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EDITORIAL FOCUS
Institute of Cardiovascular Sciences, St. Boniface Hospital, Research Centre, University of Manitoba, Winnipeg, Manitoba, Canada
The Necessity for NCX1 Regulation Considering that the cardiac Na+/Ca2+ exchanger (NCX1.1) is the predominant mechanism for transsarcolemmal Ca2+ efflux in cardiac myocytes, it is obvious that this transport system must be regulated actively. Sodium/calcium exchange removes roughly the same quantity of Ca2+ that enters through L-type Ca2+ channels on a beat-to-beat basis. Imbalances in Ca2+ influx and Ca2+ efflux cannot occur in cardiac cells over any extended time period because this would ultimately lead to contractile failure, either by intracellular Ca2+ depletion or overload. This critical requirement for dynamic Ca2+ balance requires that the Na+/Ca2+ exchanger be capable of sensing alterations in Ca2+ influx that routinely occur during normal cardiac function, since cardiac output can change dramatically to meet ever-changing body demands.
Despite the critical role of Na+/Ca2+ exchange in cardiac Ca2+ homeostasis, remarkably little is known about how this ion countertransporter accomplishes the task of balancing Ca2+ efflux and Ca2+ influx. While there is considerable information on the physiological regulation of Ca2+ entry through L-type Ca2+ channels, there is no concrete information on how Na+/Ca2+ exchange is regulated physiologically. Notably, even minor changes in the effectiveness of Na+/Ca2+ exchange function can dramatically alter cardiac contractility, exemplified by digitalis-like compounds that alter the electrochemical set point for sodium/calcium exchange to control cytoplasmic Ca2+ levels. In contrast, fairly massive changes in NCX1 expression levels (e.g., as occurs in transgenic overexpressors or various knockout mice) produce relatively minor or late developing changes in cardiac contractility and Ca2+ handling (8, 14, 20). Presumably, this latter observation reflects the fact that NCX1 is already present in large excess of that required to perform its normal function and is therefore rather insensitive to increases or decreases in expression levels. Still, we do not know how this large population of exchangers is regulated or controlled, if at all.
Current Knowledge of NCX1 Ionic Regulation: All's Well in the World of Biophysics and Structure-Function The cloning, heterologous expression, and electrophysiological characterization of sodium/calcium exchangers have provided considerable information on the structure-function relationships of these transporters. Notably, all Na+/Ca2+ exchangers studied to date are also regulated by their transport substrates, Na+ and Ca2+. These autoregulatory processes have been extensively characterized and are commonly referred to as Na+-dependent (or I1) inactivation and Ca2+-dependent (or I2) inactivation (10, 11). Intracellular pH and metabolic factors can profoundly influence both inactivation mechanisms (3, 4). Na+-dependent inactivation, the subject of the current study by Chernysh et al. (2), is apparent as the time-dependent inactivation of outward Na+/Ca2+ exchange currents that develops in the presence of high cytoplasmic Na+ levels (11). This process is analogous to the voltage-dependent inactivation of many ion channels, whereby activity leads to their subsequent inactivation. Notably, the intracellular Na+-dependence of outward current development and current inactivation are nearly identical, suggesting that both processes originate from a common exchanger conformation (11). This process is capable of dramatically reducing exchange currents anywhere between 50% and 90% of their peak value, although it is only apparent at supraphysiological intracellular Na+ concentrations. Furthermore, physiological concentrations of ATP or phosphatidylinositol 4,5-bisphosphate (PIP2) can completely antagonize the I1 inactivation process (9, 10), obscuring any comprehension of its physiological role. In fact, regulation of NCX1 function by PIP2 has grown increasingly complex because this signaling molecule also appears to play an important role in NCX1 trafficking, surface expression, and internalization in addition to its direct effects on the inactivation process (22).
