic solution. The concentrations of F–and vanadate are selected to maintain free Mg2+ in the range of 0.1–0.3 mM, and vanadate alone was usually effective. Fig. 8 describes typical results with 0.1 mM F– and 50 µM vanadate (0.5 mM total Mg2+ in nucleotide-free solution). The outward current activated by Na+ shows the usual inactivation when 40 mM cytoplasmic Na+ is applied. As shown in Fig. 8 A, 2 mM of the nonhydrolyzable ATP analogue, AMP-PNP, was without effect, while 2 mM ATP increased the current to a magnitude somewhat greater than the peak current on initial application of Na+. On removal of ATP, the current returned partially toward baseline over 2 min. The thio-analogue of ATP, ATPS (2 mM), typically had no effect, or only a small stimulatory effect (Fig. 8 B). After removal of ATPS, application of ATP stimulated the exchange current, as usual. As mentioned in the INTRODUCTION, the stimulatory effect of ATP in cardiac membrane appears to reflect the generation of PIP2 from phosphatidylinositol. PIP2 also activates some potassium channels (Hilgemann and Ball, 1997) and, with inward rectifier potassium channels, application of an antibody to PIP2 reverses the ATP effects within 1 min (Huang et al., 1998). As shown in the latter part of the record in Fig. 8 B, the same antibody to PIP2 (AB) reverses the stimulatory effect of ATP on the NCX1 exchanger; the antibody was without effect when exchange currents were stimulated by other anionic lipids (e.g., cardiolipin).
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85%. With application of 2 mM ATP, the current increased to a magnitude higher than the peak current on initial application of Na+. Notably, this result was often obtained in the absence of F– and vanadate; stimulation by ATP was observed only rarely with NCX1 in this condition. After removal of ATP, the current declines about half-way toward baseline over 1 min before it declines more slowly. After allowing current to return nearly to the control level (not shown), Na+ was removed and reapplied in the presence of 0.2 mM F– and 50 µM vanadate. The current increased to the same extent as previously upon addition of ATP, but on removal of ATP the current remained stable; presumably, the maximal currents in these records reflect a "ceiling" for simulatory effects. Reversal rates are 10x slower than without the phosphatase inhibitors. Fig. 9 B demonstrates that the PIP2 antibody is also effective in reversing the stimulatory effects of ATP on the squid exchanger. F– and vanadate are present throughout the experiment. After application of the antibody, current returned nearly to baseline in 2 min in the presence of antibody.
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S and Its Reversal by PIP2 AntibodyS (four of six results). Typical results are shown in Fig. 10. In the presence of F– and vanadate, application of a nonhydrolyzable ATP derivative, AMP-PNP (2 mM) was nearly without effect (Fig. 10 A); the small inhibition of exchange current may be due to a small increase of free Mg2+. Thereafter, application of 2 mM ATPS stimulates the exchange current roughly to the extent observed with ATP in NCX-SQ1-expressing patches. The stimulatory effect reverses within a few minutes on removal of the ATPS. As shown in Fig. 10 B, PIP2 antibody can reverse the effect of ATPS over the course of 90 s, suggesting a possible involvement of PIP2 in the effects of ATPS as well as those of ATP.
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15 pA (not shown).
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), indicating that similar molecular mechanisms might be involved.
Lack of Effect of Phosphoarginine on Outward Squid (NCX-SQ1) Na+–Ca2+ Exchange Current in Oocyte Patches
As noted in the INTRODUCTION, the high-energy compound phosphoarginine stimulates the Na+–Ca2+ exchange process in squid axons, probably by a mechanism that involves phosphorylation (DiPolo and Beaugé, 1995). As shown in Fig. 12, phosphoarginine (5 mM) was without effect on the outward exchange current in oocyte patches (four observations, applying phosphoarginine with Mg2+). In the same patch, 50 µM PIP2 was highly effective. As with all nucleotides, other phosphates, and citrate, large stimulatory effects were observed in patches when phosphoarginine was applied without added Mg2+ (not shown). The probable explanation for our results in patches is that all of these anions chelate Mg2+ and thereby relieve an inhibition of exchange current by cytoplasmic Mg2+.
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-chymotrypsin (1 mg/ml for 30 s). Current changes were negligible in patches from uninjected oocytes, using the same conditions and protocols.
