Characterization of the relationship between Na⁺-Ca²⁺ exchange rate and cytosolic calcium in trout cardiac myocytes

The whole-cell patch-clamp technique combined with rapid caffeine (CAF) applications was used to measure Na+-Ca2+ exchange (NCX) currents (INCX). The rate of Ca2+ extrusion and the amount of Ca2+ extruded from the cell upon a rapid CAF exposure were obtained from INCX and its time integral, respectively. This gave a maximal NCX rate (VNCX) of 151 amol pF-1 s-1 or 2.3 mM s-1 and a half-maximal VNCX (K0.5) at a total cellular [Ca2+] ([Ca2+]tot) of 15.4 amol pF-1. Using the same approach for the tail current induced by repolarization to -80 mV gave a K0.5 of 7.0 amol pF-1 corresponding to 108 μM total or 2-4 μM free Ca2+. The relationship between [Ca2+]tot and VNCX was linear in the physiological range. Inhibition of the SR function with cyclopiazonic acid plus ryanodine reduced the slope significantly from 23.2±1.4 to 17.6±1.6 s-1, while ryanodine alone had no effect. The relationship between [Ca2+]tot and VNCX was steeper at more negative membrane potentials, and with identical SR Ca2+ loads the maximal VNCX at -10 mV was reduced to 39.7±2.7% of the value at -90 mV. Long depolarizations caused SR Ca2+ loading through reverse-mode NCX. Between -30 and +10 mV reverse mode VNCX=Vm·0.047 amol pF-1 s-1 mV-1+2.51 amol pF-1 s-1, giving a reversal potential of -54 mV. In conclusion, the relationship between VNCX and [Ca2+]tot shows that the NCX is capable of removing a total Ca2+ transient of 60 μM at physiological heart rates, while reverse-mode NCX reloads the sarcoplasmic reticulum (SR) during depolarization. Furthermore, small alterations in the action potential configuration are predicted to change significantly the relative importance of the NCX in the regulation of cytosolic [Ca2+] and SR Ca2+ loading.