Measurement of reversal potential of Na+‐Ca2+ exchange current in single guinea‐pig ventricular cells.

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Abstract
1. To identify the Na+‐ or Ca2+‐induced current as Na+‐Ca2+ exchange current and to determine the stoichiometry of the Na+‐Ca2+ exchange, the reversal potential was measured in a wide range of external Na+ [( Na+]o) or Ca2+ [( Ca2+]o) concentrations. The Na+‐ or Ca2+‐induced current was recorded in single ventricular cells enzymatically dispersed from guinea‐pig hearts, using the technique of whole‐cell voltage clamp combined with internal perfusion. 2. In the presence of 10‐40 mM‐Na+ and 55‐803 nM‐Ca2+ in the internal solution, an increase of [Ca2+]o from 0.1 to 0.5‐20 mM or an increase of [Na+]o from 30 to 50‐140 mM induced an extra current associated with an increase in membrane conductance. The reversal potential of these extra currents was determined from an intersection of the current‐voltage (I‐V) relations obtained in the absence and presence of a Na+‐Ca2+ exchange blocker, Ni2+ (2 mM). 3. Ba2+ in the external solution failed to induce the extra current, but inhibited the background conductance having a reversal potential at around 0 mV. Thus, 1 mM‐Ba2+ was added to all external solutions, so that a change in the background current was minimized during application of Ca2+ or Ni2+. 4. The relation between [Ca2+]o and amplitude of the Ca2+‐induced current was examined in the presence and absence of Ni2+. Lineweaver‐Burk analysis revealed that the action of Ni2+ on the extra current might be a mixed type of competitive and non‐competitive inhibition. 5. During the application of Ca2+, the Ca2+‐induced outward current decayed in a time‐dependent manner, resulting in a shift of the I‐V relations towards positive potentials. This current decay was inhibited by increasing the capacity of the internal Ca2+‐buffer, using BAPTA (1,2‐bis(o‐aminophenoxy)ethane‐N,N,N',N'‐tetraacetic acid) or higher concentrations of EGTA. The result indicates that [Ca2+]i, at least under the cell membrane, changes due to ion fluxes through the Na+‐Ca2+ exchange and that control of the ion concentrations within the cell is prerequisite for measuring the reversal potential of the Na+‐Ca2+ exchange. 6. The shift of both the holding current and the I‐V relations during stimulation of the exchange was suppressed, when the membrane potential was clamped at the equilibrium potential of 3Na+:1Ca2+ exchange.(ABSTRACT TRUNCATED AT 400 WORDS)