Figures and data in Electron cryo-microscopy structure of the canonical TRPC4 ion channel

Figures
Videos
Tables
Additional files

9 figures, 2 videos, 3 tables and 1 additional file

Figures

Figure 1 with 1 supplement

Download asset Open asset

Activation of TRPC4_DR by the selective activator (-)-Englerin A.

(**a–f**) HEK293 cells heterologously expressing TRPC4_DR-EGFP (**a,c,e**) and untransfected control cells (**b,d,f**) were investigated by voltage-clamp experiments in the whole-cell configuration. The membrane potentials were clamped to values ranging from −90 to +90 mV in the absence (**a,b**) and in the presence (**c,d**) of 50 nM (-)-Englerin A. Upon addition of 50 nM of (-)-Englerin A, the current density at −60 mV increased from −3.1 ± 1.9 pA/pF (n = 6) to −16.7 ± 10.7 pA/pF (n = 6). In untransfected control cells, the current density in the absence and presence of the activator was virtually the same with values of −2.1 ± 1.2 pA/pF (V = - 60 mV, n = 5) and −1.8 ± 0.9 pA/pF (V = −60 mV, n = 5) respectively. (**e,f**) Current-voltage curves in the absence (black squares) and in the presence (red circles) of 50 nM (-)-Englerin A. Currents were normalized to the current value in the absence of (-)-Englerin A at a membrane potential of +80 mV. Note that the measurements in the absence and in the presence of (-)-Englerin A were performed on the same cells. Shown are the normalized mean currents of 6 (e) and 5 (f) different cells. Error bars are ± SEM. The measurements were performed as described in Materials and methods.

https://doi.org/10.7554/eLife.36615.002

Figure 1—figure supplement 1

Download asset Open asset

Multiple sequence alignment of TRPC4_DR, human TRPC4 and human TRPC5.

The grey-shaded regions highlight conserved residues. Helices are indicated by red bars. Domains are colored according to Figure 2b with high transparency.

https://doi.org/10.7554/eLife.36615.003

Figure 2 with 5 supplements

Download asset Open asset

Structure of TRPC4_DR.

(a) Cryo-EM density map of TRPC4_DR with each protomer colored differently and shown as side, bottom and bottom view. (b) Ribbon representation of the atomic model of TRPC4_DR. Colors are the same as in (a). (c) Topology diagram depicting the domain organization of a TRPC4_DR protomer. (d) Ribbon representation of a TRPC4_DR protomer. Each domain is shown in a different color and labeled accordingly.

https://doi.org/10.7554/eLife.36615.004

Figure 2—figure supplement 1

Download asset Open asset

Purification of TRPC4_DR and negative stain EM of TRPC4_DR in amphipols.

(a) SDS-PAGE of the peak fraction of TRPC4_DR in amphipols after size exclusion chromatography. Lane 1: molecular weight marker, lane 2: protein. (b) Size exclusion chromatography profile of TRPC4_DR in amphipols. Peak 1, 2 and 3 correspond to the void volume, tetrameric TRPC4_DR and cleaved GFP, respectively. (c) Representative negative stain electron micrograph of TRPC4_DR. Scale bar, 50 nm, (d) Representative 2-D class averages. Scale bar, 10 nm. (e) 3-D reconstruction of negatively stained TRPC4_DR shown in different orientations.

https://doi.org/10.7554/eLife.36615.005

Figure 2—figure supplement 2

Download asset Open asset

Cryo-EM structure of TRPC4_DR.

(**a-b**) Representative digital micrograph area (a) and selected 2-D class averages (b) of TRPC4_DR embedded in vitrified ice. Scale bars, 50 nm (a), and 10 nm (b). (c) The *ab initio* 3-D reconstruction obtained with RVIPER. (d) Angular distribution of the particles. (e) The cryo-EM density map of TRPC4_DR colored according to the local resolution. (f) Fourier Shell Correlation (FSC) curve between maps from two independently refined half data sets (black). The 0.143 criterion indicates an average resolution of 3.6 Å. The grey curve shows the FSC curve between the final map versus the atomic model. (g) Representative regions of the density with fitted atomic model.

https://doi.org/10.7554/eLife.36615.006

Figure 2—figure supplement 3

Download asset Open asset

Single particle processing workflow for TRPC4_DR structure determination.
https://doi.org/10.7554/eLife.36615.007

Figure 2—figure supplement 4

Download asset Open asset

Comparison of the Ca²⁺binding site in TRPM4 and TRPC4_DR.

(**a-b**) Side view of TRPM4 (a) and TRPC4_DR (b) shown in ribbon representation. Helices involved in Ca²⁺-binding are shown in red. Zoomed-in view on the Ca²⁺-binding site in TRPM4 (c) and TRPC4_DR (d). Residues involved in Ca²⁺ binding in TRPM4 and its topologically equivalent residues in TRPC4_DR are shown in stick representation. Ca²⁺ ion is shown as green sphere.

https://doi.org/10.7554/eLife.36615.008

Figure 2—figure supplement 5

Download asset Open asset

LFW motif of the pore region and cysteines involved in disulphide bridges in the TRPC4_DR structure.

