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 play 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 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 release of bioactive lipids from a critical allosteric regulatory site.Transporters and ion channels reside in biological membranes, where lipids play important structural and regulatory roles 1-3 . However, structural characterization of protein-lipid interactions is challenging in detergent-based systems, making implementation of more Author ContributionsY.G. carried out protein purification, nanodisc reconstitution, and detailed cryo-EM experiments, including data acquisition, image processing, atomic model building and refinement of TRPV1-nanodisc complexes. E.C. carried out cryo-EM experiments of TRPV1-capsazepine complex solubilized in amphipol. All authors contributed to experimental design, data analysis, and manuscript preparation. Author InformationThe 3D cryo-EM density maps of TRPV1-nanodisc complexes without low-pass filter and amplitude modification have been deposited in the Electron Microscopy Data Bank under the accession numbers EMD-8118 (TRPV1-nanodisc), EMD-8117 (TRPV1-RTX/DkTx-nanodisc), EMD-8119 (TRPV1-capsazepine-nanodisc) and EMD-8120 (TRPV1-capsazepine in amphipol). Particle image stacks after motion correction related to TRPV1-nanodisc and TRPV1-RTX/DkTx-nanodisc are deposited for download at http:// www.ebi.ac.uk/pdbe/emdb/empiar/ with identification number EMPIAR-10059. Atomic coordinates for the atomic model of TRPV1 in nanodisc, TRPV1-RTX/DkTx in nanodisc and TRPV1-capsazepine in nanodisc have been deposited in the Protein Data Bank under the accession number 5IRZ, 5IRX and 5IS0.
The TRPA1 ion channel (a.k.a the ‘wasabi receptor’) is a detector of noxious chemical agents encountered in our environment or produced endogenously during tissue injury or drug metabolism. These include a broad class of electrophiles that activate the channel through covalent protein modification. TRPA1 antagonists hold potential for treating neurogenic inflammatory conditions provoked or exacerbated by irritant exposure. Despite compelling reasons to understand TRPA1 function, structural mechanisms underlying channel regulation remain obscure. Here, we use single-particle electron cryo-microscopy to determine the structure of full-length human TRPA1 to ~4Å resolution in the presence of pharmacophores, including a potent antagonist. A number of unexpected features are revealed, including an extensive coiled-coil assembly domain stabilized by polyphosphate co-factors and a highly integrated nexus that converges on an unpredicted TRP-like allosteric domain. These findings provide novel insights into mechanisms of TRPA1 regulation, and establish a blueprint for structure-based design of analgesic and anti-inflammatory agents.
The TRPA1 ion channel (also known as the wasabi receptor) is a detector of noxious chemical agents encountered in our environment or produced endogenously during tissue injury or drug metabolism. These include a broad class of electrophiles that activate the channel through covalent protein modification. TRPA1 antagonists hold potential for treating neurogenic inflammatory conditions provoked or exacerbated by irritant exposure. Despite compelling reasons to understand TRPA1 function, structural mechanisms underlying channel regulation remain obscure. Here we use single-particle electron cryo-microscopy to determine the structure of full-length human TRPA1 to~4 Å resolution in the presence of pharmacophores, including a potent antagonist. Several unexpected features are revealed, including an extensive coiled-coil assembly domain stabilized by polyphosphate co-factors and a highly integrated nexus that converges on an unpredicted transient receptor potential (TRP)-like allosteric domain. These findings provide new insights into the mechanisms of TRPA1 regulation, and establish a blueprint for structure-based design of analgesic and anti-inflammatory agents.
Micro-sized hair-like structures, such as cilia, are abundant in nature and have various functionalities. Many efforts have been made to mimic the fluid pumping function of cilia, but most of the fabrication processes for these "artificial cilia" are tedious and expensive, hindering their practical application. In this paper a cost-effective in situ fabrication technique for artificial cilia is demonstrated. The cilia are constructed by self-assembly of micron sized magnetic beads and encapsulated with soft polymer coatings. Actuation of the cilia induces an effective fluid flow, and the cilia lengths and distribution can be adjusted by varying the magnetic bead concentration and fabrication parameters.
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