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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.

View larger version (19K): [in this window] [in a new window]   FIG. 9. Cai2+ regulatory site in the Na+/Ca2+ exchanger. A: absolute requirement of Cai2+ for the activity of the reverse (Nai+/Cao2+) exchange in a dialyzed squid axons (R. DiPolo, unpublished data). The cartoon on the right shows the three experimental conditions: 1) Ca2+ influx in the absence of Nai+, with [Ca2+]i = 10 µM (notice the low levels of Ca2+ influx); 2) Ca2+ influx in the presence of 40 mM Nai+, with a [Ca2+]i = 10 µM (notice the large activation of the reverse exchange); and 3) Ca2+ influx in the presence of 40 mM Nai+ in the absence of Cai2+ (notice the total inhibition of the reverse exchange indicating the absolute requirement of Ca2+ at the intracellular regulatory site). B: dose-response relation between the mean current density and [Ca2+]i in whole cell cardiac myocyes. The current density was obtained by measuring the difference current at 0 mV and dividing it by the membrane capacitance. The current starts to become larger at 10 nM and saturates at 50 nM. The half-maximum [Ca2+]i is 22 nM, and the Hill coefficient is 3.7. [From Miura et al. (181) by copyright permission of The Rockefeller University Press.] C: results of several axons in which the Nai+/Nao+ exchange was used to measured the affinity of the Cai2+-regulatory site. Experiments were carried out in the presence of MgATP and at a physiological pHi of 7.3. Note that the K1/2 for activation of the Cai2+ regulatory site is 400 nM. The cartoon to the right shows the experimental set up for estimating the affinity of the Cai2+ regulatory site. [From DiPolo and Beaugé (90).]

The possibility that the Ca_i²⁺ bound to the regulatory site is also extruded during the exchange cycle has also been ruled out by experiments in dialyzed squid axons. If those Ca_i²⁺ were translocated, one would expect ⁴⁵Ca efflux to increase together with Na⁺ efflux during Na_i⁺ activation of the reverse exchange (Na_i⁺-dependent Ca²⁺ influx), but this does not happen. Figure 10 shows measurements of three of the exchange reactions of the Na⁺/Ca²⁺ exchanger in dialyzed squid axons: Na_i⁺-dependent ⁴⁵Ca²⁺ influx, Na_o⁺-dependent ⁴⁵Ca²⁺ efflux, and Ca_o²⁺-dependent ⁴⁵Ca²⁺ efflux, all as a function of the [Na⁺]_i at constant [Ca²⁺]_i (81). As expected for a countertransport system, Na_o⁺/Ca_i²⁺ and Ca_o²⁺/Ca_i²⁺ exchanges (open symbols) are inhibited by intracellular Na⁺; actually, at 100 mM internal Na⁺, the Ca²⁺ efflux-dependent modes almost vanish. On the contrary, activation of the reverse exchange (Ca_o²⁺/Na_i⁺) by [Na⁺]_i increases continuously until it saturates at 300 mM Na_i⁺, and this increase is not accompanied by any Ca²⁺ efflux. Another important inference from these experiments is that internal Na⁺ does not displace Ca²⁺ from its regulatory site; otherwise, one should expect a bell-shaped curve between reverse Na⁺/Ca²⁺ exchange and [Na⁺]_i. Similar to the external monovalent cation modulation, the apparent affinity of the Ca_i²⁺ regulatory site is voltage insensitive (81).

View larger version (27K): [in this window] [in a new window]   FIG. 10. Inhibition of Nao+-Cai2+ (forward) and Cao2+-Cai2+ exchanges and activation of Cao2+-Nai+ (reverse) exchange by Nai+ at saturating [Ca2+]i. Observe that at 100 mM or higher [Na+]i forward and Ca2+-Ca2+ exchanges are very small in contrast to a still-increasing reverse exchange (see text for details). [From DiPolo and Beaugé (81), copyright 1991 New York Academy of Sciences, USA.]

View larger version (24K): [in this window] [in a new window]   FIG. 11. Deregulation of the Cai2+ regulatory site of the Na+/Ca2+ exchanger by chymotrypsin treatment on the cytosolic side. A: mammalian cardiac sarcolemmal excised giant patches. Note that -chymotrypsin abolished inhibition of the exchange current by removal of calcium. [From Hilgemann (121).] B: outward Na+/Ca2+ exchange current of NCX-SQ1 in an excised oocyte membrane patch. Note that after complete deregulation by chymotrypsin, the current is insensitive to changes of cytoplasmic free Ca2+ in the micromolar range. [From He et al. (118) by copyright permission of The Rockefeller University Press.]

View larger version (24K): [in this window] [in a new window]   FIG. 12. The Cai2+ binding domain of the Na+/Ca2+ exchanger. Top: amino acid sequence of the exchanger loop region which strongly binds Ca2+. Two acidic segments of the fragment are underlined, and amino acid residues which were examined by mutagenesis are marked by an asterisk. When the region was truncated from the NH2 terminus to amino acid 385 or from COOH terminus to amino acid 494 (arrows), strong Ca2+ binding was lost. A segment truncated at the COOH terminus to amino acid 508 (thick arrow) retained strong Ca2+ binding. Bottom: Ca2+ affinities of wild-type and mutated exchanger loops. Ca2+ binding to wild-type fusion protein 240–532 and to mutations of 240–532 are as noted. 45Ca2+ binding was measured in the presence of 1 mM MgCl2. [From Levitsky et al. (163).]

By exposing the intracellular surface to the bath medium and allowing fast changes in ion concentrations on that side, giant cardiac sarcolemmal patches provided a powerful technique to analyze the Na_i⁺ inactivation of the Na⁺/Ca²⁺ exchanger (123). In Figure 13A, with 1 µM cytosolic and 4 mM extracellular Ca²⁺ and without Na⁺ on either side, there is no exchange current. Addition of 90 mM Na⁺ to the cytosolic side produces a fast development of an outward current; almost immediately, that current starts to decay exponentially ("inactivates"), with a time constant of 4 s, to a steady state that represents 20% of the peak. Figure 13A also shows that Na_i⁺ inactivation is destroyed when the cytoplasmic side of the membrane is treated with chymotrypsin, indicating that this process takes place at the large intracellular loop. According to the model proposed by Hilgemann et al. (129) and Matsuoka et al. (179) (Fig. 13B), inactivation starts with the binding of 3 Na⁺ to their E₁3Na intracellular transport sites; this conformation can follow two routes: either face the extracellular side, E₂3Na, or become occluded into the (E_i3Na) state. The conformational change E₁3Na (E_i3Na) is governed by two rate constants: k_inac and k_back for inactivation and recovery from inactivation, respectively (175). In native cardiac patches and intact cardiac myocytes, intracellular Ca²⁺ has two effects on Na_i⁺-dependent inactivation: in the low range of concentrations (0.01–0.5 µM), the peak current is increased; at higher [Ca²⁺]_i (1–30 µM), the Na_i⁺-dependent inactivation is markedly reduced (Fig. 13C). The increase in the peak current has been attributed to the exchanger recovering from a Na_i⁺-independent inactive state (I₂; see model of Fig. 13B) that takes place from the Na_i⁺ free carrier (E₁) (129); the attenuation of the Na_i⁺-dependent inactivation at higher Ca_i²⁺ responds to a facilitation from the recovery of the Na_i⁺-dependent inactivation (I₁) (179). Similar transient and steady-state exchange currents can be predicted by the schemes of H_i⁺ + Na_i⁺ inhibition proposed by Doering and Lederer (98) and by the H_i⁺, Na_i⁺, and Ca_i²⁺ interactions suggested by DiPolo and Beaugé (90) (see below).

View larger version (15K): [in this window] [in a new window]   FIG. 13. Secondary modulation of outward exchange current: sodium-dependent inactivation and calcium-dependent activation. A: outward Na+/Ca2+ exchange current in a giant cardiac membrane patch. Left: current response under standard experimental conditions. Note decay of current during application of cytoplasmic sodium. Right: current response after treatment of the cytoplasmic membrane face with chymotrypsin. Note attenuation of the current decay. [Redrawn from Hilgemann et al. (128).] B: minimum model of cardiac Na+/Ca2+ exchanger function. The exchanger functions in a consecutive cycle. Ion translocation takes place in two steps. The exchanger can enter an inactive state whenever binding sites facing the cytoplasmic side are loaded with three sodium ions (I1) or from the empty unloaded carrier (I2) (see text for detail). [From Hilgemann (123) and Matsuoka et al. (179) copyright 1996 New York Academy of Sciences, USA.] C: secondary Na+ and Ca2+ regulation of the cardiac Na+/Ca2+ exchanger, NCX1. The left panel demonstrates Na+-dependent inactivation. The right panel demonstrates Ca2+ regulation. In this case the level of regulatory calcium in the bath was 15 µM instead of 1 µM. Two phenomena are evident. First, the Na+-dependent inactivation is eliminated in the presence of high regulatory calcium. Ca2+ modulates Na+ regulation. Second, upon removal of regulatory Ca2+, exchange activity declines to zero. Ca2+ is transported by the exchanger but also exerts a separate modulatory effect. [From Philipson and Nicoll (204), copyright 2000 by Annual Reviews.]

3. H_i⁺ and H_o⁺ effects on Na⁺/Ca²⁺ exchange activity

Inhibition of the Na⁺/Ca²⁺ exchanger by intracellular protons was first described in injected squid axons by Baker and McNaughton (14) and dialyzed squid axons (74) and further characterized in mouse heart (251, 252), dog cardiac sarcolemmal vesicles (201), reconstituted Na⁺/Ca²⁺ exchanger in liposomes (153), and myocyte excised patches (97, 98). In dialyzed squid axons there is an asymmetric effect of protons (Fig. 14A), whereas intracellular acidification strongly inhibits and internal alkalinization markedly enhances activity, similar changes in extracellular pH have no effect. The lack of effect of external pH in the squid exchanger contrasts with a recent report in patch-clamped guinea pig ventricular myocytes where extreme external alkalinization (pH_o = 10) or acidification (pH_o = 5) strongly inhibits the Na⁺/Ca²⁺ exchanger (105); to account for these results, the authors suggest that protons may interact at multiple extracellular sites of the exchanger to affect its stoichiometry.

