I.  INTRODUCTION

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Cytosolic Ca²⁺ plays a key role in intracellular signaling in virtually all types of animal cells. In many instances, as exemplified by cardiac muscle contraction and the transient release of neurotransmitter substances, the elevated cytosolic free Ca²⁺ concentration ([Ca²⁺]_i) may be required for only brief periods of time.¹ The Ca²⁺ must then be rapidly removed from the cytosol. In other situations, such as during the maintenance of tonic tension in smooth muscle, elevated [Ca²⁺]_i may be required for long periods of time. Consequently, [Ca²⁺]_i 1) must be precisely controlled, 2) must be able to be increased and decreased rapidly to provide transient signals, and 3) must be modulated so that steady-state [Ca²⁺]_i can be altered to fit cell needs. Moreover, in at least some cells (neurons are a good example), there must be spatial as well as temporal variation in [Ca²⁺]_i so that multiple Ca²⁺-regulated processes within these cells are not all indiscriminately activated simultaneously. Furthermore, it follows that abnormal cell Ca²⁺ regulation is likely to have profound pathophysiological consequences. For these several reasons, much interest has focused on the regulation of [Ca²⁺]_i in all types of cells.

Calcium is uniquely suited for these signaling functions, due, in part, to its abundance (Ca²⁺ is the most prevalent cation in the body) as well as to its unusual chemistry (the combination of ionic radius, charge density, and coordination number) (981). Furthermore, the resting (or "background") intracellular free Ca²⁺ concentration {[Ca²⁺]_i(rest)} is sufficiently low (on the order of 10⁷ M; Refs. 89, 109, 156, 939) so that a large signal-to-background ratio {[Ca²⁺]_i/[Ca²⁺]_i(rest)} can be generated with only a relatively small increment in the absolute amount of Ca²⁺ added to the cytosol (89). This "signal Ca²⁺" ([Ca²⁺]_i) can come either from the extracellular fluid, via voltage-gated or receptor-operated Ca²⁺-selective channels located in the plasma membrane (PM) or from intracellular stores [especially endoplasmic reticulum (ER) or, in muscle, the sarcoplasmic reticulum (SR)]. Calcium is released from these intracellular stores by Ca²⁺-activated or inositol trisphosphate-activated mechanisms (63, 64, 112, 883). Moreover, the amount of Ca²⁺ sequestered in the stores, and thus potentially available for release during cell activation, is largely dependent on [Ca²⁺]_i(rest). An additional critical feature of Ca²⁺ metabolism is its slow diffusion in the cytosol (102, 419, 543). This is largely a consequence of Ca²⁺ buffering by cytoplasmic proteins such as parvalbumin and vitamin D-dependent Ca²⁺-binding protein (92, 93, 156, 994) and Ca²⁺ sequestration in intracellular organelles such as the SR/ER (105, 106, 647, 739) and mitochondria (277, 404, 459, but see Refs. 157, 882). Therefore, it is possible to generate local increases in [Ca²⁺]_i, and local responses, without having to raise [Ca²⁺]_i throughout the cell. This is energetically important because only limited energy expenditure is then required to remove this "local" Ca²⁺ from the cytosol.

Calcium-dependent signaling is mediated by specific receptors for Ca²⁺ such as calmodulin, troponin C, protein kinase C (PKC), and synaptotagmin. Cells must also have mechanisms to remove Ca²⁺ from the cytosol. Two important mechanisms are the ATP-driven Ca²⁺ pumps in the ER (or SR) for resequestering Ca²⁺, and in the PM, for extruding Ca²⁺ and helping to maintain steady Ca²⁺ balance. These ATP-driven Ca²⁺ pumps bind Ca²⁺ with high affinity but have relatively low turnover rates (i.e., the number of Ca²⁺ transported per carrier per unit time), typically ~100 s¹ (155, 156, 160, 240, 242). Their capacity to transport Ca²⁺ depends on both the turnover rate and the density of transport molecules in the membrane.

The PM of most animal cells also contain another Ca²⁺ transport system, the Na⁺/Ca²⁺ exchanger, that operates in parallel with the Ca²⁺-selective channels and the ATP-driven Ca²⁺ pump. The Na⁺/Ca²⁺ exchanger can move Ca²⁺ either into or out of cells, depending on the net electrochemical driving force on the exchanger. Interestingly, this driving force, and thus the net Ca²⁺ movement mediated by the exchanger, may change direction during a cell's activity cycle, when the membrane potential (V_M) varies (e.g., during an action potential) and/or when the cytosolic Na⁺ concentration ([Na⁺]_i) or [Ca²⁺]_i is altered. As described in section IIIB6, the Na⁺/Ca²⁺ exchanger has about a 10-fold lower affinity for Ca²⁺ but 10- to 50-fold higher turnover rate than the ATP-driven Ca²⁺ pumps.

Sodium-dependent Ca²⁺ transport has also been described in other organelles, including mitochondria (156, 214, 372) and secretory vesicles (451, 538, 539, 724, 804). This review is limited, however, to an examination of the properties of the PM Na⁺/Ca²⁺ exchanger.

A puzzling feature of cell Ca²⁺ regulation concerns the apparent redundancy of the PM Ca²⁺ transport systems, since the Na⁺/Ca²⁺ exchanger operates in parallel with both the Ca²⁺-selective channels and the ATP-driven pump. As we shall see, however, the exchanger has several unique features that may help to explain the need for this redundancy as well as the exchanger's utility. For example, the high maximal turnover rate of the Na⁺/Ca²⁺ exchanger may be important in cells that need to extrude a lot of Ca²⁺ rapidly (as do cardiac muscle cells; see sect. VIA).² Indeed, the rate of Ca²⁺ mediated by the exchanger increases and decreases as [Ca²⁺]_i is raised and lowered, respectively, during the cell activity cycle so that its activity is modulated to meet cellular demands for rapid transport of Ca²⁺ and regulation of [Ca²⁺]_i. Also, the electrochemical driving force on the exchanger in excitable cells is usually altered markedly during the cell cycle. Therefore, in some instances (such as during the plateau of the cardiac action potential), the decrease in the driving force will reduce the net rate of Ca²⁺ extrusion compared with the rate expected at 80 mV. Thus the exchanger may help to maintain [Ca²⁺]_i at a relatively elevated level for an extended period of time. In addition, the efflux of Ca²⁺ via the exchanger is associated with an inward current (see sect. IIID) that will tend to prolong the cardiac action potential (367). The time course of these effects will depend on the time course of the action potential and on that of the Ca²⁺ transient. During quiescent periods, the electrochemical driving force (_Na/Ca; see below) on the exchanger can be modulated by altering V_M or [Na⁺]_i. Changes in these parameters may therefore be used to modulate quasi-steady-state [Ca²⁺]_i as well as the amount of Ca²⁺ sequestered in the intracellular stores. This may be particularly important in cells that must sustain Ca²⁺-dependent activities (such as tonic tension in certain smooth muscle cells). One feature of the exchanger that has recently come to light and that may contribute in important ways to its physiological role, is its localization in restricted portions of the PM of at least some types of cells. As discussed in section VA, certain transport proteins are not universally distributed in the PM but are localized in particular domains of the PM.

