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Sodium/Calcium Exchanger: Influence of Metabolic Regulation on Ion Carrier Interactions

Laboratorio de Permebilidad Ionica, Centro de Biofísica y Bioquímica, Instituío Venezolano de Investigaciones Científicas, Caracas, Venezuela; and Laboratorio de Biofísica, Instituto de Investigacíon Médica "Mercedes y Martin Ferreyra", Consejo Nacional de Investigaciones Científicas y Tenicas, Córdoba, Argentina

ABSTRACT

I. DISCOVERY, EVOLUTION OF THE FIELD IN TIME, AND PHYSIOLOGICAL RELEVANCE

II. TECHNIQUES USED TO UNRAVEL THE SODIUM/CALCIUM EXCHANGE TRANSPORT PROPERTIES

III. SODIUM/CALCIUM COUNTERTRANSPORT: MODES OF OPERATION, STOICHIOMETRY, ELECTROGENICITY, AND VOLTAGE DEPENDENCE

IV. PROPERTIES OF SODIUM AND CALCIUM TRANSPORT SITES OF THE SODIUM/CALCIUM EXCHANGER

A. External and Internal Sodium Transport Sites

B. External and Internal Calcium Transport Sites

C. Effect of Nontransported Monovalent Cations on Na+/Ca2+ Transport Activity

D. Na+-Ca2+ Interactions at the External and Internal Transport Sites

E. Turnover Rates, Vmax, and Density of the Na+/Ca2+ Exchangers

V. STRUCTURAL CHARACTERISTICS OF THE SODIUM/CALCIUM EXCHANGE PROTEIN

A. Topology, the {alpha}-1 and {alpha}-2 Repeats, the Intracellular Loop, and the Exchanger Isoforms

VI. REGULATION OF THE SODIUM/CALCIUM EXCHANGER

A. Ionic Regulation

1. Cai2+-dependent regulation

2. Nai+-dependent inactivation

3. Hi+ and Ho+ effects on Na+/Ca2+ exchange activity

4. Cai2+-Hi+ interactions

5. Nai+-Hi+ synergistic inhibition: kinetic model

6. Cai2+-Hi+-Nai+ interactions: kinetic models

B.Metabolic Regulation

1.Modulation by nucleotides

A) DISCOVERY OF ATP STIMULATION.

B) KINETIC EFFECTS.

C) BASIC CHARACTERISTICS OF ATP-REGULATED TRANSPORT SYSTEMS: MECHANISMS UNDERLYING ATP STIMULATION OF SODIUM/CALCIUM EXCHANGE.

D) NUCLEOTIDE SELECTIVITY.

E) METABOLIC PATHWAYS FOR ATP STIMULATION.

F) ROLES OF INTRACELLULAR MAGNESIUM.

G) PRELIMINARY BIOCHEMICAL AND FUNCTIONAL CHARACTERIZATIONS OF THE SCRP.

H) PHOSPHORYLATION OF THE SODIUM/CALCIUM EXCHANGER PROTEIN.

I) USE OF INHIBITORS AS TOOLS TO UNDERSTAND IONIC AND ATP MODULATION OF THE SODIUM/CALCIUM EXCHANGER.

2.The squid nerve: modulation of the Na+/Ca2+ exchanger by phosphoarginine

A) KINETIC EFFECTS OF PA MODULATION.

B) AN INTEGRATED KINETIC MODEL FOR IONIC, MAGNESIUM ATP, AND PA MODULATION OF THE SQUID SODIUM/CALCIUM EXCHANGER.

C) PRELIMINARY BIOCHEMICAL EXPERIMENTS ON PA REGULATION: THE SQUID SODIUM/CALCIUM EXCHANGER.

VII. SUMMARY: CONCLUSIONS ON THE RELATIVE EFFICIENCY OF THE CALCIUM EXTRUSION MECHANISMS IN SQUID AXONS

GRANTS

ACKNOWLEDGMENTS

REFERENCES

	ABSTRACT

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	I. DISCOVERY, EVOLUTION OF THE FIELD IN TIME, AND PHYSIOLOGICAL RELEVANCE

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From the classical works of Ringer in 1883 (225) and Daly and Clark in 1921 (67) it has been shown that the contraction of the frog cardiac muscle is directly related to the extracellular calcium concentration ([Ca²⁺]_o), and inversely associated with the extracellular sodium concentration ([Na⁺]_o). The first observations were made by Wilbrandt and Koller in 1948 (256) who found that contractility of the cardiac muscle was related to the ratio [Ca²⁺]_o/[Na⁺]_o². They proposed a site, whereby sodium prevents calcium entry into the cardiac cell. Ten years later, Lüttgau and Neidergerke (169) confirmed these findings and concluded that calcium competes with external sodium for external sites carrying negative charges (Ca-carrier, and Na₂-carriers). The scheme of Ca²⁺ movements in the frog heart proposed by Lüttgau and Niedergerke (169) was not a Na⁺/Ca²⁺ exchanger but rather a Na⁺-Ca²⁺ antagonism. In later developments, Reuter and Seitz (223, 224) working in heart, and the Cambridge group lead by Baker (15), working in squid giant axons, documented for the first time the presence of a Na⁺/Ca²⁺ countertransport system. The first group, by looking at ⁴⁵Ca²⁺ efflux in guinea pig auricles, discovered that calcium exit was greatly reduced when external sodium and calcium were eliminated from the external medium proposing an exchange of 2 Na⁺ for 1 Ca²⁺. The second group, working in squid axons, found a ouabain-insensitive Na⁺ efflux incompatible with the operation of the Na⁺-K⁺ pump (15, 16) and discovered ouabain-insensitive Na_i⁺/Ca_o²⁺ and Na_o⁺/Ca_i²⁺ exchanges that we know now correspond to two modes of operation of the Na⁺/Ca²⁺ exchanger. Contrary to the 2Na:1Ca stoichiometry proposed by Reuter and Seitz (224), Blaustein and Hodgkin (46) suggested a higher stoichiometry. From that time it was clear that there existed a new transport mechanism sufficiently general to encompass different evolutionary systems such as vertebrates and invertebrates. This has been confirmed in most animal cells (for review, see Ref. 43).