View larger version (11K): [in this window] [in a new window]   FIG. 14. A: extracellular and intracellular pH dependence on forward Na+/Ca2+ exchanger fluxes in dialyzed squid nerve. Notice that while extracellular changes in pH have no effect, intracellular alkalinization markedly increases the activity of the exchanger. The experiments were done at constant concentrations of intracellular Na+ and Ca2+ and in the absence of ATP. [From DiPolo and Beaugé (74) with permission from Elsevier.] B: intracellular proton inhibition of Na+/Ca2+ exchange currents in giant excised membrane patches obtained from cardiac myocytes of adult guinea pig. Preexposure to protons decreases the amplitude of the Na+/Ca2+ exchange current indicating that partial proton blocks develop in 0 mM sodium. Nevertheless, complete proton blocks do not develop in the absence of sodium. Note that the solid circle indicates the lower trace that corresponds to 30 s preexposure to pHi 6.4 in the absence of Nai+. [From Doering and Lederer (97).] C: magnitude of steady-state Na+/Ca2+ exchange current is strongly dependent on pHi particularly at the physiological range (solid symbols). This dependence on pHi is abolished with chymotrysin digestion (open symbols). [From Doering and Lederer (98).]

4. Ca_i²⁺-H_i⁺ interactions

If the effects of protons involve some kind of competition with Ca_i²⁺, that interaction might occur at two places: Ca_i²⁺ transport sites, which will render the exchanger unable to transport in the Ca²⁺ efflux mode, or at the Ca_i²⁺ regulatory site, inhibiting all transport modes of the exchanger. In either case, inhibition by H_i⁺ should be a function of [Ca²⁺]_i. Experiments carried out to explore these possibilities in giant excised membrane patches from guinea pig were not conclusive (97). This point was also explored in dialyzed squid axons by using two approaches: 1) by following the Na_o⁺-dependent Ca²⁺ efflux (forward mode) without Na_i⁺ at variable pH_i values as a function of the [Ca²⁺]_i (range from 0.7 to 1,000 µM) and 2) by determining the apparent affinity of the Ca_i²⁺-regulatory site on the basis of the Ca_i²⁺ stimulation of the Na⁺/Na⁺ exchange (see above and Fig. 15B) at different pH_i values. Figure 15A demonstrates proton inhibition associated with a proton-calcium antagonism in the absence of Na_i⁺; notice that at 1,000 µM Ca_i²⁺, protons are ineffective. An answer regarding the site(s) at which proton act is provided by the second approach. Figure 15B shows that, in the absence of ATP, the apparent affinity of the Ca_i²⁺ regulatory site decreases 60 times when pH_i goes from 8.8 to 6.9. The fact that at 1,000 µM Ca_i²⁺ the V_max of the Na²⁺/Ca²⁺ exchanger is not modified by pH_i strongly suggests that H_i⁺-Ca_i²⁺ interactions take place at, or at least mostly at, the Ca_i²⁺ regulatory site. Two additional lines of evidence point in that direction: 1) intracellular, controlled chymotrypsin digestion relieves proton inhibition and eliminates Ca_i²⁺ regulation without affecting the maximum Na⁺/Ca²⁺ exchange rate (see Figs. 14C and 11B) and 2) PCMBS, which is known to block the Ca_i²⁺ modulation of the squid exchanger, renders intracellular protons ineffective (89). Similar effects of protons on the apparent affinity of the Ca_i²⁺ regulatory site have been reported in the cardiac exchanger in which intracellular alkalinization dramatically increases the affinity of that site for Ca²⁺ (127, 129).

View larger version (19K): [in this window] [in a new window]   FIG. 15. A: Cai2+-dependent activation of forward Na+/Ca2+ exchange flux at different pHi values in the absence of Nai+ and ATP. Shown is the Nao+-dependent Ca2+ efflux at different [Ca2+]i and pH values of 6.9, 7.3, 7.7, and 8.8. The error bars indicate the SE. The mean temperature was 17°C. B: effect of acid and alkaline pHi on the Cai2+-dependent Nao+/Nai+ exchange. The figures displays the percentage Cai2+-dependent Nao+/Nai+ exchange at pH 6.9, 7.3, and 8.8 in the absence of ATP at a physiological [Na+]i. The measurements at 0.3 µM Ca2+ were obtained with BAPTA as Ca2+ chelator. All other measurements were carried out with dibromoBAPTA. The error bars indicate SE. The mean temperature was 17.5°C.

As shown initially by Doering and Lederer (97) in guinea pig cardiac cells under patch clamp, H_i⁺ inhibition is enhanced by Na_i⁺; this additional effect disappears 9 s after removal of Na_i⁺. The model proposed for H_i⁺ and H_i⁺-Na_i⁺ interactions with the intracellular side of the exchanger is shown in Figure 16A. Protons can bind to the free, calcium-loaded, and sodium-loaded carrier, but the affinity is higher in the sodium-bound form (99). In all cases, dead-end inhibitory complexes are formed. This model can also explain the observation that in dialyzed squid axons Ca²⁺/Ca²⁺ exchange (a Ca²⁺ extruding mode) is much less sensitive to pH_i than Na⁺/Ca²⁺ and Na⁺/Na⁺ exchanges, the two modes that move Na⁺ outward (76). An important consequence of this Na_i⁺-H_i⁺ interaction is that Na_i⁺ inactivation observed at or near pH 7.2 results from a combined synergistic interaction between H_i⁺ and Na_i⁺. In fact, there are similarities between Na_i⁺-dependent inactivation and H_i⁺-inhibition: both develop with a time course (seconds) and both are prevented by chymotrypsin digestion. A Na_i⁺-H_i⁺ synergism predicts that internal alkalinization should reduce Na_i⁺-dependent inactivation. This is confirmed in Figure 16B, where the fast Na_i⁺ inactivation at pH_i 6.8 is completely overcome by raising internal pH to 7.8 (129). Figure 16B also shows that intracellular alkalinization increases the steady-state level of the exchange current. Intracellular H_i⁺-Na_i⁺ synergism in the inhibition of the Na⁺/Ca²⁺ exchanger at steady state has also been observed in dialyzed squid axons. In Figure 16C, the K_0.5 for intracellular Na⁺ inhibition goes from 10 mM at pH_i 6.9 to 90 mM at pH_i 8.8 (90).

View larger version (18K): [in this window] [in a new window]   FIG. 16. A: model of the Na+/Ca2+ exchange cycle and inhibition by cytoplasmic protons. A simple consecutive transport model is assumed with five different forms corresponding to different occupancy states of the transporter with the transport sites accessible only to the extracelllular environment, and five forms corresponding to states with intracellularly accessible transport sites. [From Doering et al. (99), copyright 1996 New York Academy of Sciences, USA.] B: outward exchange current transient at pH 6.8 and 7.8, activated by 100 mM cytoplasmic sodium in the presence of 4 µM cytoplasmic calcium. [From Hilgemann et al. (127) by copyright permission of The Rockefeller University Press.] C: effect of Nai+ on the forward Na+/Ca2+ exchange at different pHi values in the absence of ATP. The data show the Nai+-dependent inhibition of forward Na+/Ca2+ exchange at different pHi values in the absence of ATP. Ordinate, percent inhibition of Nao+/Cai2+ exchange; abscissa, intracellular Na+ in millimolar. The error bars indicate SE. The mean temperature was 17°C. Notice the exquisite sensitivity of the exchange activity to Nai+ at the acidic pH. [From DiPolo and Beaugé (90).]

The topics reviewed in section VI, A1–A5, are intended to provide a detailed analysis of how the Na⁺/Ca²⁺ exchanger is regulated by ions acting on regulatory sites located at the intracellular loop of the exchanger. We have intentionally emphasized the similarities and differences between the two preparations used in most of these studies: the mammalian heart and the squid nerve. The relevant findings are summarized in Table 3. There are two points to be emphasized in the models of ionic regulation presented so far. 1) Na_i⁺ inhibits by interacting with the intracellular transport sites. This interaction of Na_i⁺ with its transport sites is implicit in Figure 16 (99) and explicitly stated in Figure 13B (123, 129, 179, 129). 2) In the Na_i⁺-dependent inactivation (Fig. 13B) and H_i⁺-Na_i⁺ synergism (Fig. 16A), the Ca_i²⁺regulatory site was left aside. To understand how the Na⁺/Ca²⁺ exchanger is modulated by metabolism (see sect. VIB), it is necessary to take into account the three ionic interactions, H_i⁺, Na_i⁺, and Ca_i²⁺, all together. Figure 17A shows a kinetic scheme that includes H_i⁺-Ca_i²⁺ competition and H_i⁺-Na_i⁺ synergism as the basis for this ionic regulation (90). For simplicity, only the intracellular ionic interactions (E₁ forms) have been considered. An important feature of this scheme, that differs from the two mentioned above, is that the inhibitory Na⁺ binding site is different from the transport sites. Its location is not known; it could reside on the intracellular loop, but in this case it should be different from the Ca_i²⁺ regulatory site, since Ca²⁺ is not displaced from that site by Na⁺ (see above). E₁ is the free carrier, Ca_i.E₁ the carrier loaded with Ca²⁺ at the regulatory site, Ca_r.E₁.Ca and C_r.E₁.3Na are the carriers loaded with 1 Ca²⁺ or 3 Na⁺ on the transporting sites ready to perform either Ca²⁺ or Na⁺ extrusion. H.E₁, H.E₁.Na, and H₂.E₁.Na are carriers binding H⁺ and Na⁺ at their inhibitory sites. Proton inhibition occurs even in the absence of Na_i⁺ (H.E₁ complex). Since, in the absence of protons (extreme alkalinization), there is no Na_i⁺-dependent inhibition, protons must bind first. This explains the reduction in the apparent affinity of the intracellular Ca²⁺ regulatory site. Na_i⁺ enhances H_i⁺ inhibition by binding to H.E₁ to produce the H.E₁.Na form that allows the binding of a second proton leading to the H₂.E₁.Na dead end complex. Therefore, inhibition of intracellular Na⁺ will have two components: H_i⁺-Na_i⁺ synergism on the regulatory site and Na_i⁺-Ca_i²⁺ competition at the transporting sites; the second effect can be dissected from the first during internal alkalinization (see Fig. 16C). From the data, and the simulations, H_i⁺-Na_i⁺ synergism is the major component at low [Na⁺]_i, whereas competition at the transport sites comes into play at high [Na⁺]_i. This new model puts the Ca_i²⁺ regulatory site at the center of the scene; as such it can explain most of the experimental data on ionic regulation of both mammalian and invertebrate exchangers. Figure 17, B and C, is a simulation of the Na⁺/Ca²⁺ exchange activity during steady state, as a function of [Ca²⁺]_i and [Na⁺]_i at different pHi values in the absence of intracellular ATP. Figure 17, D and E, shows that this new model also predicts the response of the exchanger under pre-steady-state conditions: Na_i⁺ inactivation and the relief of Na_i⁺ inactivation at high [Ca²⁺]_i and alkaline pH_i, respectively.

