Physiological temperature drives TRPM4 ligand recognition and gating

Abstract

Temperature profoundly affects macromolecular function, particularly in proteins with temperature sensitivity^1,2. However, its impact is often overlooked in biophysical studies that are typically performed at non-physiological temperatures, potentially leading to inaccurate mechanistic and pharmacological insights. Here we demonstrate temperature-dependent changes in the structure and function of TRPM4, a temperature-sensitive Ca²⁺-activated ion channel^3-7. By studying TRPM4 prepared at physiological temperature using single-particle cryo-electron microscopy, we identified a 'warm' conformation that is distinct from those observed at lower temperatures. This conformation is driven by a temperature-dependent Ca²⁺-binding site in the intracellular domain, and is essential for TRPM4 function in physiological contexts. We demonstrated that ligands, exemplified by decavanadate (a positive modulator)⁸ and ATP (an inhibitor)⁹, bind to different locations of TRPM4 at physiological temperatures than at lower temperatures^10,11, and that these sites have bona fide functional relevance. We elucidated the TRPM4 gating mechanism by capturing structural snapshots of its different functional states at physiological temperatures, revealing the channel opening that is not observed at lower temperatures. Our study provides an example of temperature-dependent ligand recognition and modulation of an ion channel, underscoring the importance of studying macromolecules at physiological temperatures. It also provides a potential molecular framework for deciphering how thermosensitive TRPM channels perceive temperature changes.

Fig. 1. The overall structures of TRPM4_cold bound to Ca²⁺ and TRPM4_warm bound to Ca²⁺, Ca²⁺ and DVT, or Ca²⁺ and ATP.

The structures are shown as a surface representation with one subunit in cartoon, viewed parallel to the membrane (top row) or from the intracellular side (bottom row).

Fig. 2. The ion-conducting pore.

a, The profiles of the ion-conducting pore (shown as a surface representation) in different functional states, viewed parallel to the membrane. The pore region (shown as a cartoon) and residues (shown as sticks) forming the gate and the selectivity filter in two subunits are depicted. b, The pore radius along the pore axis.

Fig. 3. Temperature and Ca²⁺ govern the cold-to-warm transition.

a, Comparisons between subunits of Ca²⁺-bound wild-type TRPM4 cold and warm conformations, Ca²⁺-bound TRPM4(E396A) and Ca²⁺-bound zebrafish TRPM5 (Protein Data Bank (PDB): 7MBQ), superimposed using MHR3/4 (residues 391–687 in TRPM4 and 332–627 in TRPM5). The root mean squared deviation is shown for domains. b, The Ca_warm site; interactions between Ca²⁺ and coordinating residues are indicated by grey lines. c, Comparison of the Ca_warm site in the TRPM4 cold (blue) and warm (yellow) conformations by aligning helix α13 (residues 374–382), which forms half of the site. The angle between α12, which forms the other half of the site, is indicated by the black arrow. d, Whole-cell voltage-clamped currents were measured using patch pipettes containing 1 µM free calcium in tsA cells overexpressing wild-type TRPM4 (left) and the TRPM4(E396A) variant (right) at 37 °C. A protocol was applied every 5 s to monitor current changes, initiating at −100 mV for 50 ms, ramping to +100 mV over 200 ms, then maintaining +100 mV for 50 ms; the holding potential was 0 mV. The black and orange traces represent average peak and steady-state current traces (n = 5 each for WT and E396A). e, After reaching the steady-state current in d, additional measurements were made using a multistep voltage-clamp protocol from −120 mV to 160 mV. Representative traces are shown in Extended Data Fig. 1a,b. Current amplitudes at the end of each pulse are plotted as a function of clamp voltage. n = 16 (22 °C, WT), n = 15 (37 °C, WT), n = 8 (22 °C, E396A) and n = 7 (37 °C, E396A) particles. Data are mean ± s.e.m. Statistical analysis was performed using two-way analysis of variance (ANOVA) with Bonferroni’s post hoc test; *P < 0.05, **P < 0.01, ***P < 0.001. The P values for the 60 to 160 mV steps on the left are as follows: P = 0.0015, P< 0.0001, P < 0.0001, P < 0.0001, P < 0.0001 and P < 0.0001, respectively.

