Structural insight into the assembly of TRPV channels.
Ontology highlight
ABSTRACT:
SUBMITTER: Huynh KW
PROVIDER: S-EPMC5548120 | biostudies-literature | 2014 Feb
REPOSITORIES: biostudies-literature
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Project description:Transient Receptor Potential channels from the vanilloid subfamily (TRPV) are a group of cation channels modulated by a variety of endogenous stimuli as well as a range of natural and synthetic compounds. Their roles in human health make them of keen interest, particularly from a pharmacological perspective. However, despite this interest, the complexity of these channels has made it difficult to obtain high resolution structures until recently. With the cryo-EM resolution revolution, TRPV channel structural biology has blossomed to produce dozens of structures, covering every TRPV family member and a variety of approaches to examining channel modulation. Here, we review all currently available TRPV structures and the mechanistic insights into gating that they reveal.
| S-EPMC7153993 | biostudies-literature
Project description:Transient receptor potential channels, of the vanilloid subtype (TRPV), act as sensory mediators, being activated by endogenous ligands, heat, mechanical and osmotic stress. Within the vasculature, TRPV channels are expressed in smooth muscle cells, endothelial cells, as well as in peri-vascular nerves. Their varied distribution and polymodal activation properties make them ideally suited to a role in modulating vascular function, perceiving and responding to local environmental changes. In endothelial cells, TRPV1 is activated by endocannabinoids, TRPV3 by dietary agonists and TRPV4 by shear stress, epoxyeicosatrienoic acids (EETs) and downstream of Gq-coupled receptor activation. Upon activation, these channels contribute to vasodilation via nitric oxide, prostacyclin and intermediate/small conductance potassium channel-dependent pathways. In smooth muscle, TRPV4 is activated by endothelial-derived EETs, leading to large conductance potassium channel activation and smooth muscle hyperpolarization. Conversely, smooth muscle TRPV2 channels contribute to global calcium entry and may aid constriction. TRPV1 and TRPV4 are expressed in sensory nerves and can cause vasodilation through calcitonin gene-related peptide and substance P release as well as mediating vascular function via the baroreceptor reflex (TRPV1) or via increasing sympathetic outflow during osmotic stress (TRPV4). Thus, TRPV channels play important roles in the regulation of normal and pathological cellular function in the vasculature.
| S-EPMC3134601 | biostudies-literature
Project description:Transient receptor potential (TRP) proteins are cation channels composed of a transmembrane domain flanked by large N- and C-terminal cytoplasmic domains. All members of the vanilloid family of TRP channels (TRPV) possess an N-terminal ankyrin repeat domain (ARD). The ARD of mammalian TRPV6, an important regulator of calcium uptake and homeostasis, is essential for channel assembly and regulation. The 1.7 A crystal structure of the TRPV6-ARD reveals conserved structural elements unique to the ARDs of TRPV proteins. First, a large twist between the fourth and fifth repeats is induced by residues conserved in all TRPV ARDs. Second, the third finger loop is the most variable region in sequence, length and conformation. In TRPV6, a number of putative regulatory phosphorylation sites map to the base of this third finger. Size exclusion chromatography and crystal packing indicate that the TRPV6-ARD does not assemble as a tetramer and is monomeric in solution. Adenosine triphosphate-agarose and calmodulin-agarose pull-down assays show that the TRPV6-ARD does not interact with either ligand, indicating a different functional role for the TRPV6-ARD than in the paralogous thermosensitive TRPV1 channel. Similar biochemical findings are also presented for the highly homologous mammalian TRPV5-ARD. The implications of the structural and biochemical data on the role of the ankyrin repeats in different TRPV channels are discussed.