View larger version (23K): [in this window] [in a new window]   FIG. 17. A: kinetic model of Nai+-Hi+-Cai2+ interactions. B: kinetic model simulations for the Nai+, Hi+, and Cai2+ interactions in the regulation of the squid Na+/Ca2+ exchanger in the absence of ATP. B and C are steady-state simulations (90), and D and E are pre-steady-state simulation (unpublished data). See text for details.

1.Modulation by nucleotides

A) DISCOVERY OF ATP STIMULATION.  The first indication that there was an energy-dependent regulation of the Na⁺/Ca²⁺ exchanger came from work in injected Sepia giant axons (12) with manipulations that reduced the levels of ATP. That reduction was achieved in two ways: inhibition of ATP production with cyanide or increase in ATP hydrolysis with apyrase injections. In both cases the Na_o⁺-dependent Ca²⁺ efflux was inhibited (12). That ATP was actually involved in stimulation of the exchanger was later shown in dialyzed axons from the squid Doryteuthis plei where removal of ATP from the cytosol inhibited the Na_o⁺-dependent Ca²⁺ efflux. This effect was reversed by restoring the nucleotide in the dialysis solution. At the same time, these experiments showed that the requirement for ATP was not absolute, since a sizable part of the exchange fluxes was present without the nucleotide (69, 70). Although the mechanism(s) and/or the targets of that stimulation were not known then, ATP appeared to be an important, nonessential activator. ATP stimulation of Ca²⁺ efflux has two components: the Ca²⁺ pump and the Na⁺/Ca²⁺ exchange. In this particular case (40 mM Na_i⁺, 0.7 µM Ca_i²⁺, and 3 mM MgATP), the two components are not very different. At any rate, the stimulation of the Na_o⁺-dependent exchange component is about sevenfold. The K_1/2 values for ATP are different, 20 µM for the pump and 250 µM for the exchanger (33, 70, 73).

In early experiments with sarcolemmal membrane vesicles, Caroni and Carafoli (56) reported an ATP modulation that took place via a calmodulin-dependent kinase-mediated phosphorylation step. In that report the authors proposed the existence of an associated Ca_i²⁺-stimulated phosphatase. On the other hand, work done by others failed to detect any ATP effects on the Na⁺/Ca²⁺ exchanger in mammalian heart vesicles (205, 213). With the introduction of the giant patch technique (61, 126), an MgATP stimulation of the reversal exchange mode was demonstrated in guinea pig, rabbit, and mouse myocytes. The K_m for ATP was exceedingly high, 12 mM. In the late 1990s, a consistent MgATP stimulation of the uptake of Ca²⁺ through the Na⁺/Ca²⁺ exchanger was demonstrated in bovine heart sarcolemmal vesicles, but to detect it, addition of vanadate to the extravesicular solution was required (27). In these cases, the K_m for the nucleotide is 500 µM. In recent years, ATP stimulation was also observed in whole cells, both with native exchanger and when expressing a cloned exchanger (Table 3).

B) KINETIC EFFECTS.  MgATP modulation takes place by affecting the binding affinity of several ligands. The initial findings came from studies in squid axons on the forward exchange mode. On the one hand, there was an increase in the apparent affinity for the external Na⁺ transport site, from 120–160 mM to 50 mM (12, 13, 33, 70) and for intracellular Ca²⁺, 10–5 µM to 1–3 µM (33, 75). On the other, an apparent increase in the V_max of the forward mode was also reported, but that happened only in the presence of intracellular Na⁺ (71, 75, 222) and was reduced as the [Ca²⁺]_i increased (33). MgATP stimulation was also seen on the reverse Na_i⁺/Ca_o²⁺ (73, 75), Ca²⁺/Ca²⁺ (16, 46), and Na⁺/Na⁺ exchange modes (75, 77). The initial proposal that ATP antagonizes Na_i⁺ inhibition of the forward exchange by decreasing the affinity for Na_i⁺ (33, 222) was unsatisfactory, since a corresponding change in the K_1/2 for ATP at high [Na⁺]_i was not observed (75). The ATP-dependent increase in the apparent affinity for the binding of Ca_i²⁺ to its intracellular regulatory site (77) focused for the first time on the possible interrelationship between intracellular ionic and ATP modulation of the exchanger.

In cardiac giant excised patches MgATP diminishes the extent and slows the kinetics of Na_i⁺ inactivation (Fig. 18A) (61, 128). Figure 18B shows that removal of MgATP causes an increase in the rate of Na_i⁺ inactivation and a decrease in the steady-state exchange current. Consequently, the nucleotide decreases the affinity for Na_i⁺. In addition, the nucleotide acts with low affinity, since 10 mM almost doubles the effect of 2 mM (Fig. 18C). There is a second effect: reduction of the rundown time constant of the exchanger activity after intracellular Ca²⁺ is removed (see Fig. 18, E and F). A reduction in the "off" rate constant for Ca²⁺ binding is equivalent to an increase in the affinity of that site for Ca_i²⁺. This relationship between MgATP and the Ca_i²⁺ regulatory site was confirmed and extended in bovine cardiac sarcolemmal vesicles where MgATP increases the affinity of that site for Ca_i²⁺ by 5- to 10-fold (27). Actually, antagonism between Na_i⁺ and Ca_i²⁺ had already been observed (129), but at that time there was no information about the possible nature and significance of that connection. Another important observation regarding the mammalian cardiac exchanger was that the characteristics of the MgATP upregulation are maintained when the exchanger is expressed in an heterologous cell like Xenopus oocytes (179). This has at least two relevant connotations. On the one hand, the final target for that modulation is indeed the exchanger protein and, on the other hand, the biochemical machinery involved is present both in mammalian and amphibian cells.

View larger version (24K): [in this window] [in a new window]   FIG. 18. Analysis of MgATP effect on sodium-dependent inactivation kinetics. Results are with 2 mM calcium and 150 mM sodium in the pipette. Current transients were recorded for cytoplasmic solution switches from a solution containing sodium-free cytoplasmic solution with 3 µM free calcium to one with 100 mM sodium and 3 µM free calcium, and back to the original solution. Dotted lines give the current level in the absence of cytoplasmic calcium and sodium. A: records were first obtained in the presence of 2 mM MgATP. B: ATP was then washed out and the current transients became more pronounced over 20 min. C: finally, 10 mM ATP was applied and the current transients became much less pronounced. D: the slow recovery portions of the records upon switching back to sodium-free solution were fitted to exponentials and are plotted semi-logarithmically after subtracting an asymptote. The rate constants for rundown with 0 mM, 2 mM, and 10 mM ATP are 0.09, 0.18, and 0.34 s–1, respectively. E: application and removal of 2 µM free calcium in the presence of 100 mM cytoplasmic sodium. Note that the initial rate of rise of current upon application of calcium is almost unchanged by MgATP, while the decay rate upon removal of calcium is drastically slowed. F: decay of outward exchange current upon removal of cytoplasmic calcium before and after MgATP. An asymptote was subtracted and results are plotted semi-logarithmically. [From Hilgemann et al. (127) by copyright permission of The Rockefeller University Press.]

View larger version (16K): [in this window] [in a new window]   FIG. 19. ATP relief of Nai+-Hi+ inhibition of forward Na+/Ca2+ exchange. A: Nai+-induced inhibition of Nao+-dependent Ca2+ efflux at pH 6.9 in an axon dialyzed first without ATP, and after 3 mM ATP. Notice the following: 1) the large activation in the exchange activity induced by ATP in the absence of Nai+, and 2) the relief of Nai+ inhibition. B: Nai+-dependent inhibition of forward Na+/Ca2+ exchange at an acid pH of 6.9 in the presence and absence of ATP. C: Nai+-dependent inhibition of forward Na+/Ca2+ exchange at an alkaline pH of 8.8 with and without ATP. Error bars indicate SE. [From DiPolo and Beaugé (90).]

View larger version (17K): [in this window] [in a new window]   FIG. 20. Effect of acid and alkaline pHi on the Cai+ affinity of the Cai+ regulatory site in the stimulation of Nao+/Nai+ exchange. A: steady-state Nao+-dependent Na+ efflux at pH 6.9 induced by increasing the [Ca2+]i from 0 to 200 µM in the presences of 2 mM ATP. Open circles refer to removal of extracellular Na+. B: percentage Cai+-dependent Nao+/Nai+ exchange at pH 6.9 in the presence and absence of 3 mM ATP. The error bars indicate SE. The mean temperature was 17.5. Notice the large increase in the apparent affinity of the Cai+-regulatory site induced by ATP at acidic pH. [From DiPolo and Beaugé (90).]

View larger version (28K): [in this window] [in a new window]   FIG. 21. Kinetic model for the ATP regulation of the Nai+, Hi+, and Cai2+ interactions with the Cai2+-regulatory site of the squid Na+/Ca2+ exchanger. The broken lines enclosed the intracellular regulatory loop. A–D: steady-state model simulations for the Nai+, Hi+, Cai2+, and ATP interactions in the modulation of the squid Na+/Ca2+ exchanger (see text for details). [From DiPolo and Beaugé (89).] E: pre-steady-state of forward exchange activity at pHi 6.9 in the presence and absence of ATPi. (From L. Beaugé and R. DiPolo, unpublished results.)

