How many sodium ions does it take to turn an exchanger?

Cardiac muscle contraction is mediated by an elevation of cytosolic Ca2+ in the ventricle that comes partly from influx across the plasma membrane and mostly from sarcoplasmic reticulum release. Relaxation requires that Ca2+ levels are lowered through efflux and re-sequestration, in a proportion that allows the maintenance of beat-to-beat homeostasis. The principal means of extruding Ca2+ from heart cells is via the Na+-Ca2+ exchanger (Bers, 2002). That the Na+-Ca2+ exchanger is essential for heart function and for cardiac glycoside action has been long known, but was demonstrated recently and mostly clearly in mice using a gene ablation strategy (Reuter et al. 2002). The role that the exchanger plays in modulating normal heart function or in contributing to heart dysfunction in response to pathological disturbances, on the other hand, has been the subject of much controversy (see several articles published in Lytton et al. 2002). As can be gleaned from its name, the Na+-Ca2+ exchanger operates by sequentially moving Na+ ions in one direction and Ca2+ ions in the other. Indeed, the exchanger can operate in either direction, depending upon the relative magnitude of the electrochemical gradients for Na+ and Ca2+. One of the most fundamental properties of this molecule, then, is the number of ions that bind and are subsequently transported, the so-called stoichiometry of the exchanger. As operation of the Na+-Ca2+ exchanger produces a measurable current that flows in the direction of Na+ transport, it is clear that more than two Na+ must be transported for each Ca2+ ion. But exactly how many? The answer to this question has very important implications for our understanding of the cellular electrophysiology that underlies cardiac function and dysfunction. It is generally accepted now that the Na+-Ca2+ exchanger can participate in Ca2+ entry during the early phase of systole (though the magnitude of its contribution is debated). This being so, the concentration of Na+ sensed on the inside of the membrane would have to be much higher to allow Ca2+ entry for an exchanger operating with a 4:1 stoichiometry than one with a 3:1 stoichiometry. Such a scenario is in fact compatible with recent data and current working hypotheses concerning restricted diffusional spaces in cardiac myocytes (Bers, 2002). Later on in systole, the exchanger clearly switches direction of operation and extrudes Ca2+. The current produced by the exchanger during Ca2+ extrusion is known to contribute significantly to the plateau phase of the action potential. An exchanger operating with a 4:1 stoichiometry would move twice as much charge for each Ca2+ transported compared to one operating with a 3:1 stoichiometry, and thus make a proportionately larger contribution to the action potential plateau. It is important to note that alterations to the length and shape of the ventricular action potential are at the root of many fatal cardiac diseases (Bers, 2002). Originally, Mullins (1977) used known ionic concentrations across the squid axon to hypothesize that four Na+ ions are transported for each Ca2+. Subsequently, many different experimental approaches indicated that the Na+-Ca2+ exchanger stoichiometry was actually three Na+ to one Ca2+ (Blaustein & Lederer, 1999), and it was thought that the debate was settled. Controversy re-emerged, however, 2 years ago when Matsuoka and colleagues (Fujioka et al. 2000) used a careful thermodynamic approach to calculate that the stoichiometry of the Na+-Ca2+ exchanger measured in patches torn off from guinea-pig cardiac myocytes was 4:1, an observation subsequently corroborated using a similar approach in a recombinant system (Dong et al. 2002). What, then, is the reason for these disparate results? In the current issue of The Journal of Physiology, Hinata et al. (2002) use an elegant experimental approach to provide a possible explanation, and in so doing swing the weight of the pendulum back toward 3:1. Using guinea-pig myocytes subjected to whole-cell patch-clamp, these authors first reconfirm previously published observations demonstrating that operation of the Na+-Ca2+ exchanger can itself alter ionic conditions sufficiently to invalidate the thermodynamic approach. Then, in their key experiment, they initially inhibit the exchanger with Ni2+ under conditions in which other channels and pumps are also inhibited, and examine the reversal potential (a sensitive thermodynamic parameter indicative of Na+-Ca2+ exchanger stoichiometry) as exchanger current develops subsequent to Ni2+ washout. They observe that under conditions in which the exchanger is poised to accumulate Ca2+ assuming a 3:1 stoichiometry (but should be close to equilibrium for a 4:1 stoichiometry), the measured reversal potential shifts with time until a new equilibrium is achieved. On the other hand, under conditions where the exchanger is initially poised close to its equilibrium point for a 3:1 stoichiometry (but should drive Ca2+ efflux if the stoichiometry is 4:1) the current that develops upon washout has a stationary reversal potential. These results are clearly consistent with a 3:1 but not a 4:1 stoichiometry. These findings raise the question: could erroneous assumptions regarding sub-membrane ionic conditions have confounded the earlier reports by Fujioka et al. (2000) and Dong et al. (2002), and thus accounted for the apparent measured stoichiometry of 4:1? This is the argument put forth by Hinata et al. (2002). While it is hard to explicitly rule out such a scenario, both of these studies included a series of careful controls which suggested, but did not prove, that control over submembrane ionic conditions was precise and accurate. Could the currents measured by Hinata et al. (2002) have been contaminated with ionic conductances not related to the exchanger? The inhibitors they use to define Na+-Ca2+ exchange are not particularly selective, and so while this is conceivable, again a series of controls argues against such a possibility. Where does this leave us? Certainly a 'counting of hands' approach would lead one to conclude the Na+-Ca2+ exchanger stoichiometry is 3:1. And the current work of Hinata et al. (2002) is beginning to shut the door on those outlying studies that still favour a 4:1 stoichiometry. Is there room for one more kick at the can before the crack is closed? Perhaps it would be possible to use a combination of Ca2+ flux measured by indicator dyes and whole-cell voltage-clamp to put the issue to rest at last.

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