Structures of the TRPM5 channel elucidate mechanisms of activation and inhibition

Abstract

The Ca²⁺-activated TRPM5 channel plays essential roles in taste perception and insulin secretion. However, the mechanism by which Ca²⁺ regulates TRPM5 activity remains elusive. We report cryo-EM structures of the zebrafish TRPM5 in an apo closed state, a Ca²⁺-bound open state, and an antagonist-bound inhibited state. We define two novel ligand binding sites: a Ca²⁺ site (Ca_ICD) in the intracellular domain and an antagonist site in the transmembrane domain (TMD). The Ca_ICD site is unique to TRPM5 and has two roles: modulating the voltage dependence and promoting Ca²⁺ binding to the Ca_TMD site, which is conserved throughout TRPM channels. Conformational changes initialized from both Ca²⁺ sites cooperatively open the ion-conducting pore. The antagonist NDNA wedges into the space between the S1-S4 domain and pore domain, stabilizing the transmembrane domain in an apo-like closed state. Our results lay the foundation for understanding the voltage-dependent TRPM channels and developing new therapeutic agents.

Extended Data Figure 1:. Patch-clamp analysis of Ca_ICD mutants.

Representative traces of inside-out voltage-clamp measurements (+200 mV to −200 mV) from tsA201 cells overexpressing human TRPM4 (hsTRPM4), human TRPM5 (hsTRPM5), and zebrafish TRPM5 (drTRPM5) channels. Patches were stimulated with 1, 30, 100, or 1000 μM Ca²⁺. The number of patches analyzed: a, hsTRPM4(WT): 1 μM Ca²⁺ [4], 100 μM [4], 1000 μM [4] from 3 transfections; b, drTRPM5(E337A): 1 μM Ca²⁺ [6], 30 μM [6], 100 μM [4], 1000 μM [4] from 8 transfections; c, drTRPM5(C324A): 1 μM Ca²⁺ [6], 30 μM [6], 100 μM [6], 1000 μM [6] from 4 transfections; d, drTRPM5(D333A): 1 μM Ca²⁺ [5], 30 μM [4], 100 μM [5], 1000 μM [3] from 3 transfections; e, drTRPM5(E212A): 1 μM Ca²⁺ [4], 30 μM [3], 100 μM [4], 1000 μM [3] from 3 transfections; f, drTRPM5(D336A): 1 μM Ca²⁺ [3], 30 μM [3], 100 μM [3], 1000 μM [3] from 2 transfections; g, hsTRPM5(WT): 1 μM Ca²⁺ [5], 30 μM [5] from 3 transfections; h, hsTRPM5(E351A): 1 μM Ca²⁺ [3], 30 μM [3] from 1 transfection; and i, hsTRPM5(E560A): 1 μM Ca²⁺ [5], 30 μM [3], 100 μM [3], 1000 μM [3] from 5 transfections. j–r, Mean current (50 ms) of experiments were plotted versus indicated voltage. The +200 mV clamp was chosen for normalization. Horizontal bars represent SEM. The current-voltage relation plots of drTRPM5(E337A) are identical to those presented in Fig. 3e. s–u, Individual patch clamp measurements I_−200mV / I_+200mV, I_+200mV, I_−200mV, of experiments are shown as individual points, where bars represent mean values. v, w, Currents of drTRPM5 WT and Ca_ICD mutants plotted as a function of Ca²⁺ concentration for voltage clamps of −200 mV and +200 mV. Symbols represent mean current and horizontal bar is SEM.

Extended Data Figure 2:. Cyro-EM analysis of apo–TRPM5.

a, The representative 2D class average of apo–TRPM5. b, The Fourier shell correlation (FSC) curves for the apo–TRPM5. The cryo-EM map FSC is shown in black and the model vs. map FSC is shown in red. The map resolution was determined by the gold-standard FSC at 0.143 criterion, whereas the model vs. map resolution was determined by a threshold of 0.5. c, The angular distribution of particles that gave rise to the apo–TRPM5 cryo-EM map reconstruction. d, A schematic domain organization of a single TRPM5 subunit. Secondary structures and important domains are labeled. e, The atomic model of a single TRPM5 subunit in cartoon representation. The domains are colored as in (d). The left and right panels are two different views of the same subunit rotated 180° along the central axis.

