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Structural basis of TRPV5 channel inhibition by econazole revealed by cryo-EM

Abstract

The transient receptor potential vanilloid 5 (TRPV5) channel is a member of the transient receptor potential (TRP) channel family, which is highly selective for Ca2+, that is present primarily at the apical membrane of distal tubule epithelial cells in the kidney and plays a key role in Ca2+ reabsorption. Here we present the structure of the full-length rabbit TRPV5 channel as determined using cryo-EM in complex with its inhibitor econazole. This structure reveals that econazole resides in a hydrophobic pocket analogous to that occupied by phosphatidylinositides and vanilloids in TRPV1, thus suggesting conserved mechanisms for ligand recognition and lipid binding among TRPV channels. The econazole-bound TRPV5 structure adopts a closed conformation with a distinct lower gate that occludes Ca2+ permeation through the channel. Structural comparisons between TRPV5 and other TRPV channels, complemented with molecular dynamics (MD) simulations of the econazole-bound TRPV5 structure, allowed us to gain mechanistic insight into TRPV5 channel inhibition by small molecules.

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Fig. 1: Econazole-bound TRPV5 (TRPV5ECN) structure as determined by cryo-EM.
Fig. 2: Econazole inhibition of TRPV5.
Fig. 3: Ion pore of TRPV5ECN.
Fig. 4: Pore comparison of select Ca2+-selective ion channels.
Fig. 5: Structural comparison of TRPV5ECN to TRPV6*.
Fig. 6: Schematic representation of the proposed TRPV5 inhibition mechanism by econazole.

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Acknowledgements

We thank S. Molugu for support and training of future cryo-EM microscopists at Cleveland Center for Membrane and Structural Biology. We thank D. Major for assistance with hybridoma and cell culture at Department of Ophthalmology and Visual Sciences (supported by the National Institutes of Health Core Grant P30EY11373). We thank A. I. Sobolevsky (Columbia University) for generously providing the structure of TRPV6* before its release. MD simulations were run on resources available through the Scientific Computing Facility at the Icahn School of Medicine at Mount Sinai and the Extreme Science and Engineering Discovery Environment under MCB080077 (to M.F.), which is supported by National Science Foundation grant number ACI-1053575. We acknowledge the use of instruments at the Electron Imaging Center for NanoMachines supported by the NIH (1S10RR23057 and 1S10OD018111), NSF (DBI-1338135) and CNSI at UCLA. This work was supported by grants from the National Institute of Health (R01GM103899 to V.Y.M.-B., R01GM093290 to T.R., and U24 GM116792 to Z.H.Z and V.Y.M.-B).

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Authors and Affiliations

Authors

Contributions

T.E.T.H. conducted all biochemical and cryo-EM studies, including protein purification, sample preparation, imaging, data analysis, and interpretation; D.T.L. built and refined the atomic model; K.W.H. assisted T.E.T.H. in cryo-EM data collection and analysis; A.Y., J.D.R., and T.R. performed and interpreted electrophysiological data; A.K. performed MD simulations, and M.F. helped interpret the data; A.S. trained T.E.T.H. in cryo-EM sample preparation; X.H. participated in the initial stage of this project; S.B. and S.C. trained T.E.T.H. in data analysis; Z.H.Z. supervised cryo-EM data collection and analysis; S.H. assisted with data analysis; V.Y.M.-B. designed and supervised the execution of all experiments in this manuscript; T.E.T.H. and V.Y.M.-B. drafted the manuscript; all authors reviewed the final manuscript.

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Correspondence to Vera Y. Moiseenkova-Bell.

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Integrated supplementary information

Supplementary Figure 1 TRPV5 purification, whole-cell electrophysiology, and data processing

a A size exclusion chromatogram of purified TRPV5. The inset panel depicts a SDS-PAGE image of purified TRPV5. b Whole cell patch clamp experiments were performed as described in the methods section in HEK293 cells transfected with rbTRPV5 using a ramp protocol from −100 to 100 mV; currents are plotted at -100 and 100 mV, zero current is indicated by the dashed line. Representative trace (n = 11), monovalent currents were initiated by removal of extracellular Ca2+ and Mg2+ and the application of 0.33 % DMSO and 3 μM econazole is indicted by the horizontal lines. c Concentration response curve for econazole. Error bars represent ± s.e.m. of the respective n biological replicates. d The first panel is a representative micrograph of TRPV5 incubated with econazole frozen in vitreous ice (n = 3,313). Scale bar = 25 nm. The second panel depicts 2D class averages used when reconstructing the TRPV5ECN structure. e The workflow used to reconstruct TRPV5ECN to 4.8Å. A box around a 3D class indicates that that class was taken for further processing.

Supplementary Figure 2 Resolution data for TRPV5ECN refinement

a FSC curves for masked, unmasked and corrected reconstructions. The dashed line represents an FSC of 0.143. b The angular distribution of 2D views for the final particles used for the reconstruction. High numbers of particles are represented as taller red cylinders while views with a low number of particles are shown as shorter blue cylinders. The final density map of TRPV5ECN is shown in grey. c Multiple views of the local resolution map of TRPV5ECN. Local resolution was determined using ResMap software.

