Indiscriminate activation of opioid receptors provides pain relief but also severe central and intestinal side effects. We hypothesized that exploiting pathological (rather than physiological) ...conformation dynamics of opioid receptor-ligand interactions might yield ligands without adverse actions. By computer simulations at low pH, a hallmark of injured tissue, we designed an agonist that, because of its low acid dissociation constant, selectively activates peripheral μ-opioid receptors at the source of pain generation. Unlike the conventional opioid fentanyl, this agonist showed pH-sensitive binding, heterotrimeric guanine nucleotide–binding protein (G protein) subunit dissociation by fluorescence resonance energy transfer, and adenosine 3′,5′-monophosphate inhibition in vitro. It produced injury-restricted analgesia in rats with different types of inflammatory pain without exhibiting respiratory depression, sedation, constipation, or addiction potential.
Background
The ion channel TRPV1 is mainly expressed in small diameter dorsal root ganglion (DRG) neurons, which are involved in the sensation of acute noxious thermal and chemical stimuli. Direct ...modifications of the channel by diverse signalling events have been intensively investigated, but little is known about the composition of modulating macromolecular TRPV1 signalling complexes. Here, we hypothesize that the novel adaptor protein ankyrin‐rich membrane spanning protein/kinase D interacting substrate (ARMS) interacts with TRPV1 and modulates its function in rodent DRG neurons.
Methods
We used immunohistochemistry, electrophysiology, microfluorimetry and immunoprecipitation experiments to investigate TRPV1 and ARMS interactions in DRG neurons and transfected cells.
Results
We found that TRPV1 and ARMS are co‐expressed in a subpopulation of DRG neurons. ARMS sensitizes TRPV1 towards capsaicin in transfected HEK 293 cells and in mouse DRG neurons in a PKA‐dependent manner. Using a combination of functional imaging and immunocytochemistry, we show that the magnitude of the capsaicin response in DRG neurons depends not only on TRPV1 expression, but on the co‐expression of ARMS alongside TRPV1.
Conclusion
These data indicate that ARMS is an important component of the signalling complex regulating the sensitivity of TRPV1.
Significance
The study identifies ARMS as an important component of the signalling complex regulating the sensitivity of excitatory ion channels (TRPV1) in peripheral sensory neurons (DRG neurons) and transfected cells.
Transient receptor potential vanilloid 1 (TRPV1) is a nonselective ligand-gated cation channel responding to noxious heat, protons, and chemicals such as capsaicin. TRPV1 is expressed in sensory ...neurons and plays a critical role in pain associated with tissue injury, inflammation, and nerve lesions. Transient receptor potential ankyrin 1 (TRPA1) is coexpressed with TRPV1. It is activated by compounds that cause a burning sensation (e.g., mustard oil) and, indirectly, by components of the inflammatory milieu eliciting nociceptor excitation and pain hypersensitivity. Previous studies indicate an interaction of TRPV1 and TRPA1 signaling pathways. Here we sought to examine the molecular mechanisms underlying such interactions in nociceptive neurons. We first excluded physical interactions of both channels using radioligand binding studies. By microfluorimetry, electrophysiological experiments, cAMP measurements, and site-directed mutagenesis we found a sensitization of TRPV1 after TRPA1 stimulation with mustard oil in a calcium and cAMP/protein kinase A (PKA)-dependent manner. TRPA1 stimulation enhanced TRPV1 phosphorylation via the putative PKA phosphorylation site serine 116. We also detected calcium-sensitive increased TRPV1 activity after TRPA1 activation in dorsal root ganglion neurons. The inhibition of TRPA1 by HC-030031 (1,2,3,6-tetrahydro-1,3-dimethyl-N-4-(1-methylethyl)phenyl-2,6-dioxo-7H-purine-7-acetamide, 2-(1,3-dimethyl-2,6-dioxo-1,2,3,6-tetrahydro-7H-purin-7-yl)-N-(4-isopropylphenyl)acetamide) after its initial stimulation (and the calcium-insensitive TRPA1 mutant D477A) still showed increased capsaicin-induced TRPV1 activity. This excludes a calcium-induced additive TRPA1 current after TRPV1 stimulation. Our study shows sensitization of TRPV1 via activation of TRPA1, which involves adenylyl cyclase, increased cAMP, subsequent translocation and activation of PKA, and phosphorylation of TRPV1 at PKA phosphorylation residues. This suggests that cross-sensitization of TRP channels contributes to enhanced pain sensitivity in inflamed tissues.
The mechanisms of drug–receptor interactions and the controlled delivery of drugs via biodegradable and biocompatible nanoparticulate carriers are active research fields in nanomedicine. Many ...clinically used drugs target G-protein coupled receptors (GPCRs) due to the fact that signaling via GPCRs is crucial in physiological and pathological processes and thus central for the function of biological systems. In this letter, a fast and reliable ratiometric fluorescence lifetime imaging microscopy (rmFLIM) approach is described to analyze the distribution of protein–ligand complexes in the cellular context. Binding of the fluorescently labeled antagonist naloxone to the G-protein coupled μ-opioid receptor is used as an example. To show the broad applicability of the rmFLIM method, we extended this approach to investigate the distribution of polymer-based nanocarriers in histological liver sections.
