A pentameric TRPV3 channel with a dilated pore.

Transient receptor potential (TRP) channels are a large, eukaryotic ion channel superfamily that control diverse physiological functions, and therefore are attractive drug targets1-5. More than 210 structures from more than 20 different TRP channels have been determined, and all are tetramers4. Despite this wealth of structures, many aspects concerning TRPV channels remain poorly understood, including the pore-dilation phenomenon, whereby prolonged activation leads to increased conductance, permeability to large ions and loss of rectification6,7. Here, we used high-speed atomic force microscopy (HS-AFM) to analyse membrane-embedded TRPV3 at the single-molecule level and discovered a pentameric state. HS-AFM dynamic imaging revealed transience and reversibility of the pentamer in dynamic equilibrium with the canonical tetramer through membrane diffusive protomer exchange. The pentamer population increased upon diphenylboronic anhydride (DPBA) addition, an agonist that has been shown to induce TRPV3 pore dilation. On the basis of these findings, we designed a protein production and data analysis pipeline that resulted in a cryogenic-electron microscopy structure of the TRPV3 pentamer, showing an enlarged pore compared to the tetramer. The slow kinetics to enter and exit the pentameric state, the increased pentamer formation upon DPBA addition and the enlarged pore indicate that the pentamer represents the structural correlate of pore dilation. We thus show membrane diffusive protomer exchange as an additional mechanism for structural changes and conformational variability. Overall, we provide structural evidence for a non-canonical pentameric TRP-channel assembly, laying the foundation for new directions in TRP channel research.

Capsaicin and TRPV1 Channels in the Cardiovascular System: The Role of Inflammation.

Capsaicin is a potent agonist of the Transient Receptor Potential Vanilloid type 1 (TRPV1) channel and is a common component found in the fruits of the genus Capsicum plants, which have been known to humanity and consumed in food for approximately 7000-9000 years. The fruits of Capsicum plants, such as chili pepper, have been long recognized for their high nutritional value. Additionally, capsaicin itself has been proposed to exhibit vasodilatory, antimicrobial, anti-cancer, and antinociceptive properties. However, a growing body of evidence reveals a vasoconstrictory potential of capsaicin acting via the vascular TRPV1 channel and suggests that unnecessary high consumption of capsaicin may cause severe consequences, including vasospasm and myocardial infarction in people with underlying inflammatory conditions. This review focuses on vascular TRPV1 channels that are endogenously expressed in both vascular smooth muscle and endothelial cells and emphasizes the role of inflammation in sensitizing the TRPV1 channel to capsaicin activation. Tilting the balance between the beneficial vasodilatory action of capsaicin and its unwanted vasoconstrictive effects may precipitate adverse outcomes such as vasospasm and myocardial infarction, especially in the presence of proinflammatory mediators.

Pre-pro is a fast pre-processor for single-particle cryo-EM by enhancing 2D classification.

2D classification plays a pivotal role in analyzing single particle cryo-electron microscopy images. Here, we introduce a simple and loss-less pre-processor that incorporates a fast dimension-reduction (2SDR) de-noiser to enhance 2D classification. By implementing this 2SDR pre-processor prior to a representative classification algorithm like RELION and ISAC, we compare the performances with and without the pre-processor. Tests on multiple cryo-EM experimental datasets show the pre-processor can make classification faster, improve yield of good particles and increase the number of class-average images to generate better initial models. Testing on the nanodisc-embedded TRPV1 dataset with high heterogeneity using a 3D reconstruction workflow with an initial model from class-average images highlights the pre-processor improves the final resolution to 2.82 Å, close to 0.9 Nyquist. Those findings and analyses suggest the 2SDR pre-processor, of minimal cost, is widely applicable for boosting 2D classification, while its generalization to accommodate neural network de-noisers is envisioned.

High-resolution structures of transient receptor potential vanilloid channels: Unveiling a functionally diverse group of ion channels.