The Na⁺/Ca²⁺ exchanger was first described in the late 1960s. Since then, it has been identified in most types of cells in higher animals in which assessment has been carried out; the human erythrocyte is a noteworthy exception. The properties of the exchanger appear to be very similar in most of these cells. The exchanger in vertebrate photoreceptors has some unusual ion coupling features, however, and is the product of a separate gene (see sect. IV). Therefore, after a brief historical note, we review the general properties of the exchangers found in most cells. The various modes of transport mediated by the Na⁺/Ca²⁺ exchanger are described, and a summary of the exchanger's thermodynamic and kinetic properties is presented in the context of overall cell Ca²⁺ homeostasis. We then discuss the molecular structure of the exchanger and its pharmacology. The final portion of this article is devoted to a discussion of the physiological roles of the Na⁺/Ca²⁺ exchanger (and possible pathophysiological implications) in various tissues in which the exchanger has been identified.

A number of reviews published during the past several years have covered selected aspects of the Na⁺/Ca²⁺ exchanger and its physiological role in specific tissues. Some of the more extensive reviews published within the last decade include those by DiPolo and Beaugé (250, 254) on excitable cells, by Philipson and Nicoll (721, 722) on the molecular and kinetic properties of the exchanger, by Reeves and co-workers (759-761) and Khananshvili (489) on the cardiac exchanger, by Sheu and Blaustein (845) on the cardiac and vascular smooth muscle exchangers, and by Schnetkamp (832) and Lagnado and McNaughton (547) on the vertebrate photoreceptor exchanger. In addition, many aspects have been summarized in the proceedings of three international conferences on the Na⁺/Ca²⁺ exchanger (13, 97, 415). The literature on the Na⁺/Ca²⁺ exchanger has exploded in the last few years. Major advances have been made in exploring the molecular biology of the exchanger. Moreover, there has been renewed emphasis on exchanger kinetics, mechanism, and regulation. A new comprehensive review now seems warranted to summarize and integrate the most recent literature and latest views on the properties, physiological roles, and possible pathophysiological significance of the Na⁺/Ca²⁺ exchanger. In many instances, it is not yet clear how the various observations fit together, and there are still numerous controversies. Nevertheless, we have endeavored to present a full spectrum of new concepts and observations in the context of earlier work that established the basic framework. A number of areas that are in need of further investigation are identified. We have tried to provide the reader with a current perspective that we hope will not be misleading. Perhaps our review will encourage others to prepare reviews on more specialized aspects of Na⁺/Ca²⁺ exchange that have been touched on only superficially in this very broad review.

Our review of the literature was completed in 1998. Because of the continually expanding literature on the subject of Na⁺/Ca²⁺ exchange, we are able to provide only selected citations to some of the vast literature (especially on Na⁺/Ca²⁺ exchange in the heart, about which much has been written). We regret omissions of specific citations, including some "first reports," and the citation to several reviews instead. Nevertheless, we hope that we have provided a useful perspective of the current status of many aspects of Na⁺/Ca²⁺ exchange.

	II.  HISTORICAL BACKGROUND

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In the 1880s, Ringer (785, 786) demonstrated that Ca²⁺ in the bathing medium were required for cardiac muscle contraction. Nearly 40 years later, Daly and Clark (221) drew attention to the fact that the effect of extracellular Na⁺ removal "shows a striking resemblance to the effect of strophanthidin" in its ability to enhance cardiac muscle contraction. Then, in 1948, these two observations were brought together, when Wilbrandt and Koller (980) demonstrated an interaction between Na⁺ and Ca²⁺ at the PM. They found that the strength of contraction of the frog's heart was directly related to the ratio [Ca²⁺]_o/([Na⁺]_o)², where subscript o refers to extracellular. Ten years later, Luttgau and Niedergerke (611) showed that, even in depolarized frog cardiac muscle, removal of external Na⁺ induced contractures, and in 1963, Niedergerke (690) demonstrated that frog ventricles gained Ca²⁺ when exposed to Na⁺-depleted solutions. These ventricles promptly lost Ca²⁺ and relaxed when they were returned to control (Na⁺-rich) Ringer solution. On the basis of these observations, Niedergerke (690) postulated that Ca²⁺ enters the muscle fibers via a carrier mechanism for which external Na⁺ and Ca²⁺ compete, presumably in the ratio 2 Na⁺:1 Ca²⁺.

Also in the mid 1960s, attention was directed toward the possible role of cell Na⁺ accumulation to explain the positive inotropic responses associated with cardiotonic steroids (775) and the staircase phenomenon (treppe) in the heart (550); however, no specific mechanisms were proposed. Subsequently, Langer (551) suggested that Na⁺ and Ca²⁺ might compete at intracellular membrane sites (on the sarcotubular system).

Competition between Na⁺ and Ca²⁺ at the PM was also observed in both twitch and slow skeletal muscle in the frog. In the rectus abdominis, a slow muscle, reduction of [Na⁺]_o caused sustained contractures (818). Cosmos and Harris (207) found that frog twitch muscle fibers gained Ca²⁺ when incubated in Na⁺-depleted media. This led them to conclude that in this tissue, too, Na⁺ and Ca²⁺ compete for entry sites. With hindsight, it is also possible to explain some of their other observations in terms of an Na⁺/Ca²⁺ exchange mechanism, namely, conditions that induced net Na⁺ gain (removal of external K⁺, cooling, or treatment with ouabain) also promoted net Ca²⁺ gain in frog muscle. They used radioactive tracer labeled Sr²⁺ (⁸⁹Sr) as a substitute for Ca²⁺ in efflux experiments; after ⁸⁹Sr loading, external Na⁺ promoted (net) ⁸⁹Sr efflux from this tissue.

Similarly, reports of apparent competition between Na⁺ and Ca²⁺ in mammalian smooth muscles were published in the 1960s. The key evidence was that the Ca²⁺ content of guinea pig tenia coli (362) and rabbit aorta (136) increased when [Na⁺]_o was lowered. Even earlier, Friedman and co-workers (323, 324) had suggested that blood pressure was, in part, dependent on "sodium transfer systems" that govern the influx and efflux of Na⁺ in vascular smooth muscle cells, but the importance of Ca²⁺ was not yet recognized.

