Department of Physiology, University of Texas Southwestern Medical Center at Dallas, Dallas, Texas 75390-9040
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ABSTRACT |
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excitation-contraction cycle
Recently, the Reeves group (2) has addressed the autoregulatory properties of the exchanger in a series of experiments with similarly insightful qualities. Their experiments use Chinese hamster ovary (CHO) cell lines in which NCX1 and important mutants of NCX1 have been expressed. At issue is the function of gating reactions of the exchanger that modulate its activity in dependence on cytoplasmic Ca2+. Just as in squid axon (6), we have known for many years that in cardiac myocytes the reverse mode operation of Na+/Ca2+ exchange, i.e., the movement of Ca2+ into cells in exchange for cytoplasmic Na+, requires the presence of cytoplasmic Ca2+. In giant excised patches, activation of reverse transport by cytoplasmic Ca2+ usually takes several seconds (11), but the reactions are complex because the binding of Na+ to transport sites in the reverse mode sets an inactivation process in motion that opposes the activation by Ca2+ (11, 12). As pointed out by Reeves and colleagues, the conditions under which the gating reactions have been studied most carefully (e.g., with saturating cytoplasmic Na+) are not really relevant to cardiac physiology. Thus the role of these reactions for the normal functioning of NCX1 has remained rather enigmatic. A careful characterization of NCX1-mediated Ca2+ fluxes and their time dependence, over just the physiological Ca2+ concentration range, has been missing all these years. It is therefore a most welcome and important contribution, and it is impressive that the Reeves group has accomplished this by manipulating innate mechanisms of Ca2+ homeostasis in the CHO lines employed, namely, the Ca2+ release, influx, and uptake mechanisms.
In brief, the exchanger is activating over several seconds when free Ca2+ rises just above its resting level, and the exchanger inactivates over 515 s when Ca2+ decreases back toward its resting level. Thus the exchanger shuts off after it has done its job of extruding Ca2+, and as a result, NCX1 operation does not interfere with mechanisms that may subsequently release Ca2+ or otherwise tend to increase cytoplasmic Ca2+ within 1 or 2 s. The principle is just the same as in voltage-gated K+ channels that activate slowly upon membrane depolarization, carry out the repolarization process, and then turn off again so as not to dampen excessively subsequent depolarizing influences.
In rough approximation, the experimental results of the Reeves group confirm expectations about exchanger gating reactions from analysis of the reactions under nonphysiological conditions with higher cytoplasmic Ca2+ concentrations (11). Figure 1 shows the predictions, as a fraction of maximal exchange activity, using the published equations and parameters (11). Figure 1A shows the steady-state Ca2+ dependence of the forward exchange mode in the absence of cytoplasmic Na+, as predicted for an NCX1 system that is regulated and for an NCX1 system that is not regulated (i.e., unregulated) by the Ca2+-dependent gating reactions. No clear deviations from Michaelis-Menten behavior were detected in the giant patch studies in this condition, probably because deviations are negligible when exchanger function is viewed from the perspective of its total Ca2+ extrusion capacity. As shown in Fig. 1B, however, major differences are indeed predicted for the steady-state activity of the regulated and unregulated exchange systems in the low free Ca2+ range of 0.10.3 µM. The regulated system activates steeply (i.e., as a power function) in dependence on cytoplasmic free Ca2+, and during steady-state operation, the regulatory reactions inhibit exchange activity severalfold in relation to unregulated exchangers.
