Na⁺/Ca²⁺ exchange activity is most often measured in the "reverse mode", i.e. as Ca²⁺ influx. The two methodological approaches to measuring exchange activity in transfected CHO cells involve assaying ⁴⁵Ca²⁺ uptake or measuring changes in the cytosolic Ca²⁺ concentration in cells loaded with the Ca²⁺ indicating dye, fura-2. Either approach entails interpretational problems because once Ca²⁺ enters the cell, it can be accumulated by intracellular compartments, most notably the mitochondria. To minimize this difficulty, we used Ba²⁺ as a substitute for Ca²⁺ since Ba²⁺ did not appear to be sequestered by either the endoplasmic reticulum or the mitochondria [15]. Using various preincubation protocols to modulate the cytosolic Ca²⁺ concentration, the initial rate of Ba²⁺ influx via the exchanger in transfected CHO cells was found to increase with increasing cytosolic [Ca²⁺] [16]. The half-maximal [Ca²⁺] was determined to be 44 nM, a value similar to that observed with intact cardiac myocytes (see above) but an order of magnitude lower than found with excised giant patches. This dependence on [Ca²⁺] was not observed in cells expressing a mutant exchanger which was missing much of its hydrophilic, regulatory domain and was therefore insensitive to regulation by Ca²⁺.

Activation of Na⁺/Ca²⁺ exchange activity by similarly low values of cytosolic [Ca²⁺] was also observed in studies of Ca²⁺ efflux in these cells (manuscript submitted). We used the Ca²⁺ ionophore ionomycin to release Ca²⁺ from internal stores in the presence or absence of extracellular Na⁺. In fura-2 loaded cells, the amplitude of the ionomycin-induced [Ca²⁺] transient was reduced in the presence of extracellular Na⁺, reflecting the acceleration of Ca²⁺ efflux by Na⁺/Ca²⁺ exchange. These effects of Na⁺ were not observed in parental cells which lacked Na⁺/Ca²⁺ exchange activity. The effects of Na⁺ on Ca²⁺ efflux were observed at cytosolic [Ca²⁺] values of 75 nM or less, confirming that the exchanger was active at these low values. The results were successfully simulated by a kinetic model which depicted the exchanger as a high Vmax, low Km (5 µM) transporter, with half-maximal regulatory activation by cytosolic Ca²⁺ at a concentration of 47 nM.

Thus, whether operating in a Ca²⁺ influx or Ca²⁺ efflux mode, the exchanger would appear to be nearly fully activated at "resting" cytosolic [Ca²⁺] values of 100 nM. This, of course, makes the physiological function of regulatory Ca²⁺ activation difficult to understand, since the exchanger would be "turned on" at all cytosolic [Ca²⁺] values likely to be experienced by the cell.

Hilgemann and Ball's study [9] of the effects of PIP2 on Na⁺/Ca²⁺ exchange and K-ATP channel activity ushered in a new appreciation of the modulatory effects of phosphoinositides on membrane transporters. It was suggested that a potential binding site for PIP2 might be the XIP region, a stretch of 20 amino acids containing 8 positively charged residues located immediately after the exchanger's 5th transmembrane segment. A peptide corresponding to the sequence of this region was found to inhibit Na⁺/Ca²⁺ exchange activity, hence the name XIP or eXchange Inhibitory Peptide [17]. This observation led to the concept that the XIP segment might have an autoregulatory function and inactivate the exchanger through an intramolecular interaction with an as yet unidentified docking site. In giant patch studies, certain mutations within this region were found to alter the time course and extent of Na⁺-dependent inactivation, as well as the sensitivity to activation by regulatory Ca²⁺. Interaction of PIP2 with the XIP region would be expected to disrupt an autoinhibitory configuration and provide an easily understood mechanism for activation of exchange activity. Two questions arise in consideration of this speculative scenario: (1) Do cellular PIP2 levels vary sufficiently to alter exchange activity under physiological conditions? (2) Is the XIP region involved in physiological regulation of exchange activity?

With respect to the first question, cellular PIP2 levels are regulated by an exceedingly complex interplay of pathways for the degradation and resynthesis of this important phosphoinositide [18]. When CHO cells are plated on a plastic surface coated with either fibronectin or polylysine, the cells on fibronectin flatten out and spread over the substratum within a few hours while the cells on polylysine remain round. These differing morphologies are associated with differences in the cellular PIP2 content: The cells on fibronectin contain nearly twice as much PIP2 as those on polylysine (unpublished observations). Similar effects of the extracellular matrix on PIP2 levels have been reported for other cells [19,20]. Despite these differences, the rate of Na⁺/Ca²⁺ exchange activity, measured as ⁴⁵Ca²⁺ uptake, was identical under the two conditions. Our inability to detect changes in exchange activity upon a 50% reduction in PIP2 levels suggests that, at least in CHO cells, the exchanger is not highly sensitive to variations in the amount of this phosphoinositide. Of course, it is possible that we would begin to see inhibition of exchange activity if we were able to reduce PIP2 levels by more than 50%. Even so, the physiological significance of such an effect would be questionable: PIP2 levels are highly protected in cells and transient reduction of PIP2, e.g. through G-protein mediated phospholipase C activation, leads to a rapid resynthesis of PIP2. Thus, reductions in total PIP2 of more than 50% would rarely be observed under physiological conditions.