(a) Extracellular view near the pore axis showing the loops harboring cysteine residues. The loops are shown in ribbon representation with cysteines shown in stick representation. The neighboring glutamate residue is involved in an electrostatic interaction with an arginine. (b) Zoomed-in view on the disulphide bridge region. The chain trace is shown in stick representation including the side chains. The disulphide bridge is indicated by an asterisk. Electron density in (a) and (b) is shown in wired grey mesh. (c) LFW motif region viewed along the pore axis from the extracellular side. Each protomer is colored differently. The LFW motif and interacting residues are shown in stick representation. The interface at the LFW motif is highlighted with a black dotted ellipse. (d) Zoomed-in view on the interface at the LFW motif. The density is shown in wired grey mesh with key residues highlighted.

https://doi.org/10.7554/eLife.36615.009

Figure 3

Download asset Open asset

Comparison of the VSL domain of selected TRP family members with the voltage-sensing domain of the chimeric K_V1.2-K_V2.1 channel.

(a) The voltage-sensing domain of the chimeric K_V1.2-K_V2.1 channel and the VSL domains of TRPC4_DR,TRPP1, TRPM4, TRPM8 and TRPV1 are shown in ribbon representation. The S4 helix in each case is highlighted with dark shaded color. The rest of the domains are shown in light transparent color. The residues which form the hydrophobic patch in the middle of the domain are shown in golden yellow sphere representation with light transparency. The residues involved in ion-pair interactions are shown in stick representation. (b) The structures in (a) are rotated to better view the residues important for voltage sensing in the S4 helix of the chimeric K_V1.2-K_V2.1 channel and topological equivalent residues of TRP channels are shown in stick representation and labeled.

https://doi.org/10.7554/eLife.36615.013

Figure 4

Download asset Open asset

Lipid binding sites in the TRPC4_DR structure.

(a) Side and top view of the TRPC4_DR structure with lipid densities highlighted in yellow against the model shown in ribbon representation. (**b–c**) Zoomed-in view on the cholesteryl hemisuccinate (CHS) and phosphatidic acid lipid (PA) binding sites, respectively. CHS and PA molecules are shown in yellow stick representation.

https://doi.org/10.7554/eLife.36615.014

Figure 5

Download asset Open asset

Architecture of the pore domain.

(a) Surface electrostatic Coulomb potential at the extracellular mouth of TRPC4_DR. (b) Hydrophobic surface of the pore shown in vertical cross section. Hydrophobic patches are colored orange. (c) Ion conduction pore of TRPC4_DR shown with diagonally facing protomers shown in ribbon representation. Critical residues important for gating and selection are shown in stick representation. (d) Pore radius determined along the pore axis using HOLE (Smart et al., 1996).

https://doi.org/10.7554/eLife.36615.015

Figure 6

Download asset Open asset

Interaction of the TRP domain with a conserved glycine in the S4-S5 linker.

(a) Ribbon overview indicating the interaction site between the TRP domain and the S4-S5 linker. Key helices are shown in different colors. The rest of the protein is shown in light grey. (b) Zoomed-in view on the interaction site with key residues labeled. The chain trace is shown in stick representation. (c) Structure-based sequence alignment of TRP family members. The conserved glycine in the M2 motif is highlighted by a red dotted box.

https://doi.org/10.7554/eLife.36615.017

Figure 7 with 1 supplement

Download asset Open asset

Position of the Rib helix in TRPC4_DR and TRPM4.

(**a-b**) Side and bottom view of the atomic models of TRPC4_DR (a), and TRPM4 (b). The Rib helix is highlighted in blue color and the density of the map is shown in the background with high transparency. E648 in TRPC4_DR is shown in sphere representation and colored red.

https://doi.org/10.7554/eLife.36615.018

Figure 7—figure supplement 1

Download asset Open asset

Predicted model of TRPC4_DR interaction with IP₃ receptors and calmodulin.

(a) Possible interaction of an IP₃ receptor with TRPC4_DR. The putative site of interaction (Boulay et al., 1999; Tang et al., 2001) is highlighted with a yellow star. The CIRB domain of TRPC4_DR is shown in blue. The predicted interacting region of the IP₃ receptor is highlighted in dark color. (b) Interaction of calmodulin with the CIRB domain of TRPC4_DR. Calmodulin is shown in pink. Calcium is shown as spheres. Calmodulin has been fitted to the structure of TRPC4_DR in analogy to the MLC-kinase peptide bound to calmodulin in the crystal structure (PDB-ID: 2LV6).

https://doi.org/10.7554/eLife.36615.019

Figure 8

Download asset Open asset

Comparison of the ankyrin domain arrangement of TRPC4_DR and TRPA1.