Fig. 4. Temperature determines DVT binding and modulation.

a,b, The rectification index (RI) ratio for TRPM4 wild type and mutants at the two DVT_cold sites (a) and mutants at the DVT_warm site (b). The RI is defined as the current ratio of I(−120 mV)/I(+120 mV), and the RI ratio is defined as RI(+DVT)/RI(−DVT). Statistical analysis was performed using one-way ANOVA with Bonferroni’s post hoc test (WT was compared with each mutant). The P values in b from left (R597A) to right are as follows: P = 0.0013, P > 0.9999, P = 0.0042, P = 0.0100, P = 0.0085, P = 0.0122 and P = 0.0042, respectively. Representative traces are provided in Extended Data Fig. 8. Insets: the structure of Ca²⁺/DVT–TRPM4_cold (a) and Ca²⁺/DVT–TRPM4_warm (b) as a surface representation, with one subunit as a cartoon and DVT molecules as yellow spheres. The magnified view in b shows the interactions within the DVT_warm site, which is encompassed by the MHR3/4 domain, pore domain and TRP helix of one subunit, along with the S4–S5 linker of the adjacent subunit. The DVT molecule is shown as sticks with a transparent surface, and the surrounding positively charged residues are shown as sticks. c,d, Comparisons of Ca²⁺-bound cold (blue) versus warm (yellow) conformations (c), and Ca²⁺-bound warm versus Ca²⁺/DVT-bound warm conformations (d), by aligning the tetrameric pore helix and loop (residues 958–989). A single subunit is depicted, with the centre-of-mass movement of the MHR1/2 domain indicated to represent the motion of the ICD. The centre-of-mass movement of the S1–S4 movement is also indicated. e,f, The pore domain in the Ca²⁺-bound warm (e) and Ca²⁺/DVT-bound warm (f) conformations viewed from the intracellular side. The movements of the S6 helix, TRP helix and S4–S5 linker caused by DVT binding are indicated. The side chain of Ile1040, which forms the channel gate, is shown as sticks. The DVT molecule is shown as sticks with a transparent surface.

Fig. 5. Temperature dictates inhibitor binding and action.

a, Whole-cell currents were measured in tsA cells overexpressing wild-type TRPM4 at 22 °C (left) and 37 °C (right). A protocol was applied every 5 s to monitor current changes, initiating at −100 mV for 50 ms, then ramping to +100 mV over 200 ms, and finally holding at +100 mV for 50 ms. The black and orange traces represent the average steady-state current traces measured with 1 µM free Ca²⁺ and 1 µM free Ca²⁺ plus 5 mM ATP, respectively, in the pipette solution. n = 8 (−ATP, 22 °C), n = 8 (+ATP, 22 °C), n = 11 (−ATP, 37 °C) and n = 9 (+ATP, 37 °C). b, Normalized current amplitudes in the presence and absence of ATP (at 50 ms of holding potentials of +100 and −100 mV) of the experiments in a were plotted. Each point represents a single cell and the bars represent the mean. c, The structure of Ca²⁺/ATP–TRPM4_cold (left) and Ca²⁺/ATP–TRPM4_warm (right) as a surface representation, with one subunit as a cartoon and ATP molecules as orange spheres. The dashed white circle marks the position of the ATP_cold site hypothetically mapped in the Ca²⁺/ATP–TRPM4_warm structure, with its distance to the ATP_warm site indicated. d, A magnified view of the interactions within the ATP_warm site. One subunit is coloured grey, while the adjacent subunit is coloured magenta. The ATP molecule is shown as sticks with a transparent surface, and the surrounding residues are shown as sticks.

Extended Data Fig. 1. Comparison of thermosensitive currents of wild-type TRPM4 and its E396A variant.

a,b, Steady-state whole-cell voltage-clamped currents were measured using patch pipettes containing 1 µM free calcium in tsA cells overexpressing wild-type TRPM4 (a) and the E396A variant (b) at 22 °C (blue) and 37 °C (red). Voltage clamps were imposed from −120 mV to 160 mV with a final tail pulse at −120 mV, with a holding potential of 0 mV. c,d, Whole-cell voltage-clamped currents were measured using patch pipettes containing 1 µM free calcium in tsA cells overexpressing wild-type TRPM4 (c) and the E396A variant (d) at 22 °C (left) and 37 °C (right). A protocol was applied every 5 s to monitor current changes, initiating at −100 mV for 50 ms, then ramping to +100 mV over 200 ms, and finally at +100 mV for 50 ms, with a holding potential of 0 mV. The black and orange traces represent average peak and steady-state current traces (n = 7 and n = 5 for WT at 22 °C and 37 °C, and n = 5 and n = 5 for E396A at 22 °C and 37 °C). e,f, Mean current amplitudes at the end of the +100 mV and −100 mV pulses of experiments in (c,d) were plotted as a function of time, showing the activation and desensitization of TRPM4. The number of patches is indicated in the parentheses after the construct name. Error bars represent SEM.

Extended Data Fig. 2. Thermostability test of human TRPM4 in the presence of 5 mM Ca²⁺ (top) or 5 mM EDTA (bottom) using fluorescence-detection size-exclusion chromatography.

Protein samples were incubated at various of temperatures for 10 min before loading onto a size-exclusion column.

Extended Data Fig. 3. Cryo-EM data processing workflow of TRPM4 with 5 mM Ca²⁺ at 37 °C.