The study of the Na⁺/Ca²⁺ exchanger has experienced a rapid growth since then. Until the late 1970s, most works were related to thermodynamic and kinetic mechanisms of the Na⁺/Ca²⁺ exchanger including apparent affinities for transported and nontransported ions (see below), as well as the discovery of two fundamental regulatory sites: the intracellular calcium regulatory site (72) and the nucleotide (ATP) site (12). The reasons for the large increase in the interest in the Na⁺/Ca²⁺ exchange field in the last 30 years, however, come mainly from three interconnected factors: 1) the development of powerful electrophysiological techniques for the study ion transport across plasma membranes, including the patch clamp for measuring ionic currents in single cells and the giant excised patch for looking at small currents generated by carrier-mediated electrogenic ion transporters; 2) the molecular biology approach for deducing the structure-function relationships of the exchanger (see below); and 3) the progressive finding of the involvement of the Na⁺/Ca²⁺ exchanger in several physiological functions (see Fig. 1), including cardiac muscle relaxation, control and refilling of the sarcoplasmic reticulum (SR) calcium content in the heart and in the endoplasmic reticulum (ER) of neuronal and nonexcitable cells, control of neurosecretion, excitation-contraction (E-C) coupling, and photoreception (31, 32, 36–39, 41; for review, see Refs. 6, 43, 173, 213, 28, 104). Significant attention has been focused on its role in the physiology of the heart in which 20–25% of the calcium-induced contraction results from the entrance of extracellular Ca²⁺ through the L-type Ca channels (28), whereas 75–80% is due to Ca²⁺ released from the SR. Then, for the heart [Ca²⁺]_i to be in steady state, the same amount released by the SR must be pumped back into the SR, and the remaining 20% must be extruded from the cell by the exchanger and the Ca²⁺ pump. By using rapid cooling to affect Ca²⁺ release from the SR during contraction, Bers and Bridge (29) provided a good example of how these compete for intracellular Ca²⁺. The cross-talk between the plasmalemmal Na⁺/Ca²⁺ exchanger and the intracellular Ca²⁺ stores (ER) indicates that the exchanger has a major role in control of the amplification of the Ca_i²⁺ induced by calcium entry through voltage-dependent calcium channels and activation of calcium-induced calcium release (CICR) from the ER. In hippocampus neurons and amacrine cells, the activity of the exchanger, in particular in its forward mode, has been shown to control the amount of calcium in the ER and, therefore, the amplification of the calcium signal. This, therefore, explains the role of the exchanger not only in E-C coupling, but also in the control of the neurosecretion processes (42, 43, 133). Furthermore, the Na²⁺/Ca²⁺ exchanger, working in the extrusion mode, has being recently implicated in the modulation of Ca²⁺ signal in mast cells (7). Finally, there are numerous reports that link the Na⁺/Ca²⁺ exchanger with many pathophysiological syndromes including heart arrhythmias, all salt-dependent hypertension, reperfusion injury, and cardiac ischemia, among others (34, 35, 40, 43, 173, 185, 193, 238, 241, 255).

View larger version (31K): [in this window] [in a new window]   FIG. 1. Intracellular Ca2+ homeostasis in a model cell. Role of the Na+/Ca2+ exchanger under physiological and physiopathological conditions.

	II. TECHNIQUES USED TO UNRAVEL THE SODIUM/CALCIUM EXCHANGE TRANSPORT PROPERTIES

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View larger version (37K): [in this window] [in a new window]   FIG. 2. Main experimental preparations and techniques used to characterize the Na+/Ca2+ exchange structural and functional properties (see text for details).

Finally, substantial progress has been made in the molecular biology of the Na⁺/Ca²⁺ exchanger (204). Exons coding for different regions of the intracellular loop have been used in different combinations and in different tissues. New exchangers have been cloned, and several alternative splicings have been described, which has led to the classification of the Na⁺/Ca²⁺ exchanger superfamily (204). These studies have not only produced a picture of the primary structure of the exchanger protein (204) but also, through mutagenesis and deletions of amino acids, produced a map of specific regions responsible for the transport of Na⁺ and Ca²⁺ and for regulation (see below). Antisense oligonucleotides have been useful to inhibit the Na⁺/Ca²⁺ exchange activity in myocytes and other preparations. Finally, genetic manipulations, like transgenic mice with overexpression or knock out of the exchanger, provided elements for the understanding of its physiological and pathological significance (119, 204).

