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 (I_Naca) 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 μM cytoplasmic Ca²⁺, inward I_Naca increased 2-fold at -120 mV when EGTA concentration was increased from 0.1 mM to 10 mM (37°C and 140 mM extracellular sodium). Stimulation by EGTA was decreased or abolished under conditions of attenuated exchanger turnover rate: temperature < 30°C, extracellular sodium < 70 mM, and membrane potential > + 60 mV. EGTA concentration had no effect on outward I_Naca with 100 mM cytoplasmic Na⁺ and 0.8 μM 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 I_Naca was stimulated by 2 mM cytoplasmic ATP or by phosphatidylserine. The apparent K_i for EGTA was 0.2 mM. The electroneutral calcium ionophore, A23187, activated outward I_Naca 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 I_Naca is expected due to submembrane calcium depletion by the exchanger; enhancement of the outward I_Naca at low EGTA concentrations is expected because submembrane calcium accumulates and activates I_Naca via regulatory calcium binding sites.