Hyperalgesia is a cardinal symptom of opioid withdrawal. The transient receptor potential vanilloid 1 (TRPV1) is a ligand-gated ion channel expressed on sensory neurons responding to noxious heat, ...protons, and chemical stimuli such as capsaicin. TRPV1 can be inhibited via μ-opioid receptor (MOR)-mediated reduced activity of adenylyl cyclases (ACs) and decreased cyclic adenosine monophosphate (cAMP) levels. In contrast, opioid withdrawal following chronic activation of MOR uncovers AC superactivation and subsequent increases in cAMP and protein kinase A (PKA) activity. Here we investigated (1) whether an increase in cAMP during opioid withdrawal increases the activity of TRPV1 and (2) how opioid withdrawal modulates capsaicin-induced nocifensive behavior in rats. We applied whole-cell patch clamp, microfluorimetry, cAMP assays, radioligand binding, site-directed mutagenesis, and behavioral experiments. Opioid withdrawal significantly increased cAMP levels and capsaicin-induced TRPV1 activity in both transfected human embryonic kidney 293 cells and dissociated dorsal root ganglion (DRG) neurons. Inhibition of AC and PKA, as well as mutations of the PKA phosphorylation sites threonine 144 and serine 774, prevented the enhanced TRPV1 activity. Finally, capsaicin-induced nocifensive behavior was increased during opioid withdrawal in vivo. In summary, our results demonstrate an increased activity of TRPV1 in DRG neurons as a new mechanism contributing to opioid withdrawal-induced hyperalgesia.
The patch clamp is a valuable electrophysiological technique, which allows the study of single or multiple ion channels in cells, and it is particularly useful in testing the excitable cells such as ...neurons. Activation of neuronal opioid receptors results in the modulation of various ion channels, which enables to examine the receptors' action with the patch clamp. In this chapter, we analyze the activation of the G-protein-coupled inwardly rectifying potassium channel 2 by opioids, and the capsaicin-induced transient receptor potential vanilloid 1 channel currents during opioid withdrawal, using the whole cell patch clamp in transfected human embryonic kidney 293 cells as well as in mouse and rat primary dorsal root ganglion neurons.
Salivary gland carcinosarcoma, or true malignant mixed tumor, is a very rare and extremely aggressive neoplasm. The clonality and clonal origin of this tumor are discussed controversially. We report ...a carcinsarcoma of the left parotid gland in a patient who subsequently died of cutaneous, lymphatic and pulmonary metastases. Immunohistochemical staining, electron micrograph analysis, loss of heterozygosity (LOH) analysis and sequence analysis were performed on this tumor with an adenocarcinomatous and a predominant spindle cell-like component. While smooth muscle actin was undetectable by immunohistochemistry, cytoplasmatic myoepithelial structures could be detected by electron microscopy. LOH analysis at 12 genomic locations detected complete deletion of one allele at 17p13.1, 17q21.3, and 18q21.3 indicating allelic loss in both components of the tumor. Double strand sequencing of the remaining allele of the
p53 tumor suppressor gene revealed a wild-type allele. Based on our results, we favor the hypothesis of monoclonal origin of this salivary gland carcinosarcoma with a common stem cell that could be the myoepithelial cell and an inactivated tumor suppressor gene on chromosome 17 other than
p53.
Macromolecular machines, such as the ribosome, undergo large-scale conformational changes during their functional cycles. Although their mode of action is often compared to that of mechanical ...machines, a crucial difference is that, at the molecular dimension, thermodynamic effects dominate functional cycles, with proteins fluctuating stochastically between functional states defined by energetic minima on an energy landscape. Here, we have used cryo-electron microscopy to image ex-vivo-derived human polysomes as a source of actively translating ribosomes. Multiparticle refinement and 3D variability analysis allowed us to visualize a variety of native translation intermediates. Significantly populated states include not only elongation cycle intermediates in pre- and post-translocational states, but also eEF1A-containing decoding and termination/recycling complexes. Focusing on the post-translocational state, we extended this assessment to the single-residue level, uncovering striking details of ribosome-ligand interactions and identifying both static and functionally important dynamic elements.
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•Structural analysis of actively translating ribosomes•Identification of significantly populated states at close to in vivo conditions•Functional states feature localized chemical and conformational heterogeneity•Human 80S ribosome map at near-atomic resolution reveals native interactions
Multiparticle cryo-EM analysis reveals 11 distinct functional states from a native actively translating human polysomal sample, providing insights into the configuration of the human ribosome at near-atomic resolution and highlighting the functional importance of both rigid and flexible regions.
Structural studies of translating ribosomes traditionally rely on in vitro assembly and stalling of ribosomes in defined states. To comprehensively visualize bacterial translation, we reactivated ex ...vivo-derived E. coli polysomes in the PURE in vitro translation system and analyzed the actively elongating polysomes by cryo-EM. We find that 31% of 70S ribosomes assemble into disome complexes that represent eight distinct functional states including decoding and termination intermediates, and a pre-nucleophilic attack state. The functional diversity of disome complexes together with RNase digest experiments suggests that paused disome complexes transiently form during ongoing elongation. Structural analysis revealed five disome interfaces between leading and queueing ribosomes that undergo rearrangements as the leading ribosome traverses through the elongation cycle. Our findings reveal at the molecular level how bL9's CTD obstructs the factor binding site of queueing ribosomes to thwart harmful collisions and illustrate how translation dynamics reshape inter-ribosomal contacts.
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