Stimulation of Na⁺-Ca²⁺ exchange by purified antibody against alpha-2 repeat of Na⁺-Ca²⁺ exchanger in rat cardiomyocytes

Introduction

The process of Na⁺-Ca²⁺ exchange was first identified in guinea pig atria by Reuter and Seitz in 1968^[1], and in the squid giant axon by Baker et al shortly after in 1969^[2]. The entity, the so-called Na⁺-Ca²⁺ exchanger (NCX), is an ion transport protein that catalyzes electrogenetic antitransport of Na⁺ and Ca²⁺ across the plasma membrane in a coupling ratio of 3 Na⁺:1 Ca²⁺ and exists in the plasma membrane of almost all animal cells^[3]. It is in cardiomyocytes, however, that the exchanger is highly expressed and plays an important role in Ca²⁺ homeostasis. The NCX system is the primary mechanism responsible for transarcolemmal Ca²⁺ extrusion. There is a general agreement that the majority of Ca²⁺ entry through voltage-dependent Ca²⁺ channel is transported out of the cell by NCX^[4,5]. Moreover, it has also been suggested that Ca²⁺ entry mediated by NCX in Ca²⁺ influx mode contributed directly to contraction of failing human ventricular myocytes during the early period of the cardiomyocyte action potential^[6].

The study of NCX in molecular biology started after the cloning of canine cardiac NCX by Nicoll and Philipson^[7]. Later in 1997, Schwarz and Benzer^[8] first identified the highly conserved regions in all known members of the NCX family, designated the alpha-1 and alpha-2 repeats. These regions are highly conserved among different exchangers and between one another. In cardiac NCX, the alpha-1 repeat comprises most of the putative transmembrane segment 2 and 3 (TM2 and TM3) and their connecting loop, whereas alpha-2 locates in putative TM7 and its C-terminal sequence^[9].

A recent study by Nicoll and Iwamoto demonstrated that the NCX1 had oppositely oriented reentrant loop domains in alpha-1 and alpha-2 repeats, and that these reentrant domains in the alpha-repeats might be involved in the formation of the ion transport pathway^[9]. Mutation analysis also showed that alpha-repeats were involved in the interaction of the exchanger with transport substrates (Na⁺ and Ca²⁺), Ni²⁺, Li⁺ and KB-R7943^[10–12].

Now that the alpha-repeats regions were considered important in the ion binding and translocation, it is possible that the antibodies against alpha-1 repeat and alpha-2 repeat may have a crucial action on Na⁺-Ca²⁺ exchanger activity. However, the effect of the antibody against alpha-2 repeat on I_NCX and its specificity is unclear until now. The goal of this study is to identify the effects of the antibody on Na⁺-Ca²⁺ exchanger current using a whole-cell patch-clamp technique. Furthermore, the functional specificity of the antibody was also investigated in adult rat cardiomyocytes.

Materials and methods

Ventricular myocyte isolation Single ventricular myocytes were isolated from Wistar rats (250–300 g) using an enzymatic dissociation procedure similar to that described by Mubagwa et al^[13]. In brief, rats were anesthetized with sodium pentobarbital (30 mg/kg, ip) 30 min after having received heparin (500 U, ip). The heart was quickly removed, rinsed in ice-cold Ca²⁺ free Tyrode’s solution and perfused with oxygenated Ca²⁺ free Tyrode’s solution (at 37 °C) via aorta for approximately 7–8 min to wash out the blood. The composition of Tyrode’s solution was (in mmol/L): NaCl 135, KCl 5.4, CaCl₂ 1.8, MgCl₂ 1.0, NaH₂PO₄ 0.33, HEPES (4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid) 10, glucose 10 (pH adjusted to 7.4 with NaOH). The composition of Ca²⁺-free Tyrode’s solution was the same to Tyrode’s solution except for the absence of CaCl₂. Then the perfusate was switched to enzyme solution for 8–10 min. Enzyme solution contained (in mmol/L): NaCl 135, KCl 5.4, CaCl₂ 75 µmol/L, MgCl₂ 1.0, NaH₂PO₄ 0.33, HEPES 10, glucose 10, taurine 20, collagenase P (Boehringer Mannheim, Mannheim, Germany) 100 mg/L (pH adjusted to 7.4 with NaOH). The ventricle was then separated and minced with a pair of surgical scissors in the Kraftbrühe (KB) solution. The isolated myocytes were stored in KB solution at room temperature (22 °C) at least 4 h before use. KB solution was composed of (in mmol/L): KOH 85, L-glutamic acid 50, KCl 30, MgCl₂ 1.0, KH₂PO₄ 30, glucose 10, taurine 20, HEPES 10, EGTA[ethyleneglycol-bis(β-amino-ethylether)-N,N,N´,N´-tetraacetic acid] 0.5 (pH adjusted to 7.4 with KOH 1 mol/L).

