IONIC MECHANISMS OF TRANSIENT INWARD CURRENT IN THE ABSENCE OF NA+-CA2+ EXCHANGE IN RABBIT CARDIAC PURKINJE-FIBERS

1. Membrane currents were measured with a two-microelectrode technique in voltage clamped rabbit cardiac Purkinje fibres under conditions known to cause intracellular calcium overload and to eliminate or minimize Na-Ca2+ exchange. 2. Increasing [Ca2+]o from 2.5 to 5 mM or above and substituting external sodium with either sucrose, choline or Li+ induced an oscillatory transient inward current (TI) which peaked 200-300 ms after repolarization from a previous depolarizing pulse. The TI quickly disappeared upon return to normal Tyrode solution. Both the rate and configuration of action potentials of Purkinje fibres also returned to control upon return to Tyrode solution after 30 min of high Ca2+ exposure, if the Ca2+ concentration was 30 mM or less, 3. The TI in Na+-free solution was Ca2+ dependent. Either zero or low (2.5 mM) [Ca2+]o), or replacement of [Ca2+]o by BaCl prevented induction of the TI current upon repolarization from a previous depolarizing pulse. 4. In the presence of 30 mM-CaCl2 and with choline chloride as the substitute for NaCl, TI had a distinct reversal potential (E(rev)) of -25 mV. The time-to-peak TI. either inward or outward, did not shift significantly with change in voltage. Both inward and outward TI were simultaneously abolished by exposure to 1 muM-ryanodine, suggesting they were both activated by transient release of Ca2+ from the sarcoplasmic reticulum. The occurrence of TI in the absence of [Na+]o) is not compatible with an electrogenic Na+-Ca2+ exchange mechanism. The existence of a clear-cut reversal potential suggests that an ionic channel may be responsible for the TI under these conditions. 5. Both the magnitude of peak TI and the E(rev) were affected by changes of CaCl2 concentration. (i) Under steady-state conditions, peak inward TI was significantly increased when the [Ca2+]o was elevated from 5 to 15 mM. The peak TI in the outward direction was significantly increased when [Ca2+]o was elevated from 15 to 30 mM; however, the difference in peak inward TI at 15 and 30 mM [Ca2+]o was small. (ii) Clear-cut reversals of TI were found at Ca2+ concentrations of 10 mM (E(rev) = -19.5 mV) or greater, and elevation of [Ca2+]o to 20, 30, 50 and 105 mM shifted the E(rev) to more negative potentials. (iii) In the presence of 5 mM [Ca2+]. the inward TI declined to zero at about -30 mV, and test voltages between -55 and +5 mV failed to reveal a distinct outward TI. 6. The negative shift of E(rev) with elevation of [Ca2+]o does not support the hypothesis that cations are the only charge carriers for TI. The [Ca2+]o was elevated by adding CaCl2 to the extracellular solution, therefore the effects of changing [Cl-]o on E(rev) were studied. When [Ca2+]o was fixed at 30 mM, E(rev) was similar when NaCl was substituted by either choline chloride (-25 mV) or LiCl (-21 mV). However, when [Cl-]o was changed by substituting half or all choline chloride with sucrose, E(rev) was shifted to -12.5 and +5 mV, respectively. The peak outward TI was also greatly suppressed when [Cl-]o was decreased. The magnitude of peak inward TI was not significantly different with either choline chloride or sucrose substitution. 7. Two anion transporter blockers, 4,4'-diisothiocyanatostilbene-2,2'disulphonic acid (DIDS) and 4-acetamide-4'-isothiocyanatostilbene-2,2'-disulphonic acid (SITS), which are known to block the Cl- conductance in cardiac tissues, abolished outward TI at a concentration of 10 muM for DIDS and 0.1 to 0.2 mM for SITS. 8. Our results indicate that the TI can occur in the absence of Na+-Ca2+ exchange in rabbit cardiac Purkinje fibres. In addition, under the present experimental conditions, the TI is carried by two components: (1) a Ca2+-activated cationic component which is primarily responsible for inward TI (i.e. rectifies inwardly); (2) a Ca2+-activated Cl- conductance that accounts for most or all of the outward TI, but which also contributes to inward TI. A balance of cation and anion conductances determine the magnitude of the TI plus the occurrence and value of a reversal potential.