Fig. 13 A shows current–voltage relations for outward NCX-SQ1 exchange current with 4 mM extracellular Ca2+ and no extracellular Na+. With the Ca2+-free/10 mM EGTA cytoplasmic solution employed in these experiments, Ca2+-activated Cl– conductance is zero, and the same solutions (with 20 mM Cl–) could be used as in previous measurements with cardiac membrane patches (Hilgemann et al., 1992a). Cytoplasmic Na+ was varied from 5 to 90 mM, substituting it for Cs+, and baseline current–voltage relations in the absence of Na+ were subtracted. The current–voltage relations are similar in shape and they can be scaled well to each other (not illustrated). Fig. 13 B shows the Na+ dependence of current at –60 and +60 mV; the K50 (half-maximal concentration) for Na+ is 27 mM at +60 mV and 24 mM at –60 mV, and the Hill slopes are 1.2 and 1.7, respectively. For the cardiac exchanger, by contrast, current–voltage relations become less steep with high cytoplasmic Na+, and the K50 for Na+ decreases somewhat at positive potentials (Matsuoka and Hilgemann, 1992).
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30 mV in the steepest region of the current–voltage relations. This is close to the slope expected for a single charge movement across the entire electrical field. Fig. 14, C and D, shows the Ca2+ dependencies of the inward current at –150 and –30 mV. Both data sets are well- described by Hill equations with slopes of 1, and there are small shifts of the K50's to higher concentrations at more negative potentials; 2.5–4.2 µM for NCX1, and 3.5–7.2 µM for NCX-SQ1.
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; DiPolo and Beaugé, 1990) and similar results are described for the barnacle Na+– Ca2+ exchanger (Rasgado-Flores et al., 1996). To test directly whether Ca2+ translocation is electrogenic in NCX-SQ1, ion concentration jump experiments were designed to isolate the possible charge movements of outward Ca2+ and Na+ translocation. To do so, the outward translocation of ions is initiated by moving the patch pipette tip through the interface between two solution streams in 1 ms, whereby one stream contains no substrate and the other contains a high substrate concentration. A computer-controlled Piezzo-type manipulator is used to move the patch clamp head stage together with the patch pipette (Hilgemann and Lu, 1998). The speed of the substrate concentration change occurring at the membrane surface in these experiments (150-ms time constant) is determined by diffusion from the pipette orifice to the membrane surface (20–70 µm). We point out, however, that the speed of current activation, observed upon applying a substrate, can be much faster. That is because binding sites can be saturated quickly, compared with average diffusion times, if substrate concentrations are high with respect to binding site affinity. This is the case for experiments with Na+ jumps, but it appears not to be the case for Ca2+ jumps.
Fig. 15 illustrates the major experimental results. All patches were chymotrypsin treated and it is noted that experiments were successful only in oocyte batches with high exchanger expression (approximately one batch out of six). The protocols, based on the predicted function of an alternating-access exchange model, were the same as used previously to monitor "half-cycles" of ion transport (Hilgemann et al., 1991): in the presence of substrate on the extracellular side and no substrate on the cytoplasmic side, the exchanger binding sites will orient to the cytoplasmic side and will be free of substrate. When a high concentration of substrate is applied to the cytoplasmic side, substrate will bind and the binding sites will reorient and open to the extracellular side. Substrate will be released, and binding sites will remain in the extracellular orientation, on average, if the extracellular substrate concentration is relatively low. Thus, charge movement observed during this protocol should reflect the electrogenicity of ion transport for the substrate added.
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). An outward current transient, 150 ms in duration with a peak of 3 pA, is observed on application of Na+, and a slower inward current transient with a peak of 1 pA is observed on removing Na+. The areas defined by the current transients correspond to 300,000 elementary charges, which in turn corresponds to 300 charges/µm2 with a 10-pF patch. Using the same protocols in patches expressing the squid exchanger, current transients were either absent or very small (five observations; Fig. 15 B). That these results reflect a real difference in exchanger function is supported by the observation that inward currents activated by applying cytoplasmic Ca2+ were of similar magnitude in the NCX1 and NCX-SQ1 patches (Fig. 15, C and D). We point out that the current activated by 5 µM Ca2+ corresponds to 10% of the current activated in patches from the same oocyte batches when 150 mM Na+ was included in the pipette.
Fig. 15, E and F, shows the equivalent results for jumps of cytoplasmic Ca2+ from 0 to 5 µM in the presence of 50 µM extracellular Ca2+. As described previously (Hilgemann et al., 1991), charge movements are small or absent for this protocol with NCX1. For NCX-SQ1, however, inward current transients are obtained on applying Ca2+ and outward current transients on removing Ca2+. These charge movements may correspond to a movement of negative charge when Ca2+ is moved by the exchanger. The magnitudes of charge movements in these experiments were roughly similar to those for Na+ jumps with NCX1 (Fig. 15 A). Fig. 15, G and H, shows the time courses with which outward exchange current was turned on and off with cytoplasmic Na+ jumps.
