Molecular structures of the human Slo1 K+ channel in complex with β4
- The Rockefeller University, Howard Hughes Medical Institute, United States
Figures
Figure 1 with 4 supplements
Overall structure of the open human Slo1 channel in complex with the β4 subunit.
(A) Determination of atomic structures of human Slo1 in four various states. (B) Overall structure of the human Slo1-β4 channel complex in the presence of 10 mM Ca2+ in stereo, viewed parallel to …
Figure 1—figure supplement 1
Electrophysiological studies of truncated hsSlo1 alone, co-expressed with β1 or β4 in HEK293T cells.
(A, C, E) Voltage-dependent channel activation of the truncated hsSlo1 (hsSlo1EM) alone (A), co-expressed with the β1 (C) or β4 (E) subunit in the absence of Ca2+. Representative current traces …
Figure 1—figure supplement 2
Structure determination of the open Human Slo1 channel in complex with β4 using Cryo-EM.
(A) Representative raw micrograph. (B) Selected 2D class averages. (C) Euler angle distribution of particles for the final 3D reconstruction. (D) Fourier shell correlation (FSC) curves between the …
Figure 1—figure supplement 3
Individual EM densities of the open human Slo1 channel in complex with β4.
Figure 1—figure supplement 4
Structure determination of the closed human Slo1 channel in complex with β4, the open human Slo1 channel, and the closed human Slo1 channel using Cryo-EM.
(A, E, I) Euler angle distribution of particles for the final 3D reconstruction of closed hsSlo1 in complex with β4 (A), open hsSlo1 (E), and closed hsSlo1 (I). (B, F, J) Fourier shell correlation …
Figure 2 with 1 supplement
Architecture of the β4 subunit.
(A) Stereo view of the β4 subunit monomer in ribbon representation with the extracellular side up. The N-terminal loop (‘N-loop’), TM1, extracellular domain (‘EC’) and TM2 are discretely colored …
Figure 2—figure supplement 1
Sequence alignments.
(A) Sequence alignment of the β subunit family. Secondary structures based on the cryo-EM structure of β4 are represented by ribbons (α helices), arrows (β strands), and lines (loops). Residues of …
Figure 3
Detailed interactions between Slo1 and β4.
(A) Extensive interactions between TM1, TM2 of β4 subunit and transmembrane domains of two contiguous Slo1 subunits (α1 and α2) in stereo. β4 and Slo1 are shown as ribbons and colored blue and red, …
Figure 4 with 1 supplement
Influence of β4 N-terminus on Slo1 gating.
(A) Voltage-dependent channel activation of the human Slo1 channel alone and co-expressed with the β1 or β4 subunit. Representative current traces recorded using two-electrode voltage clamp (TEVC) …
Figure 4—figure supplement 1
Identification of the β4 regions with critical functional effects with TEVC.
(A, B) Bar graphs of the activation time constant (τ_on) (A) and deactivation time constant (τ_off) (B) of all the mutants in an ascending order from left to right. The activation and deactivation …
Figure 5 with 1 supplement
Ca2+gating mechanism of human Slo1 in the absence and presence of β4 subunit.
(A) Ca2+-induced conformational changes in the pore domain of human Slo1-β4 channel complex. Superposition of the pore domain in the absence (red Cα trace) and presence (blue Cα trace) of Ca2+ is …
Figure 5—figure supplement 1
β4 has minimal impact on the Ca2+-induced open and closed conformations of hsSlo1.
(A) Overlay of Ca2+-bound human Slo1 in the absence (red) and presence (black) of β4. (B) Overlay of Ca2+-free human Slo1 in the absence (red ribbon) and presence (black) of β4. (C, D) The Ca-RCK1 …
Figure 6 with 1 supplement
Structural basis for modification of Slo1 toxin sensitivity by β subunits.
(A) CTX docked onto the Slo1 channel based on the crystal structure of the CTX-Kv1.2–2.1 paddle chimera (PDB 4JTA). Only the transmembrane domain of Slo1 is shown (gray surface) and CTX is shown as …
Figure 6—figure supplement 1
CTX docked onto the Ca2+-bound hsSlo1-β4 channel complex.