Extended Data Figure 3:. Cyro-EM analysis of TRPM5 in the presence of different concentrations of Ca²⁺, or in the presence of Ca²⁺ and NDNA.

a and d, The representative 2D class average the 5mM Ca²⁺ dataset (a) and 6 μM Ca²⁺ dataset (d), respectively. b and e, The Fourier shell correlation (FSC) curves for the 5mM Ca²⁺ dataset (b) and 6 μM Ca²⁺ dataset (e). The cryo-EM map FSC is shown in black and the model vs. map FSC is shown in red. The map resolution is determined by the gold-standard FSC at 0.143 criterion, whereas the model vs. map resolution is determined by a threshold of 0.5. c and f, The angular distribution of particles that give rise to the cryo-EM map reconstruction for 5mM Ca²⁺ dataset (c) and 6 μM Ca²⁺ dataset (f). g, The close up view of the Ca_TMD and Ca_ICD of the 6 μM Ca²⁺ dataset. From left to right, Ca_TMD of apo–TRPM5(6 μM Ca²⁺), Ca_TMD of Ca²⁺–TRPM5(6 μM Ca²⁺), Ca_ICD of apo–TRPM5(6 μM Ca²⁺), and Ca_ICD of Ca²⁺–TRPM5(6 μM Ca²⁺). The cryo-EM densities are shown in mesh representation. The expected Ca²⁺ density is indicated by a circle. h, The representative 2D class average the (Ca²⁺, NDNA)–TRPM5 dataset. i, The Fourier shell correlation (FSC) curves for the (Ca²⁺, NDNA)–TRPM5. The cryo-EM map FSC is shown in black and the model vs. map FSC is shown in red. The map resolution was determined by the gold-standard FSC at 0.143 criterion, whereas the model vs. map resolution was determined by a threshold of 0.5. j, The angular distribution of particles that gave rise to the (Ca²⁺, NDNA)–TRPM5 cryo-EM map reconstruction.

Extended Data Figure 4:. Local resolution estimation of TRPM5 structures and representative densities.

a-d, The local resolution estimation for apo–TRPM5(GDN) (a), Ca²⁺–TRPM5(GDN) (b), Ca²⁺–TRPM5(E337A)(GDN) consensus (c), (Ca²⁺, NDNA)–TRPM5(GDN) (d). For each map, a side view, a top-down view of the TMD from the extracellular side, and a focused side view of the S6 and pore helix are shown. The color bar unit is in Ångstroms. e, Representative densities from Ca²⁺–TRPM5(GDN) map. For the GDN density, one maltose group of the molecule is not resolved in the cryo-EM density map.

Extended Data Figure 5:. The gate and the selectivity filter of TRPM5.

a, Cryo-EM densities of the Ca_ICD site, contoured at 0.018. b, Cryo-EM densities of the Ca_TMD site, contoured at 0.022. c, Cryo-EM densities of the water molecule and residues in the selectivity filter, contoured at 0.023. Hydrogen bonds are shown as solid yellow lines. The “lower” water molecule is surrounded by the sidechain of Q906 and the backbone oxygen atoms of F904 and G905, forming three hydrogen bonds. d, Cryo-EM densities of I966, which forms the channel gate, contoured at 0.03. e, The selectivity filter formed by two layers of ordered water molecules (blue spheres) and backbone oxygen atoms (pink spheres) of G905. f and g, The two hydration layers the selectivity filter viewed from the extracellular side. Upper layer in (f) and lower layer in (g).

Extended Data Figure 6:. Comparison of TRPM5 with other TRPM channels.

a, A structural comparison between Ca²⁺–TRPM5 and TRPM4 (PDBID: 6BQV). A single subunit is in color and shown as a cartoon. The TRPM5 channel is more compact, but wider than, the TRPM4 channel. b, An overlap of the selectivity filter of Ca²⁺–TRPM5 (red) and TRPM4 (yellow). c, A comparison of the Ca_TMD site for the available TRPM members. From left to right, drTRPM5, hsTRPM4 (6BQV), drTRPM2 (6DRJ), nvTRPM2 (6CO7), hsTRPM2 (6PUS), and pmTRPM8 (6O77)^,,,,; Shown in parentheses are the PDBIDs. d, Comparison of the “square” helices in TRPM2 (6PUO), TRPM4 (6BQR), TRPM5, TRPM7 (5ZX5), TRPM8 (6O6A); Shown in parentheses are the PDBIDs^,,,. Only TRPM5 has a broken square helix. e, A sequence alignment of the square helix across different TRPM5 orthologs and TRPM family members. Red indicates that the α-helix is observed in structures. For TRPM1, TRPM3, and TRPM6, in which no structures are currently available, the helical annotation is based on the secondary structure prediction from PSIPRED server.

