Abstract
The transient receptor potential (TRP) channel TRPV4 participates in multiple biological processes, and numerous TRPV4 mutations underlie several distinct and devastating diseases. Here we present the cryo-EM structure of Xenopus tropicalis TRPV4 at 3.8-Å resolution. The ion-conduction pore contains an intracellular gate formed by the inner helices, but lacks any extracellular gate in the selectivity filter, as observed in other TRPV channels. Anomalous X-ray diffraction analyses identify a single ion-binding site in the selectivity filter, thus explaining TRPV4 nonselectivity. Structural comparisons with other TRP channels and distantly related voltage-gated cation channels reveal an unprecedented, unique packing interface between the voltage-sensor-like domain and the pore domain, suggesting distinct gating mechanisms. Moreover, our structure begins to provide mechanistic insights to the large set of pathogenic mutations, offering potential opportunities for drug development.
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Acknowledgements
We thank staff at APS beamlines 24-ID C/E, especially K. Rajashankar, K. Perry and N. Sukumar, for assistance at the synchrotron. This work used NE-CAT beamlines (GM103403), a Pilatus detector (RR029205) and an Eiger detector (OD021527) at the APS (DE-AC02-06CH11357). We thank the staff of the Sloan Kettering cryo-EM facility and Subangstrom LLC for assistance with cryo-EM data collection. This work was supported by startup funds from Washington University School of Medicine, the Mallinckrodt Foundation grant, American Heart Association Award 17SDG33400229, National Institutes of Health Grant R01NS099341 (all to P.Y.) and by startup funds from Memorial Sloan Kettering Cancer Center (to R.K.H.).
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Z.D. and P.Y. performed biochemical preparations and X-ray crystallography experiments. N.P. and R.K.H. conducted cryo-EM experiments and model building. G.M. and M.S.-R. conducted rubidium-flux and electrophysiology experiments. P.Y. designed and supervised the project. Z.D., G.M., C.G.N., R.K.H. and P.Y. analyzed the results and prepared the manuscript. All authors discussed the results and edited the manuscript.
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Supplementary Figure 1 Channel activation by GSK1016790A (GSK101).
Activation for the full-length wild type Xenopus TRPV4 (black filled squares, n = 4, mean ± SEM) and the truncated construct TRPV4cryst (grey filled squares, n = 4, mean ± SEM) in CosM6 cells by GSK101 at a, 0 nM, b, 40 nM, c, 200 nM and d, 1000 nM is evaluated by relative efflux of 86Rb+, in comparison with cells transfected with an empty vector (open squares, n = 4, mean ± SEM). e, Relative, apparent GSK101 activation constant as a function of GSK101 concentration, calculated from data presented in a-d. Wild type TRPV4 (filled circles) is more sensitive to GSK101 (~ 4- to 6-fold) than TRPV4cryst (open circles). Calculation and curve fitting are described in Methods.
Supplementary Figure 2 Ion permeation and blockage of Xenopus TRPV4 and TRPV4cryst.
a, Confocal images of CosM6 cells expressing full-length wild type Xenopus TRPV4 fused with EGFP (left panel) and the crystal construct TRPV4cryst fused with EGFP (right panel). TRPV4cryst shows altered expression pattern and is mainly localized to intracellular organelles. b, K+ and Cs+ conduction of wild type Xenopus TRPV4 and TRPV4cryst. Single-channel current-voltage dependence of TRPV4 in symmetrical 150 mM KCl (open circles, n = 8), in symmetrical 150 mM CsCl (open squares, n = 7), and TRPV4cryst in symmetrical 150 mM KCl (filled circles, n = 12), in symmetrical 150 mM CsCl (filled squares, n = 6). Currents were measured in inside-out excised patches upon application of GSK101 (10 nM for wild type TRPV4, 20 nM to 5 μM in KCl and 5 μM in CsCl for TRPV4cryst) from the cytoplasmic side. Wild type TRPV4 has a unitary conductance of 280 ± 11 pS