Direct inhibition of the cold-activated TRPM8 ion channel by Gαq

Activation of the TRPM8 ion channel in sensory nerve endings produces a sensation of pleasant coolness. Here we show that inflammatory mediators such as bradykinin and histamine inhibit TRPM8 in intact sensory nerves, but do not do so through conventional signalling pathways. The G-protein subunit Gα(q) instead binds to TRPM8 and when activated by a Gq-coupled receptor directly inhibits ion channel activity. Deletion of Gα(q) largely abolished inhibition of TRPM8, and inhibition was rescued by a Gα(q) chimaera whose ability to activate downstream signalling pathways was completely ablated. Activated Gα(q) protein, but not Gβγ, potently inhibits TRPM8 in excised patches. We conclude that Gα(q) pre-forms a complex with TRPM8 and inhibits activation of TRPM8, following activation of G-protein-coupled receptors, by a direct action. This signalling mechanism may underlie the abnormal cold sensation caused by inflammation

TRPA1 channels mediate acute neurogenic inflammation and pain produced by bacterial endotoxins

Producción CientíficaGram-negative bacterial infections are accompanied by inflammation and somatic or visceral pain. These symptoms are generally attributed to sensitization of nociceptors by inflammatory mediators released by immune cells. Nociceptor sensitization during inflammation occurs through activation of the Toll-like receptor 4 (TLR4) signalling pathway by lipopolysaccharide (LPS), a toxic by-product of bacterial lysis. Here we show that LPS exerts fast, membrane delimited, excitatory actions via TRPA1, a transient receptor potential cation channel that is critical for transducing environmental irritant stimuli into nociceptor activity. Moreover, we find that pain and acute vascular reactions, including neurogenic inflammation (CGRP release) caused by LPS are primarily dependent on TRPA1 channel activation in nociceptive sensory neurons, and develop independently of TLR4 activation. The identification of TRPA1 as a molecular determinant of direct LPS effects on nociceptors offers new insights into the pathogenesis of pain and neurovascular responses during bacterial infections and opens novel avenues for their treatment.Projects SAF2010-14990 and PROMETEO2010-046. Instituto de Salud Carlos III. CONSOLIDER-INGENIO 2010. ISCIII grants R006/009 (Red Heracles), the Spanish Fundación Marcelino Botín and Belgian Federal Government (IUAP P6/28 and P7/13), the Research Foundation-Flanders and the Research Council of the KU Leuven

Methodological considerations to understand the sensory function of TRP channels

9 p., and references.Transient Receptor Potential channels are exquisite molecular transducers of multiple physical and chemical stimuli, hence the raising interest to study their relevance to Sensory Biology. Here we discuss a number of aspects of the biophysical and pharmacological properties of TRP channels, which we consider essential for a clear understanding of their sensory function in vivo. By examining concrete examples extracted from recent literature we illustrate that TRP channel research is a field in motion, and that many established dogmas on biophysical properties, drug specificity and physiological role are continuously reshaped, and sometimes even dismantled.V.M.M was supported by Spanish CONSOLIDER-INGENIO 2010 CSD2007-00023. K.T. is a fellow of the Research Foundation–Flanders (Fonds voor Wetenschappelijk Onderzoek, FWO). This work was supported by grants from Interuniversity Attraction Poles Programme (Belgian Science Policy, P6/28), FWO (G.0172.03 and G.0565.07), the Research Council of the KU Leuven (GOA 2004/07) and the Flemish Government (Excellentiefinanciering, EF/95/010).Peer reviewe

Retinoic Acid Induces Hyperactivity, and Blocking Its Receptor Unmasks Light Responses and Augments Vision in Retinal Degeneration