These findings were reinterpreted and extended in the late 1960s by investigators in three different laboratories: Reuter and co-workers in Germany and Switzerland (783, 352); Baker, Blaustein, and Hodgkin and their co-workers in England (34, 35, 102); and Martin and DeLuca in the United States (625). While studying different tissues, they independently and simultaneously reached the conclusion that a coupled countertransport mechanism, involving the exchange of Na⁺ for Ca²⁺ (i.e., "Na⁺/Ca²⁺ exchange"), rather than just a competition between Ca²⁺ and Na⁺ for binding sites, may be involved in Ca²⁺ transport across the PM in mammalian cardiac muscle (352, 783), in invertebrate neurons (34, 35, 102), and in mammalian small intestinal epithelium (625). It is noteworthy that during the 1950s and 1960s experiments were also being carried out on the transport of small solutes (sugars and amino acids) that revealed a coupling between the fluxes of these other solutes and of Na⁺ (181, 182, 211, 784, 836). In these latter cases, Na⁺ was cotransported with the sugars or amino acids. This coupling of the Na⁺ electrochemical gradient (145) to the transport of other solutes (see sect. IIIB) is often referred to as "secondary active transport." It is widely recognized as a general mechanism that has been adapted in a variety of ways for the transport, against a concentration or electrochemical gradient (see sect. IIIB), of numerous ions and other solutes in virtually all types of animals cells.

In studies on guinea pig, sheep, and calf heart, Reuter and Seitz (782, 783) showed that Ca²⁺ efflux from ⁴⁵Ca-loaded auricular and ventricular muscle was largely dependent on external Na⁺ and Ca²⁺. They interpreted these observations as evidence for a Na⁺/Ca²⁺ exchange mechanism (see Fig. 1). Subsequently, Glitsch et al. (352) found that Ca²⁺ influx in mammalian cardiac muscle was proportional to ([Na⁺]_i)² over a wide range of [Na⁺]_i (see Ref. 85).

View larger version (17K): [in this window] [in a new window]   Fig. 1. Effects of removing extracellular Ca2+ concentration ([Ca2+]o) and extracellular Na+ concentration ([Na+]o) on Ca2+ efflux from a 45Ca-loaded guinea pig auricle (top) and a sheep ventricular trabeculum (bottom). Ordinate shows 45Ca efflux rate constant. Concentrations of Na+ and Ca2+ present in various external solutions at various times are indicated between the two curves; in Na+-free solutions, NaCl was replaced by isosmotic sucrose. Numbers just above each curve indicate fractional rates of 45Ca efflux in various solutions, relative to that in control Tyrode solution which contained 1.8 mM Ca2+ and 149 mM Na+. Note that most of Ca2+ efflux into Ca2+-free Tyrode is dependent on external Na+. [From Reuter and Seitz (783).]

At about the same time, Baker et al. (35) observed an external Ca²⁺-dependent, ouabain-insensitive Na⁺ efflux from squid axons (Figs. 2 and 3). In addition, Ca²⁺ influx appeared to be a function of the [Na⁺]_o/[Na⁺]_i ratio; increasing [Na⁺]_i and/or decreasing [Na⁺]_o (Fig. 3) promoted Ca²⁺ influx in this preparation (444). Similar observations were also made on crab nerve (34). The fact that the Ca²⁺ efflux from ⁴⁵Ca-injected squid axons was, in part, dependent on external Na⁺ (102) fostered the idea that Ca²⁺ extrusion involved a Na⁺/Ca²⁺ exchange mechanism. In sum, these Ca²⁺ influx and efflux data seemed consistent with a transport mechanism that could move Ca²⁺ in either direction across the PM, in exchange for Na⁺; net Ca²⁺ movement (and the Ca²⁺ gradient) appeared to depend on the prevailing Na⁺ electrochemical gradient _Na (35). The Na⁺/Ca²⁺ exchange systems in squid axons and mammalian cardiac muscle appeared to be similar, if not identical. Therefore, these findings led directly to the suggestion that the positive inotropic action of cardiac glycosides was due to a Na⁺/Ca²⁺ exchanger-mediated rise in cell Ca²⁺, as a result of Na⁺ pump inhibition and the consequent reduction in _Na across the sarcolemma (35 and see sect. VIA7). One feature that has challenged this hypothesis is the question of whether the very limited Na⁺ pump inhibition produced by low (nanomolar) concentrations of cardiotonic steroids is sufficient to account for the observed enhancement of contraction (see sect. VIA7).

View larger version (15K): [in this window] [in a new window]   Fig. 2. Effect of external cation concentration on Na+ efflux from squid giant axons bathed in media containing 105 M ouabain in presence (open symbols) and absence (closed symbols) of Ca2+. A: mean data from 5 experiments. B: external Ca2+-dependent Na+ efflux difference curves (curve 1 minus curve 2 and curve 3 minus curve 4 from A). Ordinates indicate [Na+]o or [Li+]o; at reduced alkali metal ion concentrations ([M+]o), monovalent cation was replaced by isosmotic dextrose. Curves in B were calculated from a model described in original article (35). These curves show that low [Na+]o and [Li+]o stimulate ouabain-insensitive, external Ca2+-dependent Na+ efflux and that high [Na+]o (>100 mM), but not high [Li+]o, inhibits this flux. [From Baker et al. (35).]

View larger version (15K): [in this window] [in a new window]   Fig. 3. Ca2+ influx (left) and external Ca2+ (Cao)-dependent Na+ efflux (right) in squid giant axons, graphed as a function of [Na+]o. At reduced [Na+]o, Na+ was replaced by isosmotic dextrose (dextrose-Na curves, ) or Li+ (Li-Na curves, ). All external solutions were K+ free and contained 105 M ouabain. Na+ efflux data (right) are shown as either efflux rate contants (left ordinate scale) or calculated Cao-dependent effluxes (right ordinate scale). Na+ efflux curves were calculated from a model described in original article (35). Both Ca2+ influx (left) and ouabain-insensitive, Cao-dependent Na+ efflux (right) are activated by low [Na+]o and inhibited by high [Na+]o. [From Baker et al. (35).]

Martin and DeLuca (625) examined the effect of Na⁺ in the mucosal and serosal fluids on the transepithelial transport of Ca²⁺ in rat duodenum. They found that Na⁺ was required in the serosal fluid to effect the net transfer of Ca²⁺ from mucosa to serosa. They therefore suggested that the _Na across the basolateral membrane of the columnar epithelial cells powered the extrusion of Ca²⁺ from the cytoplasm into the serosal medium via a Na⁺/Ca²⁺ exchange mechanism.

The regulation of the exchanger by ATP-dependent phosphorylation and by internal Ca²⁺ was first hinted at by the observations of Baker and Glitsch (36). Subsequently, DiPolo (237, 238) clearly demonstrated the activation of exchanger-mediated Ca²⁺ efflux by ATP, and Blaustein (88) showed that the effect was due to a shift in the cation activation kinetics. DiPolo (239, 244; see also Ref. 37) also made the unanticipated (and counterintuitive) observation that the Ca²⁺ entry (and Na⁺ exit) mode of exchange exhibited an absolute requirement for internal Ca²⁺. This observation presages the finding that intracellular Ca²⁺ catalyzes the turnover rate of the the Na⁺/Ca²⁺ exchanger (see sect. IIE4).

