Abstract
Na +− Ca 2+ exchange activity in cardiac sarcolemmal vesicles is known to to be sensitive to charged, membrane lipid components. To examine the interactions between membrane components and the exchanger in more detail, we have solubilized and reconstituted the Na +− Ca 2+ exchanger into membranes of defined lipid composition. Our results indicate that optimal Na +− Ca 2+ exchange activity requires the presence of certain anionic phospholipids. In particular, phosphatidylserine (PS), cardiolipin, or phosphatidic acid at 50% by weight results in high Na +− Ca 2+ exchange activity, whereas phosphatidylinositol and phosphatidylglycerol provide a poor environment for exchange. In addition, incorporation of cholesterol at 20% by weight greatly facilitates Na +− Ca 2+ exchange activity. Thus, for example, an optimal lipid environment for Na +− Ca 2+ exchange is phosphatidylcholine (PC, 30%)/PS (50%)/cholesterol (20%). Na +− Ca 2+ exchange activity is also high when cardiac sarcolemma is solubilized and then reconstituted into asolectin liposomes. We fractionated the lipids of asolectin into subclasses for further reconstitution studies. When sarcolemma is reconstituted into vesicles formed from the phospholipid component of asolectin, Na +− Ca 2+ exchange activity is low. When the neutral lipid fraction of asolectin (including sterols) is also included in the reconstitution medium, Na +− Ca 2+ exchange activity is greatly stimulated. This result is consistent with the requirement for cholesterol described above. Proteinase treatment, high pH, intravesicular Ca 2+ and dodecyl sulfate all stimulate Na +− Ca 2+ exchange in native sarcolemmal vesicles. We examined the effects of these interventions on exchange activity in reconstituted vesicles of varying lipid composition. In general, Na +− Ca 2+ exchange could be stimulated only when reconstituted into vesicles of a suboptimal lipid composition. That is, when reconstituted into asolectin or PC/PS/cholesterol (30:50:20), the exchanger is already in an activated state and can no longer be stimulated. The one exception was that the Na +− Ca 2+ exchanger responded to altered pH in an identical manner, independent of vesicle lipid composition. The mechanism of action of altered pH on the exchanger thus appears to be different from other interventions.
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