(**a-b**) Side and bottom view of the structures of TRPA1 (PDB-ID: 3J9P) (a) and TRPC4_DR (b) in surface representation. Each subunit is colored with unique colors. (**c–d**) Ribbon and cartoon representation of TRPA1 (c) and TRPC4_DR (d) showing two protomers in side view. The ankyrin repeats of one protomer are highlighted in brown.

https://doi.org/10.7554/eLife.36615.020

Figure 9

Download asset Open asset

Comparison of the C-terminal helix architecture in TRPA1, TRPM4 and TRPC4_DR.

(**a-c**) Each panel shows the complete tetramer in bottom and side view on the left and the zoomed-in view of the C-terminal helix alone in bottom and side view on the right. The C-terminal helix is shown in blue with one helix highlighted in shaded dark blue.

https://doi.org/10.7554/eLife.36615.021

Videos

Video 1

Download asset

posterframe for video — Overview of the TRPC4_DR structure
https://doi.org/10.7554/eLife.36615.011

Video 2

Download asset

Tables

Table 1

EM data collection and refinement statistics of TRPC4_DR

https://doi.org/10.7554/eLife.36615.010

Data collection	Data set 1 [Data set 2]
Microscope	Titan Krios (Cs corrected, XFEG)
Voltage (kV)	300
Camera	K2 summit (Gatan)
Pixel size (Å)	1.09 [1.09]
Number of frames	60 [40]
Total electron dose (e^-/Å²)	69 [74]
Number of particles	132,622
Estimated defocus range	0.844–2.931
Atomic model composition
Non-hydrogen atoms	21,412
Protein atoms	21,192
Ligand atoms	236
Refinement (Phenix)
RMSD bond	0.01
RMSD angle	1.11
Model to map fit, CC mask	0.80
Resolution (FSC@0.143, Å)	3.6
Map sharpening B-factor (Å²)	−130.0
Validation
Clashscore	3.53
Poor rotamers (%)	0.17
Favoured rotamers (%)	94.48
Ramachandran outliers (%)	0.0
Ramachandran favoured (%)	92.38
Molprobity score	1.62
EMRinger score	2.42

Table 2

Comparison of selectivity filters among TRP family members

https://doi.org/10.7554/eLife.36615.016

TRP family name	Residue	Van der waals diameter (Å)	Selectivity* PCa: PNa	Reference
TRPC4_DR	G577	3.7	7
TRPV1	G643	1.5	3.8–9.6	(Liao et al., 2013)
TRPV2	Gly604 (M606)	1.9 (1.35)	3	(Zubcevic et al., 2016)
TRPML3	G457 (D459)	2.0 (4.2)	Highly selective for Ca²⁺	(Zhou et al., 2017)
TRPML1	G470 (D471)	1.4 (2.1)	N.D.	(Schmiege et al., 2017)
TRPN (NOMPC)	G1506	3.8	N.D.	(Jin et al., 2017)
TRPA1	D915	3.2	0.8	(Paulsen et al., 2015)
TRPV5	D542	2.6	>100	(Hughes et al., 2018)
TRPV6	D541	0.9	>100	(Saotome et al., 2016)
TRPM4	G972	4.3	PNa: PCa > 100	(Guo et al., 2017a)
TRPP1 (PKD2)	Leu641 (Gly642)	1.7 (3.6)	PNa: PCa > 100	(Shen et al., 2016a)

*(Venkatachalam and Montell, 2007)

Key resources table

Reagent type (species) or resource	Designation	Source or reference	Identifiers	Additional information
Gene (Danio rerio)	TRPC4_DR	N/A	NCBI Reference sequence: NM_001289881	Genes ordered from GenScript
Cell line (HEK293 GnTI-)	HEK293 GnTI-	ATCC	CRL-3022
Cell line (HEK293)	HEK293	ATCC	CRL-1573
Cell line (Sf9)	Sf9	N/A	Cat.No.600100	Purchased from Oxford Expression Technologies Ltd (UK)
Recombinant DNA reagent	pcDNA3.1+TRPC4_DR(See Materials and methods section for details)	This paper		Cloned by GenScript in the customized pcDNA3.1 vector harbouring N_term-HIS₈tag-TEV- and C_term-HRV3C-eGFP
Recombinant DNA reagent	pEG BacMam	Eric Gouaux Lab PMID: 25299155
Recombinant DNA reagent	pEG BacMam +TRPC4_DR(See Materials and methods section for details)	This Paper		Subcloned the pcDNA construct harbouring His-TEV- TRPC4- HRV3C -eGFP into the pEG BacMam with the introduction of StrepII tag.
Chemical compound, drug	(-)Englerin A	N/A		Obtained from Lead Discovery Center GmbH, Dortmund Germany
Software, algorithm	SPHIRE software package	Moriya et al., 2017 PMID: 28570515
Software, algorithm	CrYOLO	N/A		Wagner et al., unpublished. manuscript in preparation
Software, algorithm	RosettaCM	Wang et al. (2015) PMID: 27572730
Software, algorithm	Rosetta	Wang et al. (2015) PMID: 27572730
Software, algorithm	rosettaES	Frenz et al. (2017) PMID: 28628127
Software, algorithm	Chimera	Pettersen et al. (2004) PMID:15264254