Early observations that the Ca²⁺ efflux was activated by the cooperative action of more than two Na⁺ provided a clue that the coupling ratio of the exchanger was >2 Na⁺:1 Ca²⁺ (85). This led to the view that the exchange was voltage sensitive (108, 670). Subsequently, Hume and Uehara (438, 439) observed "creep currents" in cardiac muscle, which they attributed to a rheogenic Na⁺/Ca²⁺ exchange. Kimura, Noma, and co-workers (514, 515) then clearly identified a current attributable to the Na⁺/Ca²⁺ exchanger in patch-clamped cardiac myocytes (Fig. 4). With the development of the giant membrane patch method by Hilgemann and co-workers (199, 405), rapid and accurate measurement of a current attributable to Na⁺/Ca²⁺ exchange in cardiac myocyte membranes could be made.

View larger version (22K): [in this window] [in a new window]   Fig. 4. Activation of Cao-dependent outward (Na+/Ca2+ exchange) current by Cao in a single patch-clamped guinea pig cardiac myocyte. A: voltage-clamp records showing voltage (top traces) and current (bottom traces). Ramp pulses from a holding potential of 30 to +60 mV, and then to 120 mV, at a ramp speed of 0.2 V/s, were given every 10-15 s. In first panel, patch pipette (internal solution) contained 30 mM Na+ and 42 mM EGTA (no free Ca2+), and external solution was also Ca2+ free; effect of [Ca2+]o = 1 mM (added during period indicated by bar below current trace) is shown. In second and third panels, pipette solution contained 73 nM free Ca2+; third panel shows effect of adding 1 mM free Ca2+ to external solution. External solutions all contained 1 mM BaCl2 and 2 mM CsCl to block K+ channels, 2 µM methoxyverapamil to block Ca2+ channels, and 20 µM ouabain to block Na+ pump. Pipette solutions all contained 126 mM CsCl to reduce current through K+ channels. B and C: current-voltage (I-V) curves were obtained from descending portions (+60 to 120 mV) of ramp pulses. In B, curves were obtained before (a) and during (b) superfusion with solution containing 1 mM Ca2+ under internal Ca2+-free conditions. There is no difference between these curves (i.e., no Cao-activated outward current in absence of internal Ca2+). In C, I-V relations were obtained with internal Ca2+ (Cai) equal to 73 nM before (c) and during (d) superfusion with solution containing 1 mM Ca2+. In this case, there is a Cao-activated outward current (d minus c) that is also dependent on internal Ca2+; current also depended on internal Na+. [From Kimura et al. (515).]

The apparent stoichiometry (coupling ratio) of the exchanger was established on the basis of a large variety of tracer flux and electrophysiological experiments in various tissues. These included cardiac muscle (285, 514, 726, 762, 765), squid axons (249), and barnacle muscle (757, 754). In all of these tissues, a coupling ratio of 3 Na⁺:1 Ca²⁺ was observed.

Additional properties of the exchanger were elucidated as a result of the application of other novel methods: ion-selective Ca²⁺ and Na⁺ electrodes (846, 847), release of caged Ca²⁺ (695), and electrical measurements of transport in reconstituted proteoliposomes fused to a planar lipid bilayer (289).

Experiments on vertebrate photoreceptors by Yau and Nakatani (997) demonstrated that these cells, too, also contain a PM Na⁺/Ca²⁺ exchanger. The studies of Schnetkamp et al. (830) and Cervetto, McNaughton, and co-workers (165, 643), however, revealed that the vertebrate photoreceptor exchanger, unlike the cardiac/neuronal Na⁺/Ca²⁺ exchanger (261, 996), also has an absolute requirement for K⁺ on the same side as Ca²⁺ to effect Ca²⁺ transport. The coupling ratio of this vertebrate photoreceptor exchanger was then found to be 4 Na⁺:1 Ca²⁺ + 1 K⁺ (165).

Twenty years ago, Reeves and Sutko (764) launched the biochemical approach to the exchanger with their seminal studies of Na⁺/Ca²⁺ exchange in cardiac sarcolemmal vesicles. This led to the partial purification of the cardiac exchanger by Philipson et al. (719) and to the eventual cloning of this exchanger by Nicoll et al. (688). The exchanger from bovine rod outer segments was purified by Cook and co-workers (206, 767) (and see also Ref. 575), and subsequently cloned by Reilander et al. (769). The photoreceptor exchanger and the cardiac/neuronal exchanger have very different primary sequences, and it seems clear that these two types of exchangers evolved independently. This encourages speculation about the physiological need for such exchangers and the process of evolution, a topic that is discussed in section IVI1.

Very recently, Tsoi et al. (940) cloned a novel exchanger from rat brain. The exchanger expressed from this clone is very homologous to the photoreceptor exchanger, and Ca²⁺ transport mediated by this brain exchanger, like the photoreceptor exchanger, also requires K⁺ (see also Ref. 219). Surprisingly, as discussed in sections VA and VIE, both the K⁺-independent (cardiac-type) and K⁺-dependent (vertebrate photoreceptor-type) exchangers appear to be coexpressed in some of the same neurons (465, 940). These observations open important new avenues for exploration, just as they raise critical questions about our previously conceived notions about the physiological role(s) of Na⁺/Ca²⁺ exchange.

	III.  PROPERTIES OF THE SODIUM/CALCIUM EXCHANGER

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The observations on Na⁺/Ca²⁺ exchange in nerve, muscle, and epithelia made during the late 1960s provided a starting point and a paradigm for numerous studies on Ca²⁺ transport that have been carried out during the past three decades. A Na⁺/Ca²⁺ exchanger has now been identified in most types of tissues, in both vertebrates and invertebrates. Here we describe the properties of the very widely distributed "cardiac/neuronal" type of exchanger that is found in most tissues. Some of the unusual properties of the vertebrate photoreceptor exchanger, already alluded to above, are also discussed.

Initial studies in several preparations indicated that the Na⁺/Ca²⁺ exchanger could move net Ca²⁺ either out of or into cells depending on the prevailing electrochemical driving forces (i.e.., the Na⁺ and Ca²⁺ concentration gradients and the V_M). These two modes of Na⁺/Ca²⁺ exchange (see Figs. 1 and 3) will be referred to, respectively, as "Ca²⁺ exit mode" and "Ca²⁺ entry mode" exchange. The often used terms forward mode and reverse mode will be avoided because the latter may be construed, incorrectly, as an abnormal mode of exchange. Indeed, a very important part of the exchanger's function may be to promote Ca²⁺ entry (and to reduce Ca²⁺ exit) under some normal physiological conditions. A clear-cut example is in the erythrocytes of certain mammals such as dogs and ferrets (see sect. VIF1), where the Na⁺/Ca²⁺ exchanger plays an important role in the extrusion of Na⁺.

