Na‐Ca Exchange Tail Current Indicates Voltage Dependence of the Cai Transient in Rabbit Ventricular Myocytes

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Abstract
Na‐Ca Exchange Tail Currents and Cai Transient Introduction: in mammalian cardiac myocytes, a rise of intracellular calcium (Cai) 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 (INa‐Ca) when the membrane is repolarized after a depolarization‐activated rise of Cai. If, during a voltage step, the membrane is repolarized at the time of the peak of the Ca, transient, the size of the INa‐Ca tail might he expected to reflect the magnitude of the Cai transient. Therefore, it might be possible to estimate the amplitude and voltage dependence of the Cai transient without, for instance, using fluorescent indicators that can interfere with Cai regulation. The first aim of this study was to use INa‐Ca tails to investigate the voltage dependence of the Cai 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 INa‐Ca tails varied with changes to the dialyzing Na concentration. The third aim was to test the correlation of voltage dependence of INa‐Ca tails with the voltage dependence of the Cai transient obtained using a fluorescent Ca indicator. Methods and Results: Experiments were performed at 35° to 37°C using whole cell patch clamp, and the holding potential was set at ‐40 mV. Depolarization elicited a Cai transient that peaked in 40 to 50 msec. We reasoned, therefore, that membrane repolarization after 50 msec would cause the raised level of Cai to activate an inward current on forward Na‐Ca exchange. The amplitude of INa‐Ca measured shortly (10 msec) after repolarization should reflect the peak amplitude of the Cai transient elicited by the depolarization. In cells dialyzed with 10 mM Na‐containing solution and depolarized for 50 msec to differing test potentials, the INa‐Ca tail on repolarization increased progressively after pulses to between −40 and +20 mV. The INa‐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 Cai 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 INa‐Ca tail became bell‐shaped, with a maximum at +20 mV (n = 4). Voltage dependence of the INa‐Ca tail was little affected by raising dialyzing Na from 10 to 20 mM (n = 4); but the amplitude of the INa‐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 Cai transient exhibited a similar voltage dependence to the 1Na‐Ca tail (n = 6). Conclusion: The results of this study suggest that in cells dialyzed with 10 mM Na, the voltage dependence of the Cai 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 INa‐Ca tail current measurement tracked the Cai sufficiently well to reflect the voltage dependence of the Cai transient. The data also confirm that the voltage dependence of the Cai transient in rabbit cells can be modulated by altering dialyzing Na concentration.