	III. SODIUM/CALCIUM COUNTERTRANSPORT: MODES OF OPERATION, STOICHIOMETRY, ELECTROGENICITY, AND VOLTAGE DEPENDENCE

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Early work in squid axons indicated that the Na⁺/Ca²⁺ exchanger can produce net calcium fluxes into or out of the cell depending on the electrochemical gradients for Na⁺ and Ca²⁺ (43). Figure 3 is a simplified consecutive kinetic model that shows the four basic modes of operation of the exchanger (80): 1) forward or direct mode, responsible for net Ca²⁺ extrusion (Na_o⁺/Ca_i²⁺ exchange); 2) the reverse exchange, responsible for Ca²⁺ entry (Na_i⁺/Ca_o²⁺ exchange); 3) the homologous Ca_o²⁺/Ca_i²⁺ exchange; and 4) the homologous Na_o⁺/Na_i⁺ exchange (for more information concerning the evidence for a consecutive or "Ping-Pong" mechanism of Na⁺ and Ca²⁺ translocation vs. a simultaneous transport model, see Ref. 43).

View larger version (13K): [in this window] [in a new window]   FIG. 3. Consecutive kinetic scheme of transport modalities of the Na+/Ca2+ exchanger. The carrier sites phasing the intracellular medium are labeled EI, and those phasing the extracellular solutions are labeled EO. Curved arrows indicate forward (Nao+/Cai2+) exchange, reverse (Nai+/Cao2+) exchange. Broken arrows indicate Nao+/Nai+ and Cao2+/Cai2+ exchanges.

In 1979 Mullins (183, 184) demonstrated theoretically that if the energy store in the inward [Na⁺] gradient (assuming a 3:1 stoichiometry) was greater than that in the [Ca²⁺] inward gradient, then Ca²⁺ extrusion via the exchanger should be thermodynamically favored. In mathematical terms

where n is the coupling ratio and E_Na and E_Ca are the equilibrium potentials for Na⁺ and Ca²⁺, respectively {E_x = (RT/zF)log ([X]_o/[X]_i)}. For n = 3; the reversal potential of the Na⁺/Ca²⁺ is

If V_m is more positive than E_Na/Ca, then Ca²⁺ entry is favored; when V_m is negative to E_Na/Ca, Ca²⁺ extrusion is favored. These thermodynamic equations also indicate that under equilibrium conditions the ratio of free extra- to intracellular Ca²⁺ is given by (16, 46)

On the other hand, the amplitude of the exchange current (I_Na/Ca) and the real contribution of the exchanger to the resting [Ca²⁺]_i are subject to kinetic limitations. The voltage dependence of a current generated by a channel or a transporter is not a thermodynamic but a kinetic parameter that involves assumptions about the constant electrical field. Quantitative models of I_Na/Ca have been proposed (30, 68, 120, 127, 129) in which the net I_Na/Ca is the difference between the unidirectional calcium fluxes

This leads to more elaborate equations that have been used to fit experimental data on current-voltage relationships of the I_Na/Ca (104)

where k_Na/Ca is a scaling factor and r represents the position of a single energy barrier present in the electric field across the membrane and is responsible for the steepness and symmetry of the voltage dependence (68).

Electroneutral fluxes associated with Na⁺/Na⁺ and Ca²⁺/Ca²⁺ exchanges do not produce net charge movements and cannot be measured with voltage-clamp techniques. However, this does not mean that these homologous modes are voltage insensitive; in fact, the consecutive model developed by Eisner and Lederer (107) shows that Ca²⁺/Ca²⁺ exchange can be voltage sensitive. With the use of dialyzed squid axons to measure unidirectional isotope fluxes of Na⁺ and Ca²⁺, it has been possible to determine the voltage sensitivity of the homologous Na⁺/Na⁺ and Ca²⁺/Ca²⁺ exchanges. Figure 4, A and B, summarizes several experiments showing that the voltage sensitivity of the squid exchanger under symmetrical ionic conditions is identical for the Na_o⁺/Ca_i²⁺, Na_i⁺/Ca_o²⁺, and Ca_o²⁺/Ca_i²⁺ exchanger modes, while the homologous Na_o⁺/Na_i⁺ translocation is voltage insensitive (79). A similar finding has been reported in barnacle muscle fibers (209).

View larger version (21K): [in this window] [in a new window]   FIG. 4. A and B: influence of membrane potential on the different components of the Na+/Ca2+ exchange from 16 different squid axons. A: Nao+/Cai2+ and Cao2+/Cai2+ exchanges. B: Nai+/Cao2+ and Nai+/Nao+ exchanges. In A, solid symbols, Cao2+-dependent Ca2+ efflux (Cao2+/Cai2+ exchange) in symmetrical Li+ media; open symbols, Cao2+-dependent Ca2+ efflux in symmetrical Tris solutions; half open, half solid symbols, Nao+-dependent Ca2+ efflux (forward exchange) in symmetrical Na+ solutions. In B, solid symbols, Cao2+-dependent Na+ efflux (reverse exchange) in symmetrical Li+ solutions; half open, half solid symbols, Nao+-dependent Na+ efflux (Nao+,/Nai+ exchange) in symmetrical Na+ solutions. Each symbol represents a different axon. Notice that all modes of operation of the Na+/Ca2+ exchange except the Nao+/Nai+ exchange are voltage sensitive. [From DiPolo and Beaugé (79) by copyright permission of the Rockefeller University Press.] C and E: charge movements associated with half reaction cycles of the Na+/Ca2+ exchange. Na+ is present in the pipette and Na+ is applied to the bath as indicated. D and F: Ca2+ is present in the pipette applied to the bath; no Na+ is present. Note that for the cardiac exchanger NCX1, current transients are associated with Na+ translocation (C) but not with Ca2+ translocation (D). Strikingly, the opposite is true of the squid exchanger NCX-SQ1. In this case, the electrogenic step is Ca2+ translocation (D) not Na+ translocation (F). See text for details. [From He et al. (118) by copyright permission of the Rockefeller University Press.]

