Abstract
Ca2+-activated, non-selective (CAN) ion channels sense increases of the intracellular Ca2+ concentration, producing a flux of Na+ and/or K+ ions that depolarizes the cell, thus modulating cellular Ca2+ entry. CAN channels are involved in cellular responses such as neuronal bursting activity and cardiac rhythm. Here we report the electron cryo-microscopy structure of the most widespread CAN channel, human TRPM4, bound to the agonist Ca2+ and the modulator decavanadate. Four cytosolic C-terminal domains form an umbrella-like structure with a coiled-coil domain for the ‘pole’ and four helical ‘ribs’ spanning the N-terminal TRPM homology regions (MHRs), thus holding four subunits in a crown-like architecture. We observed two decavanadate-binding sites, one in the C-terminal domain and another in the intersubunit MHR interface. A glutamine in the selectivity filter may be an important determinant of monovalent selectivity. Our structure provides new insights into the function and pharmacology of both the CAN and the TRPM families.
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Acknowledgements
Cryo-EM data was collected at the David Van Andel Advanced Cryo-Electron Microscopy Suite in the Van Andel Research Institute (VARI) and we are grateful to G. Zhao and X. Meng for technical support. We thank C. Xu for help with SerialEM, the HPC team at VARI for computational support and D. Nadziejka for proofreading. This work is supported by internal funding from VARI.
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W.L. designed the project. P.A.W., Y.H. and W.L. purified TRPM4 and performed cryo-EM data collection and processing. W.S. performed electrophysiological experiments. W.L. and J.D. analysed the data and wrote the manuscript. All the authors contributed in preparing the manuscript.
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Extended data figures and tables
Extended Data Figure 1 Cryo-EM analysis of TRPM4 in complex with agonist calcium and the modulator DVT.
a, Representative electron micrograph. b, Selected two-dimensional class averages of the electron micrographs. c, The gold-standard Fourier shell correlation curves for the electron microscopy maps are shown in black and the FSC curves between the atomic model and the final EM map are shown in blue. d, Angular distribution of particles used for refinement. e–g, Local resolution estimation. The map is coloured according to local resolution estimation.
Extended Data Figure 4 Densities of DVT1, DVT2 and neighbouring protein.
a–d, DVT1. e–h, DVT2. Protein is shown in cartoon representation, whereas DVT molecules are shown in spheres and lines. The densities are contoured at different σ levels (from left to right, 0.03 (a, e), 0.04 (b, f), 0.05 (c, g) and 0.06 (d, h)), showing strong densities of DVT molecules and clear boundaries between DVT molecules and neighbouring protein.
Extended Data Figure 5 Calcium activation and DVT modulation of TRPM4.
a, Application of 0.5 mM Ca2+ onto inside-out patches pulled from HEK-293 cells transfected with TRPM4 plasmid elicited currents at +60 mV. Flufenamic acid (FA, 100 μM) blocked the Ca2+ induced current by 98 ± 6% (mean ± s.e.m., n = 3 cells). b, 100 μM ATP4− blocked Ca2+ induced current by 81 ± 11% (n = 3 cells). c, Top, an inside-out patch showing single channel activity when exposed to 0.5 mM Ca2+. Single channel current had a mean amplitude of 1.8 pA, corresponding to a single channel conductance of 30 pS. Bottom, channel activities were not observed from non-transfected cells (n = 4 cells). d–f, After application of 0.5 mM (d), 1 mM (e) or 5 mM (f) CaCl2, alternating voltage commands were delivered (±100 mV, 1 s pulses). Once the current amplitude at both voltages stabilized, 10 μM DVT was co-applied with CaCl2. At 0.5 mM Ca2+, 10 μM DVT blocked Ca2+ induced currents by 67 ± 5% (n = 4 cells, P = 0.001) at +100 mV and did not change current amplitude at −100 mV (n = 4 cells, P = 0.148). At 1 mM Ca2+, 10 μM DVT blocked Ca2+ induced currents by 56 ± 14% (n = 4 cells, P = 0.013) at +100 mV while 10 μM DVT potentiated the currents by 322 ± 83% at −100 mV (n = 4 cells, P = 0.030). At 5 mM Ca2+, 10 μM DVT did not change current amplitude (n = 4 cells, P = 0.517) but potentiated Ca2+ induced currents at −100 mV by 520 ± 147% (n = 4 cells, P = 0.035). Two-sided t-tests were used for statistical comparisons. Observations of the inhibitory effect of DVT at 0.5 mM Ca2+ is more pronounced compared to previously reported data21, where the authors only occasionally saw inhibitory effect of DVT at positive potentials, and the extent of inhibition was less pronounced. The reason for this discrepancy is not clear; we suspect differences in construct, solution composition, solution pH, cell line, transfection methods, other variations in experimental protocols or combinations of these factors. Although the effects of DVT on current amplitude varied with calcium concentration, 10 μM DVT linearized the current–voltage relationship in all three calcium concentrations (see g–l). g–i, 500 ms voltage pulses ranging from −140 mV to 140 mV from a holding potential of 0 mV (20 mV step size) were used to obtain the current–voltage relationship. Currents recorded in the presence of 0.5 mM (g, left), 1 mM (h, left) or 5 mM (i, left) Ca2+. Currents recorded in the presence of 0.5 mM (g, right), 1 mM (h, right) or 5 mM (i, right) Ca2+ with 10 μM DVT from the same patch. j–l, Currents recorded with Ca2+ and Ca2+ and DVT (Ca2+ concentrations as in g–i) at different voltages, data are normalized to the amplitude of current recorded with Ca2+ at 140 mV. g–l, Data are mean ± s.e.m. from four (h, i, k, l) or five (g, j) cells, experiments repeated three times for each cell. Leak currents were obtained by running the same voltage command when no calcium was applied and were subtracted from the currents recorded in Ca2+ for all experiments in d–l.
Extended Data Figure 6 Structure of TRPM4.
a, TRPM4 tetramer viewed parallel to the membrane. b–d, Slice views of a, viewed from parallel to the membrane (b), from the extracellular side of the membrane (c), or from the cytosolic side (d). The four subunits are in blue, pink, green and yellow.
Extended Data Figure 7 Secondary structure prediction of human TRPM4 and sequence alignment of TRPM4, TRPM5, TRPM2, TRPM8, TRPA1 and NOMPC.
The NOMPC is from Drosophila, whereas all the other proteins are human. The secondary structure prediction of TRPM4 was done using the JPred online server. The sequences were aligned using the Clustal Omega program on the Uniprot website and coloured using BLOSUM62 score by conservation. Residues that coordinate DVT1 or DVT2 in TRPM4 are marked with filled circles or triangles, respectively. Residues that correspond to R0, Q1, R2, R3, R4, K5 and R6 in the S4 of Kvchim (PDB accession number 2R9R, Extended Data Fig. 8) are marked with 0, 1, 2, 3, 4, 5 and 6, respectively.
Extended Data Figure 8 Comparison of the voltage sensor domain (S1–S4) in TRPM4 and in Kvchim.
a, b, The S4 and S4–S5 linker in TRPM4 (a) or Kvchim (b) are in green or orange, respectively. The residues at positions R0, Q1, R2, R3, R4, K5 and R6 in Kvchim, and the corresponding residues in TRPM4 are shown as sticks. The two positively charged residues (K914 and K919) in S4–S5 linker of TPRM4 are also shown as sticks.
Extended Data Figure 9 Comparison of the pore in TRPM4 (yellow) and in TRPV1 (grey).
The P loop and S6 of two subunits are shown in cartoon representation and the side chains of restriction residues are shown as sticks. Restriction residues in TRPM4 or TRPV1 are in black or green, respectively. Purple, green, and red spheres define radii of >2.3, 1.2–2.3, and <1.2 Å, respectively.
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Winkler, P., Huang, Y., Sun, W. et al. Electron cryo-microscopy structure of a human TRPM4 channel. Nature 552, 200–204 (2017). https://doi.org/10.1038/nature24674
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DOI: https://doi.org/10.1038/nature24674
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