Atomistic Insights of Calmodulin Gating of Complete Ion Channels

Figure 1. Representation of an Eag1 channel (A). Transmembrane regions are in yellow for the Voltage Sensitive Domain (VSD), and green for the Pore Domain (PD). Only two of the four subunits are represented, with a red or blue border. The N-terminal PAS-Cap and Pas domain in orange, the C-linker in red, the CNBHD in cyan, and CaM in purple. The critical Asp 342 in S4/5 and Arg 7 and 8 are highlighted. The presumed position of the tetrameric coiled-coil is indicated with dashed lines. (B). Realistic representation of the channel views from the top using the same color scheme as in A. (C). Realistic representation of a bottom view, excluding the C-linker. Each subunit has a different color. CaM bridges two diametrically opposed subunits. (D) Structures visualized using then PDB coordinates (6PBY), rendered with Pymol 1.30 using the same color scheme as in panel A.

Figure 2. Plot of the contact surface [27] per residue in CaM (A) and Eag1 (B). Based on PDB 5K7L, 6PBX and 6PBY. “Contact surface area” between atoms A and B is defined as the area of the sphere whose center is the center of atom A and whose radius equals the sum of the Van der Waals radii or atom A and a solvent molecule. The solvent-accessible and contact surface of every atom has been computed using the software made available by Sobolev et al., http://oca.weizmann.ac.il/oca-bin/lpccsu [27].

Figure 3. Movements during gating of Eag channels. (A). Realistic representation of the rigid body torsion of the intracellular cytoplasmic based on the structure of the closed Eag1 channel and the opened hERG channel, viewed from the bottom. (B). Realistic representation of the relative motion of the C-linker (joined rectangles representing antiparallel alpha helices) and the PAS-Cap. The dashed black line represents the presumed position of the N-terminus not seen in the cryo-EM images. (C). Structure of the C-linker and PAS-cap from opened hERG (PDB 5VA1). Arg 4 (corresponding to Arg 7) and Asp 540 (corresponding to Asp 342) are represented as stick using CPU coloring scheme. The orange box indicates the presumed position of the border of the membrane.

Figure 4. Representation of SK channels. (A). Transmembrane regions are in yellow for the Voltage inSensitive Domain (ViSD), green for the PD and calmodulin in purple. Helices A and B are colored in red and run parallel to the membrane under the diametrically placed subunit. The presumed position of the tetrameric coiled-coil is indicated with dashed lines. Only two of the four subunits are represented, with a red or blue border. (B). Realistic representation of the channel view from the top using the same color scheme as in A. Each subunit has a different color. On the left, under resting conditions the N-lobe (dashes lines) wanders around and occasionally enters into the space underneath the subunit next to the C-lobe docking site, with three preferred positions. On the right, upon Ca2+ loading (red circle) the N-lobe docks into and makes contact with three different subunits. (C). Structures visualized using then PDB coordinates (6CNO), rendered with Pymol 1.30 using the same color scheme as in panel A.

Figure 5. Plot of the contact surface per residue in CaM (A) and SK4 (B) in apo (green) and loaded with Ca2+ (black). (Based on PDB 6CNM, 6CNN and 6CNO).

Figure 6. Cartoon representation of the coordinated movements upon SK4 activation. Same coloring scheme as in Figure 4. Part of the PD of the blue subunit is represented with dashed lines. Note that the complete ViSD of the red delimited subunit has not been represented to simplify the scheme. Also, S3 of the red delimited subunit, as well as the ViSD and S6 of the blue subunit have been left out. The main CaM anchoring residues Leu 185 in helix A and Trp 323 in helix B are filled with the color of its corresponding subunit when engaged with CaM. The remaining residues contact CaM as indicated in the main text, represent the axis or helix A/B rotation (Arg 316), or constitute the pore gate (Val 282). The border of CaM is colored according to the subunit engaged with the C-lobe. Note that the N-lobe of blue CaM interacts with helix C of a red subunit, and S1 of an orange subunit.

Figure 7. The N-lobe drug binding site of SK channels. Superposition of the N-lobe engaged with the target helix of TRPV5/6 (purple), SK4 (dark grey), SK2 (grey), Eag1 and KCNQ (green) on the complex of SK2 with EBIO (PDB 4G28). The position of the helices from Eag1 and KCNQ do not appear to be compatible with the presence of this drug.

Figure 8. Hypothetical PIP2 binding site near the CaM Thr 79 phosphorylation site. The pore residues (GYG) were used as a reference to align the Kir structure (PDB 3SYA) to the open configuration of SK4 (PDB 6CNO). The PIP2 molecule was manually moved maintaining the distance from the membrane to the presumed docking site, and manually adjusted to avoid obvious clashes. The docking space is delimited by S1, S2 and S4 transmembrane segment. The positively charged Arg residues are represented as spheres using the CPK coloring scheme, and CaM Thr 79 is represented as stick.

Figure 9. Representation of TRPV5/6 channels. (A). Transmembrane regions are in yellow for the ViSD, green for the PD and calmodulin in purple. Only two of the four subunits are represented, with a red or blue border. The ankyrin repeats are in orange, and the TRP helix in red. (B). Realistic representation of the channel view from the top using the same color scheme as in A. The border of each subunit has a different border color and the ankyrin repeats have been filled with its corresponding color. (C). View from the bottom. Four hemi-CaM molecules within the dome are represented. (D). Structures visualized using then PDB coordinates (6E2G), rendered with Pymol 1.30 using the same color scheme as in panel A.

Figure 10. Plot of the contact surface per residue in CaM (A) and TRPV5/6 (B). Based on PDB 6DMW, 6O20, 6E2F.

Figure 11. Ball and chain-like mechanism of TRPV5/6 channel inactivation. The influx of Ca2+ triggers CaM binding to the C-terminal tail. CaM docks within the intracellular dome in a position that favors plugging the pore with Lys 115 located in the middle of the EF3/4 linker. The ensuing decrease on Ca2+ within the dome should cause the release of CaM, followed by the re-initiation of another cycle.

Figure 12. Representation of a KCNQ channel. (A). Transmembrane regions are in yellow for the VSD, green for the PD and CaM is in purple. The cytosolic helix parallel to the membrane before S1 is labeled as hN. Helices A and B, colored in red, are at ~45° to the membrane under the VSD from the same of subunit. The border is colored red or blue to identify each subunit. To account for swapping, the VSD and PD of two different subunits are represented continuously. The presumed position of the tetrameric coiled-coil is indicated with dashed lines. (B). Realistic representation of the channel view from the top using the same color scheme as in A. Helices A and B are represented in red. On the right, upon Ca2+ loading (red dots) there is a small movement of helix A. Each subunit has a different color in this cartoon. (C). Structures visualized using then PDB coordinates (5VMS), rendered with Pymol 1.30 using the same color scheme as in panel A.