Extended Data Figure 7:. Comparison of (Ca²⁺, NDNA)–TRPM5 with apo–TRPM5 and Ca²⁺–TRPM5.

a, the chemical structure of N’-(3,4-dimethoxybenzylidene)-2-(naphthalen-1-yl)acetohydrazide (NDNA). b, Two close-up views of the cryo-EM densities of NDNA molecule. The surrounding protein structural element is shown in cartoon representation. c, Comparison of the NDNA binding site between (Ca²⁺, NDNA)–TRPM5 and apo–TRPM5 structures. The W869 is flipped in the (Ca²⁺, NDNA)–TRPM5 structure (cyan) compared to that in apo–TRPM5 structure (blue). d, Overlay of (Ca²⁺, NDNA)–TRPM5 (cyan) with apo–TRPM5 (blue) and Ca²⁺–TRPM5 (red) structures view from the intracellular side. One subunit is shown in cartoon representation and the other three subunits are in surface representation. The ICD of (Ca²⁺, NDNA)–TRPM5 adopts an intermediate state compared to the apo–TRPM5 and Ca²⁺–TRPM5 structures. e, The superimposition of the S1-S4 domain between (Ca²⁺, NDNA)–TRPM5 (cyan) and apo–TRPM5 (blue). f, The superimposition of the S1-S4 domain between (Ca²⁺, NDNA)–TRPM5 (cyan) and Ca²⁺–TRPM5 (red). g, A close-up view of the Ca_TMD site in (Ca²⁺, NDNA)–TRPM5 structure. The Q771 moved away from Ca_TMD. h and i, An overlay of the pore domain between (Ca²⁺, NDNA)–TRPM5 (cyan) with apo–TRPM5 (blue) (h) and Ca²⁺–TRPM5 (red) (i) structures viewed from the extracellular side.

Extended Data Figure 8:. Patch-clamp electrophysiology experiments of Ca_TMD mutants of zebrafish TRPM5.

a, Representative whole-cell current traces of tsA overexpressing WT and Ca_TMD mutant TRPM5 channels. Clamps were imposed from +200 mV to −200 mV. The number of cells measured were tsA201 [n = 4 cells], TRPM5(WT) [5], TRPM5(E768A) [5], TRPM5(Q771A) [4], TRPM5(N794A) [4], TRPM5(D797A) [4], and TRPM5(E994A) [4] from 2–3 transfections. b, Mean current amplitudes of experiments in (a) were measured at 50 ms and plotted as a function of clamp voltage. Horizontal bars represent SEM. c, Individual measurements at clamps of +200 mV (I_+200mV) and −200 mV (I_−200mV) of experiments in (a) are shown as individual points, with bars representing mean values.

Extended Data Figure 9:. Patch-clamp electrophysiology experiments on the NDNA binding site mutants.

a–j, Current voltage relations of whole-cell measurements in tsA cells over-expressing WT and mutant TRPM5 channels. Symbols represents mean current and horizontal bars are SEM. k–n, Individual measurements (of experiments in a–j) for clamps of +100 mV and −100 mV. Each point represents an individual cell and bars represent mean current. Cells were first measured (clamps from −100 mV to +100 mV) in bath solution and then re-measured following bath perfusion of 10 μM NDNA. See the legend of Fig 4 for the number of cells used.