The basic feature, common to all Na⁺/Ca²⁺ exchange preparations, is that the simultaneous presence of Mg²⁺ is essential for the onset of the ATP upregulation (61, 75). The K_m for ATP is variable: 250 µM in squid nerve (73), 500 µM in mammalian sarcolemmal heart (27) and nerve (26) vesicles, and in the millimolar range in giant patches of mammalian heart (27). The fractional increase in exchange fluxes varies with the preparations and also, as will be seen below, with the experimental conditions. Depending on the preparation, there are also additional requirements for ligand interactions. In dialyzed squid axons, where the four exchange modes are all stimulated by MgATP, stimulation is increased by intracellular vanadate (K_1/2 10 µM) and millimolar concentrations of inorganic phosphate and p-nitrophenylphosphate (75). The mammalian heart exchanger in native cells, or even expressed in heterologous cells, under patch-clamp conditions is stimulated by MgATP; mammalian heart sarcolemmal or brain vesicles require the addition of 100–500 µM vanadate (26, 27). Upon removal of ATP, in the presence of Mg²⁺, upregulation of the exchangers is lost (93). In excised giant sarcolemmal patches, the effect of MgATP is insensitive to the protein kinases inhibitors 1-(5-isoquinoline-sulfonyl)-2-methylenepiperazine or to inhibitor peptides of calmodulin-dependent protein kinase II, PKC, and cAMP-dependent protein kinases. Because it was also insensitive to alkaline, acid, and heart extracted protein phosphatases, it was concluded that stimulation of the exchanger did not involve protein kinases (61). This concurs with results in mammalian heart sarcolemmal vesicles where MgATP stimulation is insensitive to alkaline protein phosphatase (27).

D) NUCLEOTIDE SELECTIVITY.  The different properties of nucleotides polyphosphate can be related to 1) the nucleoside structure, 2) the capability to be hydrolyzed, 3) the number of phosphates, and 4) the addition of other elements (for instance, sulfur or chromium) to the phosphate chain. Furthermore, selectivity studies can provide important clues as to what metabolic path or paths are likely to be involved. This approach was used in the ATP stimulation of the Na⁺/Ca²⁺ exchanger.

 I) Nucleoside structure.  In squid axons stimulation of the exchanger is highly selective towards adenosine, since other nucleotides triphosphate like GTP, UTP, and ITP are ineffective (71). This constitutes a marked difference with other systems, like the Na⁺-K⁺-ATPase, where CTP, ITP, UTP, and even GTP show variable capabilities in sustaining the overall hydrolysis and also partial reaction cycles (see Ref. 54); however, by themselves, these findings do not rule out an ATPase-related regulation of the exchanger.

 II) Capability to be hydrolyzed.  The hydrolyzable ATP analog 2-deoxy-ATP and AMP-CPP can mimic ATP, while the nonhydrolyzable analogs AMP-PCP and AMP-PNP are ineffective (71, 88). Nonhydrolyzable ATP analogs are also ineffective in mammalian heart under giant patch (118) and mammalian heart and nerve vesicles (26, 27). This rules out an allosteric, nonphosphorylating, role, similar to that reported for the stimulation of the cation transporting ATPases like the Na⁺-K⁺-dependent enzyme (20, 206, 240).

 III) Number of phosphates.  Adenosine with either one (AMP) or two (ADP) phosphate(s) is unable to promote stimulation of the exchanger. The same applies for cAMP (71). On the other hand, in the presence of ATP, ADP becomes inhibitory (75).

 IV) Addition of other elements to the phosphate chain.  In adenosine 5'-O-(3-thiotriphosphate) (ATPS), the divalent oxygen of the phosphate in position gamma is replaced with sulfur. The transference of this phosphate gives rise to what is called thio-phosphorylation. In transport ATPases, ATPS leads to a reversible thio-phosphorylation of the enzyme in the presence of Mg²⁺ and the intracellular translocating cation. However, the cycle is halted after the debinding of free ADP, and the enzyme remains in the MgE₁S-P form (234). Therefore, ATPS is not a substrate for the overall cycle of transport ATPases. With kinases, after thio-phosphorylation of the substrate, the MgADP complex is released. The thio-phosphorylated substrate may lose its thio-phosphate spontaneously and/or by the action of an Mg²⁺-activated phosphatase. However, thio-phosphorylation is more stable than phosphorylation from ATP, including a lower sensitivity to phosphatase attack. This means that 1) ATPS is substrate for kinases, and 2) if a given effect depends on the stability of a kinase-phosphorylated compound, it is likely to be more marked with ATPS than with ATP, i.e., ATPS is a better phosphorylase substrate (115). In dialyzed squid axons ATPS stimulates all exchange modalities provided Mg_i²⁺ is present. With ATP, removal of the nucleotide in the presence of Mg²⁺ leads to a fast and complete deactivation of the exchanger; in the case of ATPS, after its removal the exchanger is only partially deactivated and at a slower rate (75). In addition, the fractional ATPS stimulation is consistently greater than that of ATP. This is in line with the greater stability of thio-phosphorylation compared with phosphorylation (57, 149). The major conclusion that can be drawn is that ATP stimulation in squid nerve does not occur via a transport ATPase reaction, but involves one or more kinases. The observation that injection of an unspecific alkaline phosphatase removes MgATP stimulation indicates that the likely target(s) is a protein. On the other hand, a battery of protein kinase inhibitors of PKA, PKC, TyrK, and calmodulin-dependent kinase (see Table 3) were ineffective. Therefore, the responsible kinase(s) must lie outside this group of enzymes. In mammalian cells, the effects of ATPS are dissimilar, even when the same tissue is analyzed, depending on the experimental preparation. This is intriguing, since, as will be seen below, the same metabolic pathway applies in all these instances. Thus, in giant patches of mammalian heart, the exchange stimulation seen with MgATP is not observed with ATPS (61). On the other hand, in heart sarcolemmal vesicles, ATPS is as active as MgATP, with an additional intriguing property: it does not require the presence of vanadate (27). Also interestingly, in bovine brain membrane vesicles, although ATPS does stimulate the exchanger, it is only about one-third of that obtained with MgATP (27).

The ,-bidentate chromium-ATP complex is formed by the almost irreversible binding of a chromium atom to the oxidryl forming oxygen of the - and -phosphate. In transport ATPases, the sequence starts with the chromium-phosphorylation of the enzyme; the cycle is also halted after the debinding of free ADP, and the enzyme remains in the E₁Cr-P form (250). Therefore, CrATP is not substrate for the overall ATPase reaction. In many kinases, although not in all, chromium-phosphorylation leads to the formation of the [Cr(H₂)4(ADP)(substrate-P)] complex. This complex is very stable, and its breakdown is the rate-limiting step of the kinase-CrATP reaction; that rate can be as low as one per minute (102). Consequently, CrATP cannot sustain activity in those kinase systems.

In dialyzed squid axons CrATP has the following effects on Na⁺/Ca²⁺ exchange: 1) it does not affect the "basal" exchange fluxes; 2) it does not stimulate the exchanger even at concentrations as high as 3 mM; 3) when added after MgATP has reached its steady-state stimulation, it completely reverts upregulation; and 4) when added before MgATP, it prevents stimulation. Actually, the effect of CrATP is practically irreversible, since MgATP is ineffective more than 1 h after removal of CrATP from the dialysis solution (82). Together with the results obtained with ATPS, vanadate, inorganic phosphate, pNPP, and protein phosphatases, the effects of CrATP indicate that protein kinase(s) inhibited by this complex is involved in MgATP regulation of the squid nerve Na⁺/Ca²⁺ exchanger. Interestingly, CrATP has no effect on PA modulation of the exchanger, which seems also to involve protein phosphorylation (see sect. IVB2). So far, there are no studies of CrATP on the exchanger regulation in other systems or preparations. Finally, it must be stressed that other cation exchange systems in squid axons such as Na⁺/H⁺ (47) and the Na⁺/Mg²⁺ (87) exchanges are also metabolically regulated by kinase reactions. This property of the Na⁺/Mg²⁺ exchange has also been described in barnacle muscle fibers (210).

E) METABOLIC PATHWAYS FOR ATP STIMULATION.   I) The mammalian heart: role played by phosphatidylinositides.  In 1991 and 1995, Luciani and co-workers (166, 167) found that cardiac sarcolemmal vesicles enriched in their content of phosphatidylinositol 4-phosphate [PtdIns(4)P] and PtdIns(4,5)P₂ had an increased Na⁺/Ca²⁺ exchange rate. In addition, the deacylation products of polyphosphoinositides acted as powerful inhibitors of the exchanger. That was, indeed, the first experimental evidence that these compounds are related to the exchange function. The next step was the finding that the mechanism of MgATP stimulation was actually through the production of PtdIns(4,5)P₂ (125) and involved reversion of the Na_i⁺-dependent inactivation. Part of the experimental evidence is described in Figure 22, A–C. The MgATP stimulation of the outward exchange current observed in Figure 22A is absent when the membrane has been previously depleted of the precursor phosphatidylinositol (PtdIns) by the action of a specific PtdIns-PLC (see Fig. 22B); under this condition, addition of PtdIns(4,5)P₂ produces an increment in exchange current similar to that due to MgATP. Furthermore, in a PtdIns-depleted preparation where ATP is ineffective, the inclusion of PtdIns after MgATP leads to stimulation of the exchanger (Fig. 22C). Finally, a specific PtdIns(4,5)P₂ antibody eliminated MgATP stimulation (125). Similar results were found in bovine cardiac sarcolemmal vesicles where the formation of PtdIns(4,5)P₂ occurs through a fast phosphorylation cascade: a calcium-independent synthesis of PtdIns(4)P followed by a calcium-dependent production of PtdIns(4,5)P₂ (27). This sequence of events is illustrated in the cartoon of Figure 22.

View larger version (19K): [in this window] [in a new window]   FIG. 22. Stimulation of cardiac Na+/Ca2+ exchange current by cytoplasmic ATP and its dependence on phosphatidylinositol (PI) in giant excised inside-out cardiac membrane patches. Horizontal bars indicate the time that a particular substance was applied to the cytoplasmic patch surface. A: outward Na+/Ca2+ exchange current was activated by replacement of CsCl by NaCl. After current stabilized, ATP (2 mM) activates the outward current. B: patches treated with phosphatidylinositol-specific PLC (PI-PLC) had almost no ATP effect. C: PI-PLC-treated patch recovers their ATP effect by application of PI. [From Hilgemann and Ball (125).] D: cartoon displaying ATP regulation of the cardiac Na+/Ca2+ exchanger by PIP2.

