Calcium-dependent regulation of calcium efflux by the cardiac sodium/calcium exchanger

Abstract

Allosteric regulation by cytosolic Ca2+ of Na(+)/Ca2+ exchange activity in the Ca2+ efflux mode has received little attention because it has been technically difficult to distinguish between the roles of Ca2+ as allosteric activator and transport substrate. In this study, we used transfected Chinese hamster ovary cells to compare the Ca2+ efflux activities in nontransfected cells and in cells expressing either the wild-type exchanger or a mutant, Delta(241-680), that operates constitutively; i.e., its activity does not require allosteric Ca2+ activation. Expression of the wild-type exchanger did not significantly lower the cytosolic Ca2+ concentration ([Ca2+](i)) compared with nontransfected cells. During Ca2+ entry through store-operated Ca2+ channels, Ca2+ efflux by the wild-type exchanger became evident only after [Ca2+](i) approached 100-200 nM. A subsequent decline in [Ca2+](i) was observed, suggesting that the activation process was time dependent. In contrast, Ca2+ efflux activity was evident under all experimental conditions in cells expressing the constitutive exchanger mutant. After transient exposure to elevated [Ca2+](i), the wild-type exchanger behaved similarly to the constitutive mutant for tens of seconds after [Ca2+](i) had returned to resting levels. We conclude that Ca2+ efflux activity by the wild-type exchanger is allosterically activated by Ca2+, perhaps in a time-dependent manner, and that the activated state is briefly retained after the return of [Ca2+](i) to resting levels.

Functional comparison of the reverse mode of Na+/Ca2+ exchangers NCX1.1 and NCX1.5 expressed in CHO cells

“…A similar phenomenon was observed by Chernysh [29] . However, the [Ca 2+ ] i increase was more striking in the NCX transfected cells than in the wide-type cells because of the reverse mode activity [30] .…”

Actin-dependent regulation of the cardiac Na+/Ca2+ exchanger

“…5A show the results of an experiment in which cells expressing NCX1.1 were grown on the same coverslip as nontransfected cells. The two cell types were identified at the end of the experiment by loading the cells with high [Na ϩ ] i using gramicidin and then assaying for Ca 2ϩ uptake upon applying 0.1 mM CaCl 2 in K ϩ -PSS (9). Only the cells expressing NCX1.1 accumulated Ca 2ϩ under the latter conditions.…”

“…Cells were treated for 15 min with 10 M latrunculin B, an agent that inhibits F-actin polymerization by binding to glob- A: both cell types were grown on the same coverslip and were identified at the end of the experiment by assaying for NCX activity as described previously (9). The experimental procedure was identical to that described in Fig.…”

Ionic regulation of the cardiac sodium-calcium exchanger

“…While most observers, with one exception, 41 find that high concentrations of cytosolic Ca 2+ activate exchange activity very rapidly (<50 ms 42,43 ), concentrations of 200-300 nM appear to activate activity much more slowly. 44,45 It is unclear where physiological changes of cytosolic Ca 2+ (e.g., 100-1,000 nM) fall within this spectrum of responses, but the high local sub-membrane Ca 2+ concentrations generated during Ca 2+ release from the sarcoplasmic reticulum 46 suggest that NCX activation is more likely than not to be a rapid event (but see Section V).…”