Voltage Jump-induced Charge Movements of Ca2+ Transport of the NCX-SQ1 Exchanger
For the cardiac exchanger (NCX1), charge movements of Ca2+ transport have been isolated in voltage-jump experiments (Hilgemann, 1996). The charge movements were of small magnitude and showed weak voltage dependence with rates of 5,000 s–1 at 0 mV. Results for NCX-SQ1, after chymotrypsin treatment, are shown in Fig. 16. The rationale of the experiment is that most of the voltage dependence of Ca2+ transport comes about during occlusion of Ca2+ from the cytoplasmic side. Therefore, a high concentration of Ca2+, 4 mM, is included on the extracellular side. Without cytoplasmic Ca2+, all exchangers should orient to the cytoplasmic side with empty binding sites. Voltage pulses are applied first in the absence of cytoplasmic Ca2+, and then in the presence of 2 µM cytoplasmic Ca2+ to activate the ion occlusion reaction. Charge rather than current was recorded, and the holding potential was –40 mV. Records presented in Fig. 16 A are a subtraction of records with cytoplasmic Ca2+ from records without Ca2+, whereby 16 records were acquired in alternating order with and without cytoplasmic Ca2+, and results were averaged. The charge movements show small fast components that appear as charge jumps on changing voltage, and they show slower components that saturate progressively as larger voltage pulses are applied to +160 and –200 mV. Fig. 16 B shows the voltage dependence of the charge movements, fitted to a Boltzmann relation {1/[1 + expq*(Em+E50)/26.5]}, which gives an equivalent charge (q) of 0.46 underlying the charge movement. This is twice the value obtained for NCX1 (Hilgemann, 1996). Fig. 16 C shows the voltage dependence of the rates of the charge movements, obtained by fitting the slow components of Fig. 16 A to single exponential functions. The rates have a "U-shaped" dependence on voltage, as expected for a simple reaction with voltage dependence of both the forward and reverse rates. The rates can be well-described by the sum of two exponentials, Kf · eq*Em/55 + Kb · e–q*Em/55, where Kf and Kb are the forward and backward rates at 0 mV. The fit gives an equivalent charge of 0.59. The overall rate at 0 mV is 1,600 s–1, which is about threefold lower than rates obtained in equivalent experiments with NCX1 (Hilgemann, 1996).
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), NCX2 (Li et al., 1994), and NCX3 (Nicoll et al., 1996), as well as the Drosophila Na+–Ca2+ exchanger (Schwarz and Benzer, 1997; Ruknudin et al., 1997). NCX-SQ1 is 58% identical at the amino acid level to the canine cardiac Na+–Ca2+ exchanger, NCX1, and has similar identities (51–64%) to the other NCXs. There are several features of note. Regions determined to be of functional importance in previous studies of NCX1 are well conserved. For example, we have identified specific acidic residues within the binding site for regulatory Ca2+ (Fig. 4, boxed area) that are important for Ca2+ binding (Levitsky et al., 1994; Matsuoka et al., 1995). These triple aspartate (DDD) motifs (Fig. 4, bold) are perfectly conserved in all NCX exchangers. Likewise, we have described the endogenous XIP region of NCX1 and have proposed that the XIP region is involved in Na+-dependent inactivation (Li et al., 1991; Matsuoka et al., 1997). A homologous region (Fig. 4, shaded area) is also present in NCX-SQ1 and is conserved among the exchangers.
The predicted topology of NCX-SQ1 is similar to that of the other exchangers with 11 transmembrane segments and a large intracellular loop. Sequence conservation among the exchangers is highest in the proposed transmembrane segments consistent with a catalytic role of the hydrophobic domains in ion translocation. We have described that exchanger function is especially sensitive to mutations within portions of transmembrane segments 2, 3, 8, and 9 (Nicoll et al., 1996). These regions are known as the repeats (-1 and -2). Significantly, the repeats are highly conserved in species as divergent as squid and dog, consistent with a proposed role in ion transport. Proposed transmembrane segment 11 is the least well conserved transmembrane domain among the NCX exchangers. Perhaps the COOH terminus of the protein has a lesser role in exchange function. As noted previously (Tsuruya et al., 1994), the NH2 terminus of the exchanger, which represents a signal peptide region, is poorly conserved among NCX proteins.