View larger version (21K): [in this window] [in a new window]   FIG. 23. A: detection of PtdIns(4,5)P2 bound to bovine heart Na+/Ca2+ exchanger by Western blot as a function of total membrane protein concentrations. Cardiac sarcolernmal membrane vesicles (10–60 µg) were incubated for 20 s at 37°C in the same solutions used for Ca2+ uptake experiments. The reaction was stopped by adding Laemmli SDS-sample buffer at five times the final concentration; the samples were then heated for 10 min at 37°C. Discontinuous SDS-PAGE (7.5% acrylamide) was electrotransferred to PVDF membranes for Western blots. In a first step, the blots were incubated with a mouse monoclonal antibody against PtdIns(4,5)P2 (Per Septive Biosystems) and then developed using a biotinylated streptavidin peroxidase-complex secondary antibody and the Amersham ECL Plus immunoblotting detection system. The bands recognized by specific antibodies were detected and quantified with a Storm 840 image analyzer (Molecular Dynamics) with a Scion PC (NIH) software. In a second step, the same blots were subjected to stripping as indicated by the ECL Plus manufacturer, and then reprobed with a primary guinea pig polyclonal antibody against the NH2-terminal portion of the bovine cardiac Na+/Ca2+ exchanger. The effectiveness of the stripping was consistently checked before the second step was started by incubating the PVDF membranes with ECL Plus detection reagents and exposing them to the image analyzer. The band densities obtained from the anti-PtdIns(4,5)P2 antibody (in arbitrary units) were divided by the image densities obtained from the anti-NCX1 antibody (in arbitrary units). To compare different experiments, a value of 1 was assigned to the ratio of densities obtained in control conditions (presence of 1 µM Ca2+ and absence of ATP). The experiments were done in duplicate and repeated at least three times. Note the following: 1) the amounts of PtdIns(4,5)P2 and Na+/Ca2+ protein increase in parallel with the increase in total protein, and 2) in the presence of ATP, the levels of PtdIns(4,5)P2 bound to Na+/Ca2+ exchanger are markedly increased. B: correspondence of MgATP stimulation of Na+/Ca2+ exchange fluxes (left) and of the amount of PtdIns(4,5)P2 bound to exchanger protein (right). In the case of immunoblots, the PtdIns(4,5)P2-to-NCX1 ratio with no ATP was arbitrarily given a value of 1. [Redrawn from Asteggiano et al. (8).]

Experiments with NCX1 expressed in Xenopus oocytes suggest that PtdIns(4,5)P₂ binds to or near the XIP region of the intracellular loop (116), but the intimate mechanisms of that binding are not known. In general, there are two ways by which PtdIns(4,5)P₂ can bind to a protein; it can bind to specific sites or by strong electrostatic interactions. The NCX1 does not have pleckstrin homology (PH) domains that are the acceptor regions in several proteins that bind PtdIns(4,5)P₂ (108, 172). On the other hand, NCX1 has many arginine residues (172) that could form a positively charged pocket that traps PtdIns(4,5)P₂. However, simple electrostatic interaction may not be enough, since samples treated with up to 2% SDS still show PtdIns(4,5)P₂ bound to the exchanger. The mechanism by which PtdIns(4,5)P₂ activates the exchanger remains unknown, and there is more than one possibility. For example, in cytoskeleton proteins that bind Ca²⁺, regulation by PtdIns(4,5)P₂ leads to PtdIns(4,5)P₂ hydrolysis (110), whereas PtdIns(4,5)P₂ modulation of voltage-dependent ionic channels does not (199).

 II) The squid nerve: protein phosphorylation and the need for a cytosolic soluble protein.  An initial puzzling observation was that whereas in intact and dialyzed squid giant axons the MgATP stimulated Na⁺/Ca²⁺ exchanger, that effect was not observed in membrane vesicles prepared from squid optic nerve. This coincided with the failure to detect MgATP stimulation in cardiac sarcolemmal vesicles. At that time (P. Baker, personal communication), it was advanced that perhaps a crucial membrane and/or cytosolic component was lost during vesicle preparation. The results described above indicate that this was not the case for mammalian heart. On the other hand, this is what happens with the squid nerve. When squid axons were dialyzed with porous capillaries of high molecular mass cut off (18 kDa) for more than 5 h, MgATP-stimulated exchanges fluxes progressively diminished without any effect on the basal fluxes or the Ca²⁺ pump (Fig. 24, A and B) (23, 94), i.e., the ability to exchange Na⁺ and Ca²⁺ was unimpaired since its V_max remained unchanged but metabolic regulation was lost. This finding led to the idea that a low-molecular-weight cytosolic compound lost during prolonged dialysis was essential, and efforts were started to isolate it. Initially, the starting material was axoplasm extruded from squid giant axons (23); identical results were later obtained by using optic ganglia with the advantage that more material was obtained (94). Figure 25, top, shows the present procedure used for the partial purification of this soluble cytosolic regulatory protein (SCRP). Squid nerve membrane vesicles were used to test for biological activity on the assumption that they did not respond to MgATP because they lack the required cytosolic compound(s); the assumption proved to be correct. All filtrates, except that from a 10-kDa cut-off filter, induced MgATP stimulation of the exchanger. Therefore, it was concluded that the molecular mass was between 10 and 30 kDa. Because heat denaturation and treatment with chymotrypsin rendered the required compound inactive, it was identified as a protein. The 30-kDa filtrate was lyophilized, resuspended, and passed through a Superpex FPLC column. With the use of nerve vesicles, the appearance of MgATP stimulation was observed only in the number 65 elution fraction that corresponded to a molecular mass of 13 kDa. When that fraction was injected into axons lacking the MgATP effect due to prolonged dialysis, the upregulation by the nucleotide was restored (23, 94); other fractions were not effective. Similar results were obtained with MgATPS (Berberián, DiPolo, and Beaugé, unpublished data) but not with the nonhydrolyzable ATP analogs AMP-PCP and AMP-PNP.

View larger version (22K): [in this window] [in a new window]   FIG. 24. Rundown of ATP-stimulated Nao+-dependent Ca2+ efflux (forward exchange) after prolonged intracellular dialysis. A: experiment in a single dialyzed squid axon. Ordinate, Ca2+ efflux in fmol · cm–2 · s–1; abscissa, time in minutes. , Ca2+ efflux in the presence of external Na+; , Ca2+ efflux in the absence of Na+. The arrow indicates the addition of 100 µM vanadate to the dialysis medium. B: summary of different experiments in which (%) Vmax of Nao+-dependent Ca2+ efflux ([Ca2+]i = 100 µM), ATP-stimulated Nao+-dependent Ca2+ efflux ([Ca2+]i = 0.8 µM), and Nao+-independent Ca2+ efflux (Ca pump; [Ca2+]i = continuous lines. 0.8 µM) were measured at short (<150 min) and long (>330 min) internal dialysis times. *Statistical significant difference with the other bars (P < 0.001). The number of experiments is indicated over the bars. Statistical errors are expressed as SE. [From DiPolo et al. (94). Reprinted by permission of Federation of the European Biochemical Societies.]

View larger version (49K): [in this window] [in a new window]   FIG. 25. Top: steps in the partial purification of the squid nerve soluble cytosolic regulatory protein (SCRP) required for ATP stimulation of the Na+/Ca2+ exchanger. Bottom: cartoon of the putative regulation of the squid Na+/Ca2+ exchanger by a soluble cytoplasmic regulatory protein (SCRP).

View larger version (11K): [in this window] [in a new window]   FIG. 26. Intracellular 32P labeling from [-32P]ATP of phosphoinositides in membrane vesicles from squid optic nerve and bovine heart. A: phosphoimage of 1-dimensional TLC plates. Lanes 1–3 are from nerve membrane vesicles, and lanes 5 and 6 are from bovine heart membrane vesicles. Lane 1, control; lane 2, vesicles with addition of SCRP; lane 3, nerve vesicles treated with phospholipase C (PLC)-specific phosphatidylinositol (PtdIns; 200 U/ml); lane 4, SCRP without nerve membrane vesicles; lanes 5 and 6, control or PtdIns-PLC-treated (20 U/ml) bovine heart membrane vesicles; lane 7, negative control without membranes. Positions of phosphatidylinositol 4-phosphate [ItdIns(4)P; PIP] and phosphatidylinositol 4,5-bisphosphate [Ptdlns(4,5)P2; PIP2] are indicated. B: absolute values of 32P incorporation in PtdIns(4,5)P2 spots. Data are means ± SE of triplicate determinations expressed as pmol/mg protein present in the initial solution extracted with chloroform/methanol. Note 1) the effective reduction in PtdIns(4)P and PtdIns(4,5)P2 levels in PtdIns-PLC-treated nerve and heart vesicles, 2) the lack of effect of the SCRP on PtdIns(4)P and PtdIns(4,5)P2 synthesis, and 3) the absence of phosphoinositide production in the presence SCRP only. [From DiPolo et al. (93).]

View larger version (13K): [in this window] [in a new window]   FIG. 27. Lack of effect of PtdIns(4,5)P2 antibody [Ab-PtdIns(4,5)P2] and PtdIns-PLC on MgATP-activated Na+/Ca2+ exchange in dialyzed squid axons. , Ca2+ efflux in the presence of external Na+; , Ca2+ efflux in the absence of external Na+. A: intracellular injection of Ab-PtdIns(4,5)P2 during internal dialysis after addition of ATP. B: intracellular injection of Ab-PtdIns(4,5)P2 during internal dialysis before addition of ATP. C: intracellular injection of PtdIns-PLC (final concentration in the axon of 200 units) before ATP addition. Notice the lack of effect of these compounds on the ATP stimulation of the Na+-dependent Ca2+ efflux. [From DiPolo et al. (93).]

View larger version (11K): [in this window] [in a new window]   FIG. 28. Effects of PtdIns(4,5)P2 incorporation into membrane vesicles on the Na+ gradient-dependent [45Ca]Ca2+ uptake in squid nerves (A) and bovine heart (B). Vesicles were preincubated for 5 min at 0°C and an additional minute at 20°C with PtdIns(4,5)P2 liposomes (0.25 mg/mg vesicles) protein vesicle. [45Ca]Ca2+ uptake was measured for 15 s at 20°C. Notice the following: 1) the usual MgATP stimulation is observed in both preparations, and 2) the complete lack of effect of PtdIns(4,5)P2 in the squid Na+/Ca2+ exchange fluxes in contrast to the marked stimulation in the heart. All values are means ± SE of quadruplicate determinations. [From DiPolo et al. (93).]

Finally, the involvement of cytosolic proteins in ion transporter regulation may not be exclusive for the squid Na⁺/Ca²⁺ exchanger. The NCX1 mammalian isoform has a PKC-dependent upregulation that does not lead to phosphorylation of the exchanger; to account for these results it was proposed that the signal transmission induced by PKC activators requires a cytosolic molecule (138). Also, a possible role of calcineurin in the cardiac exchanger regulation has recently been proposed by Katanosaka et al. (148), who found that the COOH terminus of calcineurin A, containing the autoinhibitory domain, binds to the beta1 repeat of the central cytoplasmic loop and inhibits the NCX1. The necessity for cytosolic regulatory proteins has also been suggested for the MgATP modulation of the NHE1 sodium/proton exchanger (2).

