Is ATP Required For Activities Of The Na+/Mg2+ Exchange?

Abstract

Cytoplasmic Mg2+ concentration ([Mg2+]i) was measured with the fluorescent indicator furaptra in rat ventricular myocytes at 25°C. In intact cells loaded with Mg2+, introduction of extracellular Na+ induced a rapid decrease in [Mg2+]i ; the initial rate (initial Δ[Mg2+]i/Δt) was thought to represent the rate of Na+-dependent Mg2+ efflux (putative Na+/Mg2+ exchange). To study whether the Mg2+ efflux depends on energy derived from ATP, in addition to transmembrane Na+ gradient, we estimated the initial Δ[Mg2+]i/Δt after metabolic inhibition. In the absence of extracellular Na+ and Ca2+, treatment of the cells with 1 μM FCCP (∼10 min) or 5 mM KCN (≥90 min) caused a increase in [Mg2+]i from ∼1 mM to ∼2.5 mM (probably due to breakdown of MgATP) and cell shortening by ∼50% (probably due to formation of rigor cross-bridges). The initial Δ[Mg2+]i/Δt was largely reduced, on average by 90% in FCCP-treated cells and 92% in KCN-treated cells. Intracellular Na+ concentration ([Na+]i) measured with a Na+ indicator SBFI was, on average, 5.0-10.5 mM within the time range for initial Δ[Mg2+]i/Δt measurements, which is lower than that required for 50% inhibition of the Mg2+ efflux (∼40 mM, Tashiro et al., Biophys J 89:3235-3247, 2005). Normalization of intracellular pH by application of 10 μM nigericin did not reverse the inhibition of the Mg2+ efflux. These results suggest that elevation of [Na+]i or intracellular acidosis are not primarily responsible for the slowed Mg2+ efflux. Instead, in seems likely that a decrease in ATP below the threshold of rigor cross-bridge formation (∼0.4 mM estimated indirectly) inhibits the Mg2+ efflux, suggesting that absolute requirement of ATP for the Na+/Mg2+ exchange.