The Ca²⁺ entry mode of exchange is usually identified as an internal Na⁺ (Na_i)-dependent Ca²⁺ influx and an external Ca²⁺ (Ca_o)-dependent, ouabain-insensitive Na⁺ efflux. A critical feature of the (net) Ca²⁺ influx mode of operation (and also the Ca²⁺ efflux mode) is that it is dependent on (nontransported) intracellular Ca²⁺ (37, 239, 244, 246, 258, 406, 514, 515, 757). The [Ca²⁺]_i required for half-maximal activation (i.e., K_0.5 for intracellular Ca²⁺) of Ca²⁺ influx mode exchange in squid axons (239, 244, 254) and cardiac myocyte giant patches (406, 412; but see Ref. 514) is on the order of 1 µM under physiological conditions. Consequently, only a small fraction of the exchangers are active at the normal resting [Ca²⁺]_i (~100 nM) in most cells. In contrast, the exchanger is fully activated when [Ca²⁺]_i is in the low micromolar range (i.e., at about the [Ca²⁺]_i expected during peak activity in many types of excitable and secretory cells). The kinetic and physiological consequences of this regulation by internal Ca²⁺ are discussed in section IIIE4.

Another surprising feature of Ca²⁺ influx mode exchange (see Figs. 2 and 3) is its activation by low concentrations of external alkali metal ions, including Na⁺ (35, 98, 314, 336); the activating monovalent cations are not transported (57). High [Na⁺]_o reduces the exchanger-mediated Ca²⁺ influx, however. The kinetics of this activation by alkali metal ions and the biphasic actions of external Na⁺ are also described in section IIIE4.

This mode of exchange is defined operationally as an external Na⁺ (Na_o)-dependent Ca²⁺ efflux and an internal Ca²⁺ (Ca_i)-dependent ouabain- and tetrodotoxin (TTX)-insensitive Na⁺ influx. Like the Na_i-dependent Ca²⁺ influx, the Na_o-dependent Ca²⁺ efflux is activated by internal alkali metal ions and is markedly inhibited by high concentrations of internal Na⁺ (88) (but see Refs. 249, 254). The Ca²⁺ efflux mode of exchange also appears to be activated by nontransported intracellular Ca²⁺ (412) (and see sect. IIIE4), even though the K_0.5 for regulation and for transport may be similar. In contrast to Ca²⁺ entry mode exchange, however, this Ca²⁺ efflux does not require extracellular Ca²⁺ for activation (88, 249); this is clear functional evidence that the exchanger is asymmetric.

The hydrolysis of ATP is not required to power net Ca²⁺ extrusion mediated by the Na⁺/Ca²⁺ exchanger, and Na⁺/Ca²⁺ exchange can be demonstrated in ATP-depleted cells (88, 102, 244). Nevertheless, as discussed in section IIIE1, cytosolic ATP does substantially alter the exchanger kinetics (88), presumably by phosphorylating critical sites on the exchanger molecules (237, 238). This is further evidence that the exchanger is asymmetric. The asymmetry apparently enables the exchanger to function optimally because of the differences in the affinities for Ca²⁺ on the two sides of the membrane and the modulation of the transport kinetics and turnover by various ligands.

The identification of both Ca²⁺ entry and Ca²⁺ exit modes of exchange demonstrated that the exchanger can mediate the bidirectional movements of both Na⁺ and Ca²⁺ across the PM. This raised the question of whether the exchanger could also mediate the coupled exchange of internal Ca²⁺ for external Ca²⁺ (i.e., Ca²⁺/Ca²⁺ exchange) and/or internal Na⁺ for external Na⁺ (Na⁺/Na⁺ exchange) (see Fig. 5). The presence of these two modes of exchange, while perhaps not functionally important in terms of net ion translocation, could provide critical insight into the transport mechanism.

View larger version (18K): [in this window] [in a new window]   Fig. 5. Effects of ionized Cai and ATP on ouabain- and bumetanide-insensitive Na+ efflux from an internally dialized squid giant axon. Concentrations of certain key components in internal (int) and external (ext) solutions are shown at top and are in mM, except for Cai, which is in µM. Arrows indicate changes in internal solutions; different symbols indicate different external solutions. All external solutions contained 300 nM tetrodotoxin (blocks Na+ channels), 0.5 mM ouabain (blocks Na+ pump), 10 µM bumetanide (blocks Na+/K+/Cl cotransport), and 1 mM cyanide (blocks mitochondrial respiration). Cai-activated increase in Na+ efflux into 0Na-10Ca (Nao-free, 10 mM Ca) medium under ATP-free conditions corresponds to activation of Ca2+ entry mode Na+/Ca2+ exchange by Cai. In absence of external Ca2+ (0Ca, open squares) and presence of 80 µM Cai, addition of Nao activated an Na+ efflux (Na+/Na+ exchange) that was not observed in absence of Cai (solid circles at start of experiment). This efflux was augmented by internal ATP, as was Na+/Ca2+ exchange (half-filled circles at right). [From DiPolo and Beaugé (246).]

The Ca²⁺/Ca²⁺ exchange is usually identified as a trans-Ca²⁺-dependent Ca²⁺ flux (i.e., Ca_o-dependent Ca²⁺ efflux and Ca_i-dependent Ca²⁺ influx), where trans refers to ions acting on opposite sides of the PM. On the basis of this criterion, a Ca²⁺/Ca²⁺ exchange that appears to be mediated by the Na⁺/Ca²⁺ exchanger has been identified in various types of cells. A few examples are squid axons (35, 88, 107, 249, 254), barnacle muscle (686, 754, 757, 800), mammalian neurons (98), and cardiac muscle (763, 863).

Two lines of evidence indicate that these Ca²⁺ fluxes are mediated by the Na⁺/Ca²⁺ exchanger. One stems from the observations that the Ca_i-dependent Ca²⁺ influx is activated by external (i.e., on the same side of the PM, or cis to Ca²⁺) alkali metal ions and is inhibited by external Na⁺ in much the same way as is Ca²⁺ entry mode exchange (35, 107, 108). In this case, however, internal alkali metal ions (e.g., K⁺ or Li⁺) are also required for activation of the exchange, and high [Na⁺]_i is also markedly inhibitory (88). Second, the Na⁺/Ca²⁺ exchange and Ca²⁺/Ca²⁺ exchange do not appear to be independent because they cannot both operate simultaneously at maximal rates. Inhibition of Ca²⁺/Ca²⁺ exchange by external Na⁺ is associated with a shift from Ca²⁺/Ca²⁺ exchange to Ca²⁺ exit mode exchange (88). Conversely, inhibition by internal Na⁺ (107, 138) is likely due to a shift in the operation from Ca²⁺/Ca²⁺ exchange to Ca²⁺ entry mode exchange.