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The kinetics of the Ca2+-dependent gating reactions are important because they may determine how the exchanger modulates Ca2+ in myocytes on a beat-to-beat basis, over several beats, and/or over longer periods. If the kinetics are fast enough, the exchanger should activate with each ECC and inactivate between beats. The inactivation would possibly prevent Ca2+ from declining too far, and the time-dependent reactivation during the Ca2+ transient might prevent the exchanger from extruding Ca2+ simultaneously with Ca2+ entry by Ca2+ channels. From results of Matsuoka and colleagues (8), we know that the exchangers gating reactions can indeed be quite fast in membrane patches taken from the surface of freshly isolated myocytes, rather than from myocytes that have been stored for hours and whose cytoskeleton is with good certainty disrupted. Furthermore, from the group of Donald Bers (27), we know that some allosteric regulation of the exchanger can in fact be defined in intact myocytes undergoing regular ECC cycles. And finally, more recently, the group of Kenneth Philipson (19) has developed a fluorescent assay to monitor NCX1 gating. Specifically, they have expressed in cultured rat myocytes a fusion protein of the Ca2+-binding regulatory domain of NCX1 with an appropriate fluorescent protein pair to generate Ca2+-dependent fluorescence resonance energy transfer (FRET). The fusion protein responds fast enough to track beat-to-beat Ca2+ changes, and such changes can indeed be monitored in beating rat myocytes. Thus it is proved that NCX1 can in principle inactivate during the diastolic period and turn on with a delay during the immediate events of ECC.
As stimulating as these developments are for our thinking about exchanger dynamics in cardiac myocytes, still other recent results from the Philipson group bring to focus a most sobering feature of exchanger function for NCX1 "affectionnados." The conditional knockout of NCX1 in murine myocytes (10), like the overexpression of NCX1 in murine myocytes (28), has rather modest effects on cardiac ECC. At first, this result seems to contradict an important role for NCX1, but it does not. When our simplest ideas about cardiac ECC are simulated, as Denis Noble and I described 15 years ago (13), changes of Na+/Ca2+ exchange activity have very little impact on the magnitude of the cytoplasmic Ca2+ transient during continuous beating, although small changes of the Na+ gradient have very large effects on ECC by changing the exchangers equilibrium. This pattern is most striking for simulations in which action potentials are short and triangular, and when sarcoplasmic reticulum uptake and release dominate ECC, rather than Ca2+ influx and extrusion, and that is really the short description of ECC in murine myocytes.
The reason for this behavior goes right back to the original Reeves and Hale finding (22). The exchanger will always tend to bring cytoplasmic free Ca2+ to its thermodynamic equilibrium, which depends primarily on membrane potential and the Na+ gradient. In doing so, changes of exchanger activity change the precise timing and the momentary magnitudes of the exchange current. However, especially in those myocytes with strong Ca2+ release and uptake function by the sarcoplasmic reticulum, the influence of NCX1 activity per se on the time course and magnitude of the Ca2+ transient is almost negligible. The exchanger tends to bring cytoplasmic Ca2+ to its equilibrium throughout the ECC cycle, and in this respect its role should be thought of as a Ca2+ buffering function, not as competition with the sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA) Ca2+ pump. In this way, the exchanger tends to damp out most influences that do not affect the exchangers equilibrium. Changes of the Na+ pump activity can have a strong influence, whereas changes of Ca2+ channel activity have a relatively weak influence. As described subsequently, the ECC system still responds strongly to changes of the sarcoplasmic reticulum Ca2+ uptake function, and this principle reiterates the experience of the field with transgenic and knockout models relevant to cardiac ECC. An increase of SERCA Ca2+ pump activity causes a large increase of Ca2+ release in ECC (7), whereas changes of NCX activity have very minor effects. From this viewpoint, then, the exchangers gating reactions can serve only to fine-tune its function, whereas factors that determine the Na+ gradient set its overall role in cardiac ECC.