	IV. PROPERTIES OF SODIUM AND CALCIUM TRANSPORT SITES OF THE SODIUM/CALCIUM EXCHANGER

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The Na⁺/Ca²⁺ exchanger is highly regulated (see below); therefore, any discussion about activation kinetics by the transported ions, sodium and calcium, must necessarily take into account all ionic and metabolic modulations (see below and Table 3). In this section we review the main characteristics of sodium and calcium transport sites; we compare different preparations, but emphasize differences and similarities of two preparations in which the Na⁺/Ca²⁺ exchanger has been most extensively studied: the cardiac myocyte and the squid giant axon.

In practically all preparations where the effects of transporting Na⁺ have been investigated (Na_o⁺-dependent Ca²⁺ efflux; forward exchange, Na_o⁺-dependent Na⁺ efflux; Na⁺/Na⁺ exchange, and Na_i⁺-dependent Ca²⁺ influx; reverse exchange), the activation curves show a sigmoid dependence with [Na], with a Hill coefficient close to 3 (43, 214). The K_Na values are 13–30 mM in cardiac sarcolemmal vesicles (146, 200, 220, 251, 252), 34–59 mM in guinea pig atria (145), 50–80 mM in dialyzed squid axons containing ATP (70, 33), and 60 mM in barnacle muscle fibers (159, 211). A typical external Na⁺ activation curve of the forward exchange currents in single ventricular cells of guinea pig in Figure 5 has a K_m of 70 mM (155) and a Hill coefficient of 3. In the heart, the K_0.5 for Na⁺ activation of Ca²⁺ transport is lower at the intracellular site (reverse exchange) than that of the external one (forward exchange) by a factor of 8. Conversely, in squid axons, the K_0.5 for Na_i⁺ activation of Ca²⁺ influx is 50–60 mM (72), which is comparable to that of the external Na⁺ sites (33). Similarly, in barnacle muscle fibers, the K_0.5 is 30 mM, only slightly less than that for outside Na⁺ (211), thus indicating no asymmetry in the sodium affinity sites in invertebrates compared with vertebrates (43). In the way this exchanger works, Na⁺ activates transport when present on membrane sides opposite to that of Ca²⁺ (forward or reverse exchange) or Na⁺ (Na⁺/Na⁺ exchange) and inhibits when present on the same side (16, 44, 113, 224). Inhibition is competitive with a Hill coefficient close to 2 (220). In terms of its translocating sites, a remarkable property of the NCX and NCKX superfamily of exchangers is that no other monovalent cation can substitute for Na⁺. This contrasts with the Na⁺/Ca²⁺ exchanger of mitochondria where Li⁺ can be transported as efficiently as Na⁺ (66). An exchanger different from the NCX and NCKX that catalyzes active Li⁺/Ca²⁺ countertransport has been recently found in pancreas, skeletal muscle, and stomach (197).

View larger version (21K): [in this window] [in a new window]   FIG. 5. A: chart record showing application of various concentrations of external Na+. Control ionic conditions are 140 mM Li+, 0 internal Na+, 1 mM external Ca2+, and 430 nM internal Ca2+. Test external Na+ concentrations are illustrated at the top of the bars, which indicate the duration of Na+ superfusion. B: difference currents between the peak activation and the control in 140 mM external Li+ before applying external Na+ were plotted against voltage. C, left: current magnitudes were plotted against the logarithm of external Na+ concentrations. Right: Hill plot of the current in B. The data could be fit with a straight line based on a half-maximum concentration (K1/2) of 70 mM and the slope (Hill coefficient) of 3. [From Kimura et al. (155).]