The Ca²⁺/Ca²⁺ exchange mode exhibits a coupling ratio of 1 Ca²⁺:1 Ca²⁺ (107) yet is (surprisingly) voltage sensitive (249) but electroneutral (562). There is no net transfer of charge during Ca²⁺/Ca²⁺ exchange, in contrast to the situation during Na⁺/Ca²⁺ exchange (see below). Whether or not the activating alkali metal ions are also transported during Ca²⁺/Ca²⁺ exchange is not known. However, the fact that cis-activating alkali metal ions (other than Na⁺) do not appear to be transported during Na⁺/Ca²⁺ exchange (57, 996) (and see below) suggests that they may not be transported during Ca²⁺/Ca²⁺ exchange.

Adenosine 5'-triphosphate also modulates the kinetics of Ca²⁺/Ca²⁺ exchange (686, 249). The Ca²⁺/Ca²⁺ mode of exchange can proceed at a very high rate in ATP-depleted cells (686). Adenosine 5'-triphosphate appears to inhibit the Ca²⁺/Ca²⁺ exchange mode, probably by increasing the affinity of the exchanger for Na⁺ (88).

In sum, all of these studies are consistent with the view that the Na⁺/Ca²⁺ exchanger can also mediate Ca²⁺/Ca²⁺ exchange.

Evidence for coupled Na⁺ influx and Na⁺ efflux mediated by the Na⁺/Ca²⁺ exchanger was first obtained in cardiac sarcolemmal preparations by Reeves and Sutko (764, 759). They described a Na⁺ efflux that was dependent on external Na⁺; activation was sigmoid, with a K_0.5 of 15 mM. DiPolo and Beaugé (246) subsequently identified, in squid axons, a ouabain-insensitive component of Na_o-dependent Na⁺ efflux that corresponds to a Na⁺/Na⁺ exchange (see Fig. 5) mediated by the Na⁺/Ca²⁺ exchanger. This view is based on the evidence that the Na_o-dependent Na⁺ efflux, like the Ca²⁺ entry mode of exchange, is 1) dependent on internal Ca²⁺ for activation, as is the Ca²⁺ entry mode of exchange; 2) activated by internal alkalinization; 3) inhibited by internal Mg²⁺; 4) activated by internal ATP and by adenosine 5'-O-(3-thiotriphosphate) which is a substrate for kinases but not ATPases (370, 793); and 5) inhibited by external Ca²⁺ under conditions that promote Ca²⁺ entry mode exchange (i.e., external Ca²⁺ shifts the exchanger from Na⁺/Na⁺ exchange mode to Ca²⁺ entry mode). A Ca_i-independent component of Na_o-dependent Na⁺ efflux was also identified; this flux had different properties and did not appear to be mediated by the Na⁺/Ca²⁺ exchanger (246).

A few attempts have been made to study half-cycles or single complete cycles of the Na⁺/Ca²⁺ exchanger to identify and characterize intermediate steps in the exchange cycle (407, 414, 693, 694). Nevertheless, there is no evidence that the exchanger can mediate significant net efflux of Ca²⁺ (or Na⁺) in the absence of external Na⁺ or Ca²⁺, or net influx of Ca²⁺ (or Na⁺) in the absence of internal Na⁺ or Ca²⁺. Thus it appears that the coupled countertransport is an obligatory feature of the mechanism: the exchanger does not mediate "uncoupled" transport of either Na⁺ or Ca²⁺. In other words, unloaded forms of the exchanger molecules apparently do not cycle or cycle very slowly.

B.  Energetics and Kinetics of Na⁺/Ca²⁺ Exchange

1.  Thermodynamic considerations

In the resting cells of higher animals, [Na⁺]_i and [Ca²⁺]_i are on the order of 10 mM and 0.1 µM (for present purposes, we refer to concentrations rather than activities; but see Ref. 85). The extracellular Na⁺ and free Ca²⁺ concentrations ([Na⁺]_o and [Ca²⁺]_o, respectively) are usually on the order of 450 mM and 3-4 mM in marine invertebrates (85) and 120-145 mM and 1-2 mM in most vertebrates and other invertebrates, respectively. Thus, with V_M values on the order of 80 to 60 mV, there is a large, inwardly directed electrochemical gradient for Na⁺ (_Na) and a very large, inwardly directed electrochemical gradient for Ca²⁺ (_Ca). Both ions tend to move into the cell passively and must be extruded by energy-dependent mechanisms. The two electrochemical gradients are defined, respectively, as

(1)

(1A)

and

(1`)

(1A`)

where E_Na and E_Ca are, respectively, the equilibrium potentials for Na⁺ and Ca²⁺, and R, T, and F are the gas constant, absolute temperature and Faraday's number, respectively.

For a transport system in which the flux of one Ca²⁺ is coupled to the counterflow of n Na⁺, the equilibrium relationship between the two electrochemical gradients is

(2)

If the fluxes of one Ca²⁺ and one K⁺ are coupled to the counterflow of n Na⁺, as in vertebrate photoreceptors, the equilibrium relationship is given by

(2A)

where _K is the electrochemical gradient for K⁺, defined as

From the definitions of the electrochemical gradients, Equations 2 and 2A can be expanded to, respectively

(3)

and

(3A)

The coupling ratio (n) is obviously a key parameter in Equation 3. We shall adopt the terminology of Läuger (559) and shall refer to the ratio of the number of transported Na⁺ to the number of transported Ca²⁺ as the "coupling ratio" (n_T). This may be different from the ratio of the number of binding sites for transported Na⁺ and transported Ca²⁺ on the exchanger which, following Läuger (559), we shall refer to as the "stoichiometry" (n_B). Numerous attempts have been made to determine the value of n_T or of the stoichiometry, including several direct measurements of the coupled fluxes. The critical problem in direct measurements of the coupling ratio is to account for all of the exchanger-mediated fluxes of Na⁺ and Ca²⁺ and to exclude all other, parallel movements of these two ions (i.e., those not mediated by the exchanger). Without a specific inhibitor of the Na⁺/Ca²⁺ exchanger, it is difficult to ascertain that all fluxes through other pathways are excluded.

The Hill coefficient corresponding to the cooperativity between external Na⁺ in activating Ca²⁺ efflux was used initially to estimate the coupling between Na⁺ and Ca²⁺ (35, 85, 800). The first direct measurement of the coupling ratio (i.e., measurement of the coupled fluxes of Na⁺ and Ca²⁺) was made in internally dialyzed, ATP-depleted squid axons. The ratio of the Ca_i-dependent Na⁺ influx to the Na_o-dependent Ca²⁺ efflux (i.e., "Ca²⁺ influx mode exchange) was 3.1:1 (107). In internally perfused, ATP-fueled barnacle muscle cells, the ratio of the Ca_o-dependent (Ca_i-activated) Na⁺ efflux to the Na_i-dependent (Ca_i-activated) Ca²⁺ influx, over a wide range of [Na⁺]_i, was 2.8-3.2:1, with an average of 3.1:1, as shown in Figure 6 (754, 757). In these barnacle muscle experiments, the exchanger-mediated fluxes were defined not only by the counter-ion dependence, but also, independently, by the activation by nontransported internal Ca²⁺ (Figs. 5 and 6, and see sect. IIIE); thus ion movements mediated by other transport mechanisms could be excluded.

