Modulation of Na+,Ca2+ exchange current by EGTA calcium buffering in giant cardiac membrane patches

Effects of calcium buffering by EGTA were examined on sodium-calcium exchange currents (INaCa) in inside-out giant cardiac membrane patches. Free calcium concentrations (Ca2+) were monitored with a calcium electrode and a fluorescent calcium indicator (Calcium Green-5N). With 1.8 microM cytoplasmic Ca2+, inward INaCa increased 2-fold at -120 mV when EGTA concentration was increased from 0.1 mM to 10 mM (37 degrees C and 140 mM extracellular sodium). Stimulation by EGTA was decreased or abolished under conditions of attenuated exchanger turnover rate: temperature < 30 degrees C, extracellular sodium < 70 mM, and membrane potential > +60 mV. EGTA concentration had no effect on outward INaCa with 100 mM cytoplasmic Na+ and 0.8 microM cytoplasmic Ca2+, conditions under which the current inactivated by about 70%. EGTA (0.1-10 mM) and BAPTA (10 mM) inhibited the current by about 80% when the outward INaCa was stimulated by 2 mM cytoplasmic ATP or by phosphatidylserine. The apparent Ki for EGTA was 0.2 mM. The electroneutral calcium ionophore, A23187, activated outward INaCa even in presence of 10 mM EGTA. Our results are consistent with EGTA acting as a simple calcium buffer with no direct effect on the exchanger. At low concentrations of EGTA, inhibition of the inward INaCa is expected due to submembrane calcium depletion by the exchanger; enhancement of the outward INaCa at low EGTA concentrations is expected because submembrane calcium accumulates and activates INaCa via regulatory calcium binding sites.