The Cardiac Na⁺−Ca²⁺ Exchanger: Relative Rates of Calcium and Sodium Movements and Their Modulation by Protonation-Deprotonation of the Carrier

The exchange cycle of the cardiac Na⁺−Ca²⁺ exchanger can be described as separate steps of Ca²⁺ and Na⁺ transport [Khananshvili, D. (1990) Biochemistry 29, 2437–2442]. In order to determine the relative rates of Na⁺ and Ca²⁺ movement during the Na⁺−Ca²⁺ and Ca²⁺−Ca²⁺ exchange modes, the ratios (R) of Na⁺−Ca²⁺/Ca²⁺−Ca²⁺ exchanges were estimated with saturating concentrations of ions at both sides of the membrane. The effect of extravesicular pH and voltage (potassium valinomycin) on the initial rates (t = 1 s) of Na⁺−Ca²⁺ and Ca²⁺−Ca²⁺ exchange were investigated by assuming that, under the conditions tested, the intravesicular pH (pH 7.4) is not affected. Na⁺- or Ca²⁺-preloaded sarcolemma vesicles were diluted rapidly in assay medium containing ⁴⁵Ca and buffer (pH 5.0–10.9), and the reaction of ⁴⁵Ca uptake was quenched by using a semi-rapid-mixing device. Under conditions in which [⁴⁵Ca]₀ = [Ca]_i = 250 µM, the pH-dependent curve of Ca²⁺−Ca²⁺ exchange shows a bell shape in the acidic range (pK_ai = 5.1 ± 0.1 and pK_a2 = 6.5 ± 0.2) followed by activation of the exchange in the alkaline range (pK_a3 = 10.0±0.2). With [⁴⁵Ca]₀ = 250 µM and [Na];= 160 mM, the Na⁺−Ca²⁺ exchange increases monotonically from pH 5.0 to 9.5 (pK_ai = 5.1 ± 0.1, pK_a2 = 7.2 ± 0.2, and pK_a3 = 9.1 ± 0.2). At pH <6.1, the ratio of Na⁺−Ca²⁺/Ca²⁺−Ca²⁺ exchange is close to unity (R≈1), while it increases to R = 3–4 in the range of pH 7.1–9.3. These data suggest that, in the absence of monovalent ions and at pH >6.5, Ca²⁺ moves severalfold faster from the extravesicular to the intravesicular side than in the opposite direction, meaning that the bidirectional movement of Ca²⁺ is an intrinsically asymmetric process. The pH-voltage curve analysis shows that at pH >6.5 the inside positive potential accelerates Na⁺−Ca²⁺ exchange by 2–3-fold. However, at pH <6.1 the voltage response of Na⁺−Ca²⁺ exchange is lost, suggesting that the deprotonation of the carrier determines a voltage response of Na⁺−Ca²⁺ exchange. In the range of pH 5.0–10.8 and Δψp = 0–200 mV, the Ca²⁺–Ca²⁺ exchange is voltage-insensitive, indicating that the Ca²⁺ transport is not affected by membrane potential. The data can be interpreted as follows: (a) When the carrier is protonated (pH <6.1), the “electroneutral” movement of Ca²⁺ from the extravesicular (intracellular) side to the intravesicular (extracellular) side limits the rates of both Na⁺−Ca²⁺ and Ca²⁺−Ca²⁺ exchanges, (b) In the deprotonated carrier (pH >6.5), the ion movements in the opposite direction (from the intravesicular side to the extravesicular side) become rate-limiting: the voltage-sensitive Na⁺ movement controls the rate of Na⁺−Ca²⁺ exchange, and a Ca²⁺ transport limits the Ca²⁺−Ca²⁺exchange rate, (c) Deprotonation of the carrier (pH >6.5) has opposite effects on Na⁺ and Ca²⁺ movements: it accelerates the voltage-sensitive and rate-limiting transport of Na⁺ during the Na⁺−Ca²⁺ exchange, while it decelerates the rate-limiting Ca²⁺ transport during the Ca²⁺−Ca²⁺ exchange.