Key maps and the mask (shown as a transparent envelope) used to subtract the intracellular domain (MHR1/2/3/4) are included. This figure also shows the percentage of the particles in warm and cold conformations at the subunit level.

Extended Data Fig. 4. Cryo-EM data processing workflow of TRPM4 with 5 mM Ca²⁺ and 2 mM DVT at 37 °C.

Key maps are included. This figure also shows the percentage of the particles in warm and cold conformations at the subunit level.

Extended Data Fig. 5. Cryo-EM data processing workflow of TRPM4 with 5 mM Ca²⁺ and 5 mM ATP at 37 °C.

Key maps and the mask (shown as a transparent envelope) used to subtract the intracellular domain (MHR1/2/3/4) are included. This figure also shows the percentage of the particles in warm and cold conformations at the subunit level.

Extended Data Fig. 6. Cryo-EM data processing workflow of TRPM4 with 5 mM Ca²⁺ at 18 °C (a), TRPM4 with 5 mM EDTA at 37 °C (b), TRPM4(E396A) with 5 mM Ca²⁺ at 37 °C (c), and TRPM4 with 5 mM Ca²⁺, 1 mM DVT, and 2 mM ATP at 37 °C (d).

e, Superimposition of the Ca²⁺/DVT/ATP–TRPM4_WARM cryo-EM map (yellow) and the Ca²⁺–TRPM4_WARM model (blue; closed state), focusing on the transmembrane domain, viewed from the intracellular side. f, Superimposition of the Ca²⁺/DVT/ATP–TRPM4_WARM cryo-EM map (yellow) and the Ca²⁺/DVT–TRPM4_WARM model (salmon; open state), focusing on the transmembrane domain, viewed from the intracellular side. g, Superimposition of the TRPM4 cryo-EM maps of Ca²⁺/DVT/ATP–TRPM4_WARM (yellow) and Ca²⁺/DVT–TRPM4_WARM (salmon), focusing on the DVT_WARM site and its surroundings.

Extended Data Fig. 7. Cryo-EM analysis of the datasets in this paper.

a, From left to right, local resolution estimation, FSC curves for map vs. map (blue) and map vs model (orange), and angular distribution of the particles that give rise to the final cryo-EM map reconstruction for the Ca²⁺–TRPM4_WARM data. b,c, Local resolution estimation and FSC curves for the cold (b) and warm (c) monomer of the Ca²⁺–TRPM4_WARM data. The representative 2D class averages of the Ca²⁺–TRPM4_WARM data are shown on the far right of panel c. d–g, Local resolution estimation, FSC curves for map vs. map (blue) and map vs model (orange), and angular distribution of the particles that give rise to the final cryo-EM map reconstruction for Ca²⁺–TRPM4_COLD (d), Ca²⁺–TRPM4(E396A) (e), EDTA–TRPM4 (f), Ca²⁺/DVT–TRPM4_WARM (g). h, Local resolution estimation and FSC curves for the warm monomer of the Ca²⁺/DVT–TRPM4_WARM data. i, Local resolution estimation, FSC curves for map vs. map (blue) and map vs model (orange), and angular distribution of the particles that give rise to the final cryo-EM map reconstruction for the Ca²⁺/ATP–TRPM4_WARM data. j, Local resolution estimation and FSC curves for the warm monomer of the Ca²⁺/ATP–TRPM4_WARM data. k, Representative densities.

Extended Data Fig. 8. DVT modulation on wild-type TRPM4 and its variants at the DVT_COLD and DVT_WARM sites.

Representative current traces activated by 5 mM Ca²⁺ in the absence (left) or presence of 10 μM DVT (middle) from membrane patches excised from tsA201 cells overexpressing wild-type TRPM4 (a), variants at the DVTCOLD site (b–i), and variants in the DVTWARM site (j–p), recorded in the inside-out patch-clamp configuration. Voltage clamps were applied from 160 mV to −120 mV (200 ms each step) with a final tail pulse at −120 mV (200 ms), with a holding potential of 0 mV. The mean current amplitudes at the end of each pulse step were plotted as a function of clamp voltage (right). The number of patches is indicated in the parentheses after the construct name. Error bars represent SEM. Notably, adding DVT appears to inhibit the currents of certain mutants. While the exact mechanism behind these phenotypes remains unclear, it is possible that some mutations do not abolish DVT binding. Instead, these mutations may alter the interaction between DVT and the channel, thereby affecting the action of DVT on the channel.

Extended Data Fig. 9. The surfaces of Ca²⁺/DVT–TRPM4_COLD (left) and Ca²⁺/DVT–TRPM4_WARM (right) coloured according to the electrostatic surface potential from –5 to 5 kT/e (red to blue).

The positions of the DVT_COLD and DVT_WARM sites are labelled.