Delayed rectifier current of bullfrog sympathetic neurons: Ion-ion competition, asymmetrical block and effects of ions on gating

1. The delayed rectifier (DR) K+ channel pore was probed using different permeant and blocking ions applied intra- and extracellularly. Currents were recorded from bullfrog sympathetic neurons using whole-cell patch-clamp techniques. 2. With intra- and extracellular Cs+ (0 K+), there were large, tetraethylammonium (TEA)sensitive currents. Adding Kf back to the extracellular solution revealed that the current with Cs-i(+) was K+ selective (permeability ratio P-Cs/P-K = 0.17 +/- 0.02, n = 4) and showed a strong anomalous mole fraction effect. 3. There were also large non-inactivating currents with Naf and Na-o(+) (0 K+). The current with was K+ selective (Na-o(+) vs. K-o(+): P-Na/P-K = 0.022 +/- 0.005, n = 5), and was TEA sensitive with K-o(+) but not with Na-o(+). 4. Permeant ions affected gating kinetics. DR currents activated faster in K+ than in Cs+, and activated faster with increasing concentrations of either K+ or Cs+ . Deactivation was slowed by increased K+ or Cs+ concentration, with no difference between K+ and Cs+. 5. The pore was also characterized using intracellular blocking ions. A wide variety of monovalent cations (TEA, N-methyl-D-glucamine, arginine, choline, CH3NH3+, Li+, Cs+ and Na+) blocked DR channels from the inside in a voltage-dependent manner: K-D at 0 mV was 2.9 mM for TEA and 134-487 mM for the others, at apparent electrical distances (delta) of 0.33-0.79. There was no detectable block by 10 mM Mg-i(2+). Apart from TEA, the organic cations did not block from the outside. Apart from TEA, the organic. 6. The permeability to Na+ in the absence of K+, and the strong anomalous mole fraction effects observed for Cs-o(+) + K-o(+) mixtures, suggest that DR channels select for K+ using ion-ion competition. The block by large intracellular cations shows that the pore is asymmetrical. The loss of high affinity TEA, block with Nat and Na-o(+), and the effects of permeant ions on gating, suggest that channel conformation may be affected by ions in the pore.