B. External and Internal Calcium Transport Sites

The measurement of the apparent affinity of the extra- and intracellular Ca²⁺ transport sites are complicated for several reasons: 1) the existence of a nontransported Ca_i²⁺-regulatory site on the cytosolic side (see below); 2) the difficulty in separating the apparent affinities of extra- and intracellular Ca²⁺ sites in isolated membrane vesicles that consist of a mixed population of inside-out and right-side-out vesicles, i.e., intra- and extracellular sites are simultaneously exposed to the extravesicular solution (200); and 3) the use of strong Ca²⁺ buffering agents in the perfusate could affect the apparent K_Ca of the Na⁺/Ca²⁺ exchanger (200). Nevertheless, if one looks at preparations in which the ambiguities of sidedness are minimized, such as dialyzed squid axons, perfused barnacle muscle fibers, voltage-clamp perfused cardiac myocytes, and cardiac giant excised patches, a remarkable difference is found in the exchanger's apparent affinities for Ca²⁺ at intra- and extracellular sites of the membrane. In squid axons fuelled with ATP, the K_m values are 1–2 µM for Ca_i²⁺ and 3,000 µM for Ca_o²⁺ (43). In embryonic chick heart cells, the external K_m is more than 100-fold higher than the cytoplasmic K_m (251). Current measurements in guinea pig cardiac myocytes provided values of 0.6 and 1,400 µM for the intra- and extracellular K_0.5 for Ca²⁺. These data indicate that, depending on the experimental conditions, the affinities for Ca²⁺ at the inward and outward faces of the transport sites of the Na⁺/Ca²⁺ exchanger differ by a factor of 10–100 (33, 79, 252, 181). In squid axons, the intra- and extracellular Ca²⁺ dependence of exchange activity was measured under otherwise symmetrical ionic and voltage conditions (79); the ratio of the K_m values for extra- and intracellular calcium transport sites is close to 10, thus indicating an asymmetry of the intra- and extracellular Ca²⁺ transport sites.

The binding of Ca²⁺ to its external transport sites is also voltage insensitive in terms of the lack of effect of membrane potential on the apparent affinity for Ca²⁺. This has been clearly demonstrated in dialyzed squid axons under symmetrical ionic conditions (79). In contrast to the high selectivity of the sodium sites for Na⁺, other divalent cations can bind to the calcium sites and be translocated in exchange for either Na⁺ or Ca²⁺ (33). In dialyzed squid axons, the order of the external apparent affinity follows the sequence Ca²⁺ > Sr²⁺ > Ba²⁺. In sarcolemmal vesicles, the order of effectiveness in stimulating Ca²⁺ efflux in the absence of external Na⁺ in the medium was Cd²⁺ > Sr²⁺ = Ca²⁺ > Ba²⁺ > Mn²⁺ (247); as pointed out above, in this preparation there is no way to establish what sites they correspond to. The use of Ba²⁺, Sr²⁺, or Cd²⁺ as a Ca²⁺ substitute has been nicely exploited to measure properties of the Na²⁺/Ca²⁺ exchanger in cell populations expressing the NCX (62, 246).

C. Effect of Nontransported Monovalent Cations on Na⁺/Ca²⁺ Transport Activity

In both mammalian heart and squid axons, all Na⁺/Ca²⁺ exchange modalities in which external Ca²⁺ is the transported species (Ca²⁺/Ca²⁺ and reverse exchange) monovalent cations, including K⁺ (at constant membrane potential), strongly activate the exchanger by increasing the affinity of the Ca_o²⁺ transport site for Ca_o²⁺ (4, 11, 243). This is shown in Figure 6A, where the Ca_o²⁺-activated Na⁺ efflux (reverse exchange) is plotted against external [Ca²⁺] in the presence of Li⁺ or N-methylglucamine (NMG⁺). Two points should be stressed: 1) the large activation of Ca²⁺ entry in the presence of Li⁺ but not NMG and 2) the increase in the apparent affinity for Ca_o²⁺ in the presence of the monovalent cation Li⁺. The order of effectiveness of monovalent cations follows the sequence Li⁺ > K⁺ = Rb⁺ NH₄⁺ > NMG⁺. Figure 6B shows that even Na_o⁺, at low concentrations, activate Ca²⁺ influx; obviously, the dose-response curve for Na_o⁺ is biphasic due to displacement of Ca_o²⁺ from the external transporting sites at high [Na⁺]_o (9). Interestingly, the Na_o⁺-dependent Ca²⁺ efflux (forward) is not affected by either K_o⁺ (at constant membrane potential) (5) or high concentrations of Li_o⁺ (which do not affect the membrane potential) (10), i.e., there is no chemical action of external monovalent cations on the exchange activity when Na_o⁺ is the transported species (10). At present, it is not known whether the generation of the monovalent site occurs after Ca_o²⁺ binds to the carrier, or if the site exists independently of the binding of Ca_o²⁺. The experiments on exchange activity in vesicles can be interpreted by a model (243) including two external binding sites. A divalent site (A) which can bind a single calcium ion or two sodium ions and a second monovalent cation site (B). When the predominant extracellular ion is Na⁺, all A and B sites will be occupied by this ion, and therefore, no monovalent cation effect will take place. The physiological importance of the external monovalent cation sites is also unknown since, under normal physiological conditions, the monovalent cation site, if it indeed exists (site B), is occupied by external Na⁺. Finally, by a still unknown reason, little effect of nontransported monovalent cations was found in cardiac giant patches (174).

