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Physiological Reviews, Vol. 79, No. 3, July 1999, pp. 763-854
Copyright ©1999 by the American Physiological Society
Departments of Physiology and Medicine and the Center for Vascular Biology and Hypertension, University of Maryland School of Medicine, and Department of Molecular Biology and Biophysics, The Medical Biotechnology Center of the Maryland Biotechnology Institute, Baltimore, Maryland
I. INTRODUCTION
II. HISTORICAL BACKGROUND
III. PROPERTIES OF THE SODIUM/CALCIUM EXCHANGER
A. Modes of Operation of the Na+/Ca2+ Exchanger
B. Energetics and Kinetics of Na+/Ca2+ Exchange
C. Controversial Kinetic and Mechanistic Topics
D. Electrogenic (or Rheogenic) Properties and Voltage Sensitivity of the Na+/Ca2+ Exchanger
E. Regulation of the Na2+/Ca2+ Exchanger by Catalytic Modulation
F. Inhibitors of the Na+/Ca2+ Exchanger
IV. MOLECULAR BIOLOGY OF THE SODIUM/CALCIUM EXCHANGER
A. Purification, Cloning, and Reconstitution of the Exchanger Protein
B. Structure/Function of the Na+/Ca2+ Exchanger
C. Alternative Splicing
D. Gene Structure (Multiple Genes)
E. Isoforms
F. Development
G. Modulation
H. The Drosophila Exchanger
I. The Na+/(Ca2+ + K+) Exchanger: NCKX1 and NCKX2
V. PHYSIOLOGICAL ROLES AND PATHOPHYSIOLOGICAL CONSEQUENCES OF SODIUM/CALCIUM EXCHANGER ACTIVITY IN CELLS AND TISSUES: GENERAL PRINCIPLES
A. Localization of the Na+/Ca2+ Exchanger: Possible Clues to Function
B. Ca2+ Extrusion Versus Ca2+ Entry
C. Control of SR/ER Ca2+ Content
VI. PHYSIOLOGICAL ROLES OF THE SODIUM/CALCIUM EXCHANGER IN VARIOUS TISSUES
A. Heart Muscle
B. Smooth Muscle
C. Vertebrate Skeletal Muscle
D. Invertebrate Skeletal Muscle
E. Nervous System
F. Na+/Ca2+ Exchange in Blood Cells: Physiology and Pathophysiology
G. Kidney
H. Intestinal Absorption of Ca2+
I. Eye
J. Secretory Cells
K. Na+/Ca2+ Exchanger in Miscellaneous Other Types of Cells
VII. EPILOGUE
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ABSTRACT |
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Blaustein, Mordecai P. and
W.
Jonathan Lederer.
Sodium/Calcium Exchange: Its Physiological Implications. Physiol. Rev. 79: 763-854, 1999.
The Na+/Ca2+
exchanger, an ion transport protein, is expressed in the plasma
membrane (PM) of virtually all animal cells. It extrudes
Ca2+ in parallel with the PM ATP-driven
Ca2+ pump. As a reversible transporter, it also mediates
Ca2+ entry in parallel with various ion channels. The
energy for net Ca2+ transport by the
Na+/Ca2+ exchanger and its direction depend on
the Na+, Ca2+, and K+ gradients
across the PM, the membrane potential, and the transport stoichiometry.
