The open gate structure of the membrane-embedded KcsA potassium channel viewed from the cytoplasmic side

Abstract

Crystallographic studies of channel proteins have provided insight into the molecular mechanisms of ion channels, even though these structures are obtained in the absence of the membrane and some structural portions have remained unsolved. Here we report the gating structure of the membrane-embedded KcsA potassium channel using atomic force microscopy (AFM). The activation gate of the KcsA channel is located on the intracellular side, and the cytoplasmic domain was truncated to clear the view of this location. Once opened, the individual subunits in the tetramer were resolved with the pore open at the center. Furthermore, AFM was able to capture the previously unsolved bulge helix at the entrance. A molecular dynamics simulation revealed that the bulge helices fluctuated dramatically at the open entryway. This dynamic behavior was observed as vigorous open-channel noise in the single-channel current recordings. The role of the bulge helices in the open gate structure is discussed.

Figure 1. Crystal structure of the closed (residue 22–124) and open (residue 30–117) conformations of the KcsA channel (pdb code: 1K4C and 3F5W).

The longitudinal length of the channel was calculated from the crystal structure as the distance between the C_α atoms furthest apart along the channel axis plus their atomic size. For the closed conformation the unresolved terminal residue (F125) is taken into considerations. The surface of the channel is colored for the charge distribution (red for negative and blue for positive).

Figure 2. KcsA images embedded in the membrane.

(A, B) AFM images of the CPD-truncated KcsA channel in lipid bilayer and the height profiles.The KcsA-reconstituted proteoliposomes were added onto the mica surface, and the liposome membrane was extended on the mica substrate, forming the flat bilayer. The cytoplasmic part of the KcsA channel protruded from the membrane surface. The pH of the overlaying buffer solution was 7.5 for A and 4.0 for B. The height profiles along the green solid lines are shown below the images. Images were recorded with the acoustic AC mode of AFM. (C) Height distribution pattern of the CPD-truncated KcsA on the mica surface. The height was evaluated as the sum of the protruded height and the membrane thickness. The blue bars indicate the histogram for the closed conformation at pH 7.5 and the red ones the open conformation at pH 4.0. The green lines indicate the results of Gaussian fitting. The height was 6.4 ± 0.3 nm at pH 7.5 (n = 170) and 4.9 ± 0.4 nm at pH 4.0 (n = 121). The blue and red arrows indicate the height values obtained from the crystallographic structures for the closed (6.3 nm) and open (4.3 nm) states. (D) Schematic representation of the membrane-embedded channel on the mica surface. The intracellular structure protruding from the membrane surface was measured with AFM. The light blue box shown on the cytoplasmic end of the open conformation represents an AFM-retrieved structural part.

Figure 3. Structural images of the cytoplasmic end of the membrane-embedded KcsA channel.

(A,B) Cytoplasmic views of the KcsA channel at pH 7.5 (A) and pH 4.0 (B) and their height profiles along the green line. Particles are resolved in an island at neutral pH, while individual particles are identified at acidic pH. (Inset) A 3-times enlarged view of the arrowed KcsA channels. (C) A further magnified view of the open KcsA channel at acidic pH. The scale bars indicate 3 nm. (D) An averaged AFM image of the open pore at acidic pH. The averaging was performed from 14 images of the open KcsA channels from Figure 3B. The scale bars indicate 3 nm.

Figure 4. Dynamics of the CPD-truncated transmembrane structure of the KcsA channel according to the MD simulation.

(A) The side view of the closed structure (the cytoplasmic side up). (B) The bottom view of the closed structure. In the MD-simulated structure, the transmembrane region is colored cyan, which is similar to the X-ray crystallographic structure. The cytoplasmic end, that missed in the X-ray crystallographic structure is shown in yellow. (C) The side view of the open structure. (D) The bottom view of the open structure. The transmembrane region is colored cyan, which is similar to the X-ray crystallographic structure. The cytoplasmic end, which is colored in yellow, constitutes the plausible structure that the AFM images detected. The structural parts, colored magenta, make up the terminal structure that the MD simulation resolved. The membrane was not shown in these figures. (E) The B-factor was calculated from the MD simulation. The red and blue lines represent the B-factor obtained from the MD simulation for the open and closed structures, respectively. The B-factors of the crystal structures in open and closed states are also shown (the broken lines). The B-factors for residues 118–125 are significantly large. The B-factor for the extracellular loop was substantially higher than the intra-membrane region. (Inset) A subunit of the open configuration is shown, assigning the B-factors for the C_α atoms with the volume of the spheres.

Figure 5. Single-channel current at +100 mV without (A) and with (B) CPD at pH 4.0.

The all-point amplitude histograms are shown with the vertical axis in the logarithmic scale, and the peaks are fitted with the Gaussian function. The shoulder of the open conductance peak is prominent for the CPD-truncated channel, indicating the open-channel noise from the highly fluctuating gating. A low conductance sub-level (the red arrowhead) was seen for the CPD-truncated channel.