Temperature-Sensitive Ca2+ Permeable TRP Channels
Transient receptor potential (TRP) channels play a multitude of important physiological roles in humans, including temperature sensing. Since their discovery in the 1990s, much effort has been directed towards elucidating the molecular mechanisms that allow TRP channels to fulfill their physiological functions. After 2016, when we reported the first single particle cryo-electron microscopy (cryo-EM) structure of the heat-sensitive TRPV2 channel (Nat Struc Mol Biol 2016), we have aimed to dissect the design principles of temperature-sensitive TRP channels through rigorous structural and functional studies.
TRP Channels and the Perception of Hot and Cold
Transient receptor potential vanilloid (TRPV) channels comprise TRPV subtypes 1-6. However, only subtypes TRPV1-TRPV4 can be opened by elevated temperatures. These have therefore been termed thermo-TRPV channels. Because of our interest in temperature sensing and calcium permeation, the thermo-TRPV channels have become a large part of the Lee Lab’s research focus.
TRPV2 is a Ca2+ permeable channel that is activated by temperatures ≥ 52 °C. Our first structural study of TRPV2 (Nat Struc Mol Biol 2016) predicted that the channel would undergo secondary structure transitions in the key transmembrane helices upon opening—from low-energy α-helix to high-energy π-helical motifs—and that these transient non α-helical motifs are important for channel gating. In 2018, we published a follow-up study to visualize the opening of the channel where we crystallized TRPV2 in Ca2+ and ligand/Ca2+-bound conformations (Nat Struc Mol Biol 2018). These remain, along with the crystal structures of TRPV6, the only available high-resolution crystal structures of TRP channels. Our results showed that the selectivity filter gate of TRPV2 assumes a highly unusual two-fold symmetric conformation upon opening. We showed that this conformation is relevant for large organic cation permeation, and that it is achieved through the rotation of subunits around π-helical hinges that appear at strategically important junctions in the transmembrane domains. This study highlights the importance of the non-α-helical elements, π- and 310–helices, and illustrates the difference in design of static and dynamic regions between TRPV channels and their better understood relatives, potassium selective voltage-gated ion channels (KV). In a follow-up study, we further investigated the symmetry transitions in TRPV2 during gating using the new Titan Krios cryo-EM at Duke. The results of this study showed that TRPV2 preferentially enters two-fold symmetric conformations during gating in lipid membranes (eLife 2019).
TRPV3 is closely related to TRPV2 and other thermo-TRPV channels. It is involved in the maintenance of skin and hair homeostasis and is activated by warm temperatures (≥ 32 °C). TRPV3 undergoes sensitization upon repeated exposure to ligands or heat. This is in contrast to most other TRP channels which rapidly desensitize upon activation. To dissect the structural determinants of sensitization, we solved the structures of the apo and sensitized human TRPV3 channel (Nat Comms 2018) and found that the S6 helix undergoes a secondary structure transition from α-helix to π-helix upon sensitization. The π-helix enables the S6 helix to bend and open, and the α-to-π transition contributes to lowering of the activation energy during sensitization. Furthermore, in a follow-up study using the new Titan Krios cryo-EM at Duke, we have found that the C-terminal domain (CTD) of TRPV3 also plays a critical role in the sensitization of the channel (eLife 2019). In the apo conformation of TRPV3, the CTD adopts a coil conformation which upon sensitization undergoes a coil-to-helix transition and facilitates the coupling of the cytoplasmic and transmembrane domains. Indeed, mutations that stabilize the helical CTD produce a TRPV3 channel that is sensitized from the outset.
Within the TRP channel superfamily, the transient receptor melastatin (TRPM) subfamily, defined by its unique N-terminal Melastatin Homology Regions (MHRs), is composed of eight members, TRPM1 to TRPM8. They are involved in diverse physiological pathways, such as temperature sensation, regulation of ion homeostasis, and redox sensation.
TRPM8: The Human sensor for Cold and Menthol
In 2017, the Lee Lab reported the first cryo-EM structure of a full-length TRPM8, which is the long-sought-after cold and menthol sensor in humans in collaboration with the Lander lab at TSRI (Science 2017). Following the initial determination of the channel architecture, we set out to investigate the molecular mechanisms of TRPM8 gating by which the channel is activated by various ligand modulators, including cooling agents such as menthol and icilin and membrane lipid phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2]. A unique feature of TRPM8 is that the channel activation requires PI(4,5)P2, which allosterically enhances the potency of cooling agents on channel activity. Using the new Titan Krios cryo-EM at Duke, we have independently determined two cryo-EM structures of TRPM8 complex structures: TRPM8-icilin-Ca2+-PI(4,5)P2 and TRPM8-WS12 (menthol analog)-PI(4,5)P2 (Science 2019). Our study revealed that the binding pocket for cooling agents is located at the voltage-sensor-like domain (VSLD) cavity and that PI(4,5)P2 binds to a previously unobserved membrane interfacial cavity assembled by key subdomains at an inter-layer nexus. These results provided a structural explanation for the allosteric coupling between PI(4,5)P2 and cooling agents, as well as a framework to understanding the molecular basis for TRPM8 gating (Science 2019). Currently, work is underway to further explore the gating transitions in TRPM8 channels.
