Regulation of Eag1 gating by its intracellular domains
- The Rockefeller University, Howard Hughes Medical Institute, United States
Figures
Figure 1
Comparison of the PAS loop in Eag1 and hErg.
(A) Structure of Eag1 (PDB: 5K7L) in the closed conformation (PAS is orange, VS is yellow, S4-S5 linker is blue, pore is green, C-linker is red, CNBHD is cyan, CaM is purple, and membrane is …
Figure 2
Role of intracellular domains in voltage-dependent gating.
(A) Voltage family current trace of WT Eag1, Eag1TM, and the Eag1/hErg chimera with the voltage-pulse protocol shown above. (B) Normalized tail current vs. depolarization voltage plot for WT Eag1 …
Figure 3 with 1 supplement
Role of Arg 7, Arg 8, and Asp 342 in voltage dependent gating.
(A) Voltage family current trace for D342A, R7A/R8A, and Δ3–9 with the voltage-pulse protocol shown above. (B) Normalized tail current vs. depolarization voltage plot of WT Eag1 (black square, n = 6)…
Figure 3—figure supplement 1
S4-S5 linker mutations.
(A) Voltage family current trace for H343A, Y344A, I345A, E346A, and Y347A with the voltage-pulse protocol shown above. (B) Normalized tail current vs. depolarization voltage plot of WT Eag1 (black s…
Figure 4 with 1 supplement
Interaction between residues 10–13, Tyr 213, and Tyr 639.
(A) Voltage family current trace for the Δ3–13, Y213A, and Y639R with the voltage-pulse protocol shown above. (B) Normalized tail current vs. depolarization voltage plot of WT Eag1 (black square, n =…
Figure 4—figure supplement 1
PAS loop deletions.
(A) Voltage family current trace for Δ3–10, Δ3–11, and Δ3–12 with the voltage-pulse protocol shown above. (B) Normalized tail current vs. depolarization voltage plot of WT Eag1 (black square, n = 6),…
Figure 5
L341 split channels.
Voltage family current trace for the L341 split (A), D342A L341 split (B), and Δ3–9 L341 split (C) with the voltage-pulse protocol shown above. (D) Normalized current vs depolarization voltage for …
Figure 6 with 4 supplements
Structure of Eag1 Δ3–13/CaM.
(A) Left, Voltage family current trace for Eag1 Δ3–13 in the presence of 1 mM CaCl2 with the voltage-pulse protocol shown above. Right, normalized current vs depolarization voltage for Eag1 Δ3–13 in …
Figure 6—figure supplement 1
Single-particle cryo-EM structure determination of Eag1 Δ3–13/CaM.
(A) SDS-PAGE gel of fractions from the final gel filtration column of Eag1 Δ3–13/CaM (molecular weight standards are in kDa). (B) Representative micrograph of Eag1 Δ3–13/CaM in vitreous ice (scale …
Figure 6—figure supplement 2
Cryo-EM density map of Eag1 Δ3–13/CaM.
Cryo-EM maps of conformation 1 (A) and conformation 2 (B) colored based on local resolution estimated by ResMap. Slices of the transmembrane domain (top panel) and intracellular domains (bottom …
Figure 6—figure supplement 3
Structure validation of the atomic model of Eag1 Δ3–13/CaM.
( A) FSC cross validation between the Eag1 Δ3–13/CaM model and the full map used for refinement for conformation 1 (green) and conformation 2 (black). (B). Collection parameters and refinement …
Figure 6—figure supplement 4
Structural superposition of the intracellular domains.
(A) Side view of conformation 1 of Eag1 Δ3–13 bound to CaM. Position of the membrane is indicated by gray lines and domain coloring is as follows: PAS-orange, VS-yellow, S5-S6-green, C-linker-red, …
Figure 7
Eag1 pore mutants.
(A) Phe 475 and Gln 477 (shown as green sticks, with red O, and blue N) are at the interface of the S6 helices (green). C-linker is shown in red. (B) Voltage family current trace for F475I/Q477R and …
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Gene (Rattus norvegicus) | Kv10.1/Eag1/Kcnh1 | Synthetic | Uniprot: Q63472 | |
| Gene (Homo sapiens) | Calmodulin | Synthetic | Uniprot: P0DP24 | |
| Cell line (Homo sapiens) | HEK293S GnTI- | ATCC | ATCC: CRL-3022 RRID:CVCL_A785 | |
| Cell line (Spodopterafrugiperda) | Sf9 | ATCC | ATCC: CRL-1711 RRID:CVCL_0549 | |
| Cell line (Cricetulus griseus) | Chinese Hamster Ovary cells | Sigma | RRID: CVCL_0213 | |
| Recombinant DNA reagent | pEG Bacmam | doi: https://doi.org/10.1038/nprot.2014.173 | ||
| Recombinant DNA reagent | pGEM-T vector | Promega | Catalog number: A1360 | |
| Software, algorithm | pClampfit 10.5 | Molecular Devices | RRID: SCR_011323 | |
| Software, algorithm | MotionCor2 | doi: 10.1038/nmeth.4193 | RRID: SCR_016499 | http://msg.ucsf.edu/em/software/motioncor2.html |
| Software, algorithm | CTFFIND4 | doi: 10.1016/j.jsb.2015.08.008 | RRID: SCR_016732 | http://grigoriefflab.janelia.org/ctffind4 |
| Software, algorithm | RELION-3 | doi: 10.1016/j.jsb.2012.09.006 | RRID: SCR_016274 | https://www2.mrc-lmb.cam.ac.uk/relion/index.php?title=Main_Page |
| Ssoftware, algorithm | ResMap | doi: 10.1038/nmeth.2727 | http://resmap.sourceforge.net | |
| Software, algorithm | Coot | doi: 10.1107/S0907444910007493 | RRID: SCR_014222 | https://www2.mrc-lmb.cam.ac.uk/personal/pemsley/coot/ |
| Software, algorithm | Phenix | doi: 10.1107/S0907444909052925 | RRID: SCR_014224 | http://phenix-online.org/ |
| Software, algorithm | Pymol | PyMOL Molecular Graphics System, Schrödinger, LLC | RRID: SCR_000305 | http://www.pymol.org/ |
| Software, algorithm | UCSF Chimera | UCSF Resource for Biocomputing, Visualization,and Bioinformatics | RRID: SCR_004097 | http://plato.cgl.ucsf.edu/chimera/ |
| Software, algorithm | HOLE | doi:10.1016/S0263-7855(97)00009-X | http://www.holeprogram.org |
Additional files
-
Transparent reporting form
- https://doi.org/10.7554/eLife.49188.015
