Madalina Condrescu

Allosteric Activation of Sodium-Calcium Exchange Activity by Calcium: Persistence at Low Calcium Concentrations

The activity of the cardiac Na+/Ca2+ exchanger is stimulated allosterically by Ca2+, but estimates of the half-maximal activating concentration have varied over a wide range. In Chinese hamster ovary cells expressing the cardiac Na+/Ca2+ exchanger, the time course of exchange-mediated Ca2+ influx showed a pronounced lag period followed by an acceleration of Ca2+ uptake. Lag periods were absent in cells expressing an exchanger mutant that was not dependent on regulatory Ca2+ activation. We assumed that the rate of Ca2+ uptake during the acceleration phase reflected the degree of allosteric activation of the exchanger and determined the value of cytosolic Ca2+ ([Ca2+]i) at which the rate of Ca2+ influx was half-maximal (Kh). After correcting for the effects of mitochondrial Ca2+ uptake and fura-2 buffering, Kh values of ∼300 nM were obtained. After an increase in [Ca2+]i, the activated state of the exchanger persisted following a subsequent reduction in [Ca2+]i to values <100 nM. Thus, within 30 s after termination of a transient increase in [Ca2+]i, exchange-mediated Ca2+ entry began without a lag period and displayed a linear rate of Ca2+ uptake in most cells; a sigmoidal time course of Ca2+ uptake returned 60–90 s after the transient increase in [Ca2+]i was terminated. Relaxation of the activated state was accelerated by the activity of the endoplasmic reticulum Ca2+ pump, suggesting that local Ca2+ gradients contribute to maintaining exchanger activation after the return of global [Ca2+]i to low values.

Ionic regulation of the cardiac sodium-calcium exchanger.

The Na+-Ca2+ exchanger (NCX) links transmembrane movements of Ca2+ ions to the reciprocal movement of Na+ ions. It normally functions primarily as a Ca2+ efflux mechanism in excitable tissues such as the heart, but it can also mediate Ca2+ influx under certain conditions. Na+ and Ca2+ ions exert complex regulatory effects on NCX activity. Ca2+ binds to two regulatory sites in the exchangers central hydrophilic domain, and this interaction is normally essential for activation of exchange activity. High cytosolic Na+ concentrations, however, can induce a constitutive activity that by-passes the need for allosteric Ca2+ activation. Constitutive NCX activity can also be induced by high levels of phopshotidylinositol-4,5-bisphosphate (PIP2) and by mutations affecting the regulatory calcium binding domains. In addition to promoting constitutive activity, high cytosolic Na+ concentrations also induce an inactivated state of the exchanger (Na+-dependent inactivation) that becomes dominant when cytosolic pH and PIP2 levels fall. Na+-dependent inactivation may provide a means of protecting cells from Ca2+ overload due to NCX-mediated Ca2+ influx during ischemia.

Regulation of Na+/Ca2+ exchange activity by cytosolic Ca2+ in transfected Chinese hamster ovary cells

Transfected Chinese hamster ovary cells stably expressing the bovine cardiac Na+/Ca2+exchanger (CK1.4 cells) were used to determine the range of cytosolic Ca2+ concentrations ([Ca2+]i) that activate Na+/Ca2+exchange activity. Ba2+ influx was measured in fura 2-loaded, ionomycin-treated cells under conditions in which the intracellular Na+concentration was clamped with gramicidin at ∼20 mM. [Ca2+]iwas varied by preincubating ionomycin-treated cells with either the acetoxymethyl ester of EGTA or medium containing 0-1 mM added CaCl2. The rate of Ba2+ influx increased in a saturable manner with [Ca2+]i, with the half-maximal activation value of 44 nM and a Hill coefficient of 1.6. When identical experiments were carried out with cells expressing a Ca2+-insensitive mutant of the exchanger, Ba2+influx did not vary with [Ca2+]i. The concentration for activation of exchange activity was similar to that reported for whole cardiac myocytes but approximately an order of magnitude lower than that reported for excised, giant patches. The reason for the difference in Ca2+regulation between whole cells and membrane patches is unknown.