F) ROLES OF INTRACELLULAR MAGNESIUM.  The intra- and extracellular transport sites of the squid Na⁺/Ca²⁺ exchanger highly discriminate between Ca²⁺ and Mg²⁺ (10, 75). However, in dialyzed axons Mg²⁺ inhibit all transport modes even at concentrations within the physiological range (52, 75, 77, 222). A plausible mechanism may involve the Ca_i²⁺ regulatory site, since "in vitro" Mg²⁺ antagonizes the binding of Ca²⁺ to that site (162).

In the ATP regulation of the squid Na⁺/Ca²⁺ exchanger, intracellular Mg²⁺ plays two substantial, and opposing roles. On the one hand, and this is common to other preparations (27, 118, 124), Mg²⁺ is essential for ATP stimulation. How much of this is due to Mg²⁺ by themselves or to the forming of the MgATP complex has not been unambiguously determined. However, as MgATP is the substrate for kinases (102), it is very likely that this is also the case for the Na⁺/Ca²⁺ exchanger. On the other hand, in squid axons, Mg²⁺ promotes deactivation of the ATP upregulated exchanger (93). At a constant ATP concentration of 4 mM, activation of the exchanger reaches its peak at 1 mM Mg²⁺, declines at higher concentrations, and is nil at 9 mM (K_0.5 4 mM). This biphasic response to [Mg²⁺]_i is in line with a coupled phosphorylation-dephosphorylation process as the basis for ATP modulation. Actually, if intracellular Mg²⁺ is removed after ATP activation has taken place, the exchanger remains upregulated for at least 1 h (93). Similar results were obtained with squid nerve membrane vesicles, but in addition, in the vesicles there was no correlation at all between the [Mg²⁺] related ATP activation and deactivation and the levels of membrane PtdIns(4,5)P₂. When explored, both preparations, mammalian heart and squid nerve, showed the same [Ca²⁺] dependence of PtdIns(4)P and PtdIns(4,5)P₂ production. (93), thus indicating that phosphoinositides are not related to MgATP modulation in the squid.

The specific process or processes implicated in this Mg²⁺ activation-deactivation is not known. One possibility is that the SCRP behaves as part of a two component signal transduction system (198); upon arrival of a given signal, it becomes phosphorylated by a membrane-bound kinase and, after playing its modulator role, it loses the phosphate through a Mg²⁺-dependent autophosphatase activity (168; see cartoon at bottom of Fig. 25). Alternatively, other phosphatases, like the protein phosphatase 2C type (186), may come into play.

G) PRELIMINARY BIOCHEMICAL AND FUNCTIONAL CHARACTERIZATIONS OF THE SCRP.  Here we provide a summary description of relevant unpublished material from our laboratories (Berberián, DiPolo, and Beaugé, unpublished data). Our initial working hypothesis proposed that the SCRP was a "response regulator" with amino acid composition similar to those described in prokaryotes and some eukaryotes (23). Unfortunately, after spending a lot of time and effort, we were unsuccessful in identifying this factor (M. Delgado, R. DiPolo, and L. Beaugé, unpublished data).

The amounts of SCRP initially obtained when the 30-kDa cut-off filtrated was passed through a FPLC Superdex-75 column was minimal. An alternative approach was to purify further the 30-kDa cytosolic fractions. When that fraction went through anionic (Dowex 1 x 8–400) and cationic (Dowex 50 x 8–400) exchange columns, biological activity was recovered only in the anionic column, indicating a net positive charge of the SCRP. Furthermore, the eluent of an anionic column retained by a 10-kDa cut-off filter was used for tests (10- to 30-kDa fraction; Fig. 25). Coomassie blue-stained gels showed several bands, two of them quite conspicuous, 25 and 13 kDa. This fraction was then exposed to 0.5 mM [³²P]ATP in the same solution used for transport assays in squid optic nerve membrane vesicles under a variety of conditions. Only in the presence of native membrane vesicles did the cytosolic fraction become phosphorylated, and phosphorylation occurred in the two major bands, one around 25 kDa and another, more prominent, around 13 kDa. This means that 1) this 10- to 30-kDa fraction has no kinase activity and 2) the kinase(s) responsible for its phosphorylation is located in the plasma membrane of the cells. Other important findings include 1) membrane vesicles alone do not show phosphorylated bands around 10–30 kDa and 2) heat denaturation of the cytosolic fraction of the membrane vesicles prevented phosphorylation. This agrees nicely with the observation that the heat-denatured fraction is unable to promote MgATP stimulation of the exchanger (23, 94).

Staurosporine, particularly at high concentrations, is a general inhibitor of kinases (212). In dialyzed axons, MgATP stimulation of the Na⁺/Ca²⁺ exchanger is insensitive to a large variety of kinases and phosphatase inhibitors, including staurosporine (94). In squid nerve vesicles, staurosporine (up to 50 µM) does not affect MgATP + SCRP stimulation or the phosphorylation of the 13-kDa cytosolic bands. At the same time, most of the phosphorylated bands from the membrane vesicles disappear. This has two important implications: 1) it supports the idea that the 13-kDa band is involved in MgATP stimulation, and 2) staurosporine eliminates a considerable number of unrelated kinases, becoming an excellent tool for further analysis of protein phosphorylation related to stimulation of the exchanger.

The SCRP phosphorylated bands were sent for amino acid sequencing (W. M. Keck Biomedical Mass Spectrometry Laboratory, Univ. of Virginia Health System). Analysis of peptides from the 25-kDa band allowed the identification of two known proteins: Calexcitin B and RAB2. The peptide from the 13-kDa band did not correspond to any known protein and is considered a de novo species. Previous results (23, 94) suggest that the 13-kDa protein is the most likely candidate for the SCRP. RAB2 does not seem relevant, since it is a GTP-binding protein associated with Ras that regulates vesicular transport from ER to Golgi membranes and shows GTPase activity (235). This does not appear compatible with the nucleotide regulation of the squid Na⁺/Ca²⁺ exchanger where GTP and GTPS are ineffective (88, 89). The 25-kDa band (although in some cases the phosphorylation of this band is minimal) will require additional studies. Calexcitin B (CE_B) is a neural calcium sensor protein that is phosphorylated by PKC and does not bind GTP. CE_B is expressed in the L. pealei optic lobe and is considered to be a new member of the family of sarcoplasmic calcium-binding proteins (114).

Finally, we found that the phosphorylated factor could stimulate the Na⁺/Ca²⁺ exchanger in squid nerve vesicles even in the absence of ATP. Native SCRP was compared with SCRP phosphorylated from MgATP phosphorylated SCRP in the presence of membrane vesicles. Phosphorylation was stopped by adding EDTA, and the vesicles were separated by centrifugation in an Airfuge. The phosphorylated SCRP of the supernatant stimulated Na⁺/Ca²⁺ exchange fluxes in a manner similar to that seen with native SCRP + MgATP. Interestingly, as happens with ATP, stimulation by the phosphorylated SCRP did not take place in the absence of millimolar [Mg²⁺]. Therefore, one can conclude the following. 1) The 10- to 30-kDa fraction not only contains the essential cytosolic factor(s), but that to exert its action, this factor must be phosphorylated. 2) Phosphorylation of the 10- to 30-kDa fraction requires at least one MgATP-dependent protein phosphorylating step. 3) Regardless of the specific biochemical reaction(s) by which the 10- to 30-kDa fraction stimulates the exchanger, either by the transfer of its phosphate to another structure, or just by binding to a target receptor, Mg²⁺ is required but MgATP is no longer needed.

H) PHOSPHORYLATION OF THE SODIUM/CALCIUM EXCHANGER PROTEIN.  We investigated whether the Na⁺/Ca²⁺ exchanger protein became phosphorylated under the condition in which MgATP stimulates exchange fluxes in both squid nerve membrane (NCXSQ1) and bovine heart sarcolemmal (NCX1) vesicles. Numerous phosphorylated membrane proteins were resolved in a 8% SDS-PAGE, including proteins at about the molecular weight corresponding to the exchangers, but neither NCX1 nor NCXSQ1 immunoprecipitated with a specific antibody showed any [³²P]P_i incorporation. In addition, in the squid, the phosphorylation pattern of the total membrane proteins is the same with or without the SCRP (Berberián, DiPolo, and Beaugé, unpublished data). In other preparations the results are variable. No phosphorylation was observed in the immunoprecipitated ATP-stimulated bovine cardiac Na⁺/Ca²⁺ exchanger transfected in CHO cells (63). In contrast, phosphorylation of NCX1, related to PKC and PKA, has been observed in canine exchanger expressed in transfected cells, isolated rat cardiac myocytes in culture (142), and cardiac and kidney NCX1 isoforms expressed in Xenopus oocytes (226, 227). These phosphorylations were associated with an increase in the activity of the exchanger, which was much smaller that those seen in squid nerve, giant cardiac patches, or cardiac sarcolemmal vesicles. Furthermore, their relationships with ions interacting with the intracellular loop were not established.

