NA-CA EXCHANGE TAIL CURRENT INDICATES VOLTAGE-DEPENDENCE OF THE CA-I TRANSIENT IN RABBIT VENTRICULAR MYOCYTES

Introduction: In mammalian cardiac myocytes, a rise of intracellular calcium (Ca-i) is well known to activate Ca extrusion via forward Na-Ca exchange, which generates an inward membrane current. This can be observed as an inward ''tail'' current (I-Na-Ca) when the membrane is repolarized after a depolarization-activated rise of Ca-i. If, during a voltage step, the membrane is repolarized at the time of the peak of the Ca-i transient, the size of the I-Na-Ca tail might be expected to reflect the magnitude of the Ca-i transient. Therefore, it might be possible to estimate the amplitude and voltage dependence of the Ca-i transient without, for instance, using fluorescent indicators that can interfere with Ca-i regulation. The first aim of this study was to use I-Na-Ca tails to investigate the voltage dependence of the Ca-i transient in whole cell patch clamped rabbit ventricular myocytes dialyzed with a ''normal'' level of internal Na. The second aim was to investigate how the voltage dependence of the I-Na-Ca tails varied with changes to the dialyzing Na concentration. The third aim was to test the correlation of voltage dependence of I-Na-Ca tails with the voltage dependence of the Ca-i transient obtained using a fluorescent Ca indicator. Methods and Results: Experiments were performed at 35 degrees to 37 degrees C using whole cell patch clamp, and the holding potential was set at -40 mV. Depolarization elicited a Ca-i transient that peaked in 40 to 50 msec. We reasoned, therefore, that membrane repolarization after 50 msec would cause the raised level of Ca-i to activate an inward current on forward Na-Ca exchange. The amplitude of I-Na-Ca measured shortly (10 msec) after repolarization should reflect the peak amplitude of the Ca-i transient elicited by the depolarization. In cells dialyzed with 10 mM Na-containing solution and depolarized for 50 msec to differing test potentials, the I-Na-Ca tail on repolarization increased progressively after pulses to between -40 and +20 mV. The I-Na-Ca tail was maximal after a +20-mV pulse and showed no decline after depolarizations to more positive potentials, up to +100 mV (P > 0.1; n = 8). This implies that the Ca-i transient has a similar amplitude for depolarizing pulses between +20 and +100 mV. When Na-free solution dialyzed the cell, the voltage dependence of the I-Na-Ca tail became bell-shaped, with a maximum at +20 mV (n = 4). Voltage dependence of the I-Na-Ca tail was little affected by raising dialyzing Na from 10 to 20 mM (n = 4); but the amplitude of the I-Na-Ca tail increased. Inhibition of the Na-K pump with strophanthidin in cells dialyzed with 10 mM Na had qualitatively similar effects to increasing dialyzing Na. In Fura-2 loaded cells dialyzed with 10 mM Na, the Ca-i transient exhibited a similar voltage dependence to the I-Na-Ca tail (n = 6). Conclusion: The results of this study suggest that in cells dialyzed with 10 mM Na, the voltage dependence of the Ca-i transient is different from the L-type Ca current, since this current declines at potentials > +20 mV. The results obtained using Fura-2 suggest that the I-Na-Ca tail current measurement tracked the Ca-i sufficiently well to reflect the voltage dependence of the Ca-i transient. The data also confirm that the voltage dependence of the Ca-i transient in rabbit cells can be modulated by altering dialyzing Na concentration.