Structure of full-length human TRPM4

Abstract

Transient receptor potential melastatin subfamily member 4 (TRPM4) is a widely distributed, calcium-activated, monovalent-selective cation channel. Mutations in human TRPM4 (hTRPM4) result in progressive familial heart block. Here, we report the electron cryomicroscopy structure of hTRPM4 in a closed, Na⁺-bound, apo state at pH 7.5 to an overall resolution of 3.7 Å. Five partially hydrated sodium ions are proposed to occupy the center of the conduction pore and the entrance to the coiled-coil domain. We identify an upper gate in the selectivity filter and a lower gate at the entrance to the cytoplasmic coiled-coil domain. Intramolecular interactions exist between the TRP domain and the S4-S5 linker, N-terminal domain, and N and C termini. Finally, we identify aromatic interactions via π-π bonds and cation-π bonds, glycosylation at an N-linked extracellular site, a pore-loop disulfide bond, and 24 lipid binding sites. We compare and contrast this structure with other TRP channels and discuss potential mechanisms of regulation and gating of human full-length TRPM4.

Keywords: cardiac arrhythmia; cryomicroscopy; ion channel; transient receptor potential channel.

Fig. 1.

Overall structure of human full-length TRPM4 in the apo state. (A and B) Side (A) and top (B) views of the cryo-EM reconstruction density map of human TRPM4 at 3.7-Å overall resolution; each subunit is color-coded. (C and D) Ribbon diagrams representing the same orientation and colors with the channel’s dimensions indicated. (E) Structural details of a single human TRPM4 subunit. (F) Linear diagram depicting the major structural domains, color-coded to match the ribbon diagram. The N-linked N992 glycosylation site (N-G) and the Cys993–Cys1011 disulfide bond are indicated.

Fig. 2.

Detailed domain interactions of human TRPM4. Interactions between (A) the TRP domain and S4–5 linker, (B) TRP domain and N terminus, (C) N terminus and pre-S1 helix, and (D) N and C termini. Residues at domain interfaces are labeled with hydrogen bonds and electrostatic interactions as dashed lines.

Fig. 3.

Aromatic interactions of human TRPM4. Ribbon diagrams showing aromatic interactions of human TRPM4: π–π bonds (A and B) and cation–π bonds (C–E). Residues and helices at these interactions are labeled.

Fig. 4.

Cytosolic domains of human TRPM4. (A) Side (Left) and top (Right) views of the C-terminal domain. (B) Top views along the enlarged central hole formed by coiled-coil helices are shown with the key residues labeled. Hydrogen bonds and electrostatic interactions are shown as dashed lines and distances are indicated. (C) Side and top views of the N-terminal domain. (D) Side views of a single subunit of the N-terminal domain. ALR, ankyrin-like repeat; ISD, isolated subdomain. The arrows in C and D are pointing to a helix of each N-terminal domain. These hydrophobic regions are anchored to the inner leaflet of the plasma membrane.

Fig. 5.

TRPM4’s ion conduction pathway. (A) The ion conduction pathway is shown as dots and mapped using HOLE. (B) Pore radius along the central axis. The side chains of Q977 to F975 form a narrow constriction at the selectivity filter, while I1040 is the most restricted site of the lower gate. Side (C) and bottom (D) views of the electrostatic map. The surface is colored according to the calculated electrostatic potential, revealing the tetrameric distribution of charge. Blue indicates positive potential, red indicates negative, with transparent white being neutral.

Fig. 6.

Putative sodium binding sites of human TRPM4. Overall (A) and enlarged (B) views of the putative Na⁺ binding sites; side views of TRPM4. Five nonprotein densities, consistent with partially hydrated Na⁺ ions, along the pore and at the entrance of the coiled-coil domain, are indicated as purple spheres and labeled as Na1 to Na5 (Top to Bottom). (B) Enlarged densities of partially hydrated Na⁺ (Na1 to Na5) are shown at the binding sites. The key amino acid residues are labeled, and interactions between the ions and binding sites are shown as dashed lines.