Structural mechanism of C-type inactivation in K(+) channels

Abstract

Interconversion between conductive and non-conductive forms of the K(+) channel selectivity filter underlies a variety of gating events, from flicker transitions (at the microsecond timescale) to C-type inactivation (millisecond to second timescale). Here we report the crystal structure of the Streptomyces lividans K(+) channel KcsA in its open-inactivated conformation and investigate the mechanism of C-type inactivation gating at the selectivity filter from channels 'trapped' in a series of partially open conformations. Five conformer classes were identified with openings ranging from 12 A in closed KcsA (Calpha-Calpha distances at Thr 112) to 32 A when fully open. They revealed a remarkable correlation between the degree of gate opening and the conformation and ion occupancy of the selectivity filter. We show that a gradual filter backbone reorientation leads first to a loss of the S2 ion binding site and a subsequent loss of the S3 binding site, presumably abrogating ion conduction. These structures indicate a molecular basis for C-type inactivation in K(+) channels.

Figure 1

Crystal structure of Open-Inactivated tKcsA-OM Δ1-20. a. Open state of KcsA with one subunit removed for clarity and oriented with the extracellular surface on top. Individual subunits are rainbow coloured from the N-to the C-terminus. b. Left, experimental electron density of the closed state (PDB 1k4c) selectivity filter . Shown is the 2F_o−F_c electron density map (blue mesh contoured at 2σ) of the filter corresponding to residues 74–80 from two diagonally symmetric subunits. And F_o−F_c omit map (magenta mesh contoured at 6σ) corresponding to ions in the filter. Right, electron density of the open state selectivity filter represented as a composite omit map (blue mesh contoured at 2.0σ) and F_o−F_c omit map (magenta mesh contoured at 6σ) of ions. The polypeptide chain is in stick representation. Ions are represented as spheres. c. Comparing the conductive and C-type inactivated selectivity filter structures. The filter conformation in the open state solved in high K⁺ is shown in stick representation (Blue, with Oxygen atoms in red). For comparison, the conductive filter in the closed state solved in high K⁺ concentration (PDB 1k4c) is shown in yellow. The filter backbone heavy atom rmsd is 1.33 Å with respect to the closed state. All molecular graphics are rendered using Pymol [http://pymol.sourceforge.net].

Figure 2

Conformational classes in Open KcsA structures. Classification of the different open KcsA structures was carried out from geometrical analysis of 15 refined open or partially open structures and the closed structure of KcsA (1K4C). The degree of hinge opening was evaluated from the angle (Ψ) between the z axis (defined by the permeant ions) and the core axis of the C-terminal half of TM2 or the angle (φ) between the N- and C-terminal halves of TM2. Including the fully closed state, we defined five structural classes for the different open and partially open structures of tKcsA-OM Δ1-20, as shown represented by their electron density maps on the top panels. These are the composite omit maps (blue mesh contoured at 2σ) of the channel corresponding to two diagonally symmetric subunits (1K4C-closed state is shown using 2Fo−Fc electron density map). And F_o−F_c omit map (magenta mesh contoured at 4–6σ) corresponding to ions in the filter. Structural classes are named after the inter-subunit Cα-Cα distances at position T112: Closed (12 Å), plus 15, 17, 23 and 32 Å openings.

Figure 3

Correlation between inner gate opening and the conformation of the selectivity filter. Each major structural class is shown as a stick representation with the G77diagonal Cα-Cα distances highlighted by a dotted line. Gate openings associated with the closed, Open 14 and Open 15 Å classes show equivalent filter conformations. After the large transition between the Open 17 and Open 23 Å classes, no major rearrangements of the selectivity filter are observed.

Figure 4

Correlation between inner gate opening and selectivity filter ion occupancy. a. Electron density maps for the different K⁺ ion occupancies at the selectivity filter and various degrees of inner bundle gate opening (measured as Cα-Cα distances at T112, top numbers). Shown, in each case, are the composite omit maps (blue mesh contoured at 2σ) of the filter corresponding to residues 74–80 from two diagonally symmetric subunits and the F_o−F_c omit map (magenta mesh contoured at 4–6σ) corresponding to ions in the filter. b. Individual one-dimensional electron density profiles along the axis of symmetry (Z-axis) for each of the distinct occupancy models, shown using Gly79-Cα as Z=0. S1, S2, S3 and S4 represent the binding sites of K⁺ ions in the selectivity filter. The cartoon channels represent the relative degree of opening in the inner bundle gate for each density profile. c. The dependence of the relative ion occupancies for each of the four K⁺ binding sites (S1–S4) with the degree of opening in the inner bundle gate.

Figure 5

Structure-based mechanism for C-type inactivation. a. The minimal K⁺ channel gating cycle highlighting the main kinetic pathway between the closed and open inactivated states (gray background) and the possibility that the hybrid partially open inactivated structures might represent an alternative kinetic pathway. b. A mechanistic model of C-type inactivation at the selectivity filter. On the left, the structural transition of selectivity filters of different structures and associated ion occupancies. The selectivity filter evolves form a conductive form of the filter (O) with two ions distributed over four sites (S1–S3, S2–S4) towards an inactivated form of the filter with ion in S1 and S4 (state I₂). This mechanism involves the sequential narrowing of the permeation pathway by pinching at G77 (states I₁ and I₂) and an expected carbonyl reorientation at V76 (state I₂), highlighted in red and sequentially identified by a yellow arrow. In state I₁, the stability of S2 is compromised (but not that of S1, S3 and S4), while state I₂ is associated with a loss of S3. In each stage, a mixed population is depicted as a way to account for the experimental one-dimensional electron density map, shown in idealized form on the right.