Transient receptor potential vanilloid (TRPV) channels are part of the superfamily of TRP ion channels and play important roles in widespread physiological processes including both neuronal and non-neuronal pathways. Various diseases such as skeletal abnormalities, chronic pain, and cancer are associated with dysfunction of a TRPV channel. In order to obtain full understanding of disease pathogenesis and create opportunities for therapeutic intervention, it is essential to unravel how these channels function at a molecular level. In the past decade, incredible progress has been made in biochemical sample preparation of large membrane proteins and structural biology techniques, including cryo-electron microscopy. This has resulted in high resolution structures of all TRPV channels, which has provided novel insights into the molecular mechanisms of channel gating and regulation that will be summarized in this review.

Molecular mechanism of TRPV2 channel modulation by cannabidiol.

Transient receptor potential vanilloid 2 (TRPV2) plays a critical role in neuronal development, cardiac function, immunity, and cancer. Cannabidiol (CBD), the non-psychotropic therapeutically active ingredient of Cannabis sativa, is an activator of TRPV2 and also modulates other transient receptor potential (TRP) channels. Here, we determined structures of the full-length rat TRPV2 channel in apo and CBD-bound states in nanodiscs by cryo-electron microscopy. We show that CBD interacts with TRPV2 through a hydrophobic pocket located between S5 and S6 helices of adjacent subunits, which differs from known ligand and lipid binding sites in other TRP channels. CBD-bound TRPV2 structures revealed that the S4-S5 linker plays a critical role in channel gating upon CBD binding. Additionally, nanodiscs permitted us to visualize two distinct TRPV2 apo states in a lipid environment. Together these results provide a foundation to further understand TRPV channel gating, their divergent physiological functions, and to accelerate structure-based drug design.

Symmetry transitions during gating of the TRPV2 ion channel in lipid membranes.

The Transient Receptor Potential Vanilloid 2 (TRPV2) channel is a member of the temperature-sensing thermoTRPV family. Recent advances in cryo-electronmicroscopy (cryo-EM) and X-ray crystallography have provided many important insights into the gating mechanisms of thermoTRPV channels. Interestingly, crystallographic studies of ligand-dependent TRPV2 gating have shown that the TRPV2 channel adopts two-fold symmetric arrangements during the gating cycle. However, it was unclear if crystal packing forces played a role in stabilizing the two-fold symmetric arrangement of the channel. Here, we employ cryo-EM to elucidate the structure of full-length rabbit TRPV2 in complex with the agonist resiniferatoxin (RTx) in nanodiscs and amphipol. We show that RTx induces two-fold symmetric conformations of TRPV2 in both environments. However, the two-fold symmetry is more pronounced in the native-like lipid environment of the nanodiscs. Our data offers insights into a gating pathway in TRPV2 involving symmetry transitions.

Combining Structural Data with Computational Methodologies to Investigate Structure-Function Relationships in TRP Channels.

Since the emergence of high-resolution three-dimensional structures of membrane proteins, and the increasing availability of state-of-the-art algorithms and high-performance-computing facilities, classical molecular dynamics (MD) simulations have become a routine device to explore the molecular behavior of these proteins. The rise of cryo-electron microscopy (cryo-EM) as a credible experimental tool to resolve structures at an atomic level has revolutionized structural biology in recent years, culminating in the disclosure of the first high-resolution three-dimensional structure of a transient receptor potential (TRP) channel, the vanilloid receptor 1 (TRPV1). As a result, the number of research articles investigating the molecular behavior of TRP channels using macromolecular simulation techniques has proliferated. This review provides an overview of the current state of this field, including our understanding of TRP channel structure, the framework of classical MD simulations, and how to perform such simulations to investigate structure-function relationships in TRP channels.

Structural insight into TRPV5 channel function and modulation.