Figure 2 illustrates the simplest possible simulations that reconstruct these ECC principles and are relevant to murine myocytes. Briefly, we have developed over the last 15 years numerous simulations of cardiac ECC, including many different formulations of Ca2+ release, cardiac ion channels, SERCA function, background Ca2+ fluxes, and NCX1 function. The principles outlined apply in our experience to all models with strong SERCA function and relatively weak Ca2+ channel contribution to ECC. For results shown in Fig. 2, the ECC is assumed to start instantly with three events: 1) membrane potential moves suddenly to +40 mV, 2) a constant fraction of Ca2+ in the sarcoplasmic reticulum is released to the cytoplasm, and 3) a constant amount of Ca2+ enters the cytoplasm as a result of Ca2+ channel activity (here, 10 µmol per liter of cell water). Cytoplasmic Ca2+ is buffered at a constant ratio of 50:1, Ca2+ is pumped back into the sarcoplasmic reticulum at a constant rate proportional to free Ca2+, the K+ conductance of the membrane is constant [i.e., K+ current is given by GK *(Em EK)], and NCX function is represented by the simple biexponential equation suggested by DiFrancesco and Noble (5). In short, all the complexities of NCX1 function are ignored, no further conductances are simulated, ion concentrations besides Ca2+ are constant, and the cytoplasmic Na+ concentration is 8 mM.
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Shown in Fig. 2C are the action potentials, exchange currents, K+ currents, total cell Ca2+, sarcoplasmic reticulum Ca2+ load, and the integral of the transmembrane Ca2+ movements [which would correspond to the extracellular Ca2+ transient in a multicellular cardiac tissue (13)]. As noted at the beginning of this article, the overall function of NCX1 in murine myocytes may usually be trimodal. Here, an initial small phase of Ca2+ influx at the action potential upstroke is absent because the Ca2+ release function is simplified to be instantaneous. Thereafter, the exchanger extrudes Ca2+ when cytoplasmic Ca2+ is high, but it shifts to import Ca2+ slowly in early diastole. This import function requires that free Ca2+ can be reduced transiently by the SERCA pump to a value that is less than the equilibrium value of NCX1, and the obvious possibility is that this occurs in domains where internal and surface membranes are in close contact.
The peak inward exchange current in the control situation is only 30 pA, which is very small compared with the peak K+ current of 250 pA that brings about repolarization. Thus, in this ECC setting, the exchangers influence on the action potential is negligible, but the NCX1 activity is still enough to overshoot its Ca2+ extrusion function. More Ca2+ is extruded than entered the myocyte at the upstroke. As the sarcoplasmic reticulum proceeds to take up Ca2+ between beats, the exchanger maintains a supply of cellular Ca2+ (see the extracellular Ca2+ simulation). When the exchanger activity is reduced by a factor of 10, the overshoot of exchanger function is much reduced, but this change has almost no influence. When SERCA activity is increased, the overshoot becomes enhanced as the sarcoplasmic reticulum Ca2+ loading increases. These basic results are not fundamentally changed by adding Ca2+ leaks in either the surface or sarcoplasmic reticulum membranes. The take-home message is that NCX1 and SERCA need not be operating as competitors but can operate in parallel to load the sarcoplasmic reticulum between beats in the murine myocyte.
When included in these simple simulations, the exchanger gating reactions controlled by cytoplasmic Ca2+ frankly have rather little influence on ECC, and experimental results from the Bers group (27) with murine myocytes are consistent with this conclusion. The fact that murine myocytes can display nearly normal ECC in the absence of NCX1 (10) strongly supports the idea that NCX1 is functioning effectively as a Ca2+ buffer with little influence on the steady-state Ca2+ transients. Are the exchanger gating reactions really irrelevant in mice? What might be the role of the second exchanger gating reaction, the strong Na+-dependent inactivation reaction (12)? The answers to these questions are not obvious from any experimental or theoretical results outlined up to now.
Another perspective on NCX1 function arises from recent findings that the exchanger stoichiometry is not entirely fixed. Although the reversal potential for Ca2+ flux lies close to that predicted for a 3:1 exchanger, just as measured originally by Reeves and Hale (22), the reversal potential for current is shifted toward that expected for a 4:1 exchanger (9, 15, 17). In some measurements, the current reversal is just a little bit closer (14), in some roughly halfway closer (9, 15), and in some nearly at the 4:1 potential (17). These deviations of the flux ratios from 3:1 appear to come about because the exchanger occasionally moves an extra Na+ across the membrane with Ca2+ (15). That the exchanger can bind Na+ and Ca2+ together is again a fundamental property of the exchanger associated with John Reeves, this time together with John Sutko (24). The consequence for cardiac physiology is that the exchanger can contribute a significant part of background Na+ influx and background inward current that determines resting free Na+ in myocytes and that supports some forms of cardiac pacemaking, respectively. Possibly, this Na+ leak function is relevant to an understanding of the role of the gating reactions in cardiac physiology.