View larger version (27K): [in this window] [in a new window]   Fig. 6. Effects of internal Na+ (Nai) on Cao-dependent Na+ efflux (; right ordinate scale) and Nai-dependent Ca2+ influx (; left ordinate scale) in internally perfused barnacle muscle cells. External solution in all experiments was Na+-free medium (Na+ replaced by isosmotic Tris) containing 0.1 mM ouabain. Intracellular Ca2+ concentration ([Ca2+]i) was either 0.01 µM (open symbols) or 1.0 µM (solid symbols). Note that ordinate scale for Ca2+ influx is expanded 3-fold, relative to scale for Na+ efflux. Solid line fits Hill equation with a Hill coefficient of 3.0, a dissociation constant for Nai of 30 mM, and maximal fluxes of 20.5 pmol·cm2·s1 for Nai-dependent Ca2+ influx and 61.5 pmol·cm2·s1 for Cao-dependent Na+ efflux. Broken line is best fit to data, with a Hill coefficient of 3.7, a dissociation constant for Nai of 30.0 mM, and a calculated JNa/Ca(Max) of 62.4 pmol·cm2·s1. These data indicate that coupling ratio of Ca2+ influx mode of Na+/Ca2+ exchange is 3 Na+:1 Ca2+. [From Rasgado-Flores et al. (757).]

Measurements of coupled Na⁺ and Ca²⁺ fluxes, from "initial flux rates," in cardiac sarcolemmal vesicles also yielded a coupling ratio of 3 Na⁺:1 Ca²⁺ (101), whereas n was reported to have a value of 4 in dog heart sarcolemmal vesicles (568) and in rat brain plasmalemmal vesicles (47, 48). In cultured rat heart cells, the ratio of the Ca_o-dependent Na⁺ efflux to the Na_i-dependent Ca²⁺ uptake was 3.1:1 (965, 966). Measurements of net Ca_o-dependent Na⁺ efflux and Ca²⁺ uptake in cardiotonic steroid-treated rabbit ventricle also yielded a Na⁺-to-Ca²⁺ flux ratio of 3:1 (131), although this was not based on initial rate data.

These direct flux measurements only address the coupling ratio between Na⁺ and Ca²⁺; they ignore possible coupled (Na⁺/Ca²⁺ exchanger-mediated) movements of other ions such as H⁺, K⁺, or Cl. However, if more than two Na⁺ are exchanged for one Ca²⁺, and the net charge is not counterbalanced by the coupled movements of other ions, then the exchange should be voltage sensitive and should involve net charge movement that ought to be detectable as an exchanger-generated current (i.e., the flux should be rheogenic).

Indirect methods for determining the coupling ratio of the Na⁺/Ca²⁺ exchanger are generally model dependent (290). Several elegant experiments were designed to determine the exchanger coupling ratio on the assumption that the only ions transported by the exchanger are Na⁺ and Ca²⁺ and that the Ca²⁺ is not accompanied by a proton, for example, to compensate for the net charge carried by Na⁺ if more than two Na⁺ are exchanged for one Ca²⁺. Reeves and Hale (762) used a null-point method (based on Eq. 2) to establish the Na⁺ and Ca²⁺ concentration gradient and V_M under conditions in which there was no net exchanger-mediated flux of Ca²⁺ into bovine cardiac sarcolemmal vesicles; their results yielded a coupling ratio of 3:1 (Fig. 7).

View larger version (30K): [in this window] [in a new window]   Fig. 7. Intravesicular Ca2+ concentrations and Na+/Ca2+ coupling ratio (n) determinations in cardiac plasmalemmal vesicles. A: [Ca2+]i graphed as a function of incubation time for membrane potential (VM) = Na+ reversal potential (ENa) = 0 mV () and for VM = 30.71 mV ( and ) with ENa = 0, 4.0, 10.6, 15.8, 19.7, and 21.1 mV. BD: data from A obtained at 1 s (B), 3 s (C), and 5 s (D) are shown as plots of Ca2+ versus ENa. For conditions where Ca2+ reversal potential (ECa) = 0 (i.e., at Ca2+ = 0), coupling ratio (n) can be calculated from the relationship n = 2/(1 ENa/VM), because ENa then exactly compensates for VM so there is no net loss or gain of Ca2+ via Na+/Ca2+ exchanger. This is based on components of the thermodynamic driving force for exchanger (see Eqs. 2 and 6'): Na/Ca = n × Na  Ca = (n  2) VM  nENa + 2ECa. Calculated values for n (based on intersections of regression lines with points of 0 net Ca2+ flux) were 3.10, 3.02, and 2.99. [From Reeves and Hale (762).]

Kimura et al. (514) determined the reversal potential (E_Na/Ca; see Eqs. 5 and 6) for the exchanger-mediated inward and outward currents in single, internally perfused guinea pig cardiac myocytes. They found that E_Na/Ca was very close to the value expected for a coupling ratio of 3 and very different from that predicted for n = 4 (Fig. 8). This result was confirmed and extended by Ehara et al. (285).

View larger version (16K): [in this window] [in a new window]   Fig. 8. I-V curves of Cao-induced INa/Ca in guinea pig cardiac muscle. A: currents induced by [Ca2+]o = 1 and 4 mM. Ni2+ (5 mM) completely blocked INa/Ca; thus curve obtained with 2 mM Ca2+ + 5 mM Ni2+ shows only residual current. B: I-V curves of Ni2+-sensitive (INa/Ca) currents. Reversal potentials (ENa/Ca) for these currents, about 13 mV for [Ca2+]o = 1 mM, and 53 mV for [Ca2+]o = 4 mM, are very close to those expected for an exchanger with a coupling ratio of 3 Na+:1 Ca2+, and very different from values expected for a 4:1 coupling ratio. [From Kimura et al. (515).]

Bridge et al. (132) compared the intergal of the inward, nifedipine-sensitive Ca²⁺ current evoked by depolarization in cardiac myocytes (i.e., net entering Ca²⁺, which should equal the amount of Ca²⁺ that must be extruded during recovery) with the integral of the inward Na⁺/Ca²⁺ exchange current after repolarization (Fig. 9). The integral of Ca²⁺ current (I_Ca) was twice as large as the integral of I_Na/Ca. This, too, is consistent with a 3:1 coupling ratio.