I) USE OF INHIBITORS AS TOOLS TO UNDERSTAND IONIC AND ATP MODULATION OF THE SODIUM/CALCIUM EXCHANGER.  In recent years a series of organic compounds that inhibit Na⁺/Ca²⁺ exchange have been synthesized. Some of them seem to be particularly relevant for the study of ionic and ATP regulation of the exchanger. For instance, it has been suggested that 2-[2-[4-(4-nitrobenzyloxy)phenyl]ethyl]isothiourea (KB-R7943) and the more potent 2-[4-[(2,5-difluorophenyl)methoxy]phenoxy]-5-ethoxyaniline (SEA0400) (Fig. 29, A and B) reduce the reverse exchange mode (Na_i⁺-dependent Ca²⁺ influx) by acting from the extracellular surface (134, 135, 143, 160, 244, 253). Amino acid mapping indicates that Gly 833, on the -2 repeat region, is critically important for inhibition (136). In fibroblasts expressing NCX1 and excised membrane patches of Xenopus oocytes expressing NCX1.1, SEA favors the transition of the exchangers into the Na_i⁺-dependent inactive state; expressed exchangers with the XIP region either mutated or deleted become resistant to the inhibitor (48). Another newly synthesized compound, [2-[4-(-4-nitrobenzyloxy)benzyl]thiazolidine-4-carboxylic acid ethyl ester (SN-6), also inhibits the reversal exchange in a manner that required the integrity of the XIP region, suggesting that its effect is related to Na_i⁺ inactivation (134). In these cases NCX1 was more sensitive than NCX2 and NCX3. An important additional finding with SN-6 was that ATP depletion enhanced inhibition (see Fig. 29A) (134). In dialyzed squid axons SEA inhibits all transport modes of the exchanger from both membrane sides, but its potency is increased when applied internally (19). Other characteristics of SEA inhibition included 1) increase of the synergic (Na_i⁺ + H_i⁺) inactivation, 2) enhancement by intracellular acidification even in the absence of Na⁺, and 3) its prevention by MgATP and by intracellular alkalinization (see Fig. 30). These findings can be explained by the model recently proposed for the squid Na⁺/Ca²⁺ exchange regulation (90). According to that scheme SEA would act on the (H_i⁺ + Na_i⁺)-inactivation process by increasing the affinity of H⁺ and Na⁺ binding to their inhibitory sites. Protection by ATP would come from its ability to antagonize (H_i⁺ + Na_i⁺) synergic inhibition by reducing the binding affinities for H⁺ and Na⁺; protection by alkalinization would occur because protonation of the carrier is required for Na_i⁺ to get attached to its inhibiting site (19). An additional important point supporting this hypothesis is that in the squid the SEA effects are poorly reversible or nonreversible, whereas the Na⁺ + H⁺ inhibition enhanced by SEA is fully reversible (19, 90).

View larger version (23K): [in this window] [in a new window]   FIG. 29. A: effects of ATP depletion on the inhibition of Nai+-dependent 45Ca2+ uptake by SN-6 in cells expressing wild-type NCX1 and K229Q and F223E mutants. The initial rates of 45Ca2+ uptake into cells were measured in the presence or absence of indicated concentrations of SN-6. The uptake rates into control transfectants were 13, 17, and 7.3 nmol · mg–1 · 30 s–1 for NCX1, K229Q, and F223E, respectively. Cells were pretreated with 10 mM D-glucose (control) or 2.5 µg/ml oligomycin and 10 mM 2-deoxy-D-glucose (minus ATP) during the last 20 min of Na+ loading. Data are means ± SE of three independent experiments. [From Iwamoto et al. (134).] B: effect of SEA-0400 and KB-R7943 on NCX. Concentration-response relationships for the inhibitory effects of SEA-0400 and KB-R7943 on inward and outward NCX current measured at –80 and +30 mV, respectively. Symbols with bars indicate means ± SE. from three or four experiments. [From Tanaka et al. (244), copyright 2002 Macmillan Publishers Ltd.]

View larger version (16K): [in this window] [in a new window]   FIG. 30. Intracellular pH dependence of SEA inhibition of Nao+/Cai2+ (forward) exchange in the presence and absence of Nai+. Collected data from 15 squid axons showing the pHi dependence of 3 µM intracellular SEA inhibition of the Nao+-dependent Ca2+ efflux (forward exchange) in the presence (solid circles) and absence (open circles) of Nai+. Ordinate, percent inhibition of Nao+-dependent Ca2+ efflux; abscissa, pHi. The [Na+]i, [Ca2+]i, [ATP]i, and mean temperature are given in the inset. The bars on the points represent ±SE. The solid triangle is the mean of two experiments showing that in the presence of 40 mM Nai+ and at pH 7.3, 2 mM MgATP prevents SEA-0400 inhibition. [Modified from Beaugé and DiPolo (19).]

Several biologically active molecules act through what is called a "group transfer potential"; chemical groups that, when transferred to another molecule, lead to a change in the chemical potential (or free energy). Acetyl, methyl, or phosphate groups can play this role. Actually, ATP "releases" energy from the transfer of its gamma phosphate. In addition to ATP, other molecules include 1) those with transfer potentials lower than ATP (in descending order): phosphoarginine; acetyl-S-esters, glycylglycine, glutamine, glucose 6-phosphate, glycerol 1-phosphate; and 2) those with transfer potentials higher than ATP (in ascending order): acetyl-S-CoA, glycine-O-ester, CoA-S-phosphate, acetyl phosphate, creatine phosphate, enol pyruvate phosphate, and acetyl imidazol. The usual ATP concentration in normal cells is in the millimolar range (1–4 mM). Phosphoarginine (PA), in mollusks and other invertebrates, and phosphocreatine (PCr), in vertebrates, are also present in millimolar amounts in the cytosol. These last two compounds differ only in a methyl group; their group transfer potential is quite different though and is related to a phosphate attached to a guanidine residue. The main function of PA and PCr is to buffer [ATP] by phosphorylating ADP through the action of arginine and creatine kinases.

A nonnucleotide potential group transfer molecule like acetyl phosphate can act as substrate for the Na⁺-K⁺-ATPase (or Na⁺ pump). It can replace, and competes with, ATP in its phosphorylating role, leading to the same phosphointermediates, but it does not do so in the ATP regulatory site (17, 22, 249). On the other hand, PA does not fuel the Na⁺-K⁺ pump in the squid (51). In squid axons, acetyl phosphate does not affect the Na⁺/Ca²⁺ exchanger (DiPolo and Beaugé, unpublished data), whereas PA is a potent upregulator. This effect is fully reversible with a K_0.5 of 7–8 mM, close to the normal 10 mM PA concentration in the axoplasm. The experimental evidence to be reviewed below shows that PA does not simply replace ATP, but it has a role of its own; it acts independently and through a metabolic pathway different from, and in parallel with, that of the nucleotide. In addition, the kinetics consequences are also different (85, 87, 90).

Figure 31A describes the stimulation by 5 mM PA of the forward Na⁺/Ca²⁺ exchange (almost 5-fold in this case) in a dialyzed squid axon. Upon removal of external Na⁺, the efflux of Ca²⁺ drops to leak values, indicating that PA does not stimulate any other Ca²⁺ extruding systems, i.e., it does not affect the Ca²⁺ pump. At the end of the experiment the measurement of ATP concentration in the dialysis effluent gave values below 1 µM, showing that no ATP regeneration occurred after it was washed out of the axoplasm; in other words, ADP was also effectively removed by dialysis. Another important observation shown in Figure 31B is that neither 5 mM arginine nor 5 mM PCr reproduces the stimulation seen with 5 mM PA. In the case of arginine, the results were negative with and without the simultaneous presence of 5 mM P_i (data not shown). Furthermore, and in contrast with ATP, Figure 31C shows that PA stimulation does not require a cytosolic factor: running down of the ATP effect by prolonged dialysis does not prevent PA stimulation (notice that the MgATP stimulation of the Ca²⁺ pump is unchanged). An analogous PA stimulation of the exchanger is obtained in squid nerve membrane vesicles with and without the addition of the SCRP (see below). Taking aside the kinetic consequences, other differences between the phosphagen and the nucleotide are as follows: 1) while CrATP completely blocks the MgATP effect, it does not influence PA activation; and 2) neither vanadate nor the phosphatase substrate p-nitrophenylphosphate, which stimulate the exchanger in the presence of MgATP, influences the exchange fluxes in the presence of PA alone. In addition, the absolute PA stimulation is the same in the absence and in the presence of saturating concentrations of ATP, i.e., the PA and ATP effects are additive. On the other hand, MgATP and PA upregulations have two things in common: 1) both are insensitive to a series of phosphatase (okadaic acid, microcystin) and kinase (staurosporine, H-7, tyrphostin, genistein, lavendustin) inhibitors, and 2) their effects are fully inhibited by unspecific alkaline phosphatases. The later observations indicate that both processes are mediated by protein phosphorylation.

View larger version (14K): [in this window] [in a new window]   FIG. 31. A: effect of phosphoarginine (PA) on Ca2+ efflux in the virtual absence of ATP. All concentrations are in millimolar (mM) unless specified. , Ca2+ efflux in the presence of Nao+ and Cao2+; , Ca2+ efflux in the absence of Nao+ and Cao2+. The [ATP] in the axoplasm at the end of the experiment was <1 µM. The arrows indicate the addition of 5 mM PA. Notice the lack of effect of PA on the Ca2+ pump and hence the absence of ATP production from ADP. [From DiPolo and Beaugé (85).] B: lack of effect of arginine and phosphocreatine of the squid Na+/Ca2+ exchanger. Notice the large activation induced by PA on the Nao+-dependent Ca2+ efflux (forward exchange) (DiPolo and Beaugé, unpublished results). C: effect of prolonged dialysis on the ATP and PA stimulated Nao+-dependent Ca2+ efflux. All concentrations are given as millimolar. The arrows indicate the removal or addition of compounds to the dialysis medium. , Ca2+ efflux in the presence of Nao+; , Ca2+ efflux in the absence of Nao+. This axon was dialyzed with a standard dialysis medium containing no ATP over 4.3 h prior to the addition of 2 mM ATP. Note the absence of ATP-dependent, Nao+-dependent Ca2+ efflux. Only the Ca2+ pump component remains and is inhibited by the addition of 100 µM vanadate (Van). Note that addition of 5 mM PA (after 5.7 h) causes its usual activation of the Nao+-dependent Ca2+ efflux. [From DiPolo and Beaugé (87).]

View larger version (25K): [in this window] [in a new window]   FIG. 32. Phosphoarginine stimulates preferentially (16-fold) the forward Nao+/Cai2+ exchange over all other exchange modes. Notice (see bottom cartoon) that PA activates mostly the forward Nao+/Cai2+ exchange (80%) compared with only 20% of the Cao2+/Cai2+ exchange. [Redrawn from DiPolo et al. (91).]

View larger version (19K): [in this window] [in a new window]   FIG. 33. Kinetic parameters of the squid Na+/Ca2+ reaction cycle modified by PA. A: lack of effect on the Hi+-Nai+ inhibitory synergism. B: marked increased in the affinity of the Cai2+ transport site by PA. C: increase in the affinity of Cai2+ by PA enhances inhibition of the Nai+ transport site. [From DiPolo et al. (91).]