Electrophysiological measurement Voltage-clamp recording was carried out in the whole-cell configuration of the patch-clamp method^[14] using a Patchclamp Amplifier (Axopatch-200A, Axon Instruments, Foster City, CA, USA). Patch electrodes were made from thin-walled glass capillaries and the electrodes with resistance of 2–4 MΩ were filled with the pipette solution. Cell capacitance was measured by the method described by Coetzee et al^[15]. Analysis was carried out using pClampfit 8.0 software (Axon Instruments).

For the measurement of Na⁺-Ca²⁺ exchange current (I_Na/Ca), the extracellular (test) solution contained (in mmol/L): NaCl 140, CaCl₂ 2.0, MgCl₂ 2.0, glucose 10, HEPES 5.0 (pH adjusted to 7.4 with CsOH). In addition, the Na⁺-K⁺ pump, K⁺ channel and Ca²⁺ channel were blocked with ouabain (Sigma Chemical, St Louis, MO, USA) 20 µmol/L, BaCl₂ 1.0 mmol/L, CsCl 2.0 mmol/L and nicardipine (Sigma) 1.0 µmol/L. The pipette solution contained (in mmol/L): EGTA 42, CaCl₂ 29, MgCl₂ 13, aspartate, K₂ATP 10, Na₂-cretinephosphate 5.0, TEA (tetraethylammonium) (Sigma) 20, HEPES 5.0 (pH adjusted to 7.4 with CsOH). To measure L-type Ca²⁺ current (I_Ca,L), the extracellular (test) solution contained (in mmol/L): NaCl 135, CaCl₂ 1.8, MgCl₂ 1.0, KCl 5.4, glucose 10, NaH₂PO₄ 0.33, HEPES 10 (pH adjusted to 7.4 with NaOH). The pipette solution contained (in mmol/L): EGTA 10, KCl 140, Na₂ATP 2.0, HEPES 5.0, 4-AP 5.0, MgCl₂ 1.0 (pH adjusted to 7.3 with KOH). To record voltage-gated Na⁺ current (I_Na), the extracellular (test) solution contained (in mmol/L): NaCl 60, CsCl 5.0, CdCl₂ 0.1, MgCl₂ 2.5, glucose 10, 4-AP 5.0, HEPES 5.0, saccharose 80 (pH adjusted to 7.4 with NaOH). The pipette solution contained (in mmol/L): EGTA 11, KCl 130, Na₂ATP 5.0, HEPES 10, MgCl₂ 2.0, CaCl₂ 1.0, 4-AP 5.0 (pH adjusted to 7.2 with CsOH). For the measurement of delayed rectifier K⁺ current (I_K), the extracellular (test) solution contained (in mmol/L): NaCl 145, KCl 4.0, MgCl₂ 1.0, HEPES 10, glucose 5.0, CaCl₂ 0.l, CdC1₂ 0.1 (pH adjusted to 7.4 with NaOH). The pipette solution contained (in mmol/L): KCl 130, MgCl₂ 2.0, CaCl₂ 1.0, EGTA 11, MgATP 5, K₂ATP 5.0, HEPES 10 (pH adjusted to 7.4 with KOH).

Antibody preparation The antibody against alpha-2 repeat of Na⁺-Ca²⁺ exchanger was prepared in our laboratory^[16]. Briefly, peptide corresponding to alpha-2 repeat (815 TFASKVAATQDQYADASIGNVTGSN 839) in cardiac NCX was synthesized by CL (Xi’an) Bioscientific Incorporation. Then the rats were randomly divided into two groups: control and immunized groups. Rats in immunized groups were immunized with the synthesized alpha-2 repeat emulsified in equal volume of Freund’s adjuvant (CFA, Sigma). The positive antiserum with high titer (≥1:640 by enzyme-linked immunosorbent assay [ELISA]) were affinity-purified using a Mab Trap Kit (Amersham Pharmacia Biotech, Uppsala, Sweden). The concentration of the purified antibody was determined using the method described by Bradford^[17]. The control group received the same disposal as described above except that the peptide was substituted for saline solution.

Data analysis Results were expressed as mean±SD, and analyzed with least-significant difference (LSD) test of ANOVA in SPSS 11. P<0.05 was considered significant. EC₅₀ values were determined using GraphPad Prism 4 software.