(A, B) Stereo side view (A) and top view (B) of CTX docked onto the Ca2+-bound hsSlo1-β4 channel complex based on the crystal structure of the CTX-Kv1.2–2.1 paddle chimera (PDB 4JTA). The hsSlo1-β4 …
Tables
Table 1
Structure refinement and validation, related to Figure 1 and Figure 5.
| hsSlo1 + β4 Open | hsSlo1 + β4 Closed | hsSlo1 Open | hsSlo1 Closed | |
|---|---|---|---|---|
| Data acquisition | ||||
| Microscope/Camera | Titan Krios/Gatan K2 Summit | |||
| Voltage (kV) | 300 | |||
| Defocus range (μM) | 0.7 to 2.0 | 0.7 to 2.0 | 0.8 to 2.4 | 0.8 to 2.4 |
| Pixel size (Å) | 1.04 | 1.3 | 1.3 | 1.3 |
| Total electron dose (e-/Å2) | 74 | 89 | 89 | 89 |
| Exposure time (s) | 10 | 15 | 15 | 15 |
| Reconstruction | ||||
| Particle number | 117,791 | 42,842 | 28,073 | 53,961 |
| Resolution (unmasked, Å) | 3.7 | 4.0 | 4.2 | 4.4 |
| Resolution (masked, Å) | 3.2 | 3.5 | 3.8 | 4.0 |
| RMS deviation | ||||
| Bond length (Å) | 0.007 | 0.007 | 0.007 | 0.01 |
| Bond angle (°) | 0.801 | 0.946 | 0.950 | 0.94 |
| Ramachandran plot | ||||
| Favored (%) | 95.55 | 91.70 | 91.72 | 90.43 |
| Allowed (%) | 4.45 | 8.30 | 8.16 | 9.46 |
| Outliers (%) | 0.00 | 0.00 | 0.12 | 0.11 |
| MolProbity | ||||
| Clash score | 5.83 | 6.07 | 8.56 | 5.36 |
| Rotamer outliers (%) | 0.21 | 0.30 | 1.56 | 0.63 |
Table 2
List of the β4 mutants for TEVC studies, related to Figure 4.
| ShortName | β4 sequence | β1 sequence | ShortName | β4 sequence | β1 sequence | |
|---|---|---|---|---|---|---|
| Slo1 | N/A | N/A | m27 | 1-10,20-190,206-210 | 10-18,178-191 | |
| β1 | N/A | 1-191 | m28 | 11-193 | 1-9,181-191 | |
| β4 | 1-210 | N/A | m29 | 11-198 | 1-9,181-186 | |
| m2 | 7-210 | N/A | m30 | 49-210 | 1-47 | |
| m3 | 1-205 | N/A | m31 | 1-163 | 151-191 | |
| m4 | 10-210 | N/A | m32 | 1-10,49-210 | 10-47 | |
| m5 | 14-210 | 1-12 | m33 | 1-163,206-210 | 151-191 | |
| m6 | 1-34,40-210 | 34-38 | m34 | 1-10,49-163 | 10-47,151-191 | |
| m7 | 1-190,197-210 | 178-183 | m35 | 1-28,49-210 | 28-47 | |
| m8 | 1-10,14-210 | 10-12 | m36 | 1-163,181-210 | 151-167 | |
| m9 | E9A | N/A | m37 | 1-19,30-210 | 19-28 | |
| m10 | 1-18,20-210 (R19C) | 18 | m38 | 1-180,191-210 | 168-177 | |
| m11 | R19L | N/A | m39 | 1-10,30-210 | 10-28 | |
| m12 | E14A | N/A | m40 | 1-180,206-210 | 168-191 | |
| m13 | 1-42,44-210 (A43Y) | 42 | m41 | 1-10,20-210 | 10-18 | |
| m14 | 1-164,168-210 | 152-154 | m42 | 1-190,206-210 | 178-191 | |
| m16 | 7-205 | N/A | m43 | 11-210 | 1-9 | |
| m17 | 1-42,44-164,168-210 | 42,152-154 | m44 | replace β4EC (49-166) with "GGSGGGSG" | N/A | |
| m18 | 1-11,13-14,16-18,20-210 (E12R, D15T, R19C) | 11,14,18 | m45 | replace β1EC (41-153) with "GGSGGGSG" | N/A | |
| m19 | 1-48,164-210 | 48-150 | m46 | replace β1EC (48-153) with "GGSGGGSG" | N/A | |
| m20 | 1-45,167-210 | 45-153 | m47 | 1-47,151-210 | 49-163 | |
| m21 | 49-163 | 1-47,151-191 | m48 | 1-44,154-210 | 46-166 | |
| m22 | 1-10,49-163,206-210 | 10-47,151-191 | m49 | 1-6,49-210 | 6-47 | |
| m23 | 1-10,49-163,196-210 | 10-47,151-182 | m50 | 1-6,30-210 | 6-28 | |
| m24 | 1-28,49-163,181-210 | 28-47,151-167 | m51 | 1-2,30-210 | 2-28 | |
| m25 | 1-19,30-180,191-210 | 19-28,168-177 | m52 | 1-6,20-210 | 6-18 | |
| m26 | 1-10,30-180,206-210 | 10-28,168-191 | m53 | 1-2,20-210 | 2-18 |
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information | |
|---|---|---|---|---|---|
| Gene (Homo sapiens) | hsSlo1 (human_KCNMA1) | synthetic | accession: Q12791.2 GI: 46396283 | synthesized at GeneWiz | |