The squid Na+–Ca2+ exchanger has previously been reported to be stimulated by phosphorylation reactions and by phosphoarginine (DiPolo and Beaugé, 1994; DiPolo et al., 1997) and we analyzed the NCX-SQ1 sequence for potential phosphorylation sites. Potential sites for phosphorylation by protein kinases A and C, Ca2+/ calmodulin-dependent kinase, and tyrosine kinases are shown in Fig. 4. The PKC site (threonine 184) between transmembrane segments 3 and 4 and the tyrosine kinase site in the Ca2+ binding region (tyrosine 462) are unique among the exchangers. Phosphorylation at either site might be expected to have functional effects.
As shown in Fig. 4, there is a deletion of 47 amino acids in the large intracellular loop of NCX-SQ1 in comparison with NCX1. This region of NCX1 displays extensive alternative splicing (Nakasaki et al., 1993; Kofuji et al., 1994; Lee et al., 1994; Quednau et al., 1997). Six small exons are used in different combinations in a tissue-specific manner. The first two of these exons (exons A and B) are mutually exclusive. The NCX1 splice variant in Fig. 4 is NCX1.1 (Quednau et al., 1997) using exons A and C–F. Of the six small exons, NCX-SQ1 apparently uses only exon A. The homologous exons A of NCX-SQ1 and NCX1 are shaded in Fig. 4. We performed reverse transcriptase–PCR using optic lobe and stellate ganglion RNA to detect other splice variants of NCX-SQ1. Nine clones were sequenced, but no other splicing isoforms were detected.
Regulation of Na+–Ca2+ exchange by intracellular Ca2+ was first described in the squid axon (Baker and McNaughton, 1976; DiPolo, 1979). Micromolar levels of free intracellular Ca2+ are required to activate function. That is, in addition to transporting Ca2+, the exchanger is separately regulated by Ca2+. This secondary effect of intracellular Ca2+ has been analyzed at the molecular level for the cloned cardiac exchanger NCX1 (Levitsky et al., 1994; Matsuoka et al., 1995). Using 45Ca2+ fluxes, we find that Ca2+ regulation is apparently intrinsic to the NCX-SQ1 exchanger protein. Chelation of intracellular Ca2+ with EGTA prevents Na+i-dependent Ca2+ uptake (Fig. 6). This is not surprising since, as noted above, the regulatory Ca2+ binding site of NCX1 is conserved in the sequence of NCX-SQ1.
Regulatory Mechanisms Acting on NCX-SQ1 in Oocyte Giant Patches
Given the sequence similarities to NCX1, it is not surprising that many of the regulatory properties of NCX-SQ1 are similar to those of NCX1. This includes the properties of Na+-dependent inactivation, secondary activation by cytoplasmic Ca2+, and deregulation by chymotrypsin (Fig. 7). Furthermore, similarities include stimulation by ATP and PIP2, and reversal of the stimulatory effects of ATP by a PIP2 antibody (Figs. 8 and 9). However, there are also evident differences between NCX-SQ1 and NCX1. In the absence of phosphatase inhibitors, NCX-SQ1 exchange current is usually stimulated by ATP, which is not the case with NCX1. Either the squid exchanger has higher affinity for PIP2 or it is modulated by an additional ATP-dependent reaction. Consistent with this possibility, the thioester of ATP, ATPS, stimulates NCX-SQ1 in oocyte patches but not NCX1 current (Figs. 8 and 10). An attractive speculation is that we are observing phosphorylation of NCX-SQ1 at one of its consensus phosphorylation sites. We cannot, however, completely eliminate a possibility that effects of the ATPS preparations employed reflect contaminating ATP and an increased sensitivity of the squid to PIP2.
The reversal of ATP effects on Na+–Ca2+ exchange current by a PIP2 antibody is reported here for the first time. The fact that the PIP2 antibody can reverse stimulatory effects of ATPS on the squid exchanger indicates that PIP2 is involved. Perhaps phosphorylation increases the affinity of NCX-SQ1 for PIP2. Precedents for such a mechanism come from recent work with inward rectifier potassium channels. With GIRK-type channels, activation by G-protein β subunits is accompanied by an increase of affinity for PIP2 (Huang et al., 1998), and, with ROMK-type channels, activation by cAMP- dependent protein kinase is accompanied by an increase of the apparent affinity for PIP2 (Dr. C.L. Huang, personal communication). Since ATP increases the apparent affinity for Ca2+ at exchanger regulatory sites (DiPolo and Beaugé, 1987; Collins et al., 1992), a primary effect of phosphorylation on Ca2+ affinity is also a possibility. In contrast to our results, it is reported that the PIP2 antibody is without effect on Na+–Ca2+ exchange in squid giant axons, either with or without ATP (DiPolo and Beaugé, 1998). Either the antibody does not reach the membrane in axons or the exchanger is regulated in a fundamentally different way in the squid axon environment. The fact that phosphoarginine does not stimulate the cloned squid exchanger presumably reflects the absence of appropriate phosphoarginine- dependent kinases in oocyte giant membrane patches.
