NMDG⁺ currents recorded from HEK cells expressing Kv1.5 R487Y/V or Kv1.5 W472F/R487Y mutant channels. (A) Sequence alignment of the P-loop region of Kv1.5, Kv3.1, and Kv3.2b channels. The asterisks mark the amino acid differences between the three sequences. An arrow indicates an Arg residue at the outer pore of Kv1.5, which is replaced by Tyr in Kv3.1 and Kv3.2b channels and is the positional homologue of Shaker T449. (B and C) Current recordings from HEK cells stably expressing Kv1.5 R487Y (B) or R487V (C) mutant channels. To record the currents, the cells were held at −100 mV and depolarized for 12 ms at 0.5 Hz to potentials of −20, −10, 0, and +80 mV. The experimental solutions are similar to those in Fig. 1 C. The upward current deflections during the depolarizations are on-gating currents. (D) Gating current recording from transiently expressed Kv1.5 W472F/R487Y channels with pulses from −60 to +80 mV in 10-mV steps. The holding potential was −100 mV, and pulses were applied at 0.5 Hz. (E) Averaged ratio of Q_off/Q_on as a function of the pulse potential. The amount of charge displaced during depolarization and repolarization (Q_on and Q_off, respectively) was obtained from the time integral of the currents during depolarization and repolarization shown in B, C, and D as indicated by the label on each graph (n = 5). Kv1.5 WT data are included for comparative purposes.