Structural and functional analyses of a GPCR-inhibited ion channel TRPM3.

G-protein coupled receptors (GPCRs) govern the physiological response to stimuli by modulating the activity of downstream effectors, including ion channels. TRPM3 is an ion channel inhibited by GPCRs through direct interaction with G protein (Gßγ) released upon their activation. This GPCR-TRPM3 signaling pathway contributes to the analgesic effect of morphine. Here, we characterized Gßγ inhibition of TRPM3 using electrophysiology and single particle cryo-electron microscopy (cryo-EM). From electrophysiology, we obtained a half inhibition constant (IC50) of ∼240 nM. Using cryo-EM, we determined structures of mouse TRPM3 expressed in human cells with and without Gßγ and with and without PIP2, a lipid required for TRPM3 activity, at resolutions of 2.7-4.7 Å. Gßγ-TRPM3 interfaces vary depending on PIP2 occupancy; however, in all cases, Gßγ appears loosely attached to TRPM3. The IC50 in electrophysiology experiments raises the possibility that additional unknown factors may stabilize the TRPM3-Gßγ complex.

Structural and functional basis of the selectivity filter as a gate in human TRPM2 channel.

Transient receptor potential melastatin 2 (TRPM2), a Ca2+-permeable cation channel, is gated by intracellular adenosine diphosphate ribose (ADPR), Ca2+, warm temperature, and oxidative stress. It is critically involved in physiological and pathological processes ranging from inflammation to stroke to neurodegeneration. At present, the channel's gating and ion permeation mechanisms, such as the location and identity of the selectivity filter, remain ambiguous. Here, we report the cryo-electron microscopy (cryo-EM) structure of human TRPM2 in nanodisc in the ligand-free state. Cryo-EM map-guided computational modeling and patch-clamp recording further identify a quadruple-residue motif as the ion selectivity filter, which adopts a restrictive conformation in the closed state and acts as a gate, profoundly contrasting with its widely open conformation in the Nematostella vectensis TRPM2. Our study reveals the gating of human TRPM2 by the filter and demonstrates the feasibility of using cryo-EM in conjunction with computational modeling and functional studies to garner structural information for intrinsically dynamic but functionally important domains.

N-glycosylation state of TRPM8 protein revealed by terahertz spectroscopy and molecular modelling.

TRPM8 member of the TRP superfamily of membrane proteins participates to various cellular processes ranging from Ca2+ uptake and cold sensation to cellular proliferation and migration. TRPM8 is a large tetrameric protein with more than 70% of its residues located in the cytoplasm. TRPM8 is N-glycosylated, with a single site per subunit. This work focuses on the N-glycosylation of TRPM8 channel that was previously studied by our group in relation to proliferation and migration of tumoral cells. Here, experimental data performed with deglycosylating agents assess that the sole glycosylation site contains complex glycans with a molecular weight of 2.5 kDa. The glycosylation state of TRPM8 in cells untreated and treated with a deglycosylating agent was addressed with Terahertz (THz) spectroscopy. Results show a clear difference between cells comprising glycosylated and deglycosylated TRPM8, the first presenting an increased THz absorption. Human TRPM8 was modelled using as templates the available TRPM8 and other TRPM channels structures. Glycosylations were modelled by considering two glycan structures with molecular weight close to the experiment: shorter and branched at the first sugar unit (glc1) and longer and unbranched (glc2). Simulation of THz spectra based on the molecular dynamics of unglycosylated and the two glycosylated TRPM8 models in lipid membrane and solvation box showed that glycan structure strongly influences the THz spectrum of the channel and of other components from the simulation system. Only spectra of TRPM8 with glc1 glycans were in agreement with the experiment, leading to the validation of glc1 glycan structure.

Architecture of the TRPM2 channel and its activation mechanism by ADP-ribose and calcium.

Transient receptor potential melastatin 2 (TRPM2) is a calcium-permeable, non-selective cation channel that has an essential role in diverse physiological processes such as core body temperature regulation, immune response and apoptosis1-4. TRPM2 is polymodal and can be activated by a wide range of stimuli1-7, including temperature, oxidative stress and NAD+-related metabolites such as ADP-ribose (ADPR). Its activation results in both Ca2+ entry across the plasma membrane and Ca2+ release from lysosomes8, and has been linked to diseases such as ischaemia-reperfusion injury, bipolar disorder and Alzheimer's disease9-11. Here we report the cryo-electron microscopy structures of the zebrafish TRPM2 in the apo resting (closed) state and in the ADPR/Ca2+-bound active (open) state, in which the characteristic NUDT9-H domains hang underneath the MHR1/2 domain. We identify an ADPR-binding site located in the bi-lobed structure of the MHR1/2 domain. Our results provide an insight into the mechanism of activation of the TRPM channel family and define a framework for the development of therapeutic agents to treat neurodegenerative diseases and temperature-related pathological conditions.

Structure of a TRPM2 channel in complex with Ca2+ explains unique gating regulation.

Transient receptor potential melastatin 2 (TRPM2) is a Ca2+-permeable cation channel required for immune cell activation, insulin secretion, and body heat control. TRPM2 is activated by cytosolic Ca2+, phosphatidyl-inositol-4,5-bisphosphate and ADP ribose. Here, we present the ~3 Å resolution electron cryo-microscopic structure of TRPM2 from Nematostella vectensis, 63% similar in sequence to human TRPM2, in the Ca2+-bound closed state. Compared to other TRPM channels, TRPM2 exhibits unique structural features that correlate with its function. The pore is larger and more negatively charged, consistent with its high Ca2+ selectivity and larger conductance. The intracellular Ca2+ binding sites are connected to the pore and cytosol, explaining the unusual dependence of TRPM2 activity on intra- and extracellular Ca2+. In addition, the absence of a post-filter motif is likely the cause of the rapid inactivation of human TRPM2. Together, our cryo-EM and electrophysiology studies provide a molecular understanding of the unique gating mechanism of TRPM2.

