Quantitation of Na/Ca exchanger function in single ventricular myocytes.

ABSTRACT

A Na/Ca exchange current can be elicited in voltage clamped single ventricular myocytes by the abrupt removal of extracellular Na+ by means of a rapid switcher device. We measured this reverse Na/Ca exchange current in isolated mouse ventricular myocytes from wild-type mice, and from transgenic mice with hearts overexpressing the Na/Ca exchanger. In mouse ventricular myocytes, the current was sensitive to nickel, and was eliminated by removal of intracellular Na+. It was not influenced by 3 m m ouabain, and thus not contaminated by Na pump currents. The magnitude of the current reached a plateau within 10-15 min after obtaining a whole cell patch with the pipettes containing EGTA, to buffer [Ca2+]i and in zero extracellular K+ concentration to completely inhibit the Na pump, and allow equilibration of pipette Na+ with subsarcolemmal [Na+]. The magnitude of the current increased with increases in pipette [Na+]. Comparison of the current magnitudes in wild-type and transgenic myocytes showed a 2.5 and 2.7 fold increase in the current in transgenic myocytes at pipette [Na+] of 10 and 20 m m. The magnitude of this increase in Na/Ca exchanger currents in single transgenic myocytes compares well with the reported 2.5 fold increase in Na+-dependent 45Ca2+ uptake measured in ventricular sarcolemmal vesicles obtained from transgenic animals. With this approach, we found variation in exchanger current densities in different species, with values for mouse>rat>rabbit>dog>human. This technique should also be useful in quantifying changes in Na/Ca exchanger current density as a consequence of pathologic processes, and exposure to drugs.