TRPV5 (transient receptor potential vanilloid 5) is a unique calcium-selective TRP channel essential for calcium homeostasis. Unlike other TRPV channels, TRPV5 and its close homolog, TRPV6, do not exhibit thermosensitivity or ligand-dependent activation but are constitutively open at physiological membrane potentials and modulated by calmodulin (CaM) in a calcium-dependent manner. Here we report high-resolution electron cryomicroscopy structures of truncated and full-length TRPV5 in lipid nanodiscs, as well as of a TRPV5 W583A mutant and TRPV5 in complex with CaM. These structures highlight the mechanism of calcium regulation and reveal a flexible stoichiometry of CaM binding to TRPV5.

Time course and expression pattern of the neuronal markers in the developing human spinal cord.

The aim of this study was to examine the spatio-temporal appearance of different neuronal cell subtypes by analyzing expression patterns of several neuronal markers (calretinin, neurofilament 200 (NF200), vanilloid receptor 1(VR1) and calcitonin gene-related peptide (CGRP)) of the embryonic human spinal cord (SC). Developing human SCs from 11 human conceptuses beetwen 5-10 developmental weeks (DW) were examined by light and electron microscopy and immunofluorescence. Light and electron microscopy revealed different embryonic stages of recognizable structure of the SC. NF200, CGRP and VR1 positive cells were observed in SCs during 5th-6th DW. NF200 was predominantly expressed in the ventral part, indicating presence of motoneurons. As development advanced, NF200 was mainly expressed in the marginal zone. Expression of CGRP was intense during all of the investigated periods, predominantly during the 5th-6th DW pointing to neural sensory differentiation, as opposed to the last DW when reduced expression of CGRP in the marginal layer indicated the terminations of the sensory afferents. Expression of VR1 was highest in the intermediate zone, at the beginning and at the end of the investigated periods, pointing to VR1 spatial pattern in the visceral afferents in the grey matter, while the first signs of calretinin were found in the 9th-10th DW ventrally. Delineating the relationships between factors involved in processes of neuronal differentiation as well as spatial and temporal arrangement of SC interrelated neurons can provide a useful information about normal SC development as well as the insight in possible causes of anomalies and disorders during embryonic life.

Conformational ensemble of the human TRPV3 ion channel.

Transient receptor potential vanilloid channel 3 (TRPV3), a member of the thermosensitive TRP (thermoTRPV) channels, is activated by warm temperatures and serves as a key regulator of normal skin physiology through the release of pro-inflammatory messengers. Mutations in trpv3 have been identified as the cause of the congenital skin disorder, Olmsted syndrome. Unlike other members of the thermoTRPV channel family, TRPV3 sensitizes upon repeated stimulation, yet a lack of structural information about the channel precludes a molecular-level understanding of TRPV3 sensitization and gating. Here, we present the cryo-electron microscopy structures of apo and sensitized human TRPV3, as well as several structures of TRPV3 in the presence of the common thermoTRPV agonist 2-aminoethoxydiphenyl borate (2-APB). Our results show α-to-π-helix transitions in the S6 during sensitization, and suggest a critical role for the S4-S5 linker π-helix during ligand-dependent gating.

Opening of the human epithelial calcium channel TRPV6.

Calcium-selective transient receptor potential vanilloid subfamily member 6 (TRPV6) channels play a critical role in calcium uptake in epithelial tissues. Altered TRPV6 expression is associated with a variety of human diseases, including cancers. TRPV6 channels are constitutively active and their open probability depends on the lipidic composition of the membrane in which they reside; it increases substantially in the presence of phosphatidylinositol 4,5-bisphosphate. Crystal structures of detergent-solubilized rat TRPV6 in the closed state have previously been solved. Corroborating electrophysiological results, these structures demonstrated that the Ca2+ selectivity of TRPV6 arises from a ring of aspartate side chains in the selectivity filter that binds Ca2+ tightly. However, how TRPV6 channels open and close their pores for ion permeation has remained unclear. Here we present cryo-electron microscopy structures of human TRPV6 in the open and closed states. The channel selectivity filter adopts similar conformations in both states, consistent with its explicit role in ion permeation. The iris-like channel opening is accompanied by an α-to-π-helical transition in the pore-lining transmembrane helix S6 at an alanine hinge just below the selectivity filter. As a result of this transition, the S6 helices bend and rotate, exposing different residues to the ion channel pore in the open and closed states. This gating mechanism, which defines the constitutive activity of TRPV6, is, to our knowledge, unique among tetrameric ion channels and provides structural insights for understanding their diverse roles in physiology and disease.