Structure of the human TRPM4 ion channel in a lipid nanodisc.

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.

Structure of the cold- and menthol-sensing ion channel TRPM8.

Transient receptor potential melastatin (TRPM) cation channels are polymodal sensors that are involved in a variety of physiological processes. Within the TRPM family, member 8 (TRPM8) is the primary cold and menthol sensor in humans. We determined the cryo-electron microscopy structure of the full-length TRPM8 from the collared flycatcher at an overall resolution of ~4.1 ångstroms. Our TRPM8 structure reveals a three-layered architecture. The amino-terminal domain with a fold distinct among known TRP structures, together with the carboxyl-terminal region, forms a large two-layered cytosolic ring that extensively interacts with the transmembrane channel layer. The structure suggests that the menthol-binding site is located within the voltage-sensor-like domain and thus provides a structural glimpse of the design principle of the molecular transducer for cold and menthol sensation.

Structures of the calcium-activated, non-selective cation channel TRPM4.

TRPM4 is a calcium-activated, phosphatidylinositol-4,5-bisphosphate (PtdIns(4,5)P2) -modulated, non-selective cation channel that belongs to the family of melastatin-related transient receptor potential (TRPM) channels. Here we present the electron cryo-microscopy structures of the mouse TRPM4 channel with and without ATP. TRPM4 consists of multiple transmembrane and cytosolic domains, which assemble into a three-tiered architecture. The N-terminal nucleotide-binding domain and the C-terminal coiled-coil participate in the tetrameric assembly of the channel; ATP binds at the nucleotide-binding domain and inhibits channel activity. TRPM4 has an exceptionally wide filter but is only permeable to monovalent cations; filter residue Gln973 is essential in defining monovalent selectivity. The S1-S4 domain and the post-S6 TRP domain form the central gating apparatus that probably houses the Ca2+- and PtdIns(4,5)P2-binding sites. These structures provide an essential starting point for elucidating the complex gating mechanisms of TRPM4 and reveal the molecular architecture of the TRPM family.

Electron cryo-microscopy structure of a human TRPM4 channel.

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.

Three-dimensional Imaging Reveals New Compartments and Structural Adaptations in Odontoblasts.

In organized tissues, the precise geometry and the overall shape are critical for the specialized functions that the cells carry out. Odontoblasts are major matrix-producing cells of the tooth and have also been suggested to participate in sensory transmission. However, refined morphologic data on these important cells are limited, which hampers the analysis and understanding of their cellular functions. We took advantage of fluorescent color-coding genetic tracing to visualize and reconstruct in 3 dimensions single odontoblasts, pulp cells, and their assemblages. Our results show distinct structural features and compartments of odontoblasts at different stages of maturation, with regard to overall cellular shape, formation of the main process, orientation, and matrix deposition. We demonstrate previously unanticipated contacts between the processes of pulp cells and odontoblasts. All reported data are related to mouse incisor tooth. We also show that odontoblasts express TRPM5 and Piezo2 ion channels. Piezo2 is expressed ubiquitously, while TRPM5 is asymmetrically distributed with distinct localization to regions proximal to and within odontoblast processes.

Four Ca2+ ions activate TRPM2 channels by binding in deep crevices near the pore but intracellularly of the gate.

TRPM2 is a tetrameric Ca(2+)-permeable channel involved in immunocyte respiratory burst and in postischaemic neuronal death. In whole cells, TRPM2 activity requires intracellular ADP ribose (ADPR) and intra- or extracellular Ca(2+), but the mechanism and the binding sites for Ca(2+) activation remain unknown. Here we study TRPM2 gating in inside-out patches while directly controlling intracellular ligand concentrations. Concentration jump experiments at various voltages and Ca(2+) dependence of steady-state single-channel gating kinetics provide unprecedented insight into the molecular mechanism of Ca(2+) activation. In patches excised from Xenopus laevis oocytes expressing human TRPM2, coapplication of intracellular ADPR and Ca(2+) activated approximately 50-pS nonselective cation channels; K(1/2) for ADPR was approximately 1 microM at saturating Ca(2+). Intracellular Ca(2+) dependence of TRPM2 steady-state opening and closing rates (at saturating [ADPR] and low extracellular Ca(2+)) reveals that Ca(2+) activation is a consequence of tighter binding of Ca(2+) in the open rather than in the closed channel conformation. Four Ca(2+) ions activate TRPM2 with a Monod-Wymann-Changeux mechanism: each binding event increases the open-closed equilibrium constant approximately 33-fold, producing altogether 10(6)-fold activation. Experiments in the presence of 1 mM of free Ca(2+) on the extracellular side clearly show that closed channels do not sense extracellular Ca(2+), but once channels have opened Ca(2+) entering passively through the pore slows channel closure by keeping the "activating sites" saturated, despite rapid continuous Ca(2+)-free wash of the intracellular channel surface. This effect of extracellular Ca(2+) on gating is gradually lost at progressively depolarized membrane potentials, where the driving force for Ca(2+) influx is diminished. Thus, the activating sites lie intracellularly from the gate, but in a shielded crevice near the pore entrance. Our results suggest that in intact cells that contain micromolar ADPR a single brief puff of Ca(2+) likely triggers prolonged, self-sustained TRPM2 activity.