| gene (Homo sapiens) | hsbeta4 (human_KCNMB4) | synthetic | accession: NP_055320.4 GI: 26051275 | synthesized at GeneWiz | |
| gene (Homo sapiens) | hsbeta1(human_KCNMB1) | synthetic | accession: Q16558.5 GI: 292495100 | synthesized at GeneWiz | |
| Recombinant DNA reagent | pEG BacMam | DOI: 10.1038/nprot.2014.173 | |||
| Recombinant DNA reagent | pGEM | https://www.addgene.org/vector-database/2835/ | |||
| Cell line (Homo sapiens) | HEK293S GnTI- | ATCC | CRL-3022 | cells purchased from ATCC and we have now confirmed there is no mycoplasma contamination | |
| Cell line (Homo sapiens) | HEK293T | ATCC | CRL-3216 | cells purchased from ATCC and we have now confirmed there is no mycoplasma contamination | |
| Cell line (Spodoptera frugiperda) | Sf9 | ATCC | CRL-1711 | cells purchased from ATCC and we have now confirmed there is no mycoplasma contamination | |
| Strain, strain background (Escherichia coli) | DH10Bac | ThermoFisher | 10361012 | MAX Efficiency DH10Bac Competent Cells | |
| Biological sample (Xenopus laevi) | oocyte | Xenopus laevi | |||
| Chemical compound, drug | Freestyle 293 medium | Gibco | 12338018 | ||
| Chemical compound, drug | sf-900 II SFM medium | Gibco | 10902088 | ||
| Chemical compound, drug | 2,2-didecylpropane-1,3-bis-β-D-maltopyranoside (LMNG) | Anatrace | NG310 | ||
| Chemical compound, drug | Cholesteryl hemisuccinate (CHS) | Anatrace | CH210 | ||
| Chemical compound, drug | Digitonin | Sigma-Aldrich | D141 | ||
| Chemical compound, drug | Cellfectin II | Invitrogen | 10362100 | ||
| Chemical compound, drug | FuGENE HD transfection reagent | Promega | E2312 | ||
| Chemical compound, drug | Collegenase type II | Gibco | 17107–0125 | ||
| Chemical compound, drug | Gentamicin sulphate | Sigma-Aldrich | A0752 | ||
| Commercial assay or kit | CNBr-activated sepharose beads | GE Healthcare | 17043001 | ||
| Commercial assay or kit | Superose 6, 10/300 GL | GE Healthcare | 17517201 | ||
| Commercial assay or kit | mMESSAGE mMACHINE T7 Transcription Kit | ThermoFisher | AM1344 | ||
| Commercial assay or kit | AmpliCap-MaxT7 high yield message maker kit | CELLSCRIPT | C-ACM04037 | ||
| Commercial assay or kit | R1.2/1.3 400 mesh Au holey carbon grids | Quantifoil | 1210627 | ||
| Software, algorithm | SerialEM | DOI: 10.1016/j.jsb.2005.07.007 | http://bio3d.colorado.edu/SerialEM | ||
| Software, algorithm | MotionCor2 | DOI: 10.1038/nmeth.4193 | https://msg.ucsf.edu/software | ||
| Software, algorithm | Gctf | DOI: 10.1016/j.jsb.2015.11.003 | https://www.mrc-lmb.cam.ac.uk/kzhang/ | ||
| Software, algorithm | Gautomatch | other | https://www.mrc-lmb.cam.ac.uk/kzhang/ | ||
| Software, algorithm | cryoSPARC | DOI: 10.1038/nmeth.4169 | http://www.cryosparc.com | ||
| Software, algorithm | RELION-3 | DOI: 10.7554/eLife.18722 | http://www2.mrc-lmb.cam.ac.uk/relion | ||
| Software, algorithm | FrealignX | DOI: 10.1016/j.jsb.2013.07.005 | http://grigoriefflab.janelia.org/frealign | ||
| Software, algorithm | COOT | DOI: 10.1107/S0907444910007493 | https://www2.mrc-lmb.cam.ac.uk/personal/pemsley/coot/ | ||
| Software, algorithm | PHENIX | DOI: 10.1107/S2059798318006551 | https://www.phenix-online.org | ||
| Software, algorithm | UCSF Chimera | DOI: 10.1002/jcc.20084 | https://www.cgl.ucsf.edu/chimera | ||
| Software, algorithm | Pymol | PyMOL Molecular Graphics System, Schrödinger, LLC | http://www.pymol.org | ||
| Software, algorithm | HOLE | DOI: 10.1016/s0263-7855(97)00009-x | http://www.holeprogram.org | ||
| Software, algorithm | pClamp | Axon Instruments, Inc |