View larger version (26K): [in this window] [in a new window]   FIG. 34. Effect of ATP and PA on the Na+ gradient-dependent 45Ca2+ uptake in control and chymotrypsin-digested squid optic nerve membrane vesicles. A: Na+ gradient-dependent 45Ca2+ uptake. Basal refers to Ca2+ uptake in the absence of ATP and PA. SCRP refers to the inclusion of the 13-kDa soluble regulatory cytosolic protein (SCRP). Ordinate, Na+ gradient-dependent 45Ca2+ uptake; abscissa, control and chymotrypsin-treated vesicles. B: PAGE membrane protein patterns and Western blots. Aliquots of initial 40 µg total protein of control and chymotrypsin-treated vesicles (1 mg/ml chymotrypsin for 20 min at 20°C) were separated on 4–12% Bis-Tris gel stained with Coomassie blue (I) or immunoblotted with antibodies against the loop portion of the squid (NCXSQ1) (II) and the NH2-terminal portion of the mammalian heart (NCX1) exchangers (III). The antibody against the mammalian heart exchanger cross-reacts with that of the squid nerve. Lanes 1, 4, and 7 are control vesicles; lanes 2, 5, and 6 are vesicles treated with chymotrypsin. Lane 3 corresponds to molecular mass standards (top to bottom, in kDa: 210, 135, 82, 38.7, 31.9, 18.10, 7.4). An arrow on the right side indicates the position of NCXSQ1. Bars indicate means ± SE from three different experiments. [From DiPolo et al. (91).]

Another interesting property of the squid clone expressed in frog oocytes is its insensitivity to PA (118). This PA insensitivity could result from a lack of a phosphagen regulatory mechanism in the clone, a biochemical machinery of the cell unable to handle PA and/or to the difference in the biochemical environment surrounding the exchanger.

B) AN INTEGRATED KINETIC MODEL FOR IONIC, MAGNESIUM ATP, AND PA MODULATION OF THE SQUID SODIUM/CALCIUM EXCHANGER.  Based on the results reviewed above, we have expanded the original model for ionic and MgATP regulation of the squid Na⁺/Ca²⁺ exchanger (90) by including a pathway for PA. The model proposes different structures and/or structural organization as targets for MgATP and for PA modulations (Fig. 35, top) (91). MgATP-ionic interactions occur on the regulatory loop, considered as the "ATP region," whereas the "PA region" is related to the intracellular transporting sites for Na⁺ and Ca²⁺. For simulations, the equilibrium constants for H_i⁺, Na_i⁺, and Ca²⁺ binding and the way they are influenced by ATP are those used previously (89). Within the exchange cycle, the PA region includes Ca_rE₁, Ca_rE₁Ca_t, and Ca_rE₁Na₃ complexes, and the effects are to increase the affinity of the intracellular transport sites for Ca²⁺ (20 times) and Na⁺ (50%). Another characteristic of the system, which has been experimentally verified (91), is that PA does not affect the maximal rates of Ca²⁺ and Na⁺ translocations. In addition, the model assumes that all ions bind instantaneous (rapid random equilibrium) and that 3 Na_i⁺ bind simultaneously. The scheme predicts all the experimental results, and some of the simulations are included in Figure 35, A–C. These are as follows: 1) PA has no effect on ionic interactions located on the regulatory loop (Fig. 35A); 2) PA increases, 10-fold, the apparent affinity for the Ca_i²⁺ transport site (Fig. 35B) but scarcely modifies that for Na_i⁺; as a consequence the phosphagen pushes the exchanger into transport modes favoring Ca²⁺ extrusion; and 3) inhibition, by Ca_i²⁺, of the exchange modes involving Na_i⁺ extrusion is increased in the presence of PA (see Fig. 35C). Obviously this follows the increased ability of Ca_i²⁺ to compete with Na_i⁺ for the inner transport sites.

View larger version (30K): [in this window] [in a new window]   FIG. 35. Integrated kinetic model for ATP and PA modulation of ionic interaction with the squid Na+/Ca2+ exchanger. Broken lines separate two regions: the regulatory intracellular loop and the transmembrane transport sites. A: lack of effect of PA on the Hi+-Nai+ synergic inhibition. B and C: increase in the affinity of the intracellular Ca2+ transport sites due to PA. Note that in this case the Cai2+ regulatory site is saturated at pHi of 8.8 at all Cai2+ concentrations. [Modified from DiPolo et al. (91).]

Three other aspects of the PA phosphorylation of the small NF were investigated: 1) specificity, 2) stability, and 3) staurosporine.

With regard to specificity, phosphorylation is specific for PA because it is attenuated by cold PA but not by ATP, AMP-PCP, or P_i.

With regard to stability, the phosphorylated 60–70 NF is acid-stable, labile at alkaline pH, and even at pH 7.6, and insensitive to hydroxylamine. These characteristics are consistent with phosphoserine and/or phosphothreonine residues (182). Actually, if PA does phosphorylate the exchanger, and P_i incorporation is as labile as that of the 60–70 kDa NF, it is still possible that it was missed during its immunoprecipitation.

With regard to staurosporine, PA phosphorylation of the NF is not affected by staurosporine even at concentrations as high as 100 µM. This result is in agreement with the staurosporine insensitivity of PA stimulation of the exchanger in dialyzed squid axons (87) and squid nerve membrane vesicles (data not shown).

At this stage, we do not know if the small NF indeed plays a role in the PA stimulation of the exchanger; a great deal of work has to be done to elucidate that question. However, the fact that PA is able by itself to phosphorylate a protein related to membrane and cytoskeleton structures opens a relevant field for research on the metabolic function of this phosphagen, which may go beyond the regulation of the Na⁺/Ca²⁺ exchanger.

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

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Two Ca²⁺ extrusion mechanisms work in parallel in the plasma membrane of squid nerve cells: a high-affinity, low-capacity Ca²⁺ pump and a low-affinity, high-capacity Na⁺/Ca²⁺ exchanger (80). The Ca²⁺ pump has a K_m for Ca²⁺ of 0.2 µM and a maximal rate of extrusion of 250 fmol · cm^–2 · s^–1 (73); as with all cation pumps, it does not work without MgATP. On the other hand, whereas the Na⁺/Ca²⁺ exchange can function without MgATP and PA, both compounds markedly influence its activity. MgATP acts by antagonizing the H_i⁺-Na_i⁺ synergistic inhibition and increasing the apparent affinity of the Ca_i²⁺ regulatory site, without affecting the transport site's affinities for Ca_i²⁺ or Na_i⁺ or the maximal translocation rate. Conversely, PA does not influence the ionic interactions with the Ca_i²⁺ regulatory site, but does increase, 20-fold, the affinity of the intracellular Ca²⁺ transport sites with only a slight increase (no more than 50%) of the affinity of those sites for Na_i⁺. As with MgATP, PA does not affect the maximal rate of translocation. The basal affinity observed for transporting sites was 200 µM for Ca_i²⁺ and 50 mM for Na_i⁺. The resting [Ca²⁺]_i in the squid nerve is about, or less than, 100 nM. During synaptic transmission, the peak concentration in microdomains of the synaptic region may be as high 300 µM (239). Therefore, it is likely that [Ca²⁺]_i near the exchanger is also high, particularly if this mechanism is near the microdomains just mentioned.

As a final step of this review, it seemed interesting to explore the relevance of the Ca²⁺ pump and the Na⁺/Ca²⁺ exchanger in the extrusion of Ca²⁺ as a function of [Ca²⁺]_i. To that purpose we performed simulations based on squid data (Fig. 36). The concentrations of Ca²⁺ are indicated on Figure 36; other intracellular concentrations relevant to the simulations were 40 mM Na⁺, pH 7.3, none or 3 mM ATP, and none or 10 mM PA. Figure 36A is a simulation of the forward exchange from 0 to 10 µM Ca_i²⁺ plotted in a linear scale, and Figure 36B is a semi-logarithmic plot from 1 to 300 µM Ca_i²⁺. Figure 36A shows, distinctly, the dominant role of the Ca²⁺ pump at concentrations of cytosolic Ca²⁺ below 1 µM. Above 1 µM Ca²⁺ the role of the Na⁺/Ca²⁺ exchanger becomes more and more prominent as the concentration of Ca²⁺ increases. It is interesting to consider what to expect in axons without and with the two high-energy metabolic compounds. In the absence of both ATP and PA, 10 µM Ca²⁺ is needed in order for the exchanger to match the Ca²⁺ pump. In the presence of ATP alone, although the exchanger works better, still it only surpasses the pump activity above 7 µM Ca²⁺, and at 10 µM is just 50% more productive. On the other hand, the simultaneous presence of ATP and PA results in an extremely active exchanger that at concentrations of Ca²⁺ as low as 2 µM is twice as efficient as the Ca²⁺ pump. The importance of PA regulation of the Na⁺/Ca²⁺ exchanger is even more dramatically shown in Figure 36B. Above 50 µM Ca_i²⁺ the pumped Ca²⁺ efflux is just a minor component of the total efflux, but, more important, the PA-stimulated exchanger dominates the scene, and it does it in such a way that ATP modulation is almost irrelevant. Actually, the PA regulated Na⁺/Ca²⁺ exchanger is so efficient in extruding Ca²⁺ that it can operationally be considered a genuine Ca²⁺ pump.

View larger version (21K): [in this window] [in a new window]   FIG. 36. Role of ATP and PA on Ca2+ extrusion kinetics by the Ca2+ pump and the Na+/Ca2+ exchange in dialyzed squid axons. Notice the extreme efficiency both in terms of apparent affinity for Cai2+ and the maximum rate of transport attained when the Na+/Ca2+ exchanger operates in the presence of ATP and PA.

	GRANTS

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	ACKNOWLEDGMENTS

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We thank Hector Rojas, Magaly Ramos, Daniel Delgado (IVIC), and Myriam Siravegna (Instituto M. and M. Ferreyra) for technical support and Jorge Rivas (Photography Department, IVIC) for the art work. We also thank Fundación Polar (Venezuela) and Fundación Interior Argentina for their constant support to science. We are very grateful to Profs. Mordecai Blaustein, Donald Hilgemann, Kenneth Philipson, and John Reeves for their critical revision and suggestions on the manuscript.

Addresses for reprint requests and other correspondence: R. DiPolo, Laboratorio de Permebilidad Ionica, Centro de Biofisica y Bioquimica, IVIC, Apartado 1827, Caracas 1020A, Venezuela (e-mail: dipolor{at}ivic.ve); L. Beaugé, Laboratorio de Biofisica, Instituto de Investigacion Medica "Mercedes y Martin Ferreyra," Casilla de Correo 389, 5000 Cordoba, Argentina (e-mail: lbeauge{at}immf.uncor.edu).

	REFERENCES

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