Structural basis for pharmacological modulation of the TRPC6 channel.

TRPC6 cloning and expression An N-terminally truncated human TRPC6 (residue 73–931) and its variant V867T/L868T were each cloned into a pORBMam vector with an N-terminal Strep tag. The recombinant baculoviruses were generated in Sf9 cells following a conventional protocol. P2 virus was used to infect HEK293 cells lacking N-acetylglucosaminyltransferase I (GnTI − ) at 3.5 × 10 6 cells/ml. After 12 hr of culture at 37°C, 10 mM Sodium butyrate was added to the suspension and the temperature was lowered to 30°C. Cells were harvested 48 hr post infection. TRPC6 purification and nanodisc reconstitution All purification steps were done at 4°C and in the presence of 1 μM antagonist or agonist. The cell pellet was resuspended in buffer A (150 mM NaCl, 20 mM Tris pH 8.0) supplemented with 0.5% (v/v) protease inhibitor cocktail. Cells were disrupted in an Microfluidizer and membrane fractions were isolated with two-step centrifugations. Membranes were first homogenized in buffer A and then solubilized with 1% (w/v) lauryl maltose neopentyl glycol (LMNG) and 0.1% (w/v) cholesteryl hemisuccinate (CHS) at 4°C for 2 hr. Insoluble material was removed by centrifugation at 40,000 g for 1 hr and the supernatant was mixed with Strep Tactin resin at 4°C for overnight. The resin was collected on a gravity column, washed with buffer A plus 0.06% (w/v) digitonin. The bound protein was eluted with 5 mM Desthiobiotin, concentrated and further purified by size exclusion chromatography using a Superose 6 Increase column equilibrated in buffer A plus 0.06% digitonin and 2 mM Tris(2-carboxyethyl)phosphine (TCEP). For nanodisc reconstitution, peak fractions were collected, concentrated to 1 mg/ml, and mixed with MSP2N2 and soybean lipid extract at a molar ratio of 1:3:225 for 1 hr. To remove detergents, two batches of fresh Bio-Beads SM2 were added at a concentration of 20 mg/ml with 4 hr in between. After overnight incubation, the sample was filtered and loaded onto a Superose 6 Increase column equilibrated in buffer A plus 2 mM TCEP. Peak fractions were pooled and concentrated to 1.5 mg/ml in the presence of 30 μM antagonist or agonist.

Cryo-EM sample preparation and data collection

Grid preparation was performed at 100% humidity and 10°C using a Mark IV Vitrobot (FEI). 3.5 μl of TRPC6 in nanodiscs was applied onto a glow-discharged Quantifoil R1.2/1.3 300-mesh copper holey carbon grid. Grids were blotted for 6 s at a force setting of 1 before being plunged into liquid ethane. Images were recorded on a 300 kV Titan Krios (FEI) microscope with a K2 summit detector (Gatan). Serial EM ( Mastronarde and Held, 2017 ) was used for automated image acquisition with a binned pixel size of 0.832 Å. For the antagonist-bound TRPC6 dataset, 10,009 movies were collected, and each movie was dose-fractionated to 30 frames with a total exposure time of 6 s and a total dose of ~50 electrons/Å ( Venkatachalam and Montell, 2007 ). For the agonist-bound TRPC6 dataset, 9517 movies were collected, and each movie was dose-fractionated to 31 frames with a total exposure time of 6.2 s and a total dose of ~50 electrons/Å ( Venkatachalam and Montell, 2007 ).

Cryo-EM data processing

Beam-induced motion was corrected in MotionCor2 ( Zheng et al., 2017 ). Contrast transfer function (CTF) parameters were estimated non-doseweighted micrographs on using CTFFIND4 ( Rohou and Grigorieff, 2015 ). All other data processing steps were performed using Relion-3 ( Zivanov et al., 2018 ). Initially, 20,000 particles were autopicked using Laplacian-of-Gaussian method. After 2D classification, 8 class averages were selected for reference-based autopicking on the full dataset. The extracted particles were binned to a pixel size of 4.16 Å and subjected to two rounds of 2D classification. For the antagonist-bound TRPC6 dataset, 547,081 good particles were sorted out and used for subsequent 3D classification and refinement. The initial reference map was generated ab initio and lowpass filtered to 40 Å. One good 3D class out of three, containing 90,014 particles, were re-extracted to a pixel size of 1.248 Å. 3D refinement with C4 symmetry yielded a 3.26 Å map. After CTF-refinement and Bayesian-polishing, the final resolution was improved to 3.08 Å. For the agonist-bound TRPC6 dataset, 341,431 particles belonging to good 2D class averages were selected. 3D classification further sorted out 68,553 particles. After CTF-refinement and Bayesian-polishing, the final 3D refinement yielded a 2.84 Å map. Model building The antagonist-bound TRPC6 model was built in Coot ( Emsley et al., 2010 ) using the TRPC6 cryo-EM structure ( Tang et al., 2018 ) (PDB 5Y × 9) as a guide. The model was subjected to real space refinement against sharpened map in Phenix ( Afonine et al., 2012 ) with secondary structure restraints. The refined model of antagonist-bound TRPC6 was used as a reference to build the agonist-bound TRPC6 model. Local resolution was estimated using ResMap ( Kucukelbir et al., 2014 ). Validation of geometries was performed in MolProbity ( Chen et al., 2010 ). All the structure figures were generated in Chimera ( Pettersen et al., 2004 ), Pymol (The PyMOL Molecular Graphics System) and HOLE ( Smart et al., 1996 ). FLIPR assay TRPC6 Ca 2+ channel activity was measured using a FLIPR (fluorescence imaging plate reader) Tetra system from Molecular Devices and the BD PBX Calcium Assay Kit (Becton Dickinson #640177). HEK293T cells were maintained in DMEM high glucose +10% FBS + 1X NEAA (Invitrogen #11965) and were transiently transfected with TPRC6 WT or variant expression plasmids. Site directed mutagenesis to create TRPC6 variants was carried out by Genewiz (South Plainfield, NJ) and variants were verified by DNA sequencing. Expression plasmids were prepared for transfection using Lipofectamine 3000 (Invitrogen) and added to cells. 15,000 cells/well were plated in a 384-well black poly-D-lysine coated plate (Corning #356663). 24 hr post transfection, cells were loaded with calcium sensitive fluorescent dye utilizing the BD PBX Calcium Assay kit following the manufacturer’s protocol and incubated for 2 hr in the dark at room temperature. Compound plates were prepared in assay buffer containing 10 mM HEPES pH = 7.2 @25°C, 4 mM MgCl 2 , 120 mM NaCl, 5 mM KCl, 0.1% BSA, 2 mM CaCl 2 . Compound addition to cells was automated on the FLIPR Tetra and fluorescent imaging was captured following the manufacturer’s protocol (Molecular Devices). Data were analyzed using GraphPad Prism 7 software.

Additional files Transparent reporting form