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Project description:BACKGROUND AND PURPOSE:Heat-sensitive transient receptor potential vanilloid (TRPV) channels are expressed in various epithelial tissues regulating, among else, barrier functions. Their expression is well established in the distal nephron; however, we have no data about their presence in podocytes. As podocytes are indispensable in the formation of the glomerular filtration barrier, we investigated the presence and function of Ca2+ -permeable TRPV1-4 channels in human podocyte cultures. EXPERIMENTAL APPROACH:Expression of TRPV1-4 channels was investigated at protein (immunocytochemistry, Western blot) and mRNA (Q-PCR) level in a conditionally immortalized human podocyte cell line. Channel function was assessed by measuring intracellular Ca2+ concentration using Flou-4 Ca2+ -indicator dye and patch clamp electrophysiology upon applying various activators and inhibitors. KEY RESULTS:Thermosensitive TRP channels were expressed in podocytes. The TRPV1-specific agonists capsaicin and resiniferatoxin did not affect the intracellular Ca2+ concentration. Cannabidiol, an activator of TRPV2 and TRPV4 channels, induced moderate Ca2+ -influxes, inhibited by both tranilast and HC067047, blockers of TRPV2 and TRPV4 channels respectively. The TRPV4-specific agonists GSK1016790A and 4?-phorbol 12,13-didecanoate induced robust Ca2+ -signals which were abolished by HC067047. Non-specific agonists of TRPV3 channels induced marked Ca2+ transients. However, TRPV3 channel blockers, ruthenium red and isopentenyl diphosphate only partly inhibited the responses and TRPV3 silencing was ineffective suggesting remarkable off-target effects of the compounds. CONCLUSION AND IMPLICATIONS:Our results indicate the functional presence of TRPV4 and other thermosensitive TRPV channels in human podocytes and raise the possibility of their involvement in the regulation of glomerular filtration barrier.
| S-EPMC5715976 | biostudies-literature
Project description:The Vanilloid thermoTRP (TRPV1-4) subfamily of TRP channels are involved in thermoregulation, osmoregulation, itch and pain perception, (neuro)inflammation and immune response, and tight control of channel activity is required for perception of noxious stimuli and pain. Here we report voltage-dependent modulation of each of human TRPV1, 3, and 4 by the endogenous intracellular polyamine spermine. As in inward rectifier K channels, currents are blocked in a strongly voltage-dependent manner, but, as in cyclic nucleotide-gated channels, the blockade is substantially reduced at more positive voltages, with maximal blockade in the vicinity of zero voltage. A kinetic model of inhibition suggests two independent spermine binding sites with different affinities as well as different degrees of polyamine permeability in TRPV1, 3, and 4. Given that block and relief occur over the physiological voltage range of action potentials, voltage-dependent polyamine block may be a potent modulator of TRPV-dependent excitability in multiple cell types.
| S-EPMC10038874 | biostudies-literature
Project description:Transient receptor potential (TRP) cation channel genes code for an extensive family of conserved proteins responsible for a variety of physiological processes, including sensory perception, ion homeostasis, and chemical signal transduction. The TRP superfamily consists of seven subgroups, one of which is the transient receptor potential vanilloid (trpv) channel family. While trpv channels are relatively well studied in adult vertebrate organisms given their role in functions such as nociception, thermoregulation, and osmotic regulation in mature tissues and organ systems, relatively little is known regarding their function during embryonic development. Although there are some reports of the expression of specific trpv channels at particular stages in various organisms, there is currently no comprehensive analysis of trpv channels during embryogenesis. Here, performing in situ hybridization, we examined the spatiotemporal expression of trpv channel mRNA during early Xenopus laevis embryogenesis. Trpv channels exhibited unique patterns of embryonic expression at distinct locations including the trigeminal ganglia, spinal cord, cement gland, otic vesicle, optic vesicle, nasal placode, notochord, tailbud, proctodeum, branchial arches, epithelium, somite and the animal pole during early development. We have also observed the colocalization of trpv channels at the animal pole (trpv 2/4), trigeminal ganglia (trpv 1/2), and epithelium (trpv 5/6). These localization patterns suggest that trpv channels may play diverse roles during early embryonic development.