The second type of ionic regulation is referred to as Ca2+-dependent (or I2) inactivation (10). Ca2+-dependent regulation of NCX1 describes the requirement for low concentrations (submicromolar to micromolar) of cytoplasmic Ca2+ to be present on the intracellular surface of the exchanger in order for currents to develop. In the absence of this "regulatory" Ca2+, the exchanger enters into the I2 inactive state. Giant excised patch-clamp studies have also shown that Na+-dependent inactivation can be completely antagonized by increases in cytoplasmic Ca2+ levels, suggesting two distinct roles for intracellular Ca2+ regulation (10). It is attractive to speculate that Ca2+-dependent (I2) regulation provides the signal for matching Ca2+ influx to Ca2+ efflux, because the concentration dependency for this phenomenon is poised ideally between diastolic and systolic Ca2+ levels observed in cardiac tissue. Thus, reducing intracellular Ca2+ would shut off exchangers, whereas elevations in intracellular Ca2+ would increase the active exchange population. While this proposed role for Ca2+-dependent regulation is simple and attractive, no experimental evidence exists to support this notion.
At present, there is considerable information on the specific protein domains involved in the two ionic regulatory mechanisms. For example, the XIP region of the exchanger plays a critical role in Na+-dependent inactivation, and mutations within this region can augment or eliminate this process (18). Two such mutants, namely, F223E and K229Q, are used in the Chernysh et al. study (2). Two separate Ca2+-binding domains (CBD1 and CBD2) involved in the Ca2+-dependent regulatory process have been identified, and their crystal and solution structures were recently solved (1, 6). Mutations within either CBD can dramatically alter the properties of Ca2+ regulation, although the nature of their interactions and/or functional differences are only beginning to emerge (1, 6). It is tempting to speculate that the two CBDs may subserve the distinct roles of mediating Ca2+-dependent (I2) regulation and alleviation of I1 regulation independently, although current knowledge suggests a more complex interaction between these domains (1). However, even though biophysical and structure-function studies of this nature are progressing rapidly, we are still left with the unanswered, and possibly more important, question of what these regulatory mechanisms do in cells.
So What About Cells? Chernysh et al. (2) focus directly on identifying the occurrence, prevalence, and characteristics of sodium-dependent ionic regulation of the cardiac Na+/Ca2+ exchanger, NCX1.1, and various mutants expressed in Chinese hamster ovary cells. The investigators attempt to reconcile the functional characteristics of various exchangers observed from giant excised patch-clamp experiments with the behavior of these Na+/Ca2+ exchangers operating within an intact cellular system. While much of their data confirm the anticipated results obtained from giant patch studies, numerous important differences or discrepancies were also observed.
Chernysh et al. (2) examined wild-type (WT) NCX1.1 and the two representative XIP mutations, F223E and K229Q (with augmented and ablated Na+-dependent inactivation, respectively), over a large range of experimental conditions. In many respects, both the WT and mutant exchangers behaved as expected, the details of which will not be reiterated here. The most striking difference observed in their study, compared with previous giant patch-clamp studies, was the remarkable resistance of the WT exchanger to enter into the Na+-dependent inactive state. Even at exceedingly high levels of intracellular Na+ (140 mM), little difference was observed between the WT and K229Q (I1 resistant) exchangers. As predicted from earlier patch-clamp studies (4), I1 inactivation became far more prominent when intracellular pH was lowered, particularly for the I1-enhanced F223E mutant. Furthermore, elevations in intracellular Na+ appeared to have little influence on the apparent Kh for allosteric Ca regulation of NCX1 in cells, whereas considerable differences were observed for both the WT and F223E exchangers in patch studies (10, 18). Finally, Na+-dependent inactivation was largely insensitive to depletion of PIP2 levels under many conditions for both the WT and K229Q exchangers.
It is difficult to directly compare electrophysiological data and kinetic parameters obtained by giant patch clamping with that obtained from fluorometric measurements as conducted by Chernysh et al. (2). Difficult, but worthwhile. Despite the vastly different approaches of giant patching and cell-based fluorescence measurements, a substantial portion of the current studies confirmed the regulatory behavior identified by electrophysiological techniques. Now, it becomes critical to understand why concordance was not observed for other expected behaviors and to continue exploring regulatory mechanisms in intact cellular systems or at the whole animal level using knock-in approaches.