View larger version (19K): [in this window] [in a new window]   FIG. 6. A: effect of [Ca2+]o on Na+ efflux in axons injected with (solid symbols) or without (open symbols) intracellular Na+ in the presence (circles) or absence (triangles) of external Li+. B: effect of external Na+ or Li+ on Ca2+ influx in injected squid axons. [From Baker et al. (16).] C: effect of external K+ on the Nai+-dependent Ca2+ influx (reverse Na+/Ca2+ exchange) at constant 0 mV membrane potential. D: effect of internal Ca2+ on the 86Rb-labeled K influx in the presence of 50 mM external K+. Notice that activation of the exchange by Cai2+ does not promote Rb+ influx. [From Condrescu et al. (64), with permission from Elsevier]. E: relative size and voltage dependence of outward Na+/Ca2+ exchange current in the absence of extracellular Na+ depends importantly on the Na substitute; 0.5 mM Cao2+-activated current in the presence of 145 mM [Na+]o (open circles), and after replacing all of the Na+ with 145 mM N-methylglucanine (NMG; solid circles). Free [Ca2+]pip = 50 nM; [Na+]pip = 100 mM; cell capacitance = 189 pF. F: 0.25 mM Cao2+-activated current in the presence of 145 mM [Na+] (open circles), and after replacing all of the Na+ with 145 mM Li+ (solid circles). Free [Ca2+]pip = 50 nM; [Na+]pip = 100 mM; cell capacitance = 102 pF. [From Gadsby et al. (112), copyright 1991 New York Academy of Sciences, USA.]

Intracellular nontransported monovalent cations (notincluding intracellular protons; see sect. VIA) also stimulate the Na⁺/Ca²⁺ exchanger. Intracellular K⁺ and Li⁺, at constant membrane potential, are powerful activators of the forward exchange mode (79, 96). The K_0.5 for K_i⁺ is 90 mM in dialyzed squid axons; as this activation is independent of [Na⁺]_i, it rules out any K_i⁺-Na_i⁺ competition. Monovalent cation activation was explored further in dialyzed squid axons by measuring the Ca_i²⁺-dependent Ca²⁺ efflux in the presence and absence of intracellular Li⁺ with and without Li⁺ in the extracellular solution. An intriguing and still unexplained result was the lack of effect of intracellular Li⁺ when the external monovalent cation site was vacant (18); on the other hand, the exchange fluxes do not display a biphasic relationship with [Na⁺]_i (as happens with the Na_o⁺-dependent Ca²⁺ efflux). Actually, Na_i⁺ inhibits through a sigmoid dose-response curve. Perhaps the monovalent activating cation binding sites are different in the external and internal surfaces. Nevertheless, the large activation of the exchanger, in all its modes, by intracellular K⁺ should be considered when analyzing the physiological role of the Na⁺/Ca²⁺ exchanger in cases where intracellular K⁺ is substituted with NMG⁺, Tris⁺, or other cations.

D. Na⁺-Ca²⁺ Interactions at the External and Internal Transport Sites

Dialyzed squid axons were used to analyze Na⁺ and Ca²⁺ interactions at the external surface of the membrane by measuring the influx of Ca²⁺, at 0.5 and 10 mM external Ca_o²⁺, at various [Na⁺]_o. In agreement with results in injected squid axons (see Fig. 6B), Ca²⁺ influx as a function of [Na⁺]_o is always biphasic. A model that accounts for these Na_o⁺-Ca_o²⁺ interactions reproduces the data obtained in intact injected and dialyzed squid axons (18). The model assumes that all external Na⁺ and Ca²⁺ interactions with the Na⁺/Ca²⁺ exchanger are based on Na⁺-Ca²⁺ competition for the transporting site plus a positive cross-reactivity between the monovalent activating and the transporting sites. In addition, the occupancy of the external monovalent activating site by Na_o⁺ increases the maximal rate of exchange. The fitting of the data requires that the binding of Ca²⁺ to its transporting site increases the affinity of the monovalent cation site by about fivefold. The occupancy by Na_o⁺ of the monovalent activating site increases the affinity of the transport site for Ca_o²⁺ by the same magnitude. In addition, the binding of Na_o⁺ to the external transporting site must increase the affinity of the monovalent activating site by twofold, i.e., in the squid exchanger Na_o⁺ and Ca_o²⁺ interactions display competition together with a positive activating cross-reactivity between the monovalent activating and the transporting sites.

The interactions of Na⁺ and Ca²⁺ with intracellular transport sites can be adequately explained by the simple model that is basically a kinetic scheme of random equilibrium conditions with Na_i⁺ and Ca_i²⁺ competition (18).

E. Turnover Rates, V_max, and Density of the Na⁺/Ca²⁺ Exchangers

The Na⁺/Ca²⁺ exchanger can have a very high rate of transport when it is fully activated. For instance, in rod outer segments, flux and current measurements indicate Ca²⁺ fluxes of 40–50 pmol · cm^–2 · s^–1 (231); these figures correspond to changes in [Ca²⁺]_i of >30 µM/s. In squid axons, the V_max can reach values up to 20–40 pmol · cm^–2 · s^–1 (91; and unpublished results). In cardiac cells, current (155) and flux (58) measurements produced values as high as 30 pmol · cm^–2 · s^–1. Based on kinetic analysis in reconstituted proteoliposomes, an estimate turnover value of 1,000 s^–1 has been obtained for the cardiac Na⁺/Ca²⁺ exchanger (59). From giant patch measurements at saturating intracellular Ca²⁺, currents of 30 pA/F were recorded for I_NaCa with a density of about 400 exchanger molecules/µm². If the surface of the myocytes is close to 25 x 10³ µm², then a maximum turnover rate of 5,000 s^–1 can be estimated (130). Similarly, from channel-like noise analysis and charge movements in cardiac giant excised patches, it has been possible to detect turnover rates of 5,000 s^–1 for the transport of both Na⁺ and Ca²⁺ using nonsaturating ion concentrations (122).

