Evidence of Na Current Contribution to the Transient Outward Current in Cardiac Ventricular Myocytes

BACKGROUND: To study the transient outward current (I(to)) investigators often use sodium-free external solution to minimize the possible contamination of I(to) by sodium current. Removal of extracellular sodium creates reversal of sodium gradient and thus possibly contributing to I(to) mainly at positive potentials. METHODS AND RESULTS: To address this issue, whole-cell I(to) was recorded in sodium-free choline chloride and cobalt solutions, from rat ventricular myocytes known to exhibit a prominent I(to). Depolarizing pulse to 40 mV from -100 mV holding potential every 10 seconds elicited a fast activating and time-dependent inactivating components. The activation of I(to) was fast and complete within 10 ms at 40 mV, and the decay was rapid over the first 100 ms of the pulse and slower thereafter. External superfusion of the cell with 50 μM tetrodotoxin reversibly reduced I(to) amplitude by 25% from 1.47 +/- 0.2 to 1.1 +/- 0.3 nA (P <.04, n = 9). When sea anemone toxin (ATXII), known to selectively enhanced I(Na) by causing a delay in the inactivation gate, is applied to the cell, I(to) amplitude increased in a time- and dose-dependent manner (EC(50) =.86.4 nM). ATXII (100 nM) dramatically increased I(to) amplitude at all voltages between -20 and 60 mV (from 1.51 +/- 0.4 to 3.35 +/- 0.8 nA at 40 mV, P <.003, n = 12). Superfusion of cells with 5 mM 4-AP resulted in 82% reduction in I(to) amplitude at 40 mV (from 1.95 +/- 0.5 to 0.37 +/- 0.2 nA, P <.02, n = 8). Addition of ATXII to 4-AP containing solution increased peak I(to) by 965% (from 0.37+/-0.2 to 3.95 +/- 0.9, n = 8, P <.0003). However, in 11 other cells, addition of tetrodotoxin (50 μM) to the ATXII-containing solution blocked ATXII-induced outward current (from 3.51 +/- 0.64 nA to 1.60 +/- 0.17 nA, P <.05). The conductance (G(Ito)) was calculated by dividing peak I(to) by (Vm-E(K)), with an E(K) of -75 mV. G(Ito) was increased at all voltages (greater than -40 mV). Normalized G(Ito) was fitted by Boltzmann equation and ATXII did not significantly modify V(0.5) and k (from -20.5 +/- 3.9 to -17.0 +/- 3.5 mV for V(0.5), and 12.2 +/- 2.6 to 13.4 +/- 2.1 mV for k, n = 4). Also, atropine (1 μM) did not have any significant effect on I(to) (from 1.92 +/- 0.15 nA to 1.85 +/- 0.25 nA, n = 5). CONCLUSIONS: The results showed that, in sodium-free external solution I(to) is tetrodotoxin but not atropine sensitive. ATXII-induced I(to) increase is 4-aminopyridine insensitive but tetrodotoxin sensitive. These data suggest that outward Na current due to reversal of Na gradient in the absence of external Na contributes to I(to). Caution must be taken when studying kinetics and pharmacology of I(to) in external sodium-free solutions.