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Calcium- and Sodium-dependent Regulation of Sodium-calcium Exchange in Transfected
Chinese Hamster Ovary Cells

Olga Chernysh
Pharmacology and Physiology

M.S. 1994 Kyiv Shevchenko University, Ukraine

Thesis Advisor: John P Reeves, PhD
Department of Pharmacology and Physiology

Main Conference Room
Department of Pharmacology and Physiology

Friday, October 3, 2008
12:00 p.m.


Activity of the cardiac Na+/Ca2+ exchanger (NCX1.1) is regulated by Na+ and Ca2+, the ions that comprise the main determinants of the exchanger’s driving forces. High concentrations of cytosolic Na+ are known to induce time-dependent inactivation of exchange currents, whereas cytosolic Ca2+ acts as an allosteric activator, interacting with two well-defined binding sites within the central hydrophilic domain of the exchanger molecule. Allosteric Ca2+ regulation of Na+/Ca2+ exchange has been studied primarily in Ca2+ influx mode. The Ca2+ efflux activity of the exchanger has received little attention because it has been technically difficult to distinguish between the roles of Ca2+ as an allosteric activator and a transport substrate. Most information about properties of Na+-dependent inactivation and allosteric Ca2+ regulation comes from excised patch studies. It remains unclear, however, what role these modes of Na+/Ca2+ exchange regulation may play in the heart.
In this study, we addressed the following issues: (1) Ca2+-dependent regulation of Ca2+ efflux by the cardiac Na+/Ca2+ exchanger, and (2) Na+-dependent inactivation of Na+/Ca2+ exchanger expressed in Chinese hamster ovary (CHO) cells. We found that Ca2+ efflux activity by the wild-type exchanger is allosterically activated by cytosolic Ca2+, perhaps in a time-dependent manner, and that the activated state persists for several tens of seconds after the return of [Ca2+]i to resting levels. These findings suggest that Na+/Ca2+ exchange activity in the heart may be regulated over multiple heartbeats, not on beat-to-beat basis. We also found that Na+-dependent regulation of the exchanger expressed in CHO cells differs in several respects from the inactivation process measured in excised patches. Thus, the wild-type NCX1.1 was remarkably resistant to inactivation at high Na+ concentrations, even after extensive PIP2 depletion. Inhibition of the exchange activity by high Na+ became evident only at low cytosolic pH. The refractoriness of the wild-type 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 the exchanger-mediated Ca2+ influx during ischemia.

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