Results

Measurement of the Na⁺-Ca²⁺ exchange current (I_Na/Ca) I_Na/Ca was measured as the current sensitive to 5.0 mmol/L Ni^2+[18] by the voltage protocol shown at the top of Figure 1. Ramp voltage-clamp pulse from 60 to –120 mV (90 mV/s) was applied from a holding potential of –40 mV. The current-voltage relationship was constructed from the declining slope of the ramp pulse. After application of Ni²⁺ at the concentration of 5.0 mmol/L, the current immediately decreased, at both positive and negative potentials (Figure 1). The difference between current–voltage relationships in the absence and presence of Ni²⁺ reflected I_Na/Ca (Ni²⁺-sensitive current). Significant run-down of the Ni²⁺-sensitive current was not observed during the experiment.

Figure 1 Measurement of Ni²⁺-sensitive Na⁺-Ca²⁺ exchange current in rat ventricular myocytes. The voltage protocol is shown in the top panel (see text for details). (A) Current-voltage relationship before (trace a) and after (trace b) application of 5.0 mmol/L NiCl₂. (B) Ni²⁺-sensitive Na⁺-Ca²⁺ exchange current (numerical subtraction of a–b).

Stimulating effect of antibody against alpha-2 repeat on Na⁺-Ca²⁺ exchange current (I_Na/Ca) Antibody against alpha-2 repeat had a stimulating effect on performance of Na⁺-Ca²⁺ exchange, as demonstrated by the present study. It was shown that this antibody increased both the outward current and inward current concentration-dependently with EC₅₀ values of 27.9 nmol/L and 24.7 nmol/L, respectively (Figure 2, Table 1). The stimulating effects on both outward and inward current of I_Na/Ca were abolished when the antibody was incubated with synthesized alpha-2 repeat before it was applied to cells.

Figure 2 Representative traces showing effect of antibody against alpha-2 on Na⁺-Ca²⁺ exchange current in rat ventricular myocytes. (A) Current-voltage relationship before (trace a, control) and after application of antibody against alpha-2 at 10 nmol/L (trace b), 20 nmol/L (trace c), 40 nmol/L (trace d), 80 nmol/L (trace e) and 160 nmol/L (trace f), respectively. Trace g was recorded after application of 5 mmol/L Ni²⁺. (B) Ni²⁺-sensitive Na⁺-Ca²⁺ exchange current before (a–g) and after application of antibody against alpha-2 at concentration of 10 nmol/L (b–g), 20 nmol/L (c–g), 40 nmol/L (d–g), 80 nmol/L (e–g) and 160 nmol/L (f–g), respectively. The voltage protocol is shown in the top panel (see text for details).

Table 1 Stimulating effect of antibody against alpha-2 repeat of Na⁺-Ca²⁺ exchanger on I_Na/Ca. Values were expressed as mean±SD. ^bP<0.05, ^cP<0.01 vs control. Membrane current density is expressed as membrane current (pA) per cell capacitence (pF).
Full table

Effects of the antibody against alpha-2 repeat on I_{Ca, L}, I_Na and I_K To evaluate the functional selectivity of the antibody against alpha-2 repeat, its effects on I_Ca,L, I_Na in adult rat hearts and I_K in adult guinea-pig hearts were also observed. It was shown that this antibody could also increase I_Ca,L in a concentration-dependent manner and EC₅₀ was 33.6 nmol/L. After washing with Tyrode solution, the effect could be partly abolished. Nicardipine could inhibit the above inward current completely, which proved that the current was I_Ca,L (Figure 3, Table 2). Furthermore, the current-voltage (I–V) relationship curve did not shift after application of the 40 nmol/L antibody, although peak Ca²⁺ current increased at +10 mV (Figure 4). Effects of the antibody on I_Na (Figure 5) and I_K (Figure 6) were not observed in the present study.

Figure 3 Representative traces showing effect of antibody against alpha-2 on L-type Ca²⁺ current in rat ventricular myocytes. Trace a, control. Traces b, c, d, e and f, after application of antibody against alpha-2 at 10 nmol/L, 20 nmol/L, 40 nmol/L, 80 nmol/L and 160 nmol/L, respectively. Trace g, after application of 1 µmol/L nicardipine. The voltage protocol is shown in the top panel.

Table 2 Effect of antibody against alpha-2 repeat of Na⁺-Ca²⁺ exchanger on I_Ca,L. Values are presented as mean±SD. ^bP<0.05, ^cP<0.01 vs control. Membrane current density is expressed as membrane current (pA) per cell capacitence (pF).
Full table

Figure 4 Current traces and current-voltage (I–V) relationship of L-type Ca²⁺ current. (A, B) Original Ca²⁺ current traces in the absence (A) and presence (B) of antibody against alpha-2 repeat (40 nmol/L). The currents were measured in response to depolarizing voltage clamp steps of 500 ms in the voltage range between –40 and 50 mV from a holding potential of –40 mV. (C) Current-voltage (I–V) relationship of Ca²⁺ currents in the absence and presence of 40 nmol/L antibody against alpha-2 repeat. n=13 cells from 10 hearts.

