Mechanism of activation gating in the full-length KcsA K+ channel

Abstract

Using a constitutively active channel mutant, we solved the structure of full-length KcsA in the open conformation at 3.9 Å. The structure reveals that the activation gate expands about 20 Å, exerting a strain on the bulge helices in the C-terminal domain and generating side windows large enough to accommodate hydrated K(+) ions. Functional and spectroscopic analysis of the gating transition provides direct insight into the allosteric coupling between the activation gate and the selectivity filter. We show that the movement of the inner gate helix is transmitted to the C-terminus as a straightforward expansion, leading to an upward movement and the insertion of the top third of the bulge helix into the membrane. We suggest that by limiting the extent to which the inner gate can open, the cytoplasmic domain also modulates the level of inactivation occurring at the selectivity filter.

Fig. 1.

Crystal structure of FL KcsA in the open conformation. (A) Final model of the open conformation of FL KcsA with the two Fab molecules bound with a twofold noncrystallographic symmetry to the canonical four-helix bundle portion of the cytoplasmic domain. The left inset highlights a surface representation of the open conformation showing the opening of the lower gate and the creation of associated side windows for ion flow. (Right Inset) Illustrates the expansion of the Bulge helix, comprised of residues 118–135. (B) Superposition of the closed and the open conformations of FL KcsA in cartoon representation (gray), with the Bulge helix region colored orange and blue, respectively. (Left) The permeation pathway radius profile calculated with the program HOLE (18) showing the radius profile along the z axis for the closed (orange) and open (blue) conformations of FL KcsA.

Fig. 2.

Influence of the C-terminal domain on the degree of opening of the inner bundle gate. Representative macroscopic current traces elicited in response to jumps of pH from 7.0 to pH 3.0. Currents were recorded under symmetrical 200 mM K⁺ at a holding membrane potential of +100 mV under conditions that putatively increase the degree of conformational flexibility at the inner bundle gate. In decreasing order: C-terminal truncated KcsA, full-length (WT) KcsA, and full-length KcsA in complex with a C-terminal bound Fab.

Fig. 3.

Spectroscopic analysis of the C-terminal domain conformational rearrangements. Structural rearrangements underlying channel opening. (A) Residue-specific environmental parameter profiles obtained in the open (red) and closed (gray) conformations for the C-terminal end of FL-KcsA: mobility parameter (Upper), NiEdda accessibility parameter ΠNiEdda (Lower). (B) The normalized difference for each environmental parameter was mapped onto the FL-open channel structure, where increases in local dynamics or water accessibility are depicted in shades of red, whereas decreasing changes as shades of blue according to the color spectrum below. On the left, the frame highlights the large changes in local dynamics at the inner face of the bulge helix. On the right, the bar and arrow suggest that the region immediately below the activation gate might embed into the membrane upon opening. (C) A cartoon model depicting the conformational transitions of the gate and C-terminal domain in full-length KcsA upon gating. Two diagonally-related subunits are shown. Blue model, closed state; red model, open state. The gray bars represent the approximate limits of the plasma membrane.