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Opening of the human epithelial calcium channel TRPV6

Nature volume 553, pages 233–237 (2018)Cite this article

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Abstract

Calcium-selective transient receptor potential vanilloid subfamily member 6 (TRPV6) channels play a critical role in calcium uptake in epithelial tissues1,2,3,4. Altered TRPV6 expression is associated with a variety of human diseases5, including cancers6. TRPV6 channels are constitutively active1,7,8 and their open probability depends on the lipidic composition of the membrane in which they reside; it increases substantially in the presence of phosphatidylinositol 4,5-bisphosphate7,9. Crystal structures of detergent-solubilized rat TRPV6 in the closed state have previously been solved10,11. Corroborating electrophysiological results3, these structures demonstrated that the Ca2+ selectivity of TRPV6 arises from a ring of aspartate side chains in the selectivity filter that binds Ca2+ tightly. However, how TRPV6 channels open and close their pores for ion permeation has remained unclear. Here we present cryo-electron microscopy structures of human TRPV6 in the open and closed states. The channel selectivity filter adopts similar conformations in both states, consistent with its explicit role in ion permeation. The iris-like channel opening is accompanied by an α-to-π-helical transition in the pore-lining transmembrane helix S6 at an alanine hinge just below the selectivity filter. As a result of this transition, the S6 helices bend and rotate, exposing different residues to the ion channel pore in the open and closed states. This gating mechanism, which defines the constitutive activity of TRPV6, is, to our knowledge, unique among tetrameric ion channels and provides structural insights for understanding their diverse roles in physiology and disease.

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Figure 1: Function and cryo-EM of hTRPV6.
Figure 2: Structure of hTRPV6.
Figure 3: Open and closed ion channel pore.
Figure 4: Activation-related lipid binding pocket.
Figure 5: TRPV6 channel gating mechanism.

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Acknowledgements

We thank T. Rohacs for advice on electrophysiological recordings, J. Frank for comments on the manuscript, H. Kao for computational support and members of the E.C. Greene laboratory for assistance with their fluorimeter. L.L.M. and E.C.T. are supported by the NIH (T32 GM008224 and F31 NS093838, respectively). A.I.S. is supported by the NIH (R01 CA206573, R01 NS083660), and Amgen Young Investigator and Irma T. Hirschl Career Scientist awards. Data were collected at the Columbia University Medical Center cryo-EM facility and at the Simons Electron Microscopy Center and National Resource for Automated Molecular Microscopy located at the New York Structural Biology Center, supported by grants from the Simons Foundation (349247), NYSTAR, and the NIH (GM103310).

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Author notes

  1. Luke L. McGoldrick and Appu K. Singh: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Biochemistry and Molecular Biophysics, Columbia University, 650 West 168th Street, New York, 10032, New York, USA

    Luke L. McGoldrick, Appu K. Singh, Kei Saotome, Maria V. Yelshanskaya, Edward C. Twomey, Robert A. Grassucci & Alexander I. Sobolevsky

  2. Integrated Program in Cellular, Molecular, and Biomedical Studies, Columbia University, 650 West 168th Street, New York, 10032, New York, USA

    Luke L. McGoldrick & Edward C. Twomey

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Contributions

L.L.M., A.K.S. and A.I.S. designed the project, built models and analysed data. L.L.M. and A.K.S. carried out Fura-2 experiments, cryo-EM data collection and processing. L.L.M., A.K.S., K.S. and M.V.Y. developed expression and purification protocols. L.L.M., A.K.S. and K.S. designed constructs and prepared protein samples. M.V.Y. carried out electrophysiology experiments. E.C.T advised on cryo-EM workflow. R.A.G and E.C.T assisted with microscope operation. A.I.S. supervised the project. L.L.M., A.K.S., K.S., M.V.Y., E.C.T. and A.I.S. wrote the manuscript.

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Correspondence to Alexander I. Sobolevsky.

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Reviewer Information Nature thanks R. Gaudet, X. Li and Y. Mori for their contribution to the peer review of this work.

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Extended data figures and tables

Extended Data Figure 1 Functional characterization of wild-type and mutant hTRPV6 channels.