Supplementary Figure 3 TRPV5 model validation

a Various helices of the TRPV5ECN model (cartoon) overlaid with the TRPV5ECN density map (mesh). Select residues are shown as sticks to illustrate the accuracy of the model. All helices shown are within the 3.5–4.0 Å region of the structure. b Other domains of the TRPV5ECN model (cartoon) overlaid with the final TRPV5ECN density map (mesh). The different regions of the TRPV5ECN model are colored based on the diagram in Fig. 1b.

Supplementary Figure 4 Binding sites among TRPV subfamily members

a Density maps of econazole (ECN) bound rabbit TRPV5, capsazepine (CPZ) bound rat TRPV1 solved in nanodiscs and apo rat TRPV1 solved in nanodiscs. Densities attributed to lipids in each structure are colored in blue. The lipid to which each density was ascribed is listed below each structure. b Density maps of econazole (ECN) bound rabbit TRPV5, capsazepine (CPZ) bound rat TRPV1 solved in nanodiscs. ECN densities are shown in yellow. Capsazepine densities are shown in orange.

Supplementary Figure 5 Econazole-binding pocket

a Molecular structure of econazole. The asterisk indicates the location of a chiral center. b Zoomed in view of the econazole binding pocket manually fitted with R-econazole (yellow). The grey mesh represents the cryo-EM density attributed to econazole. c Zoomed in view of the econazole binding pocket manually fitted with S-econazole (yellow). The grey mesh represents the cryo-EM density attributed to econazole. d-e An electrostatic map of TRPV5ECN was calculated via APBS software. The zoomed views depict the econazole binding pocket with R-econazole or S-econazole shown in yellow.

Supplementary Figure 6 Econazole effect on mutant TRPV5

Two electrode voltage clamp (TEVC) experiments were performed similar to that described in Fig. 2 on Xenopus laevis oocytes injected with cRNA encoding the wild type or various mutants of rabbit TRPV5. a-e Representative traces for WT and various TRPV5 mutants (n = 10, n = 7, n = 4, n = 3, n = 7, respectively), currents are shown at −100 and +100 mV, zero current is indicated by the dashed line, the applications of 2.2 % DMSO (solvent) and 20 μM Econazole (ECN) are indicated by the horizontal lines. f Summary of the effects of 20 μM econazole and 2.2 % DMSO. *** p < 0.001 indicates a difference between econazole inhibition in WT (first black column). ##p<0.01, ###p<0.001 indicates a difference from the effect of DMSO on WT channels (first grey column); one way analysis of variance with Bonferroni post hoc comparison. Error bars represent ± s.e.m. of the respective n biological replicates.

Supplementary Figure 7 Comparison of ligand binding pockets within the TRPV subfamily

a The transmembrane domain (TMD) of TRPV5ECN. Bound econazole (ECN) is represented as yellow sticks. The regions of TRPV5ECN are colored based on the diagram in Fig. 1b. b The TMD of rat TRPV1 solved in nanodiscs in the absence of added ligand (purple, PDB: 5IRZ). Bound phosphatidylinositol, a lipid cofactor of TRPV1, is shown in grey sticks. c The TMD of rat TRPV1 solved in nanodiscs in the presence of the inhibitor capsazepine (orange, PDB: 5IS0). The capsazepine molecule (CPZ) is shown in grey. d The TMD of rat TRPV1 solved in nanodiscs in the presence of the potent agonists, resiniferatoxin (RTX) and double-knot toxin (DkTx) is shown in red (PDB: 5IRX). The pocket depicted coordinates an RTX molecule (grey).

Supplementary Figure 8 Econazole flexibility during MD production run

a Time evolution of the RMSD of R-econazole (labeled ECN_1 to ECN_4) with respect to the initial manual docking into the assigned electron density within the four monomers of TRPV5 (monomers A to D, respectively) during the 25 ns production run. b Time evolution of the RMSD of S-econazole (labeled ECN_1 to ECN_4) with respect to the initial manual docking into the assigned electron density within the four monomers of TRPV5 (monomers A to D, respectively) during the 25 ns production run.

Supplementary Figure 9 TRPV5 stability during MD production run

Time evolution of a RMSD of Cα-atoms of TM helices S1 to S6 (blue) and all Cα atoms of the TRPV5 tetramer (red) bound to R-econazole, and b Cα RMSF for each monomer during the 25 ns production run of the TRPV5 complex bound to R-econazole. Panels c and d show the time evolution of RMSD and RMSF values for the TRPV5 complex bound to S-econazole.

Supplementary Figure 10 Flexibility of econazole molecules during MD production run

Binding modes of four R-econazole molecules (grey carbons; labeled ECN_1 to ECN_4 in panels a to d sampled during the 25 ns production run and compared to the initial manually docked pose of R-econazole (yellow carbons) into the assigned electron density. Each panel a to d shows five conformations of R-econazole within the four monomers of TRPV5 (monomers A to D colored blue, red, green, and dark grey, respectively) at simulation times 5 ns, 10 ns, 15 ns, 20 ns, and 25 ns.

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Hughes, T.E.T., Lodowski, D.T., Huynh, K.W. et al. Structural basis of TRPV5 channel inhibition by econazole revealed by cryo-EM. Nat Struct Mol Biol 25, 53–60 (2018). https://doi.org/10.1038/s41594-017-0009-1

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