Extended Data Figure 10:. Ca²⁺–TRPM5(E337A) in GDN detergent.

a, The representative 2D class average of Ca²⁺–TRPM5(E337A). b, The FSC curve for the consensus map of Ca²⁺–TRPM5(E337A). The map resolution was determined by the gold-standard FSC at 0.143 criterion. c, The angular distribution of particles that give rise to the consensus map of Ca²⁺–TRPM5(E337A). d, The FSC curve for apo–TRPM5(E337A) (left) and Ca²⁺–TRPM5(E337A) (right). For each panel, the cryo-EM map FSC curve is shown in black and the model vs. map FSC is shown in red. The map resolution was determined by the gold-standard FSC at 0.143 criterion, whereas the model vs. map resolution was determined by a threshold of 0.5.

Figure 1:. Zebrafish TRPM5 current.

a, Representative traces of Ca²⁺-activated currents from membrane patches excised from tsA201 cells overexpressing zebrafish TRPM5 recorded in the inside-out patch-clamp configuration. Ca²⁺ of 1, 30, 100, and 1000 μM were superfused and voltage clamps were imposed from +200 mV to −200 mV with a final tail current pulse at −140 mV. Background currents were subtracted by interleaved measurements with a calcium-free solution. b, c, Current amplitudes (at 50 ms) of experiments in (a) were plotted as a function of clamp voltage for unnormalized (b) and +200 mV normalized current values (c). Replicates consists of: 1 μM Ca²⁺ [n = 12 patches], 30 μM [n = 6], 100 μM [n = 4], 1000 μM [n = 3] from 12 transfections. Error bars represent SEM. Tail current analysis was also performed (see Supplementary Figure 6). Source data for b and c are available online.

Figure 2:. The overall architecture.

a, The cryo-EM map of Ca²⁺–TRPM5 viewed parallel to the membrane. One subunit is highlighted in red. The unsharpened reconstruction is shown as transparent envelope in. b, The atomic model of Ca²⁺–TRPM5 condition in the same view as (a). The Ca²⁺ ions are shown as green spheres. c, The atomic model of (Ca²⁺, NDNA)–TRPM5 viewed parallel to the membrane. One subunit is highlight in cyan. The NDNA molecule is colored in orange. d–f, The ion-conducting pore in apo–TRPM5 (d), Ca²⁺–TRPM5 (e), and (Ca²⁺, NDNA)–TRPM5 (f) viewed parallel to the membrane. Purple, green, and red spheres define radii of >2.3, 1.2–2.3, and <1.2 Å, respectively. The pore region (shown in cartoon), residues (shown in sticks) forming the gate, and the selectivity filter in two subunits are depicted. Lower right panel: the channel gate viewed from the intracellular side; the distance between the Cα atoms of adjacent I966 residues is labeled. Upper right box: a cartoon representing two subunits of the apo state. The unoccupied/occupied Ca²⁺ sites are shown as unfilled/filled circles, respectively. The membrane area is shown as a gray background. g, Plot of pore radius along the pore axis.

Figure 3:. Ca²⁺ binding sites.

a, b, The Ca_TMD (a) and Ca_ICD (b) sites. Ca²⁺ is shown as a green sphere. The coordinating residues and water molecules are shown in sticks and spheres, respectively. Polar interactions are indicated by yellow bars. c, Sequence alignment of the Ca_TMD (top) and Ca_ICD site (bottom) among zebrafish TRPM5 (drM5), human TRPM5 (hsM5), human TRPM4 (hsM4), human TRPM2 (hsM2), and human TRPM8 (hsM8). The Ca²⁺ coordinating residues in zebrafish TRPM5 are indicated by asterisks, and conserved coordinating residues are in red. The residue numbers are according to zebrafish TRPM5 (UniProtID: S5UH55). Sequence segments are separated by vertical bars. d, Remodeling of the Ca_ICD site upon Ca²⁺ binding. Apo–TRPM5 and Ca²⁺–TRPM5 are in blue and red, respectively. Black arrows indicate the movement of E212 and D336. The MHR1/2 domains in apo–TRPM5 and Ca²⁺–TRPM5 are represented by blue and red surfaces, respectively, showing the movement of MHR1/2. e, Voltage-clamped current amplitudes were measured (as performed in Fig. 1) from inside-out patches pulled from tsA cells overexpressing E337A mutant channels. The number of patches are (1 μM Ca²⁺ [n = 7 patches], 30 μM [n = 7], 100 μM [n = 5], 1000 μM [n = 5] from 8 transfections). Error bars represent SEM. See Extended Data Fig. 1b for representative traces. f, The superimposition of Ca_ICD site in TRPM5 (red) and TRPM4 (yellow, PDBID: 6BQR), by aligning the helix α11 and its equivalent in human TRPM4 (residues 396–403). The coordinating residues are shown as sticks. Equivalent structural elements and residues in TRPM4 are labeled with a prime symbol. The orientation of helix α12 and its equivalent (α12’) in human TRPM4 (residues 372–386) are indicated by colored 3D arrows. The differences between α11 and α11’, and between E337 and E396’, are indicated by black arrows. Source data for e are available online.