Figure 5 Representative traces showing effect of antibody against alpha-2 on voltage-gated Na⁺ current at 10 nmol/L, 20 nmol/L, 40 nmol/L, 80 nmol/L and 160 nmol/L, respectively in rat ventricular myocytes.

Figure 6 Traces showing effect of antibody against alpha-2 on delayed rectifier K⁺ current at 10 nmol/L, 20 nmol/L, 40 nmol/L, 80 nmol/L and 160 nmol/L, respectively in guinea pig ventricular myocytes.

Discussion

Under physiological conditions, NCX operates mainly in Ca²⁺ efflux mode (Na⁺ influx), and only a very small quantity of Ca²⁺ enters the cell during the very early rising phase(1–4 ms) of the action potential via NCX in Ca²⁺ influx mode^[19]. This is not the case, however, in pathological conditions such as heart failure (HF). It was shown that NCX could bring a larger amount of Ca²⁺ into the cell in HF than in normal conditions during the action potential, which partly compensated for the downregulated SR Ca-ATPase function and supported contraction in the patients with HF^[20–22]. Because the antibody against alpha-2 repeat was proven to increase both I_Na/Ca and I_Ca,L in the present study, it may have the therapeutic potential to improve systolic function in HF patients by increasing Ca²⁺ entry via NCX and also the L-type Ca²⁺ channel.

Our investigation showed that the antibody against alpha-2 repeat could also increase I_Ca,L besides enhance­ment of Na⁺-Ca²⁺ exchange current. Moreover, the current-voltage (I–V) relationship curve of I_Ca,L was not shifted by the antibody (40 nmol/L) and EC₅₀ of I_Ca,L was similar to that of I_Na/Ca. An early study has shown that in cardiac muscle where Ca influx across the sarcolemma is essential for contraction, the L-type Ca²⁺ channel has four homologous domains (I–IV), each comprising six transmembrane segments (S1–S6)^[23]. Mutational analysis indicated that S5–S6 linkers were highly conserved in domains I–IV^[23] and contributed to formation of the ion pore^[24]. Residues 1079–1110 were supposed to be located on the S5–S6 linker in domain III^[25]. Meanwhile, mutation analysis showed that residues 815–839 of alpha-2 repeat in NCX were involved in the interaction of the exchanger with Na⁺ and Ca^2+[9–12]. We compared the amino acid alignment of the alpha-2 repeat in NCX with the residues 1079–1110 of the L-type Ca²⁺ channel using EMBOSS Pairwise Alignment Algorithms (European Bioinformatics Institute), which showed that the degree of amino acid similarity was 28.1% between these two functional segments (Figure 7), providing a clue for the non-specific action of the antibody on I_Ca,L.

Figure 7 Amino acid alignment of alpha-2 repeat of Na⁺-Ca²⁺ exchanger (upper) and pore region of L-type Ca²⁺ channel (lower). Residues identical between two segments are in bold. Alignment insertions are indicated with a dash.

From the genetic and evolutionary points of view, intramolecular repeats are thought to arise from intra-genetic duplications, and can not survive throughout evolution unless they are essential to protein function^[8]. As we know, alpha-repeats exist in all known members of the NCX family, which implies that alpha-repeats arose early in the evolutionary history and furthermore, their existing as a tandem pair is essential for the protein to operate properly. In the present study, we first observed the effect of the antibody against alpha-2 repeat on I_Na/Ca in adult rat cardiomyocytes with the whole-cell patch clamp technique. Our results showed that antibody against alpha-2 repeat was a stimulating antibody because it increased I_Na/Ca in a concentration-dependent manner, which provided supplemental evidence that alpha-2 repeat was essential to translocation of sodium and calcium by NCX.

Recent topological research showed that the alpha-2 repeat comprised putative transmembrane 7 and its C-terminal segment and formed a domain mostly acces-sible from the cytoplasm^[26]. However, our results in this study indicated that the antibody against alpha-2 repeat could stimulate Na⁺-Ca²⁺ exchange from the external side of the cardiomyocytes. Then why did the antibody play its role from the extracelluar side? One possibility is that the interaction between antibody against alpha-2 repeat and NCX might lead to conformation alteration of the exchanger molecule, just as what happens to KB-R7943^[27].

The present study showed that the antibody against alpha-2 repeat of Na⁺-Ca²⁺ exchanger was a stimulating antibody to NCX and could also increase I_Ca,L in a concentration-dependent manner, whereas it did not have an obvious effect on I_Na and I_K.

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