cryoSPARC: algorithms for rapid unsupervised cryo-EM structure determination.

Single-particle electron cryomicroscopy (cryo-EM) is a powerful method for determining the structures of biological macromolecules. With automated microscopes, cryo-EM data can often be obtained in a few days. However, processing cryo-EM image data to reveal heterogeneity in the protein structure and to refine 3D maps to high resolution frequently becomes a severe bottleneck, requiring expert intervention, prior structural knowledge, and weeks of calculations on expensive computer clusters. Here we show that stochastic gradient descent (SGD) and branch-and-bound maximum likelihood optimization algorithms permit the major steps in cryo-EM structure determination to be performed in hours or minutes on an inexpensive desktop computer. Furthermore, SGD with Bayesian marginalization allows ab initio 3D classification, enabling automated analysis and discovery of unexpected structures without bias from a reference map. These algorithms are combined in a user-friendly computer program named cryoSPARC (http://www.cryosparc.com).

Clustering and Functional Coupling of Diverse Ion Channels and Signaling Proteins Revealed by Super-resolution STORM Microscopy in Neurons.

The fidelity of neuronal signaling requires organization of signaling molecules into macromolecular complexes, whose components are in intimate proximity. The intrinsic diffraction limit of light makes visualization of individual signaling complexes using visible light extremely difficult. However, using super-resolution stochastic optical reconstruction microscopy (STORM), we observed intimate association of individual molecules within signaling complexes containing ion channels (M-type K+, L-type Ca2+, or TRPV1 channels) and G protein-coupled receptors coupled by the scaffolding protein A-kinase-anchoring protein (AKAP)79/150. Some channels assembled as multi-channel supercomplexes. Surprisingly, we identified novel layers of interplay within macromolecular complexes containing diverse channel types at the single-complex level in sensory neurons, dependent on AKAP79/150. Electrophysiological studies revealed that such ion channels are functionally coupled as well. Our findings illustrate the novel role of AKAP79/150 as a molecular coupler of different channels that conveys crosstalk between channel activities within single microdomains in tuning the physiological response of neurons.

DeepPicker: A deep learning approach for fully automated particle picking in cryo-EM.

Particle picking is a time-consuming step in single-particle analysis and often requires significant interventions from users, which has become a bottleneck for future automated electron cryo-microscopy (cryo-EM). Here we report a deep learning framework, called DeepPicker, to address this problem and fill the current gaps toward a fully automated cryo-EM pipeline. DeepPicker employs a novel cross-molecule training strategy to capture common features of particles from previously-analyzed micrographs, and thus does not require any human intervention during particle picking. Tests on the recently-published cryo-EM data of three complexes have demonstrated that our deep learning based scheme can successfully accomplish the human-level particle picking process and identify a sufficient number of particles that are comparable to those picked manually by human experts. These results indicate that DeepPicker can provide a practically useful tool to significantly reduce the time and manual effort spent in single-particle analysis and thus greatly facilitate high-resolution cryo-EM structure determination. DeepPicker is released as an open-source program, which can be downloaded from https://github.com/nejyeah/DeepPicker-python.

Molecular dynamics-based refinement and validation for sub-5 Å cryo-electron microscopy maps.