Figure 4:. Effect and binding site of antagonist NDNA.

a, Voltage-clamped (+200 mV to −200 mV) calcium activated whole-cell currents from tsA201 cells over-expressing zebrafish WT TRPM5 were suppressed upon super-fusion of 10 μM NDNA. b, IC₅₀ of NDNA, 2.4 nM, was determined by plotting (I_{+200 mV}, _NDNA / I_{+200 mV}, _bath) using various NDNA concentrations (1 fM, 10 pM, 100 pM, 1 nM, 100 nM, 0.5 μM, 10 μM). Concentration is log (M). Each point represents the mean, and bars indicate SEM. Number of cells is indicated in brackets. From non-linear fitting, the Hill Slope is −0.5, and the 95% CI is 0.5 – 23 nM. c, The pore domain of (Ca²⁺, NDNA)–TRPM5 viewed from the extracellular side. The four bound NDNA molecule is shown in orange. Transmembrane helices surrounding a copy of NDNA is labeled. Prime symbol indicates the adjacent subunit. Ca_TMD is shown in green sphere. d and e, Two close-up views for the detailed interactions mediated by the NDNA. One TRPM5 subunit is colored in cyan, whereas the adjacent subunit is colored in light cyan. Polar interactions between NDNA and residues are indicated by black lines. g, h, Ratio of whole-cell current amplitudes in the presence and absence of NDNA (10 μM) for various TRPM5 mutants. Each point represents a single cell and bars denote mean value. The number of cells analyzed were: tsA (n = 3 cells, 2 transfections), WT (n = 4, 4 transfections), C796A (n = 3 cells, 3 transfections), I849A (n = 3 cells, 1 transfection), E853A (n = 5 cells, 4 transfections), I836A (n = 5 cells, 5 transfections), W793A (n = 3 cells, 2 transfections), V852A (n = 3 cells, 2 transfections), L833A (n = 6 cells, 4 transfections), W869A (n = 3 cells, 2 transfections). Source data for b, f and g are available online.

Figure 5:. The structures of the Ca_ICD-deficient mutant E337A.

a, The upper panel shows the consensus map obtained from the Ca²⁺–TRPM5(E337A) data. The cartoons in the lower panel represent the two conformations that have distinct occupancies at the Ca_TMD site, obtained by single subunit analysis of the same data: apo–TRPM5(E337A) in magenta and Ca²⁺–TRPM5(E337A) in cyan. The unoccupied Ca²⁺ sites are shown as unfilled circles; occupied Ca²⁺ sites are shown as green circles. The cell membrane is represented in gray. b–e, the Ca_ICD site (b) and Ca_TMD site (d) in apo–TRPM5(E337A), and the Ca_ICD site (c), and Ca_TMD site (e) in Ca²⁺–TRPM5(E337A). The cryo-EM densities are shown in black mesh. The Ca²⁺ density is shown as a green sphere. Unoccupied sites are indicated by a dashed gray circle. f, The superimpositions of the S1-S4 domain of the apo–TRPM5(E337A) (magenta) and the Ca²⁺–TRPM5(E337A) (cyan) structures. g, The superimpositions of the S1-S4 domain of the apo–TRPM5(E337A) (magenta) and the apo–TRPM5 (blue) structures. h, The superimpositions of the S1-S4 domain of the Ca²⁺–TRPM5(E337A) (cyan) and the Ca²⁺–TRPM5 (red) structures. i, Pore radius plot along the pore axis. j, k, Remodeling of the Ca_ICD site upon Ca²⁺ binding in TRPM5 (j) and TRPM4 (k). Apo–TRPM5 and Ca²⁺–TRPM5 are in blue and red, respectively (j). Apo–TRPM4 (PDBID: 6BQR) and Ca²⁺–TRPM4 (PDBID: 6BQV) are in gray and yellow, respectively. (k). The Cα atoms of key residues and the Ca²⁺ are shown as spheres. Shown in parentheses are the distances of the root-mean-square-deviation (RMSD) between S2 (residues 767–772 in TRPM5 and 827–832 in TRPM4) and S3 (residues 793–798 in TRPM5 and 864–869 in TRPM4), and the distances of the Cα movements of E994 in TRPM5 and E1068 in TRPM4.

