Structural basis for C-type inactivation in a Shaker family voltage-gated K + channel

Fig. 1.. C-type inactivation in the Kv1.2-2.1 channel. (A) Gating mechanisms in Kv channels. The gating processes in a Kv channel of activation (1), deactivation (2), C-type inactivation (3), and recovery from inactivation (4) are depicted. TM4 in the VSD is colored red. Voltage gating involves the movement of the TM4 helix, while C-type inactivation involves structural changes at the selectivity filter. (B) The pore domain of the Kv1.2-2.1 channel (pdb: 2r9r). Two opposite subunits are shown, with the selectivity filter colored red and the K+ ions bound at the selectivity filter depicted as purple spheres. (C) Close-up view of the selectivity filter of the Kv1.2-2.1 channel. Two opposite subunits are shown in stick representation. The ion binding sites in the selectivity filter (S1 to S4) and the 0′ to 4′ carbonyl bonds are labeled. (D) C-type inactivation in the Kv1.2-2.1, Kv1.2-2.1-W362F, and Kv1.2-2.1-3m channels. Time course of current elicited by stepping the voltage from −80 mV holding potential to 40 mV with 100 mM external K+. Initial spikes observed are the capacitance transients. Currents are presented without leak subtraction, and the zero current level is indicated by a…

Fig. 2.. Changes in the selectivity filter on C-type inactivation. (A) The pore helix of the Kv1.2-2.1-3m channel. The 2Fobs-Fcalc electron density map contoured at 1.4σ is shown with residues 361 to 367 as sticks. The W362F and S367T substitutions present in the Kv1.2-2.1-3m channel are indicated. (B) The selectivity filter of the Kv1.2-2.1-3m channel. The 2Fobs-Fcalc electron density map contoured at 0.9σ is shown with residues 368 to 375 as sticks, K+ ions as purple spheres, and water molecule as a red sphere. (C and D) Comparison of the selectivity filter of the Kv1.2-2.1-3m (C) and the Kv1.2-2.1 channel (D). Residues 370 to 375 are shown as sticks. The distance (in angstroms) between the carbonyl oxygens and the Thr370 side-chain hydroxyl group in the opposite subunits is indicated by dotted lines. (E to H) Close-up of the selectivity filter and the pore helix showing the Y373 side chain in the Kv1.2-2.1-3m (E) and Kv1.2-2.1 channel (F) and the D375 side chain in the Kv1.2-2.1-3m (G) and Kv1.2-2.1 channel (H).

Fig. 3.. The outer pore region in the C-type inactivated channel. (A) Superposition of the outer pore region of the Kv1.2-2.1-3m (yellow) and the Kv1.2-2.1 channel (cyan). Two opposite subunits are shown. Some of the positions at which fluorescence probes introduced detected structural changes on C-type inactivation are indicated (green spheres in Kv1.2-2.1-3m and magenta spheres in Kv1.2-2.1). (B and C) Top view of the extracellular mouth of the pore in the Kv1.2-2.1-3m (B) and the Kv1.2-2.1 channel (C) shows changes in conformation of D375 and M376. The H-bond interaction between D375 and T377 in adjacent subunits of the Kv1.2-2.1-3m channel is shown. (D and E) H-bond interactions of E346 in Kv1.2-2.1-3m (D) and Kv1.2-2.1 channel (E) are shown.

Fig. 4.. Structure of the selectivity filter in the Kv1.2-2.1-2m channel. (A) C-type inactivation in the Kv1.2-2.1-2m (W362F and S367T) channel. Time course of the current elicited by stepping the voltage from −80 mV holding potential to 40 mV in 100 mM external K+. (B) Electron density of the selectivity filter of Kv1.2-2.1-2m. The 2Fo-Fc electron density map contoured at 1.3σ is shown with residues 367 to 375 as sticks, and the K+ ions in the selectivity filter shown as purple spheres. (C) Superposition of the pore helix and filter region (residues 362 to 375) of the Kv1.2-2.1-2m and the Kv1.2-2.1 channel.

Fig. 5.. C-type inactivation in a Kv channel. (A) A model for the selectivity filter in the conductive and the C-type inactivated state. The conductive state of the selectivity filter is observed in the Kv1.2-2.1 channel (pdb: 2r9r), while the C-type inactivated state is based on the structure of the selectivity filter in the Kv1.2-2.1-3m channel. Two opposite subunits are shown. (B) Structure of the selectivity filter of the Shaker W434F channel (pdb: 7sj1). Residues T441 to D447 from two opposite subunits are shown in stick representation. (C) Superposition of the protein backbone of the selectivity filter of the Kv1.2-2.1-3m (residues 369 to 375, yellow) and the Shaker W434F channels (residues 441 to 447, green) in shown. (D) Superposition of residues 369 to 391 of the Kv1.2-2.1-3m channel (yellow) with residues 441 to 463 of the Shaker W434F channel (green). Residues D375/447, M376/448, and T377/449 are shown as sticks with the K+ ions in the selectivity filter shown as purple spheres. Residues numbering in italics correspond to the Shaker channel.