We approached the second question by expressing a mutant exchanger in which all the positively charged residues (R and K) in the exchanger's XIP region (219-RRLLFYKYVYKRYRAGKQR) were changed to alanines. The ability of the XIP peptide to act as an exchange inhibitor was shown to be critically dependent upon the presence of the positively charged residues [21], and a mutation in one of these residues (K229Q) was shown to eliminate Na⁺-dependent inactivation [22]. These changes might therefore be expected to eliminate any interaction between the XIP region and either PIP2 or an intramolecular docking site. The behavior of CHO cells expressing this mutant was remarkably similar to that of cells expressing the wild-type exchanger. In particular, the initial rate of Ba²⁺ influx was found to increase with cytosolic [Ca²⁺] over the same concentration range as for the wild-type exchanger. Other properties of exchange activity in these cells are currently being investigated, and it is possible that subsequent studies will reveal more extensive alterations in exchanger function in this mutant. Nevertheless, these preliminary results suggest that modulatory effects on exchange activity involving the XIP region are either very subtle, or are not manifest in the intracellular environment provided by CHO cells.

Is Na⁺/Ca²⁺ exchange regulated? It is hard to imagine that it would not be, considering its general importance in Ca²⁺ homeostasis in many cells and its specific importance in cardiac physiology. However, the results discussed above raise serious questions about our understanding of this issue. Our results, and the electrophysiologic data obtained with intact cardiac myocytes, suggest that the exchanger is substantially activated at "resting" levels of cytosolic [Ca²⁺], generally considered to be about 100 nM. Thus, there would appear to be very little room for regulatory modulation of exchange activity over the range of cytosolic [Ca²⁺] values where the exchanger is most active, i.e. following Ca²⁺ release from the sarcoplasmic reticulum, when cytosolic [Ca²⁺] increases into the µM range. Perhaps regulatory modulation of exchange activity is physiologically more efficient during diastole than during systole. The brief duration of the [Ca²⁺] transient following Ca²⁺ release from the sarcoplasmic reticulum may not provide sufficient time for efficient regulatory activation of the exchanger, which occurs with a time constant of 0.2 - 1 s [10,23]. Changes in the diastolic [Ca²⁺] might therefore preset the exchanger at an appropriate degree of activation to handle the higher levels of cytosolic [Ca²⁺] attained during the subsequent systolic interval.

What accounts for the difference in the exchanger's sensitivity to [Ca²⁺] between intact cells and excised patches? In intact cells, the local concentration of Ca²⁺ beneath the plasma membrane appears to be much greater than the bulk cytosol, as recently disclosed with plasma membrane targeted aequorins [16,24,25]. The disparity between submembrane and bulk Ca²⁺ concentrations might be substantially smaller under the experimental conditions of the excised patch studies, with the result that higher Ca²⁺ concentrations would appear to be required to activate exchange activity.

Do PIP2 and Na⁺-dependent inactivation regulate exchange activity in intact cells? This has always been an issue with this mode of regulation since cytosolic Na⁺ levels are generally quite low in cardiac cells (< 12 mM) and ATP levels are high, conditions that would appear to minimize Na⁺-dependent inactivation. Perhaps Na⁺-dependent inactivation merely serves a protective role, turning off exchange activity when ATP levels fall and cytosolic Na⁺ levels rise, as in ischemia. More speculative considerations, based on an increased Na⁺ concentration in a submembrane "fuzzy space" [26], could provide scenarios whereby Na⁺-dependent inactivation plays a more prominent role in exchanger regulation. As mentioned above, total cellular PIP2 levels are highly protected in intact cells, and large changes in total PIP2 seem unlikely. However, local changes in the vicinity of the exchanger could conceivably occur and modulate function, as recently demonstrated for K-ATP channels [27,28],

None of these speculative considerations is particularly satisfying. Compartmentalization and local changes of [Ca²⁺] and PIP2 are, quite literally, a "fact of life" in the intracellular environment, but without experimental access to such local effects, it is far easier to formulate hypotheses than to test them. Is Na⁺/Ca²⁺ exchange regulated? Probably. Do we understand the physiological mechanisms involved? Probably not.

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