Two structure determination methods, based on the molecular dynamics flexible fitting (MDFF) paradigm, are presented that resolve sub-5 Å cryo-electron microscopy (EM) maps with either single structures or ensembles of such structures. The methods, denoted cascade MDFF and resolution exchange MDFF, sequentially re-refine a search model against a series of maps of progressively higher resolutions, which ends with the original experimental resolution. Application of sequential re-refinement enables MDFF to achieve a radius of convergence of ~25 Å demonstrated with the accurate modeling of ß-galactosidase and TRPV1 proteins at 3.2 Å and 3.4 Å resolution, respectively. The MDFF refinements uniquely offer map-model validation and B-factor determination criteria based on the inherent dynamics of the macromolecules studied, captured by means of local root mean square fluctuations. The MDFF tools described are available to researchers through an easy-to-use and cost-effective cloud computing resource on Amazon Web Services.

TRPV1 structures in nanodiscs reveal mechanisms of ligand and lipid action.

When integral membrane proteins are visualized in detergents or other artificial systems, an important layer of information is lost regarding lipid interactions and their effects on protein structure. This is especially relevant to proteins for which lipids have both structural and regulatory roles. Here we demonstrate the power of combining electron cryo-microscopy with lipid nanodisc technology to ascertain the structure of the rat TRPV1 ion channel in a native bilayer environment. Using this approach, we determined the locations of annular and regulatory lipids and showed that specific phospholipid interactions enhance binding of a spider toxin to TRPV1 through formation of a tripartite complex. Furthermore, phosphatidylinositol lipids occupy the binding site for capsaicin and other vanilloid ligands, suggesting a mechanism whereby chemical or thermal stimuli elicit channel activation by promoting the release of bioactive lipids from a critical allosteric regulatory site.

Structure of the full-length TRPV2 channel by cryo-EM.

Transient receptor potential (TRP) proteins form a superfamily Ca(2+)-permeable cation channels regulated by a range of chemical and physical stimuli. Structural analysis of a 'minimal' TRP vanilloid subtype 1 (TRPV1) elucidated a mechanism of channel activation by agonists through changes in its outer pore region. Though homologous to TRPV1, other TRPV channels (TRPV2-6) are insensitive to TRPV1 activators including heat and vanilloids. To further understand the structural basis of TRPV channel function, we determined the structure of full-length TRPV2 at ∼5 Šresolution by cryo-electron microscopy. Like TRPV1, TRPV2 contains two constrictions, one each in the pore-forming upper and lower gates. The agonist-free full-length TRPV2 has wider upper and lower gates compared with closed and agonist-activated TRPV1. We propose these newly revealed TRPV2 structural features contribute to diversity of TRPV channels.

Cryo-electron microscopy structure of the TRPV2 ion channel.

Transient receptor potential vanilloid (TRPV) cation channels are polymodal sensors involved in a variety of physiological processes. TRPV2, a member of the TRPV family, is regulated by temperature, by ligands, such as probenecid and cannabinoids, and by lipids. TRPV2 has been implicated in many biological functions, including somatosensation, osmosensation and innate immunity. Here we present the atomic model of rabbit TRPV2 in its putative desensitized state, as determined by cryo-EM at a nominal resolution of ∼4 Å. In the TRPV2 structure, the transmembrane segment 6 (S6), which is involved in gate opening, adopts a conformation different from the one observed in TRPV1. Structural comparisons of TRPV1 and TRPV2 indicate that a rotation of the ankyrin-repeat domain is coupled to pore opening via the TRP domain, and this pore opening can be modulated by rearrangements in the secondary structure of S6.

Cryo-electron microscopy for structural biology: current status and future perspectives.

Recently, significant technical breakthroughs in both hardware equipment and software algorithms have enabled cryo-electron microscopy (cryo-EM) to become one of the most important techniques in biological structural analysis. The technical aspects of cryo-EM define its unique advantages and the direction of development. As a rapidly emerging field, cryo-EM has benefitted from highly interdisciplinary research efforts. Here we review the current status of cryo-EM in the context of structural biology and discuss the technical challenges. It may eventually merge structural and cell biology at multiple scales.