Figure 3 outlines the significance of the exchanger Na+ leak function for cardiac pacemaking by the sinus node atrial cells. This simulation contains just the minimum mechanisms needed to allow pacemaking and to maintain the homeostasis of Ca2+, Na+, and K+. Sarcoplasmic reticulum function is left out. Also, the hyperpolarization-activated cation channels are left out. These channels are expected to prevent excessive hyperpolarization, thereby keeping the pacemaker system robust, and to mediate faster pacemaking with a rise of cyclic AMP (26), but their presence is not necessary to generate basal pacemaking. The simulation includes a simple L-type Ca2+ channel that, for simplicity, has instantaneous activation. It includes a simple HERG-type K+ conductance that, for simplicity, has an instantaneous inactivation function. It includes a simple model of Na+ pump function that predicts accurately Na+ pump current-voltage relations and that assumes a free energy of ATP hydrolysis of 16 kcal/mol. And finally, it includes the NCX1 system, formulated exactly as published (15) and as needed to describe accurately NCX1 ion flux coupling and current-voltage relations.
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The current generated by a perfect 3Na+:1Ca2+ exchange process naturally decays as Ca2+ is extruded, and it therefore would presumably become irrelevant late in the diastolic period. What are the depolarizing influences during late diastole of the sinus node cells? This is an old and still unresolved question. It could be the hyperpolarization-activated cation current, or it could be a novel nifedipine-sensitive voltage-activated Na+ current (3). However, it may also be the contribution of NCX1 that may carry inward current late into the diastolic period by two means. A new proposal is that Ca2+ is released by sarcoplasmic reticulum spontaneously in sinus node cells with a certain delay after the previous action potential (18). The rise of cytoplasmic Ca2+ causes an increased Ca2+ extrusion by Na+/Ca2+ exchange in the diastole period and with that a "delayed" Na+/Ca2+ exchange current that promotes generation of the next action potential. Quantitatively, the possible contribution of this mechanism, if present, cannot be of large magnitude, because inhibition of sarcoplasmic reticulum Ca2+ recycling by ryanodine has only modest negative chronotropic effects, 19%, in true pacemaker cells (16). The second possibility, then, concerns the exchangers Na+ leak function, which is predicted to continue unabated through the diastolic period (15). This has been illustrated in Fig. 3 by plotting the total NCX1 current and the current generated specifically by 3:1 Na+/Ca2+ exchange. Although the real Na+/Ca2+ exchange ratio under maximized transport conditions is only
5% different from 3:1, the extra Na+ current generated is about the same magnitude when the exchanger is operating close to equilibrium. It is therefore a very significant component of background Na+ influx, and the corresponding inward current is completely appropriate to support diastolic depolarization up to the onset of the next action potential. The Ca2+-dependent gating reaction of the exchanger would naturally control this background current as follows: When the average free Ca2+ is increased in the sinoatrial node, as will be the case with catecholamines, the number of active exchangers will naturally increase, and the diastolic depolarization rate will naturally be increased via both the increased Ca2+ transport current and the Na+ leak function of the exchanger.
What about the rest of the myocardium? A speculative possibility is that the exchanger plays a significant role in control of the myocyte Na+ gradient by mediating background Na+ influx, independent of Ca2+ extrusion. This Na+-promoting influence would be controlled and countered by the exchangers Na+-dependent inactivation reaction, which depends on complete loading of the exchangers transport sites and therefore has a third-power dependence on cytoplasmic Na+ (12). In short, activation of Na+/Ca2+ exchangers by any relevant regulatory influence would tend to increase cytoplasmic Na+, and this Na+-loading function of NCX1 would be opposed by the exchangers Na+-dependent inactivation reaction. One would expect NCX1 overexpression to cause a rise in cytoplasmic Na+. It must be admitted that this was not detected in initial studies (28), but more detailed analysis seems justified. Up to now, no clear explanation for the Na+-dependent inactivation function of NCX1 has been offered that makes cellular sense, that can tested in experiments, and that can be analyzed in simulations of ECC.
