The mechanism by which aminopyridines (APs) block cloned Kv1.1 K+ channels expressed either in the mammalian Sol-8 muscle cell line or in Xenopus oocytes was investigated using whole-cell patch-clamp and two-electrode voltage-clamp techniques. When Sol-8 cells were exposed to 4-AP (30 microM to 1 mM) or 3-AP (300 microM to 10 mM) for 1-2 min at a holding potential of -80 mV, delayed rectifier K+ currents activated by the first depolarization to +40 mV showed a small reduction in peak amplitude but exhibited a rapid decay phase that was absent in control records. However, currents elicited by subsequent pulses showed maximal block throughout the pulse. These results suggest that 4-AP requires the channel to be activated before block can occur readily. After a 10-min washout of 4-AP in the absence of channel activation, the current elicited at the beginning of the first depolarizing pulse was similar to that observed during maximal block. However, during the pulse the current increased in an exponential manner, to an amplitude similar to that seen in the absence of 4-AP. Subsequent pulses elicited currents with profiles similar to that observed in controls. These results suggest that channel activation is also required for unblock (i.e., 4-AP is trapped when the channel closes). Block of Kv1.1 channels by 4-AP was voltage dependent. The magnitude of block decreased progressively as the membrane potential was depolarized to voltages more positive than -20 mV. The concentration producing half-maximal inhibition (IC50) of Kv1.1 currents in Sol-8 cells at +40 mV at physiological pH (7.2) was 89 microM for 4-AP (pKa = 9.2) and 2.2 mM for 3-AP (pKa = 6.0). However, when the intracellular pH was lowered to 6.0 the IC50 for 3-AP decreased to 290 microM. These results are consistent with the charged form of the APs being the active species.
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