A novel method for direct application of phospholipids to giant excised membrane patches in the study of sodium-calcium exchange and sodium channel currents

Effects of membrane phospholipids on Na⁺-Ca²⁺ exchange and Na⁺ channel currents were studied in giant excised cardiac sarcolemmal patches. Phospholipids were suspended in an inert vehicle of -tocopherol acetate and hexane and were then directly applied to the side of patch electrodes at a short distance from the tip during current recording. Phosphatidylserine strongly stimulated outward Na⁺-Ca²⁺ exchange current and altered the kinetics of cytoplasmic Na⁺- and Ca²⁺-dependent secondary modulation. This effect was partially reversed by phosphatidylcholine. Prolonged treatment with phosphatidylserine eliminated the inactivation transient normally observed upon rapid application of cytoplasmic Na⁺ but left cytoplasmic Ca²⁺ dependence largely intact. In such cases, subsequent chymotrypsin treatment removed cytoplasmic Ca²⁺ dependence, but had no further stimulatory effect, indicating maximum alleviation of inactivation by phosphatidylserine. While these results indicate that phosphatidylserine acts on a cytoplasmic, protease-sensitive regulatory domain of the exchanger, phosphatidylserine also stimulated the exchange current after deregulation by chymotrypsin, indicating an effect on the exchange mechanism itself. As in other myocyte preparations, cardiac Na⁺ currents in giant patches undergo a time-dependent negative shift in the voltage dependence of steady-state inactivation. Loss of phosphatidylserine from the cytoplasmic leaflet (i.e. loss of transbilayer asymmetry of phosphatidylserine distribution) does not appear to be the underlying cause, since phosphatidylserine did not reverse this shift, despite stimulation of Na⁺-Ca²⁺ exchange current in the same patches.