In summary, we are now learning a lot about how the Na+/Ca2+ exchanger gating reactions are working in live cells, reactions that have remained very enigmatic up to now. The Reeves group (2) has provided new insights into the function and kinetics of the Ca2+-dependent activation function by using physiological mechanisms to change Ca2+ in a cell line. These insights could not have been accomplished with electrophysiology. The Philipson group (19) has provided clear new information about the kinetics of the actual Ca2+-sensing domain of the exchanger in intact myocytes, with the outcome that the Ca2+-dependent regulation may be fast enough to modulate NCX1 function in the time course of each ECC. The Philipson group (10) has also generated transgenic and knockout models that challenge our understanding of NCX1 function in cardiac ECC. The simplest possible models of cardiac ECC are entirely consistent with the outcome that substantial changes of NCX1 activity have little influence on cardiac ECC. Yet, small changes of the Na+ gradient can have the largest effect of any cellular parameter on cardiac ECC via modulation of the NCX1 function. It seems possible that important control mechanisms for cytoplasmic Na+ exist in heart and are still waiting to be discovered. One possibility is that the exchangers Na+- and Ca2+-dependent gating reactions are fine-tuning the Na+ gradient by controlling background Na+ influx. Clearly, we still have a long way to go to understand many important details of NCX1 function and Na+ homeostasis in heart. The recent studies of NCX1 outlined here provide new and important clues, and they invite future NCX1 research along multiple fruitful paths.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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REFERENCES |
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2. Chernysh O, Condrescue M, and Reeves JP. Calcium-dependent regulation of calcium efflux by the cardiac sodium-calcium exchanger. Am J Physiol Cell Physiol 287: C797C806, 2004.
3. Cho HS, Takano M, and Noma A. The electrophysiological properties of spontaneously beating pacemaker cells isolated from mouse sinoatrial node. J Physiol 550: 169180, 2003.
4. Demir SS, Clark JW, Murphey CR, and Giles WR. A mathematical model of a rabbit sinoatrial node cell. Am J Physiol Cell Physiol 266: C832C852, 1994.
5. DiFrancesco D and Noble D. A model of cardiac electrical activity incorporating ionic pumps and concentration changes. Philos Trans R Soc Lond B Biol Sci 307: 353398, 1985.[ISI][Medline]
6. DiPolo R and Beauge L. Asymmetrical properties of the Na-Ca exchanger in voltage-clamped, internally dialyzed squid axons under symmetrical ionic conditions. J Gen Physiol 95: 819835, 1990.[Abstract]
7. Frank K and Kranias EG. Phospholamban and cardiac contractility. Ann Med 32: 572578, 2000.[ISI][Medline]
8. Fujioka Y, Hiroe K, and Matsuoka S. Regulation kinetics of Na+-Ca2+ exchange current in guinea-pig ventricular myocytes. J Physiol 529: 611623, 2000.
9. Fujioka Y, Komeda M, and Matsuoka S. Stoichiometry of Na+-Ca2+ exchange in inside-out patches excised from guinea-pig ventricular myocytes. J Physiol 523: 339351, 2000.
10. Henderson SA, Goldhaber JI, So JM, Han T, Motter C, Ngo A, Chantawansri C, Ritter MR, Nicoll DA, Frank JS, Jordan MC, Roos KP, Ross KS, and Philipson KD. Functional adult myocardium in the absence of Na+-Ca2+ exchange: cardiac specific knockout of NCX1. Circ Res August 12, 2004; doi:10.1161/01.RES.0000142316.08250.68.
