Structure of the human TRPM4 ion channel in a lipid nanodisc

Abstract

Transient receptor potential (TRP) melastatin 4 (TRPM4) is a widely expressed cation channel associated with a variety of cardiovascular disorders. TRPM4 is activated by increased intracellular calcium in a voltage-dependent manner but, unlike many other TRP channels, is permeable to monovalent cations only. Here we present two structures of full-length human TRPM4 embedded in lipid nanodiscs at ~3-angstrom resolution, as determined by single-particle cryo-electron microscopy. These structures, with and without calcium bound, reveal a general architecture for this major subfamily of TRP channels and a well-defined calcium-binding site within the intracellular side of the S1-S4 domain. The structures correspond to two distinct closed states. Calcium binding induces conformational changes that likely prime the channel for voltage-dependent opening.

Figure 1. Cryo-EM structure of hTRPM4

(A and B) Unsharpened (in transparent light blue) and sharpened cryo-EM density map of hTRPM4 in nanodiscs in side (A) and top (B) views with each subunit colored differently. (C) Atomic model of the tetrameric hTRPM4 in ribbons, in the same orientation and colors as the density map in A. (D) Atomic model of the hTRPM4b monomer in ribbons with domains labeled. (E) bottom view of the hTRPM4 structure showing interactions between neighboring subunits. (F) A schematic representation of the major structural components in hTPPM4b. Dashed lines denote regions where density was insufficient for model building. Each domain is labeled and color-coded to match the domain representation in (D).

Figure 2. The Ca²⁺ binding site

(A) Coordination of the bound Ca^2+-ion by Glu828, Gln831, Asn865 and Asp868, with distances labelled. The density of the CaCl₂ structure (gray mesh) is contoured at σ = 5 and overlaid with the difference density (blue mesh) between the CaCl₂ structure and the EDTA structure, contoured at σ = 14. (B) Ca²⁺ binding site within the S1–S4 domain in the absence of bound ion. (C) The same site with a bound ion. Side chains are labeled. The bound Ca²⁺ ion is shown in magenta.

Figure 3. Ion permeation pore

(A) The solvent-accessible pathway along the ion permeation pore represented by dots between two opposing monomers shown in ribbons colored yellow and blue. Residues aligning the pathway are shown in sticks and labelled. (B) The pore radius of the hTRM4 EDTA (black) and CaCl₂ (blue) structures overlaid with the pore radius of TRPV1 in its closed (purple) and open (orange) states. (C) A close-up view of the pore helix and pore loop. The two single-turn π-helices are marked with red in the middle of S6 and the pore helix. The putative selectivity filter is marked with orange. The insert represents the density of a bound lipid, fitted with the atomic model of CHS, which forms a tripartite complex with S1 from one monomer and the pore helix of the neighboring monomer (‘Pore helix).

Figure 4. Membrane embedded helical segments surround the S1–S4 domain

(A) Side view of the hTRPM4 monomer with membrane embedded helical segments labeled and in solid color. (B) The pre-S1 elbow (gold) and pre-S1 shoulder (yellow). Lipid density between the pre-S1 elbow and S1 is modeled with CHS. Side chains interacting with the CHS are shown as sticks. (C) Same as (B), viewed from the opposite direction. (D) Cytoplasmic domain MHR3 interacts with the S2–S3 linker. (E) The CH1 helix after the TRP domain. Charged residues in the pre-S1 shoulder (C) and CH1 (E) are shown in sticks. Missing links are shown in dashed lines in (A) and (D).