	V. STRUCTURAL CHARACTERISTICS OF THE SODIUM/CALCIUM EXCHANGE PROTEIN

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A. Topology, the -1 and -2 Repeats, the Intracellular Loop, and the Exchanger Isoforms

Since 1990, molecular biology techniques applied to the study of the Na⁺/Ca²⁺ exchanger have given abundant and crucial information on its structure-function relationships. The canine cardiac NCX1 exchanger was the first to be purified (202) and cloned (1, 190). Further subtypes that are products of different genes (NCX2 and NCX3) were later found in the brain (204, 245). Also, during the biosynthesis of the carrier, an NH₂-terminal hydrophobic segment of 32 amino acids ("signal peptide") (see Fig. 7 for details) is cleaved off to produce a mature protein (103). The full-length, mature cardiac exchanger contains 938 amino acids (204). Initially, hydropathy analysis suggested that the exchanger had 11 transmembrane segments; the NH₂ terminus is glycosylated, located extracellularly, and does not seem to be involved in exchange activity (111, 132, 229). Presently, it is believed (Fig. 7) that transmembrane segment 6 is part of the large intracellular loop (light shaded cylinder) and that the old transmembrane segment 9, although hydrophobic, does not span the membrane but forms a P-loop type structure similar to the pore-forming region of ion channels. The middle of this segment 9 has a GIG sequence resembling the motif characteristic of K⁺ channels. Thus, in the latest version, the Na⁺/Ca²⁺ exchanger contains nine transmembrane segments, five from the NH₂ terminus up to the large intracellular loop and four from that loop up to the COOH terminus (65, 141, 164, 191). The large intracellular cytosolic loop has 550 amino acids (204). Although this loop contains important regulatory sites, it does not appear to be required for transport, since a mutant lacking most of this loop (240–679) still retains exchange activity (177). Toward the NH₂- and COOH-terminal portions of the exchanger there are two repeat sequences of 40 amino acids called -1 and -2 repeats (Fig. 7) (236). They are conserved in all members of the NXC family and other cation exchangers (204, 236) and are thought to play an important role in ion transport. In the NCX, the -1 repeat includes part of transmembrane segments 2 and 3 and a loop connecting them; the -2 repeat consists of a portion of the transmembrane segment 7 and the GIG nontransmembrane part in the COOH-terminal region. By cysteine substitutions in these loops and the use of externally or internally applied sulfhydryl reagents, evidence has been gathered indicating that they are part of the ion translocation pathway (140, 141). Also, mutagenesis of transmembrane segment 2 markedly changes ion selectivity (100); as an example, substitution of threonine at position 103 with valine changes the high selectivity for Na⁺, resulting in an exchanger that can countertransport Li⁺ with Ca²⁺ (100, 189). Recently, Philipson and co-workers (195) have addressed the importance of the -1 repeat in ion translocation through the exchanger. In a series of experiments in which mutations of the reentrant loop and transmembrane segment 3 (TMS3) were performed, Ottolia et al. (195) conclude that TMS3, and not the reentrant loop is involved in Na⁺ binding and transport. The Na⁺ binding site associated with TMS3 appears to be unique and not involved in Ca²⁺ binding. Furthermore, they found a highly specific requirement for an asparagine or aspartate residue at position 143, indicating a crucial role of Asn¹⁴³ in Na⁺ translocation.

View larger version (21K): [in this window] [in a new window]   FIG. 7. Model of the Na+/Ca2+ exchanger. The exchanger is now modeled to have nine transmembrane segments as shown. The NH2 terminus is glycosylated and extracellular. Earlier models predicted 11 transmembrane segments. Former transmembrane segment 6 is likely to be a portion of the large intracellular loop (light shaded cylinder). Hydrophobic segment 9 does not span the membrane and is speculatively modeled to form a P loop-like structure (light shading). The -repeat regions in transmembrane segments 2, 3, and 7 are shaded. Shown on the large intracellular loop are the endogenous XIP region, the binding site for regulatory Ca2+, and the region where extensive alternative splicing occurs. The large intracellular loop is not drawn to scale but encompasses almost 550 amino acids, more than one-half the length of the protein. [Modified from Philipson and Nicoll (204).]

The most extensive studies of the Na⁺/Ca²⁺ exchanger have been carried out in cardiac membrane (patch-clamp and membrane vesicles) and squid nerve (injected, dialyzed axons, and membrane vesicles). Therefore, it seemed pertinent to compare them. Some basic properties, like stoichiometry of the exchanging cations and regulation by Ca_i²⁺, Na_i⁺, H_i⁺, and ATP, are present in both system's preparations (see Ref. 89); nevertheless, the presence of fundamental differences between them may provide insight into their structure-function relationships (see sect. VIB) and will certainly facilitate the development of models of the regulatory processes that control the activity of the Na⁺/Ca²⁺ countertransport. Figure 8 and Table 1 show the amino acid homology between squid nerve and the canine heart Na⁺/Ca²⁺ exchangers (data calculated from Ref. 118). As a whole, NCX-SQ1 is 58% identical to the cardiac exchanger and possesses overall identity (41–64%) with other exchangers (118). The regions that have functional importance such as the Ca_i²⁺-regulatory binding domain (46% homology), Na_i⁺ inactivation (56% homology), and COOH terminal (70% homology) are well conserved. Furthermore, the -1 and -2 repeats involved in ion translocation are well conserved in the two preparations (see Fig. 8). There are, however, substantial functional differences, mostly related to regulation, in particular, their voltage dependence (strong voltage dependence of Ca²⁺/Ca²⁺ but not Na⁺/Na⁺ exchange in the squid as compared with strong voltage dependence of the Na⁺ translocation branch in the cardiac) and metabolic regulation (see sect. VIB). Combination of molecular biological and electrophysiological methods may provide important information to understand the structure-function relationships of this superfamily of countertransport systems.