Figure 6:. The signal transduction from ICD to TMD.

a, The superimposition of apo–TRPM5 (blue) and Ca²⁺–TRPM5 (red) structures by aligning the coiled-coil poles in the C-terminal domain (CTD), viewed parallel to the membrane. One subunit is also shown in cartoon representation. Ca²⁺ is shown as green spheres. b, The superimposition of the MHR1-4 domains of apo–TRPM5 (blue) and Ca²⁺–TRPM5 (red) by aligning the MHR3/4 domain, viewed parallel to the membrane. The rotation of the MHR1/2 relative to the MHR3/4 domain upon Ca²⁺ binding is indicated. The surfaces are outlined in blue for apo–TRPM5 and filled with red for Ca²⁺–TRPM5. c, Superimposition of the MHR3/4 domain and the CTD rib and pole helices of apo–TRPM5 (blue) and Ca²⁺–TRPM5 (red) by aligning the CTD coiled-coil poles, viewed from the intracellular side. One subunit in both structures are shown in blue (apo–TRPM5) or in red (Ca²⁺–TRPM5). The rotation of the rib helices is indicated. d, The superimposition of the ICD–TMD interface of apo–TRPM5 (blue) and Ca²⁺–TRPM5 (red) by aligning the CTD coiled-coil poles (not shown), viewed from the intracellular side. The rotations of helices square_N and square_C are indicated. The green circle highlights the location of the intersubunit interface and ICD–TMD interface, as detailed in panels (e, f). e, f, The conformational rearrangement at the intersubunit interface and the ICD–TMD interface in apo–TRPM5 (e) to Ca²⁺–TRPM5 (f), viewed parallel to the membrane. Prime symbol indicates residues or structural elements from the adjacent subunit. Interactions are shown in yellow bars. The single headed arrows indicate the movement of the square and TRP helices. The double headed arrow indicates the angle between S6 and TRP. The positions of the intersubunit interface are shown by green circles.

Figure 7:. The channel opening.

a, The superimposition of the TMD of a single subunit in apo–TRPM5 (blue) and Ca²⁺–TRPM5 (red) by aligning their S1-S4 domain, viewed parallel to the membrane. The center-of-mass movement of the pore domain is indicated. b, The superimposition of the pore domain in apo–TRPM5 (blue) and Ca²⁺–TRPM5 (red) by aligning their S1-S4 domain, viewed from the intracellular side. The pore domain of Ca²⁺–TRPM5 is shown in surface representation and the S5-S6 domain of one subunit from each structure is shown as a cartoon. The relative movements of helices S5 and S6 are indicated. c, d, Close-ups of the circled area in (a), viewed from the intracellular side. The remodeling of the Ca_TMD site from apo–TRPM5 (c) to Ca²⁺–TRPM5 (d). The movements of S2, S3, S4, and TRP helices are indicated by arrows. Interactions are shown in yellow bars. e, f, Close-ups of the boxed area in (a). W984 on the TRP helix switches its interaction partner from P847 and G846 in apo–TRPM5 (e) to I841 in Ca²⁺–TRPM5 (f). Interactions are shown in yellow bars. The movement of W984 is indicated. The contact area between the TRP helix and the S4-S5 linker is highlighted in grey. The segment between I836 and I841 turns into a 3₁₀-helix in Ca²⁺–TRPM5. The inset shows the view along the axis of the S4 helix. g, A cartoon scheme of the activation and inhibition mechanism of TRPM5. Conformational changes initialized from both Ca²⁺ sites cooperatively open the ion-conducting pore. The antagonist NDNA wedges into the space between the S1-S4 domain and pore domain, stabilizing the TMD in an apo-like closed state. The movements of individual structural elements are indicated by arrows.