a–d, Whole-cell patch-clamp recordings from HEK 293 cells expressing wild-type hTRPV6 (a), hTRPV6(R470E) (b), hTRPV6(Q483A) (c) and hTRPV6(A566T) (d). Leak-subtracted currents (blue) are shown in response to voltage ramp protocols illustrated above the recordings. Although the shapes of the currents for wild-type and mutant hTRPV6 channels were similar, their amplitudes were different. The average current amplitudes at −60-mV membrane potential (mean ± s.e.m.) were 3,171 ± 767 pA (n = 11) for wild-type hTRPV6; 918 ± 267 pA (n = 9) for hTRPV6(R470E); 2,239 ± 398 pA (n = 7) for hTRPV6(Q483A); and 145 ± 52 pA (n = 5) for hTRPV6(A566T). e–h, Kinetics of calcium uptake using Fura-2 AM ratiometric fluorescence measurements. Representative fluorescence curves are shown for wild-type hTRPV6 (e), hTRPV6(R470E) (f), hTRPV6(Q483A) (g) and hTRPV6(A566T) (h) in response to application of 2 mM Ca2+ (arrow). Exponential fits are shown in red, with the time constants indicated. Over five measurements, the time constants (mean ± s.e.m.) were 4.2 ± 0.5 s for hTRPV6; 47 ± 13 s for hTRPV6(R470E); 18.9 ± 0.8 s for hTRPV6(Q483A); and 121 ± 12 s for hTRPV6(A566T). At n = 5 and P = 0.05, the time constant values for wild-type and mutant channels were statistically different (two-sided t-test). i, j, Fluorescence curves for wild-type hTRPV6 (i) and hTRPV6(R470E) (j) in response to application of 2 mM Ca2+ after pre-incubation of cells in different concentrations of 2-APB. These experiments were repeated independently three times with similar results. k, Dose–response curves for 2-APB inhibition calculated for wild-type hTRPV6 (black) and hTRPV6(R470E) (red) (n = 3 for all measurements). The changes in the fluorescence intensity ratio at 340 and 380 nm (F340/F380) evoked by addition of 2 mM Ca2+ after pre-incubation with various concentrations of 2-APB were normalized to the maximal change in F340/F380 after addition of 2 mM Ca2+ in the absence of 2-APB. Curves through the data points are fits with the logistic equation, with the mean ± s.e.m. values of half maximal inhibitory concentration (IC50), 274 ± 27 μM and 85 ± 5 μM, and the maximal inhibition, 72.6 ± 2.7% and 50.3 ± 1.1%, for hTRPV6 and hTRPV6(R470E), respectively. The leftward shift of the 2-APB dose–response curve of hTRPV6(R470E), when compared to the dose–response curve of wild-type hTRPV6, indicates an increased affinity of the channel for 2-APB. This is likely to result from the R470E mutation reducing the affinity of the channel for an activating lipid ligand. On the other hand, the reduced maximum inhibition of hTRPV6(R470E) at high concentrations of 2-APB, when compared to that of wild-type hTRPV6, indicates a reduced efficacy of 2-APB that could be a result of the R470E mutation disrupting the mechanism by which 2-APB binding is allosterically coupled to channel gating.

Extended Data Figure 2 Overview of single-particle cryo-EM for hTRPV6 in nanodiscs.

a, Example cryo-EM micrograph for hTRPV6 in nanodiscs with example particles circled in red. b, Orientations of particles that contributed to the final 3.6 Å reconstruction. Longer red rods represent orientations with more particles. c, Local resolution mapped on density at 0.013 threshold level (UCSF Chimera) calculated using Resmap and two unfiltered half maps, with the highest resolution observed for the channel core. d, FSC curve calculated between half maps. e, Cross-validation FSC curves for the refined model versus unfiltered half maps (only half map1 was used for PHENIX refinement) and the unfiltered summed map.

Extended Data Figure 3 Overview of single-particle cryo-EM for hTRPV6 in amphipols and comparison to the reconstruction in nanodiscs.

a, Example cryo-EM micrograph for hTRPV6 in amphipols with example particles circled in red. b, Reference-free 2D class averages of hTRPV6 in amphipols illustrating different particle orientations. c, Local resolution mapped on density at 0.01 threshold level (UCSF Chimera) calculated using Resmap and two unfiltered half maps, with the highest resolution observed for the channel core. d, Orientations of particles that contribute to the final 4.0 Å reconstruction. Longer red rods represent orientations that comprise more particles. e, FSC curve calculated between half maps. f, Cross-validation FSC curves for the refined model versus unfiltered half maps (only half map1 was used for PHENIX refinement) and the unfiltered summed map. g, h, Comparison of putative lipid densities for hTRPV6 in amphipols (g) and nanodiscs (h), filtered to the same (4.0 Å) resolution and shown at 3.5σ as purple mesh.

Extended Data Figure 4 Cryo-EM density for hTRPV6 in nanodiscs.

a, Cryo-EM density at 4σ for a single hTRPV6 subunit, with the protein shown in ribbon and coloured according to domains. b–g, Fragments of the hTRPV6 transmembrane domain with the corresponding cryo-EM densities.

Extended Data Figure 5 Fitting lipids into cryo-EM density.

a–c, Molecules of phosphatidylethanolamine (PE, a), phosphatidylcholine (PC, b) and phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2, c) fitted into the site 4 lipid density shown at 3.5σ as purple mesh. d–f, Molecules of cholesterol (d), CHS (e) and PtdIns(4,5)P2 (f) fitted into the site 2 putative activating lipid density shown at 5.3σ.

Extended Data Figure 6 Overview of single-particle cryo-EM for hTRPV6(R470E) in amphipols.

a, Example cryo-EM micrograph for hTRPV6(R470E) in amphipols with example particles circled in red. b, Reference-free two-dimensional class averages of hTRPV6(R470E) in amphipols illustrating different particle orientations. c, Local resolution mapped on density at 0.017 threshold level (UCSF Chimera) calculated using Resmap and two unfiltered half maps, with the highest resolution observed for the channel core. d, Orientations of particles that contribute to the final 4.2 Å reconstruction. Longer red rods represent orientations that comprise more particles. e, FSC curve calculated between half maps. f, Cross-validation FSC curves for the refined model versus unfiltered half maps (only half map1 was used for PHENIX refinement) and the unfiltered summed map.