View larger version (14K): [in this window] [in a new window]   Fig. 9. A: relationship between integral of inward Ca2+ current (ICa; abcissa) and integral of inward Na+/Ca2+ exchange current (INa/Ca; ordinate) in guinea pig cardiac myocytes. Data fit regression line that corresponds to an exchanger coupling ratio of n = 3, but not n = 4 or n = 2. B: integral of ICa was obtained by integrating 10 µM nifedipine-sensitive current which was assumed to reflect Ca2+ entry. C: INa/Ca was integrated for 2 s beneath dotted baseline. This included all of transient exchange and was assumed to reflect Ca2+ extrusion. [From Bridge et al. (132).]

Yasui and Kimura (996) (and see Ref. 283) tested the possibility that K⁺ might be transported by the cardiac exchanger, as in the vertebrate photoreceptor (see below). But, again, E_Na/Ca fit a coupling ratio of 3 Na⁺:1 Ca²⁺, irrespective of the external K⁺ concentration.

The exchanger in squid axons also has a reversal potential close to that predicted for a 3 Na⁺:1 Ca²⁺ coupling ratio (249). Furthermore, neither the squid axon exchanger (204, 249) nor the barnacle muscle exchanger (755) requires external K⁺.

Taken together, these numerous measurements of the coupling ratio, using several different methods, all lead to the same conclusion: the cardiac/neuronal type Na⁺/Ca²⁺ exchanger has a coupling ratio of 3 Na⁺:1 Ca²⁺ when operating in either the Ca²⁺ influx or Ca²⁺ efflux mode.

Two groups (420, 546, 997) measured the net charge carried by the Na⁺/Ca²⁺ exchanger in amphibian rod photoreceptor outer segments (ROS) during net extrusion of Ca²⁺ when V_M and the internal and external ion concentrations were varied. In both studies, the charge movement associated with the Na_o-dependent efflux of Ca²⁺ suggested that one of the Na⁺ entered as an uncompensated positive charge. They concluded that Na⁺/Ca²⁺ exchange in ROS was consistent with a fixed coupling ratio of 3 Na⁺:1 Ca²⁺. Schnetkamp et al. (830) (see also Refs. 320, 575, 687, 831), however, observed that Na⁺/Ca²⁺ exchange in ROS was halted in the absence of K⁺, and they concluded that the exchanger in ROS also transports K⁺. Cervetto et al. (165) (see also Ref. 548) then showed that E_Na/Ca in the amphibian photoreceptor ROS is consistent with a coupling ratio of 4 Na⁺ to 1 Ca²⁺ and 1 K⁺ (Fig. 10). With hindsight, this difference in coupling ratio between the exchanger in ROS and that present in many other tissues does not seem surprising. The [Na⁺]_i in vertebrate photoreceptors is much higher than in many other cells, being on the order of 40 mM. This high [Na⁺]_i is due to the "dark current" (inward Na⁺ current observed in the absence of light). Thus there is insufficient energy in ROS _Na for an exchanger with a coupling ratio of 3 Na⁺:1 Ca²⁺ to be able to extrude Ca²⁺ against the large Ca²⁺ gradient. In contrast, invertebrate photoreceptors employ a different type of phototransduction mechanism, and they do not have a dark current or a relatively high [Na⁺]_i. The exchanger in invertebrate photoreceptors therefore does not require K⁺ and appears to be more like the cardiac/neuronal type exchanger, with a coupling ratio of 3 Na⁺:1 Ca²⁺ (22, 225, 657, 659) (and see sect. VII).

View larger version (23K): [in this window] [in a new window]   Fig. 10. Na+/Ca2+ exchanger currents in salamander rod outer segments. A: currents measured during exposure to a solution containing 0.5 mM K+, 1 mM Ca2+, and indicated Na+ concentrations; after 20 s, solution was switched to one containing 110 mM Na+ and 5 mM K+. B: relation between charge flow on return to second solution and [Na+]o during first 20-s period. Intersection of the horizontal broken line and the continuous diagonal line extrapolated from the data indicates that there is no (net) charge transfer at [Na+]o = 62 mM. Arrows show predicted crossing points for exchange stoichiometries of n Na+:1 K+, where n = 3, 4, or 5. C: data from a similar experiment in which [Ca2+]o and [Na+]o were changed simultaneously. In this case, rod outer segment (ROS) was maintained in a solution containing 110 mM Na+, 5 mM Ca2+, and 5 mM K+; [Ca2+]o was reduced to 1 mM, and [Na+]o was changed to concentration shown on abscissa for 100 s. Ordinate shows charge transferred upon return to original solution. Continuous line, which was drawn by eye, crosses dashed line (no net charge transfer) at [Na+]o = 72 mM. Arrows show expected crossing points for exchange stoichiometries of n Na+:1 Ca2+, where n = 3, 4, or 5. D: effect of a simultaneous change in VM and [K+]o on charge transfer by ROS. ROS was maintained in a solution containing 110 mM Na+, 1 mM Ca2+, and 5 mM K+, and [K+]o was reduced to 2 mM for 40 s; depolarizing pulses from a holding potential of 14 mV coincided with these solution changes. Graph shows charge transfer (ordinate) as a function of pulse amplitude (abscissa). Arrow indicates change in VM required to compensate for a 2.5-fold change in [K+]o if one charge exchanges for 1 K+. The only coupling ratio consistent with all of these data is 4 Na+:(1 Ca2+ + 1 K+). [From Cervetto et al. (165).]

Recently, a K⁺-dependent Na⁺/Ca²⁺ exchanger was identified in human platelets (517). Whether this exchanger mediates a 4 Na⁺:(1 Ca²⁺ + 1 K⁺) exchange (i.e., 4 Na⁺ are exchanged for 1 Ca²⁺ + 1 Na⁺), and whether it has sequence homology to the vertebrate photoreceptor exchanger is not yet known. Also, transcripts of a clone that is homologus to the bovine ROS exchanger are expressed in rat brain; the protein encoded by this cDNA is 45% identical to the bovine ROS exchanger (940). The expressed protein mediates K⁺-dependent Na⁺/Ca²⁺ exchange.

As discussed in section IVI, there is little amino acid sequence homology between the vertebrate ROS exchanger and the cardiac/neuronal type Na⁺/Ca²⁺ exchanger. Thus the ROS Na⁺/Ca²⁺ exchanger probably evolved de novo, and it appears that there are two different molecules with very similar functions. This implies that there was evolutionary (and physiological) pressure to develop a transport system of this type (i.e., an Na⁺/Ca²⁺ exchanger, in contrast to an ATP-driven Ca²⁺ pump) to fit the needs of the vertebrate photoreceptor. Detailed structural comparison of the cardiac-type and ROS-type Na⁺/Ca²⁺ exchangers should provide novel information about the nature of the Na⁺ and Ca²⁺ binding sites on these exchangers.