Figure 5.

Probing N-type inactivation using unnatural amino acid mutagenesis with Kv1.4 channels expressed in mammalian cells. (a) Comparison of macroscopic currents recorded from HEK293 cells expressing wild-type Kv1.4, Kv1.4⋆ (Kv1.4-M109L), Kv1.4⋆-Y19F or Kv1.4⋆-Y19W channels. Untransfected HEK293 cells displayed a small, background current (0.50 ± 0.03 nA, n = 5). Inserts in each panel show the side-chain structures of amino acids at position 19. (b) Constructs for expressing Kv1.4 through amber suppression. The GFP-TAG reporter plasmid was used as an indicator for transfection. (c) Potassium currents recorded from HEK293 cells expressing Kv1.4⋆-TAG channels through amber suppression using the indicated orthogonal tRNA-synthetase pair. Only background currents were recorded for Kv1.4⋆-19TAG alone (0.40 ± 0.05 nA, n = 5) or Kv1.4⋆-TAG with the EctRNA-OmeTyrRS pair in the absence of OmeTyr (0.42 ± 0.07 nA, n = 4). The Kv1.4⋆-19OmeTyr and Kv1.4⋆-19DanAla mutants exhibited significantly slower inactivation than Kv1.4⋆ and Kv1.4⋆-19Tyr. Inserts in each panel are the side chain structures. (d) Peak current plotted as a function of voltage for the indicated Kv1.4 channels. (e) Current (black trace) elicited by a 1-s (Kv1.4⋆) or 4-s (Kv1.4⋆-19OmeTyr) voltage step to +20 mV were fit with a sum of two exponentials (red line) relaxing to a small steady-state current with the indicated time constants. (f) Quantitative analysis of inactivation kinetics. Bar graphs show the average time constants (τ₁, τ₂) for the fast and slow inactivation components, current amplitudes (A1, A2) for the exponentials and steady-state current (C). Specific values are in Table 1. Asterisks indicate statistical difference from Kv1.4⋆ (P < 0.05). (g) Western blot analysis of Kv1.4⋆-19OmeTyr and Kv1.4⋆-19DanAla. The full-length Kv1.4⋆, Kv1.4⋆-19Tyr, and a truncated Kv1.4⋆ with the first 19 amino acid residues deleted were run as controls.