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

Abstract

Background: To study the transient outward current (Ito) 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 enhance 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 (EC₅₀ = 86.4 nM). ATXII (100 nM) dramatically increased I_toamplitude 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 V₀.₅. 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-aminopyddine insensitive but tetrodotoxin sensitive. These data suggest that outward Na current due to reversal of Na gradient in the absence of external No contributes to I_to. Caution must be taken when studying kinetics and pharmacology of I_to in external sodium-free solutions.