| S-EPMC6319392 | biostudies-literature
Project description:During mitosis and meiosis, microtubule (MT) assembly is locally upregulated by the chromatin-dependent Ran-GTP pathway. One of its key targets is the MT-associated spindle assembly factor TPX2. The molecular mechanism of how TPX2 stimulates MT assembly remains unknown because structural information about the interaction of TPX2 with MTs is lacking. Here, we determine the cryo-electron microscopy structure of a central region of TPX2 bound to the MT surface. TPX2 uses two flexibly linked elements ('ridge' and 'wedge') in a novel interaction mode to simultaneously bind across longitudinal and lateral tubulin interfaces. These MT-interacting elements overlap with the binding site of importins on TPX2. Fluorescence microscopy-based in vitro reconstitution assays reveal that this interaction mode is critical for MT binding and facilitates MT nucleation. Together, our results suggest a molecular mechanism of how the Ran-GTP gradient can regulate TPX2-dependent MT formation.
| S-EPMC5679754 | biostudies-literature
Project description:Apparent blockage of monovalent cation currents by the permeating blocker Ca(2+) is a physiologically essential phenomenon relevant to cyclic nucleotide-gated (CNG) channels. The recently determined crystal structure of a bacterial homolog of CNG channel pores, the NaK channel, revealed a Ca(2+) binding site at the extracellular entrance to the selectivity filter. This site is not formed by the side-chain carboxylate groups from the conserved acidic residue, Asp-66 in NaK, conventionally thought to directly chelate Ca(2+) in CNG channels, but rather by the backbone carbonyl groups of residue Gly-67. Here we present a detailed structural analysis of the NaK channel with a focus on Ca(2+) permeability and blockage. Our results confirm that the Asp-66 residue, although not involved in direct chelation of Ca(2+), plays an essential role in external Ca(2+) binding. Furthermore, we give evidence for the presence of a second Ca(2+) binding site within the NaK selectivity filter where monovalent cations also bind, providing a structural basis for Ca(2+) permeation through the NaK pore. Compared with other Ca(2+)-binding proteins, both sites in NaK present a novel mode of Ca(2+) chelation, using only backbone carbonyl oxygen atoms from residues in the selectivity filter. The external site is under indirect control by an acidic residue (Asp-66), making it Ca(2+)-specific. These findings give us a glimpse of the possible underlying mechanisms allowing Ca(2+) to act both as a permeating ion and blocker of CNG channels and raise the possibility of a similar chemistry governing Ca(2+) chelation in Ca(2+) channels.
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Project description:Transient receptor potential vanilloid (TRPV) channels play a critical role in calcium homeostasis, pain sensation, immunological response, and cancer progression. TRPV channels are blocked by ruthenium red (RR), a universal pore blocker for a wide array of cation channels. Here we use cryo-electron microscopy to reveal the molecular details of RR block in TRPV2 and TRPV5, members of the two TRPV subfamilies. In TRPV2 activated by 2-aminoethoxydiphenyl borate, RR is tightly coordinated in the open selectivity filter, blocking ion flow and preventing channel inactivation. In TRPV5 activated by phosphatidylinositol 4,5-bisphosphate, RR blocks the selectivity filter and closes the lower gate through an interaction with polar residues in the pore vestibule. Together, our results provide a detailed understanding of TRPV subfamily pore block, the dynamic nature of the selectivity filter and allosteric communication between the selectivity filter and lower gate.
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Project description:Phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] is an important cofactor for ion channels. Affinity for this lipid is a major determinant of channel inhibition by depletion of PI(4,5)P2 upon phospholipase C (PLC) activation. Little is known about what determines PI(4,5)P2 affinity in mammalian ion channels. Here we report that two members of the Transient Receptor Potential Vanilloid (TRPV) ion channel family, TRPV5 and TRPV6 lack a positively charged residue in the TM4-TM5 loop that was shown to interact with PI(4,5)P2 in TRPV1, which shows high affinity for this lipid. When this positively charged residue was introduced to either TRPV6 or TRPV5, they displayed markedly higher affinities for PI(4,5)P2, and were largely resistant to inhibition by PI(4,5)P2 depletion. Furthermore, Ca(2+)-induced inactivation of TRPV6 was essentially eliminated in the G488R mutant, showing the importance of PLC-mediated PI(4,5)P2 depletion in this process. Computational modeling shows that the introduced positive charge interacts with PI(4,5)P2 in TRPV6.
| S-EPMC4904367 | biostudies-literature