Extended Data Figure 7 Overview of single-particle cryo-EM for rTRPV6 in CNW11 nanodiscs.

a, Example cryo-EM micrograph for rTRPV6 in CNW11 nanodiscs with example particles circled in red. b, Reference-free 2D class averages of rTRPV6 in CNW11 nanodiscs illustrating different particle orientations. c, Local resolution mapped on density at 0.011 threshold level (UCSF Chimera) calculated using Resmap and two unfiltered half maps, with the highest resolution observed for the channel core. d, Orientations of particles that contribute to the final 3.9 Å reconstruction. Longer red rods represent orientations that comprise more particles. e, FSC curve calculated between half maps. f, Cross-validation FSC curves for the refined model versus unfiltered half maps (only half map1 was used for PHENIX refinement) and the unfiltered summed map.

Extended Data Figure 8 Comparison of cryo-EM and crystal structures of rTRPV6, cryo-EM structures of hTRPV6(R470E) and rTRPV6 and regions in hTRPV6 and hTRPV6(R470E) encompassing D489 and T581.

a–c, Superimposed are the transmembrane domain of a single subunit (a), and the pore-forming region viewed parallel to the membrane (b) or intracellularly (c) from the cryo-EM (green) and crystal (orange) structures of rTRPV6. Only two of four rTRPV6 subunits are shown in b, with the front and back subunits omitted for clarity. Residues lining the selectivity filter and gate are shown as sticks. d, e, Superposition of the P loop and S6 in cryo-EM structures of hTRPV6(R470E) (blue) and rTRPV6 (green), viewed parallel to the membrane (d) and intracellularly (e). In d, only two of four subunits are shown, with the front and back subunits removed for clarity. The residues lining the pore are shown as sticks. f, g, Regions in hTRPV6 (f) and hTRPV6(R470E) (g) encompassing D489 and T581. The closest distance between D489 and T581 is indicated by dashed lines. Note, M485 and M577 either surround the potentially interacting D489 and T581 (f, hTRPV6) or reside between these residues (g, hTRPV6(R470E)), apparently preventing their interaction. Blue mesh shows cryo-EM density at 4σ.

Extended Data Figure 9 Structural superposition and sequence alignment of the pore domain in tetrameric ion channels.

a–i, Pairwise superposition of the pore domain in hTRPV6 with rat TRPV118 (a, PDB ID: 5IRX; r.m.s.d. = 2.065 Å); rabbit TRPV221 (b, PDB ID: 5AN8; r.m.s.d. = 3.757 Å); rat TRPV224 (c, PDB ID: 5HI9; r.m.s.d. = 4.399 Å); human TRPA123 (d, PDB ID: 3J9P; r.m.s.d. = 1.429 Å); human PKD225 (e, PDB ID: 5T4D; r.m.s.d. = 2.676 Å); KcsA from Streptomyces lividans47 (f, PDB ID: 1BL8; r.m.s.d. = 2.708 Å); MthK from Methanothermobacter thermautotrophicum48 (g, PDB ID: 1LNQ; r.m.s.d. = 2.947 Å); rat Shaker49 (h, PDB ID: 2A79; r.m.s.d. = 2.487 Å); and rat GluA2 AMPA-subtype iGluR28 (i, PDB ID: 5WEO; r.m.s.d. = 2.044 Å). j, Sequence alignment for the pore region of human TRPV3–TRPV6, TRPA1 and PKD2, rat TRPV1, 2, and 6, Shaker and GluA2, rabbit TRPV2 and bacterial K+ channels KcsA and MthK. The selectivity filter residues in K+ channels and gating hinge residues in S6 (M3 in GluA2) are coloured red. k, Aligned sequence logos for TRPV channels in S6, generated by WebLogo50 from 1,200 TRPV1–TRPV6 sequences. The red rectangle and arrow indicate the position of the alanine gating hinge in TRPV6. The relatively small side chain residues threonine or alanine next to the gating hinge alanine position in TRPV5 and TRPV6, instead of the bulky hydrophobic phenylalanine or tyrosine in TRPV1–TRPV4, might be critical for the α-to-π-helical transition in S6 during channel opening.

Extended Data Table 1 Cryo-EM data collection, refinement and validation statistics
Full size table

Supplementary information

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Conformational changes between closed and open states of human TRPV6

A morph between closed and open states of human TRPV6 represented by the hTRPV6-R470E and wild type hTRPV6 structures, respectively. Shown is the pore domain viewed intracellularly and illustrating the iris-like pore opening or viewed parallel to the membrane, with the front and back subunits disappearing for clarity, and illustrating the α-to-π helical transition in S6 during channel opening. Residues around the gate and critical for permeation aspartates D542 are shown in stick representation. (MP4 18935 kb)

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McGoldrick, L., Singh, A., Saotome, K. et al. Opening of the human epithelial calcium channel TRPV6. Nature 553, 233–237 (2018). https://doi.org/10.1038/nature25182

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