Light responses are initiated in photoreceptors, processed by interneurons, and synaptically transmitted to retinal ganglion cells (RGCs), which send information to the brain. Retinitis pigmentosa (RP) is a blinding disease caused by photoreceptor degeneration, depriving downstream neurons of light-sensitive input. Photoreceptor degeneration also triggers hyperactive firing of RGCs, obscuring light responses initiated by surviving photoreceptors. Here we show that retinoic acid (RA), signaling through its receptor (RAR), is the trigger for hyperactivity. A genetically encoded reporter shows elevated RAR signaling in degenerated retinas from murine RP models. Enhancing RAR signaling in healthy retinas mimics the pathophysiology of degenerating retinas. Drug inhibition of RAR reduces hyperactivity in degenerating retinas and unmasks light responses in RGCs. Gene therapy inhibition of RAR increases innate and learned light-elicited behaviors in vision-impaired mice. Identification of RAR as the trigger for hyperactivity presents a degeneration-dependent therapeutic target for enhancing low vision in RP and other blinding disorders

TRPA1 channels mediate acute neurogenic inflammation and pain produced by bacterial endotoxins

This work is licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 3.0 Unported License.-- et al.Gram-negative bacterial infections are accompanied by inflammation and somatic or visceral pain. These symptoms are generally attributed to sensitization of nociceptors by inflammatory mediators released by immune cells. Nociceptor sensitization during inflammation occurs through activation of the Toll-like receptor 4 (TLR4) signalling pathway by lipopolysaccharide (LPS), a toxic by-product of bacterial lysis. Here we show that LPS exerts fast, membrane delimited, excitatory actions via TRPA1, a transient receptor potential cation channel that is critical for transducing environmental irritant stimuli into nociceptor activity. Moreover, we find that pain and acute vascular reactions, including neurogenic inflammation (CGRP release) caused by LPS are primarily dependent on TRPA1 channel activation in nociceptive sensory neurons, and develop independently of TLR4 activation. The identification of TRPA1 as a molecular determinant of direct LPS effects on nociceptors offers new insights into the pathogenesis of pain and neurovascular responses during bacterial infections and opens novel avenues for their treatment. © 2014 Macmillan Publishers Limited. All rights reserved.O.F. and C.F.P. were predoctoral students of the Generalitat Valenciana. S.T., B.D., V.M. and J.A.M. were supported by predoctoral fellowships from the Spanish MINECO. Research was supported by Spanish public funds projects SAF2010-14990 and PROMETEO2010-046 to F.V., BFU2007-61524 to J.R.L.L., BFU2010-15898 to M.T.P.G., Instituto de Salud Carlos III PI12/00586 to R.S., BFU2005-08741 and CONSOLIDER-INGENIO 2010 CSD2007-00023 to C.B., ISCIII grants R006/009 (Red Heracles), the Spanish Fundación Marcelino Botín and Belgian Federal Government (IUAP P6/28 and P7/13), the Research Foundation-Flanders (F.W.O. G.0565.07, G.0686.09, G.A022.11N and G.0702.12), and the Research Council of the KU Leuven (GOA 2009/07, EF/95/010, PFV/10/006, OT/12/091 and GOA 14011).Peer Reviewe

How Azobenzene Photoswitches Restore Visual Responses to the Blind Retina

Azobenzene photoswitches confer light sensitivity onto retinal ganglion cells (RGCs) in blind mice, making these compounds promising candidates as vision-restoring drugs in humans with degenerative blindness. Remarkably, photosensitization manifests only in animals with photoreceptor degeneration and is absent from those with intact rods and cones. Here we show that P2X receptors mediate the entry of photoswitches into RGCs, where they associate with voltage-gated ion channels, enabling light to control action-potential firing. All charged photoswitch compounds require permeation through P2X receptors, whose gene expression is upregulated in the blind retina. Photoswitches and membrane-impermeant fluorescent dyes likewise penetrate through P2X receptors to label a subset of RGCs in the degenerated retina. Electrophysiological recordings and mapping of fluorescently labeled RGC dendritic projections together indicate that photosensitization is highly selective for OFFRGCs. Hence, P2X receptors are a natural conduit allowing cell-type-selective and degeneration-specific delivery of photoswitches to restore visual function in blinding disease

