Measurement of reversal potential of Na+-Ca2+ exchange current in single guinea-pig ventricular cells.
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)
Ehara
T
T
Department of Physiology, Faculty of Medicine, Kyushu University, Fukuoka, Japan.
Matsuoka
S
S
Noma
A
A
D000818:Animals; D001464:Barium / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism / Q000494:pharmacology*; D004305:Dose-Response Relationship, Drug; D004533:Egtazic Acid / Q000494:pharmacology / Q000031:analogs & derivatives; D006168:Guinea Pigs; D006321:Heart / Q000502:physiology*; D066298:In Vitro Techniques; D008564:Membrane Potentials / Q000187:drug effects; D009532:Nickel / Q000494:pharmacology; D012964:Sodium / Q000378:metabolism / Q000494:pharmacology*
D001464:Barium; D004533:Egtazic Acid; D009532:Nickel; D012964:Sodium; C025603:1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid; D002118:Calcium