View larger version (84K): [in this window] [in a new window]   FIG. 8. Amino acid comparison of the squid NCX-SQ1 and the canine NCX1 exchanger. Putative transmembrane segments, predicted by hydropathy analysis, are underlined and numbered. Highlighted in bold lettering are a potential signal peptidase site (SigPase), potential N-linked glycosylation sites (NXS/T), and potential phosphorylation sites (RTIK, protein kinase C; TRKLT, cAMP-dependent kinase and Ca2+/calmodulin-dependent kinase; DEHFY and DDEEEY, tyrosine kinase). The two potential phosphorylation sites marked with an asterisk are unique to NCX-SQ1. The endogenous exchanger inhibitory peptide (XIP) region and exon A are shaded, and the binding domain for regulatory Ca2+ is boxed. The triple aspartate motifs involved in Ca2+ binding are in bold. Dots in the NCX1 sequence indicate amino acids identical to those of NCX-SQ1. [From He et al. (118) by copyright permission of The Rockefeller University Press.]

	VI. REGULATION OF THE SODIUM/CALCIUM EXCHANGER

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1. Ca_i²⁺-dependent regulation

In intact, injected squid axons, addition of a Ca²⁺ chelator such as EGTA or quin 2 into the cytosol reduced the Ca²⁺ influx and the Ca_o²⁺-dependent Na⁺ efflux (3, 9), suggesting that Ca_i²⁺ could somehow regulate the exchanger. The use of dialyzed squid axons showed, unambiguously, that the Na_i⁺-dependent Ca²⁺ influx (reverse exchange) required cytoplasmic Ca²⁺ as an essential factor (72, 92). One of these experiments is illustrated in Figure 9A. In this case, Ca²⁺ influx in the absence of Na_i⁺ is quite small ([Ca²⁺]_i = 0.7 µM), indicating the presence of little Ca²⁺/Ca²⁺ exchange (cartoon on the right in Fig. 9A). The addition of Na_i⁺ in the presence of Ca_i²⁺ in the dialysis medium produces a large reverse exchange (cartoon 2, right of Fig. 9A). The key observation is that removal of Ca_i²⁺ completely inhibits the reverse exchange (cartoon 3, right of Fig. 9A). Figure 9B shows data from a series of experiments in intact cardiac myocytes in which the inward Na⁺/Ca²⁺ (reverse) exchange current is plotted against the intracellular calcium concentration (in the patch pipette); the apparent Ca_i²⁺ affinity is 50 nM (181). It must be stressed that estimating the affinity of the Ca_i²⁺ regulatory site is not easy. In most measurements of Na⁺/Ca²⁺ exchange current, the intracellular transport and regulatory Ca²⁺ sites coexist, and distinction between them is not straightforward. One way around this problem is to look at an exchange mode involving only transport of Na⁺, that is, the Na⁺/Na⁺ exchange. In this case, the Ca_i²⁺ activation would represent Ca_i²⁺ binding to its regulatory site. Figure 9C summarizes such experiments in dialyzed squid axons. The apparent affinity of the Ca_i²⁺ regulatory site in the squid is close to 400 nM. This stimulatory effect of Ca_i²⁺, also called "allosteric," has been demonstrated in several preparations including cardiac sarcolemmal vesicles (218), barnacle muscle fibers, intact myocytes, cardiac giant excised patches, and NCX1 clones expressed heterologously in alien cells (165, 179, 189, 190, 228). The values of the apparent Ca_i²⁺ affinities vary within preparations and are summarized in Table 2. These variations can be accounted for by differences in experimental conditions (see below), considering that intracellular protons and ATP affect the Ca_i²⁺ regulatory site. An interesting observation performed by Reeves and Condrescu (216) in CHO cells expressing the bovine cardiac Na⁺/Ca²⁺ exchanger was that the time course of Ca_i²⁺ stimulation on the regulatory site had a pronounced lag period, whereas the exchanger remained activated after reduction in [Ca²⁺]_i. This, which was later confirmed in intact myocytes (254), was proposed to be important for the function of the heart, since the NXC1 might play an integrating role of [Ca²⁺]_i transients over multiple beats as opposed to a beat to beat regulation (216). On the other hand, in the Drosophila melanogaster Na⁺/Ca²⁺ exchanger clone (Calx), binding of Ca_i²⁺ to its regulatory site reduces exchange activity (131). At present, it is unknown whether alternatively spliced variants of vertebrate exchangers also show this anomalous Ca_i²⁺ modulation.