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

Abstract

SUMMARY1. 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+]0) or Ca2+ ([Ca2+]0) 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+]. 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. Under these conditions, the Ni2+- 7. We conclude that the external-Ca2+-or external-Na+-induced current is generated by the Na+-Ca2+ exchange system and that the stoichiometry is 3Na+: lCa2+.

The role of the Na(+)‐Ca2+ exchanger in the rate‐dependent increase in contraction in guinea‐pig ventricular myocytes.

“…7D) is similar to the voltage-dependence of the Na+-Ca2+ exchanger (e.g. Ehara, Matsuoka & Noma, 1989). If this component of outward current is carried by the Na+-Ca2+ exchanger, it would be expected to increase progressively as aia rises during high frequency stimulation (i.e.…”

Mechanism of inhibition of the sodium current by bepridil in guinea‐pig isolated ventricular cells

“…Single guinea-pig ventricular myocytes were isolated by the enzymatic dissociation technique (Powell et al, 1980;Ehara et al, 1989). Sodium currents of single ventricular cells were recorded by the whole cell clamp technique.…”

Hypocretin/Orexin Innervation and Excitation of Identified Septohippocampal Cholinergic Neurons

“…] i ϭ 97 nM) as calculated for a 3:1 stoichiometry of the Na ϩ -Ca 2ϩ exchanger using the equation E Na-Ca ϭ 3 E Na Ϫ 2 E Ca (Kimura et al, 1987;Ehara et al, 1989; exchanger, also blocked the Hcrt-induced inward current in the entire voltage range tested. e, As expected for the Na ϩ -Ca 2ϩ exchanger, Hcrt response is increased at all potentials in ACSF with no added Ca 2ϩ ("zero" Ca 2ϩ ).…”

“…Moreover, the calculated reversal potential is predicted to shift to more depolarizing potentials as internal Ca 2ϩ levels rise in the presence of Hcrt (Keele et al, 1997). For an accurate measurement of the reversal potential of the Na ϩ -Ca 2ϩ exchanger, the precise values for external sodium and calcium and internal sodium and calcium are needed (Ehara et al, 1989). Under our experimental conditions, in the presence of an agonist, internal Ca 2ϩ levels are rising and not static and thus are hard to predict or measure (Keele et al, 1997).…”