In most cells, three Na+ are exchanged for one
Ca2+. In vertebrate photoreceptors, some neurons, and
certain other cells, K+ is transported in the same
direction as Ca2+, with a coupling ratio of four
Na+ to one Ca2+ plus one K+. The
exchanger kinetics are affected by nontransported Ca2+,
Na+, protons, ATP, and diverse other modulators. Five genes
that code for the exchangers have been identified in mammals: three in
the Na+/Ca2+ exchanger family (NCX1,
NCX2, and NCX3) and two in the
Na+/Ca2+ plus K+ family
(NCKX1 and NCKX2). Genes homologous to
NCX1 have been identified in frog, squid, lobster, and
Drosophila. In mammals, alternatively spliced variants of
NCX1 have been identified; dominant expression of these
variants is cell type specific, which suggests that the variations are
involved in targeting and/or functional differences. In cardiac
myocytes, and probably other cell types, the exchanger serves a
housekeeping role by maintaining a low intracellular Ca2+
concentration; its possible role in cardiac excitation-contraction coupling is controversial. Cellular increases in Na+
concentration lead to increases in Ca2+ concentration
mediated by the Na+/Ca2+ exchanger; this is
important in the therapeutic action of cardiotonic steroids like
digitalis. Similarly, alterations of Na+ and
Ca2+ apparently modulate basolateral K+
conductance in some epithelia, signaling in some special sense organs
(e.g., photoreceptors and olfactory receptors) and
Ca2+-dependent secretion in neurons and in many secretory
cells. The juxtaposition of PM and sarco(endo)plasmic reticulum
membranes may permit the PM Na+/Ca2+ exchanger
to regulate sarco(endo)plasmic reticulum Ca2+ stores and
influence cellular Ca2+ signaling.
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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 10
7 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 s
1 (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 (VM) 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
VM 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.
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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).
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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).
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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.
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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.
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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.
A. Modes of Operation of the Na+/Ca2+ Exchanger
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 VM). 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+.
1. Ca2+ entry mode exchange
The Ca2+ entry mode of exchange is usually identified as an internal Na+ (Nai)-dependent Ca2+ influx and an external Ca2+ (Cao)-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., K0.5 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.
2. Ca2+ exit mode exchange
This mode of exchange is defined operationally as an external Na+ (Nao)-dependent Ca2+ efflux and an internal Ca2+ (Cai)-dependent ouabain- and tetrodotoxin (TTX)-insensitive Na+ influx. Like the Nai-dependent Ca2+ influx, the Nao-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 K0.5 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.
3. Ca2+/Ca2+ exchange mediated by the Na+/Ca2+ exchanger
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.
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The Ca2+/Ca2+ exchange is usually identified as a trans-Ca2+-dependent Ca2+ flux (i.e., Cao-dependent Ca2+ efflux and Cai-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 Cai-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.
4. Na+/Na+ exchange mediated by the Na+/Ca2+ exchanger
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 K0.5 of 15 mM. DiPolo and Beaugé (246) subsequently identified, in squid axons, a ouabain-insensitive component of Nao-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 Nao-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 Cai-independent component of Nao-dependent Na+ efflux was also identified; this flux had different properties and did not appear to be mediated by the Na+/Ca2+ exchanger (246).
5. "Uncoupled" transport of Na+ or Ca2+ mediated by the Na+/Ca2+ exchanger
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 VM
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) |
|
(1`) |
|
(1A`) |
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) |
|
(2A) |

K is the electrochemical gradient
for K+, defined as
|
|
|
(3) |
|
(3A) |
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2. The coupling ratio of the cardiac/neuronal-type Na+/Ca2+ exchanger
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" (nT). 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" (nB). Numerous attempts have been made to determine the value of nT 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 Cai-dependent Na+ influx to the Nao-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 Cao-dependent (Cai-activated) Na+ efflux to the Nai-dependent (Cai-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.
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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 Cao-dependent Na+ efflux to the Nai-dependent Ca2+ uptake was 3.1:1 (965, 966). Measurements of net Cao-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 VM 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).
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Kimura et al. (514) determined the reversal potential (ENa/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 ENa/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).
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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 (ICa) was twice as large as the integral of INa/Ca. This, too, is consistent with a 3:1 coupling ratio.
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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, ENa/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.
3. Coupling ratios in the vertebrate photoreceptor
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 VM
and the internal and external ion concentrations were varied. In both
studies, the charge movement associated with the Nao-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 ENa/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).
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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.
4. Equilibrium conditions
The sum of these varied types of observations, carried out on numerous different tissues and species under a wide variety