Rate-Limiting Mechanisms of Exchange Reactions in the Cardiac Sarcolemma Na⁺-Ca²⁺ Exchanger

The effects of temperature, pH, voltage and K⁺ were tested on Na⁺-Ca²⁺ and Ca²⁺-Ca²⁺ exchanges with a goal to elucidate the rate-limiting mechanisms. The initial rates (t = 1 s) of Na_i- and Ca_i-dependent ⁴⁵Ca uptakes were measured in the sarcolemma vesicles. At pH 7.4 the Ca²⁺-Ca²⁺ exchange shows a bell-shaped temperature curve with a maximum at 27-29 °C. This effect is not caused by irreversible inactivation of the exchanger. The increase of pH from pH 6.0 to 7.4 in the K⁺-free medium decelerates the Ca²⁺-Ca²⁺ exchange 1.5-2.0-fold, while the addition of K⁺ accelerates the Ca²⁺-Ca²⁺ exchange 2.0-3.0-fold. Therefore, the accelerating effect of K⁺ opposes the decelerating effect of deprotonation. Temperatures increase (6-45 °C) in the K⁺-free medium (pH 7.4) elevates the Na⁺- Ca²⁺/Ca²⁺-Ca²⁺ exchange ratio from 0.8 to 5.0. With varying temperatures (6-37 °C) and pH 5.0-9.7, K⁺ has no considerable effect on Na⁺-Ca²⁺ exchange but accelerates the Ca²⁺-Ca²⁺ exchange 2-3-fold. At 6-45 °C and fixed pH 7.4, the inside-positive potential (∆Ψ ≥ +200 mV) accelerates the Na⁺-Ca²⁺ exchange 1.7-2.0-fold, suggesting that the same rate-limiting reaction controls the Na⁺- Ca²⁺ exchange at various temperatures. It is concluded that (a) At pH > 6.5 (6-45 °C and 0-100 mM K⁺) the voltage-sensitive Na⁺ efflux limits the Na⁺-Ca²⁺ exchange, while the Ca²⁺ efflux limits the Ca²⁺-Ca²⁺ exchange, (b) At pH < 6.1 (6-45 °C and 0-100 mM K⁺) the voltage-insensitive Ca²⁺ influx limits both Na⁺-Ca²⁺ and Ca²⁺-Ca²⁺ exchanges (this may represent a reduced voltage sensitivity of Na⁺-Ca²⁺ exchange and the similar rates of Na⁺-Ca²⁺ and Ca²⁺-Ca²⁺ exchanges), (c) The bellshaped temperature curve of Ca²⁺-Ca²⁺ exchange cannot be described by a simple reversible reaction involving two species (the exchange has to involve at least three species), (d) K⁺ interacts with a deprotonated species (pH >6.1) accelerating the rate-limiting Ca²⁺ efflux of Ca²⁺-Ca²⁺ exchange.