TRPA1 channels are early sensors of gram-negative bacterial endotoxins

Resumen del trabajo presentado al 12th World Congress on Inflammation, Symposium 18: Ion Channels and Inflammation-Sponsored by IRN-Canada; celebrado en Boston (US) del 8 al 12 de agosto de 2015.-- et al.TRPA1 are calcium permeable, non-selective cation channels expressed in sensory endings of somatic and visceral nociceptors and many non-neuronal cells, including fibroblasts, endothelial and glial cells. These ion channels are critically involved in the biological response to physical stimuli (temperature and pressure) and natural and synthetic environmental irritants, including many electrophiles, reactive oxygen species and endogenous inflammatory mediators produced following tissue damage. Abnormal activation of TRPA1 has been linked to the pathogenesis of neuropathic pain and itch, and a number of systemic inflammatory diseases, including atopic dermatitis, irritable bowel syndrome and asthma. Gram-negative bacterial infections are accompanied by inflammation and somatic or visceral pain. These symptoms are generally attributed to sensitization of nociceptors by inflammatory mediators released by immune cells. Nociceptor sensitization during inflammation by lipopolysaccharide (LPS), a toxic byproduct of gramnegative bacterial lysis, is thought to occur through activation of pattern-recognition receptors, in particular the Toll-like-receptor 4 (TLR4) signaling pathway. Using a combination of experimental techniques, including electrophysiological recordings and cellular calcium imaging, we found that LPS exerts fast (seconds) activation of mouse somatic and visceral nociceptors. These actions were mediated by opening of TRPA1 channels leading to neuronal depolarization, firing of action potentials and rapid elevation of intracellular calcium levels. Human TRPA1 channels showed a similar sensitivity to LPS. LPS, injected intradermally, produced a rapid (minutes) inflammatory response that was accompanied by pain and acute vascular reactions, including neurogenic inflammation and CGRP release (measured by enzyme immunoassay). These responses were severely blunted in TRPA1 KO mice and developed independently of TLR4 activation. Moreover, the capacity of various forms of LPS, purified from different pathogens, to activate TRPA1 in vitro correlated with their ability to induce inflammatory responses in vivo. In summary, we identified TRPA1 channels as key molecular determinants of rapid LPS effects on sensory neurons and their terminals, leading to acute neurogenic inflammation and pain. These results suggest that TRPA1 channels may play a previously unrecognized role in the first line of immune defense against microbial pathogens. Pharmacological targeting of TPA1 channels could represent a novel therapeutic avenue for treatment of complications derived from gram negative bacterial infections, including septic shock.Financial support provided by project SAF2013-45608-R of MINECO.Peer reviewe

How Azobenzene Photoswitches Restore Visual Responses to the Blind Retina

Azobenzene photoswitches confer light sensitivity onto retinal ganglion cells (RGCs) in blind mice, making these compounds promising candidates as vision-restoring drugs in humans with degenerative blindness. Remarkably, photosensitization manifests only in animals with photoreceptor degeneration and is absent from those with intact rods and cones. Here we show that P2X receptors mediate the entry of photoswitches into RGCs where they associate with voltage-gated ion channels, enabling light to control action potential firing. All charged photoswitch compounds require permeation through P2X receptors, whose gene expression is upregulated in the blind retina. Photoswitches and membrane-impermeant fluorescent dyes likewise penetrate through P2X receptors to label a subset of RGCs in the degenerated retina. Electrophysiological recordings and mapping of fluorescently-labeled RGC dendritic projections together indicate that photosensitization is highly selective for OFF-RGCs. Hence P2X receptors are a natural conduit allowing cell type-selective and degeneration-specific delivery of photoswitches to restore visual function in blinding disease