During depolarization, extrusion of Ca2 from sarcoplasmic reticulum through forward-mode Na-Ca2 exchange was studied in the rat ventricular myocytes patch-clamped in whole-cell configuration. In order to confine the Ca2 responses in a micro-domain by limiting the Ca2 diffusion time, rat ventricular myocytes were dialyzed with high (14 mM) EGTA. K current was suppressed by substituting KCl with 105 mM CsCl and 20 mM TEA in the pipette filling solution and by omitting KCl in the external Tyrode solution. Cl current was suppressed by adding 0.1 mM DIDS in the external Tyrode solution. During stimulation roughly mimicking action potential, the initial outward current was converted into inward current, 47⁑1% of which was suppressed by 0.1 mM CdCl2. 10 mM caffeine increased the remaining inward current after CdCl2 in a cAMP-dependent manner. This caffeine-induced inward current was blocked by 1μM ryanodine, 10μM thapsigargin, 5 mM NiCl2, or by Na and Ca2 omission, but not by 0.1μM isoproterenol. The I∼V relationship of the caffeine-induced current elicited inward current from 45 mV to 3 mV with the peak at 25 mV. Taken together, it is concluded that, during activation of the rat ventricular myocyte, forward-mode Na-Ca2 exchange extrudes a fraction of Ca2 released from sarcoplasmic reticulum mainly by voltage-sensitive release mechanism in a micro-domain in the t-tubule, which is functionally separable from global Ca2i by EGTA.