Transient Receptor Potential Ankyrin 1 (TRPA1) is a calcium-permeable channel acting as one of the primary sensors of environmental irritants and noxious substances, including wasabi, cinnamon, garlic and mustard. Here, by utilizing cryo-electron microscopy and electrophysiology, we reported three high-resolution structures of human TRPA1 channel, and proposed a model for the mechanism of promiscuous electrophile sensing by the highly reactive cysteine C621. Our work provides a platform for future drug and development of TRPA1.
TRPM2: A Redox- and Warmth-Sensing Ion Channel
TRPM2 plays a key role in redox sensation in many different cell types. In addition, it is responsible for sensing warm temperatures and regulating body temperature in humans. TRPM2 interacts with the endogenous agonist ADP-ribose (ADPR) as well as Ca2+ ions, both of which are required for channel activation.
In our recent work (BioRxiv 2019), we aimed to delineate how binding of ADPR and Ca2+ ions, respectively, contributes to channel activation. Using cryo-EM we solved structures of the apo TRPM2, TRPM2 bound to Ca2+, and TRPM2 bound to ADPR and Ca2+. Interestingly, we found that TRPM2 transitions through two-fold (C2) symmetric intermediate states to accommodate substantial conformational rearrangements during ligand-dependent channel activation. In the apo form, the channel assumes an almost completely four-fold (C4) symmetry. However, addition of Ca2+ or ADPR/Ca2+ induces distinct C2 symmetric conformations in the homo-tetrameric channel. We propose that TRPM2 possesses an intrinsic flexibility that enables it to enter two-fold symmetric intermediates which reduce the energetic barrier going from the closed to open state. We are presently conducting cryo-EM studies to map out the gating cycle of TRPM2 in more detail.
Organellar Ca2+ Channels
The Lee Lab also researches organellar Ca2+ ion channels that regulate the flow of Ca2+ into mitochondria and lysosomes—a function critical for cell function and survival.
TRPML3: Lysosomal Calcium Release
Transient receptor potential mucolipin (TRPML) channels are a subfamily of the TRP family cation channels. They are the major Ca2+ permeable channels localized in late endosomes and lysosomes (LEL) and regulate Ca2+ release from those organelles, which is crucial for signal transduction and organelle trafficking. It has been shown that TRPML channels can be activated by a low abundance, LEL-localized lipid phosphatidylinositol 3,5-bisphosphate [PI(3,5)P2], whereas PI(4,5)P2, which is plentiful in the plasma membrane, inhibits channel activity.
To understand the mechanism of lipid-dependent channel gating, we reported the first single particle cryo-EM structure of a TRPML3 channel from Callithrix jaccus determined to 2.9 Å (Nature 2017). From the structure, together with electrophysiology and isothermal titration calorimetry (ITC), we showed that a cytosolic domain—which we term the mucolipin domain—is responsible for the binding of PI(3,5)P2 and the lipid-dependent channel opening.
Mitochondrial Calcium Uniporter: Mitochondrial Calcium Entry
Mitochondria play a fundamental role in eukaryotic cellular calcium signaling and regulation. Mitochondrial Ca2+ transport is critical for shaping the dynamics of intracellular calcium signaling, regulating energy metabolism, generating reactive oxygen species, and modulating cell death. The mitochondrial calcium uniporter (MCU) is a Ca2+-selective ion channel that is the primary mediator for Ca2+ uptake into the mitochondrial matrix.
In 2018, we presented the cryo-electron microscopy (cryo-EM) structure of the full-length MCU homolog from Neurospora crassa to an overall resolution of ~3.7 Å. Our structure reveals the distinct tetrameric architecture of MCU, wherein the soluble and the transmembrane domains adopt different symmetric arrangements within the channel. Furthermore, our structure, together with mutagenesis, shows how the conserved “WDXXEPVTY” sequence motif of MCU forms a unique selectivity filter that provides the structural basis of Ca2+ recognition by this channel family.