Implications and Related Matters Understanding how the cardiac sodium/calcium exchanger is regulated both physiologically and under pathophysiological conditions has numerous important implications. For example, in the majority of studies examining heart failure, including that which occurs in humans, the expression levels of NCX1 are approximately doubled. This occurs coincidently with a decline in the contribution of the sarcoplasmic reticulum to the intracellular Ca2+ transient. On the basis of studies in transgenic mice overexpressing NCX1, one would predict that this change in expression level would be relatively innocuous, although even these animals behave very differently under stress (21). In the setting of heart failure, where intracellular Na+ levels are also increased, this change in NCX1 expression is thought to be a major contributor in supporting cardiac inotropy, even though it also predisposes to arrhythmogenesis (19, 23). Why do we see an increase in NCX1 in heart failure if there are already too many exchangers under normal conditions? Does the elevation in intracellular Na+ play a more important role than exchanger number in supporting inotropy in this disease? Does Na+-dependent regulation of NCX1 play any role whatsoever in this process?
In the setting of ischemia-reperfusion injury, it is clear that the reverse mode of Na+/Ca2+ exchange contributes prominently to the resultant Ca2+ overload based on pharmacological studies and through genetic manipulation of exchanger levels (12, 13, 15). Two pharmacological agents, SEA0400 and KB-R7943, which inhibit the cardiac exchanger, can dramatically reduce cardiac injury occurring in this setting. Interestingly, both of these agents appear to stabilize the Na+-dependent inactive state of NCX1.1, resulting in an apparent transport mode selectivity for these agents (16). The study of Chernysh et al. (2) demonstrates that Na+-dependent inactivation is highly resistant to intracellular Ca2+ levels but highly responsive to changes in pH. Possibly, a more compelling role for Na+-dependent inactivation is beginning to emerge. Limiting reverse-mode Na+/Ca2+ exchange in the setting of Na+ overload is a potentially life-saving mechanism. NCX1 appears resistant to Na+-dependent inactivation under normal conditions. However, the low pH occurring during ischemia would greatly augment the development of this inactive state. Since the novel sodium/calcium exchange inhibitors, such as SEA0400 and KB-R7943, facilitate or stabilize the Na+-dependent inactive state even further, they would augment this protective feature. Ultimately, however, the use of transgenic knock-in animals expressing various NCX1 mutants may become necessary toward understanding this process in sufficient detail to guide pharmaceutical development.
What Now? While the Chernysh et al. study (2) and earlier reports (17) have been able to document the operation of Na+-dependent inactivation in cellular systems, it is becoming increasingly popular to posit that there is no obvious physiological role for this mechanism. Yet nature has targeted these regulatory mechanisms for manipulation. Alternative splicing of NCX1 directly and dramatically alters the relationships between Na+-dependent and Ca2+-dependent regulation (5). Moreover, these splice variants are frequently expressed in a tissue-specific manner. The identified regulatory domains are also highly conserved throughout the entire exchanger family. It is unlikely that ionic regulation was targeted by evolution to manipulate physiologically irrelevant inactivation mechanisms. Another fall-back position has become that Na+-dependent inactivation may only occur under conditions of greatly elevated intracellular Na+, such as that which occurs during ischemia-reperfusion. Here, this mechanism would reduce the occurrence of reverse-mode Na+/Ca2+ exchange and would therefore limit pathological Ca2+ entry. The Chernysh et al. study provides reasonably strong, albeit indirect, support for this hypothesis. Alternatively, Na+-dependent inactivation may simply represent a biophysical curiosity. More likely, however, the physiological role for this mechanism has yet to be discovered. The current study by Reeves' group goes to the very heart of this matter by demonstrating its operation and alterations in a cell-based system. Continued integration of biophysical, structural, and genetic manipulation studies are ultimately required in order for us to understand what regulates Na+/Ca2+ exchange.
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FOOTNOTES |
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