11. Hilgemann DW, Collins A, and Matsuoka S. Steady-state and dynamic properties of cardiac sodium-calcium exchange. Secondary modulation by cytoplasmic calcium and ATP. J Gen Physiol 100: 933961, 1992.[Abstract]
12. Hilgemann DW, Matsuoka S, Nagel GA, and Collins A. Steady-state and dynamic properties of cardiac sodium-calcium exchange. Sodium-dependent inactivation. J Gen Physiol 100: 905932, 1992.[Abstract]
13. Hilgemann DW and Noble D. Excitation-contraction coupling and extracellular calcium transients in rabbit atrium: reconstruction of basic cellular mechanisms. Proc R Soc Lond B Biol Sci 230: 163205, 1987.[ISI][Medline]
14. Hinata M, Yamamura H, Li L, Watanabe Y, Watano T, Imaizumi Y, and Kimura J. Stoichiometry of Na+-Ca2+ exchange is 3:1 in guinea-pig ventricular myocytes. J Physiol 545: 453461, 2002.
15. Kang TM and Hilgemann DW. Multiple transport modes of the cardiac Na+/Ca2+ exchanger. Nature 427: 544548, 2004.[CrossRef][ISI][Medline]
16. Li J, Qu J, and Nathan RD. Ionic basis of ryanodines negative chronotropic effect on pacemaker cells isolated from the sinoatrial node. Am J Physiol Heart Circ Physiol 273: H2481H2489, 1997.
17. Lytton J and Dong H. Rat heart NCX1.1 stoichiometry measured in a transfected cell system. Ann NY Acad Sci 976: 137141, 2002.
18. Maltsev VA, Vinogradova TM, Bogdanov KY, Lakatta EG, and Stern MD. Diastolic calcium release controls the beating rate of rabbit sinoatrial node cells: numerical modeling of the coupling process. Biophys J 86: 25962605, 2004.
19. Ottolia M, Philipson KD, and John S. Conformational changes of the Ca2+ regulatory site of the Na+/Ca2+ exchanger detected by FRET. Biophys J 87: 899906, 2004.
20. Philipson KD, Nicoll DA, Ottolia M, Quednau BD, Reuter H, John S, and Qiu Z. The Na+/Ca2+ exchange molecule: an overview. Ann NY Acad Sci 976: 110, 2002.
21. Quednau BD, Nicoll DA, and Philipson KD. The sodium/calcium exchanger family-SLC8. Pflügers Arch 447: 543548, 2004.[CrossRef][ISI][Medline]
22. Reeves JP and Hale CC. The stoichiometry of the cardiac sodium-calcium exchange system. J Biol Chem 259: 77337739, 1984.
23. Reeves JP and Sutko JL. Sodium-calcium exchange activity generates a current in cardiac membrane vesicles. Science 208: 14611464, 1980.[ISI][Medline]
24. Reeves JP and Sutko JL. Competitive interactions of sodium and calcium with the sodium-calcium exchange system of cardiac sarcolemmal vesicles. J Biol Chem 258: 31783182, 1983.
25. Reuter H and Seitz N. The dependence of calcium efflux from cardiac muscle on temperature and external ion composition. J Physiol 195: 451470, 1968.[ISI][Medline]
26. Stieber J, Hofmann F, and Ludwig A. Pacemaker channels and sinus node arrhythmia. Trends Cardiovasc Med 14: 2328, 2004.[CrossRef][ISI][Medline]
27. Weber CR, Ginsburg KS, Philipson KD, Shannon TR, and Bers DM. Allosteric regulation of Na/Ca exchange current by cytosolic Ca in intact cardiac myocytes. J Gen Physiol 117: 119131, 2001.
28. Yao A, Su Z, Nonaka A, Zubair I, Lu L, Philipson KD, Bridge JH, and Barry WH. Effects of overexpression of the Na+-Ca2+ exchanger on [Ca2+]i transients in murine ventricular myocytes. Circ Res 82: 657665, 1998.