Voltage Dependence of NCX-SQ1 in Oocyte Giant Patches
The ion dependencies of the NCX-SQ1 exchange currents, determined here for the cytoplasmic side (Figs. 13 and 14), are consistent with those determined in dialyzed squid axons (DiPolo, 1989) and are only slightly different from results for the NCX1 exchanger. As described in Figs. 13 and 14, the voltage dependencies of outward and inward exchange currents of NCX-SQ1 are stronger than for NCX1, and the isolation of charge movements of Ca2+ transport for the squid exchanger (Figs. 15 and 16) verifies that electrogenic reactions are indeed different in the squid exchanger. Na+ transport is relatively less electrogenic, while Ca2+ transport is more electrogenic (Figs. 15 and 16). An important observation, which allows interpretation of the current–voltage relations of the squid exchanger, is that Na+–Na+ exchange by the squid exchanger is substantially greater than Na+–Ca+ or Ca2+–Ca2+ exchange (DiPolo et al., 1989; DiPolo and Beaugé, 1990; Dr. L. Beaugé, personal communication). Thus, Ca2+ transport may in general be rate limiting and the voltage dependence of Ca2+ transport will determine the overall voltage dependence of transport current. Consistent with this interpretation, the rates of Ca2+-dependent charge movements determined for the squid exchanger at 0 mV and 33°C are substantially less (1,700 s–1) than those determined for the NCX1 exchanger (5,000 s–1; Hilgemann et al., 1991; Hilgemann, 1996).
Our interpretation is tempered to some extent because an alternating access model, or "consecutive" exchange mechanism, has not been rigorously verified for the squid exchanger. In a consecutive mechanism, the apparent affinity of one transported ion should increase as the concentration of the countertransported ion is decreased. For the squid exchanger, it has been reported that the Na+ dependence of Ca2+ efflux does not change as the cytoplasmic Ca2+ concentration is reduced (DiPolo, 1989). Thus, further work is required on transport properties of the squid exchanger; the application of techniques to photo-release Ca2+ in giant patches within microseconds (Kappl and Hartung, 1996), rather than 100 ms, will allow much better resolution of these issues.
The differences in the charge-moving reactions of NCX1 and NCX-SQ1 may provide an important key to elucidating the physical basis of exchanger electrogenicity. Ion occlusion reactions may result from the movement of charged residues anywhere in the exchanger protein, but only residues that enter or leave the membrane electrical field together with ions will generate charge movements. Evidently, more than two negative charges must move into the membrane electrical field when Ca2+ is occluded by the squid exchanger from the cytoplasmic side. Studies of chimeras of the two exchangers, and ultimately point mutation studies, should be able to define the involvement of specific groups and provide an understanding of the conformational changes underlying ion transport.
In summary, the squid Na+–Ca2+ exchanger, NCX-SQ1, has been cloned and expressed in Xenopus oocytes, and its function has been characterized electrophysiologically in giant membrane patches. The differences between the sequence and functional properties of NCX-SQ1 and the mammalian NCX1 now provide a new basis to elucidate both regulatory and transport properties of Na+–Ca2+ exchange. NCX-SQ1 is strongly activated by the anionic phospholipid, PIP2, and the presence of phosphorylation sites, not present in NCX1, may correlate with stimulation of the squid exchanger by thioester derivatives of ATP. The molecular basis of differences in the voltage dependence of cardiac and squid exchangers can now be pursued by the combined methods of molecular biology and electrophysiology.
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ACKNOWLEDGMENTS |
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This work was supported by the National Institutes of Health (HL-5132302 to D.W. Hilgemann and HL-48509 and HL-49101 to K.D. Philipson).
Submitted: 4 December 1997
Accepted: 20 March 1998
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). (C) Comparison of current transients obtained on activating exchange current before (control) and after (+PIP2) applying PIP2. The time constant (t) of inactivation increases from 3.9 to 8.2 s. Results are from a different patch.
