Modulation of Intracellular Na⁺ and Ca²⁺ Induced by the Cardiac Na⁺-Ca²⁺ Exchanger in Guinea Pig Ventricular Myocytes

Abstract

The Na⁺-Ca²⁺ exchanger current was measured in single guinea pig ventricular myocytes, using the whole-cell voltage-clamp technique, and intracellular free calcium concentration ([Ca²⁺]_i) was monitored simultaneously with the fluorescent probe Indo-1 applied intracellularly through a perfused patch pipette. In external solutions, which have levels of Ca²⁺ (∼66 μM Ca²⁺) thought low enough to inhibit exchanger turnover, the removal of external Na⁺ (by replacement with Li⁺) induced both an outward shift of the holding current and an increase in [Ca²⁺]_i, even though the recording pipette contained 30 mM bis(O-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid (BAPTA), sufficient to completely block phasic contractions. The effects of Na⁺ removal were blocked either by the extracellular application of 2 mM Ni²⁺ or by chelating extracellular Ca²⁺ with 1 mM EGTA. In the presence of 10 μM Ryanodine, the effects of external Na⁺ substitution with Li⁺ on both membrane current and [Ca²⁺]_i were attenuated markedly in amplitude and at a much slower time course. Reversal potentials were estimated by using ramp pulses and by defining exchange currents as the Ni²⁺-sensitive components. The experimental values of the reversal potential and [Ca²⁺]_i were used to calculate cytosolic Na⁺ ([Na⁺]_i) by assuming an exchanger stoichiometry of 3Na⁺ : 1Ca²⁺. These calculations suggested that in the nominal absence of external Ca²⁺ (∼66 μM under our experimental conditions), the exchanger operates at −40 mV as though ∼40 mM Na⁺ had accumulated in the vicinity of the intracellular binding sites. We conclude that under the conditions of low extracellular Ca²⁺ and high intracellular Ca²⁺ buffering, the Na⁺-Ca²⁺ exchanger can still generate sufficient Ca²⁺ influx on the removal of external Na⁺ to markedly increase cytosolic free Ca²⁺.