Sodium-dependent inactivation of sodium/calcium exchange in transfected Chinese hamster ovary cells

High concentrations of cytosolic Na(+) ions induce the time-dependent formation of an inactive state of the Na(+)/Ca(2+) exchanger (NCX), a process known as Na(+)-dependent inactivation. NCX activity was measured as Ca(2+) uptake in fura 2-loaded Chinese hamster ovary (CHO) cells expressing the wild-type (WT) NCX or mutants that are hypersensitive (F223E) or resistant (K229Q) to Na(+)-dependent inactivation. As expected, 1) Na(+)-dependent inactivation was promoted by high cytosolic Na(+) concentration, 2) the F223E mutant was more susceptible than the WT exchanger to inactivation, whereas the K229Q mutant was resistant, and 3) inactivation was enhanced by cytosolic acidification. However, in contrast to expectations from excised patch studies, 1) the WT exchanger was resistant to Na(+)-dependent inactivation unless cytosolic pH was reduced, 2) reducing cellular phosphatidylinositol-4,5-bisphosphate levels did not induce Na(+)-dependent inactivation in the WT exchanger, 3) Na(+)-dependent inactivation did not increase the half-maximal cytosolic Ca(2+) concentration for allosteric Ca(2+) activation, 4) Na(+)-dependent inactivation was not reversed by high cytosolic Ca(2+) concentrations, and 5) Na(+)-dependent inactivation was partially, but transiently, reversed by an increase in extracellular Ca(2+) concentration. Thus Na(+)-dependent inactivation of NCX expressed in CHO cells differs in several respects from the inactivation process measured in excised patches. The refractoriness of the WT exchanger to Na(+)-dependent inactivation suggests that this type of inactivation is unlikely to be a strong regulator of exchange activity under physiological conditions but would probably act to inhibit NCX-mediated Ca(2+) influx during ischemia.

Molecular and Functional Studies of the Cardiac Sodium-Calcium Exchanger

In heart cells, the Na+-Ca2+ exchange system is the predominant mechanism for mediating Ca2+ efflux.s2 Therefore, understanding the Na+ -Ca2+ exchange reaction at a molecular level has obvious importance for understanding the regulation of cardiac contractility and perhaps in devising new therapeutic approaches to the treatment of heart disease as well. Recently the cardiac exchanger has been purified, cloned, and seq~enced.~ The cDNA sequence yields a predicted molecular weight of 108 kDa for the exchange protein. This represents a major breakthrough in our understanding of Na+ -CaZ+ exchange, and, because of the power and precision of molecular biological techniques, it is certain to stimulate a great deal of new research in this area. In this contribution, we will describe a new method for the purification of the bovine cardiac Na+-Ca2+ exchangee and its use in obtaining sequence information on the mature exchange protein and in examining its functional activity under pre-steady-state conditions.

Sodium-Dependent Inactivation of NCX1.3: Aortic Smooth Muscle Cells Versus Transfected CHO Cells

Despite the importance of Na/Ca exchange (NCX) activity in Ca homeostasis in blood vessels, there have been few detailed studies of the regulation of NCX activity in smooth muscle cells. Here we investigated the regulation of NCX by allosteric Ca activation and Na-dependent inactivation in rat aortic smooth muscle cells (ASMC) and compared the results with those of the smooth muscle isoform NCX1.3 expressed in CHO cells. Fura-2 loaded ASMC or transfected CHO cells were treated with ATP + thapsigargin to release Ca and prevent subsequent Ca re-accumulation in internal stores. Reverse exchange activity was initiated by applying 0.1mM Ca in Li-PSS or K-PSS. In both ASMC and transfected CHO cells treated with 1 mM ouabain, Ca uptake occurred following a 10-20sec lag period attributable to the positive feedback of allosteric Cai activation. To examine the Na-dependence of NCX activity, cells were treated with gramicidin and preincubated in 10-140 mM Na-PSS before activity was initiated by applying 0.1 mM CaCl2. In ASMC, NCX activity peaked at 20 mM Na but declined at higher Na concentrations; essentially no Ca uptake was seen at 140 mM Na. In contrast, robust activity was seen throughout that entire Na concentration range in transfected CHO cells. At 20 mM Na, Ca uptake in ASMC increased to a peak value and then declined sharply. After removing Ca with EGTA in 20 mM Na, subsequent pulses with 0.1 mM Ca revealed no activity until a 10 min recovery period had occurred. In contrast, NCX activity in the transfected CHO cells recovered within 30 s after EGTA treatment. We conclude that NCX activity in ASMC is much more susceptible to inactivation at high concentrations of Na (Na-dependent inactivation) than in transfected CHO cells.