and 191 ± 8 pS at −100 mV membrane potential in symmetrical 150 mM KCl and symmetrical 150 mM CsCl respectively. Under the same conditions, TRPV4cryst has a unitary conductance of 272 ± 13 pS and 185 ± 9 pS in symmetrical 150 mM KCl and symmetrical 150 mM CsCl respectively. c, Representative traces of activation of TRPV4cryst by GSK101 in symmetrical 150 mM KCl (top panel, 20 nM GSK101) and in 150 mM CsCl (bottom panel, 5 μM GSK101). Membrane potential is −120 mV. d, Ca2+ and Ba2+ permeation of wild type Xenopus TRPV4. Inside-out membrane patches were excised in symmetrical BaCl2 (75 mM) or CaCl2 (75 mM) buffers. Wild type TRPV4 currents (Ba2+, black; Ca2+, grey) were induced by perfusion of the bath (cytoplasmic side) with Kint containing 10 nM GSK101. Reversal potentials are 27 mV for BaCl2/KCl and 33 mV for CaCl2/KCl, respectively. After liquid junction potential correction, the corresponding permeability ratios were calculated to be PBa/PK = 3.4 (n = 8) and PCa/PK = 4.8 (n = 5). Basal currents in the absence of GSK101 were very low and only noticeable at extreme potentials (−100 and 100 mV). e, Gd3+ blockage of TRPV4cryst. GSK101 activation (1 μM) of TRPV4cryst (filled squares, n = 3, mean ± SEM) in CosM6 cells was assessed by relative efflux of 86Rb+. Basal 86Rb+ efflux was measured in cells without GSK101 stimulation (empty circles, n = 3, mean ± SEM). GSK101-induced 86Rb+ efflux was inhibited in the presence of 1 mM GdCl3 (empty squares, n = 3, mean ± SEM). f, GSK2193874 (GSK219) inhibition of Xenopus TRPV4 and TRPV4cryst. GSK101 activation (40 nM) of wild type TRPV4 (black diamonds, n = 3, mean ± SEM) and TRPV4cryst (black squares, n = 3, mean ± SEM) in CosM6 cells was assessed by relative efflux of 86Rb+. Basal 86Rb+ efflux was measured in cells transfected with empty vector and treated with GSK101 (40 nM) (black circles, n = 3, mean ± SEM). GSK219 (10 μM) partially inhibits GSK101-induced 86Rb+ effluxes for both wild type Xenopus TRPV4 (grey diamonds, n = 3, mean ± SEM) and TRPV4cryst (grey squares, n = 3, mean ± SEM).
Supplementary Figure 3 Cryo-EM reconstruction of Xenopus TRPV4cryst.
a, Flowchart of cryo-EM data processing. b, Fourier shell correlation plot for half-maps calculated in FREALIGN. The overall resolution is estimated to be 3.84 Å on the basis of the FSC = 0.143 cut-off criterion. c, Angular distribution plot for particles in the reconstruction. d, Sharpened cryo-EM density map colored according to local resolution using ResMap.
Supplementary Figure 4 Model building into the cryo-EM density map.
a, Representative density fragments with the refined TRPV4 model. b, Fourier shell correlation plots for refined model and half-map 1 (FSC work, red), refined model and half-map 2 (FSC free, blue) and refined model and full map (FSC sum, black).
Supplementary Figure 5 Sequence alignment of TRPV channels.
Sequences of Xenopus tropicalis TRPV4 (xTRPV4, Gene ID: 100496204), Homo sapiens TRPV4 (hTRPV4, Gene ID: 59341), Homo sapiens TRPV1 (hTRPV1, Gene ID: 7442), Homo sapiens TRPV2 (hTRPV2, Gene ID: 51393), Homo sapiens TRPV3 (hTRPV3, Gene ID: 162514), Homo sapiens TRPV5 (hTRPV5, Gene ID: 56302), and Homo sapiens TRPV6 (hTRPV6, Gene ID: 55503) were aligned. Secondary structure elements of xTRPV4 are shown above the sequence, and dashed lines indicate unresolved regions in the cryo-EM structure. Disease mutations discussed in the main text are highlighted in the same colors as in Fig. 6a.
Supplementary Figure 6 Superposition of the S1–S4 domains.
Orthogonal views of superposition of TRPV4 (green) and TRPV1 (grey) S1–S4 domains (PDB: 3J5P).
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Deng, Z., Paknejad, N., Maksaev, G. et al. Cryo-EM and X-ray structures of TRPV4 reveal insight into ion permeation and gating mechanisms. Nat Struct Mol Biol 25, 252–260 (2018). https://doi.org/10.1038/s41594-018-0037-5
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DOI: https://doi.org/10.1038/s41594-018-0037-5