Search

Resumen del póster presentado al 4th International TRP Meeting, celebrado virtualmente del 15 al 17 de septiembre de 2021., TRPA1 channels play a fundamental role in chemonociception, as molecular transducers of reactive irritants, stress and tissue damage. Their role in cold and mechanical nociception has also being described. Sigma-1 receptor (σ-1R) is a molecular chaperone that modulates both trafficking and function of various ion channels. In mice, the σ-1R antagonist, S1RA, is able to reduce the symptoms of neuropathic pain. Since the molecular determinants of this antinociceptive effect remain unknown, we studied a possible modulation of TRPA1 by σ-1R. We performed calcium imaging and patch-clamp experiments in HEK293 cells transfected with hTRPA1 tagged with tGFP (hTRPA1-tGFP). Incubation of these cells with S1RA decreased the amplitude of [Ca2+]i responses and of the membrane currents evoked by AITC. Similar results were obtained with BD1086, another σ-1R antagonist. FRET experiments in cells transfected with hTRPA1-tGFP and mCherry- σ-1R revealed that both proteins are localized at a distance compatible with a physical interaction that was supported by co-immunoprecipitation experiments. Treatment of these cells with S1RA decreased FRET levels, suggesting an impairment or conformational change of this putative interaction. Finally, TIRF-FRAP experiments indicate that S1RA reduces the trafficking of TRPA1 towards the plasma membrane, resulting in a reduction of TRPA1 expression at the plasma membrane which was confirmed by cell surface biotinylation assays. These results suggest a role TRPA1 in the anti-nociceptive effects of σ-1R antagonists., Funding source: SAF2016-77233-R, PID2019-108194RB-100, co-financed by ERDF, Severo Ochoa Program SEV-2017-0723 and GRISOLIA/2015/034.

Resumen del póster presentado al 49th Annual Meeting of the Society for Neuroscience, celebrado en Chicago (USA) del 19 al 23 de octubre de 2019., TRPA1 channels expressed in mammalian nociceptor endings play a critical role in chemonociception as a molecular sensors of reactive irritants, stress and tissue damage. Additionally, TRPA1 has been implicated in noxious cold and mechanical pain sensation. Sigma-1 receptor (σ-1R) is a chaperone mainly located in endoplasmic reticulum membrane, acting as an interoganelle signaling modulator that regulates the trafficking and function of different ionic channels. In the context of pain, pharmacological treatment of mice with the σ-1R antagonist, E-52862 (provided by ESTEVE PHARMACEUTICALS SA), produces antinociceptive effects, and has been shown to reduce the symptoms of neuropathic pain but the molecular mechanism is still unresolved. We studied the possible role of σ-1R in the modulation of TRPA1 channels and the result of this modulation on pain in mice. Measurement of intracellular calcium changes [Ca2+]i (FURA-2), and patch-clamp recordings were carried out in HEK-293 cells transfected with human TRPA1 (HEK-hTRPA1), and in cultured mouse DRG sensory neurons responsive to 50 µM AITC, a TRPA1 agonist. In both systems, incubation (4-24h) with E-52862 decreased significantly, in a dose dependent manner, the amplitude of [Ca2+]i responses evoked by AITC. Whole-cell membrane currents in HEK-hTRPA1 cells in response to AITC were also reduced, following incubation with the drug. In DRG sensory neurons the firing of action potentials, recorded in cell-attached mode, the membrane depolarization and the number of action potentials recorded in whole-cell current-clamp configuration evoked by AITC, were also significantly reduced following incubation with E-52862. In mice, we found that pain behaviors (licking, lifting and flickering) associated with TRPA1 activation by intraplantar injection of 10 mM AITC, decreased following intraperitoneal injection 24 h before of (40mg/kg) E-52862. These results implicate TRPA1 channels in the anti-nociceptive effects of σ-1R antagonists. All experimental procedures were carried out according to Spanish Royal Decree 1201/2005 and the ECC directive 2010/63/EU., Esteve Pharmaceuticals SA; SAF2016-77233-R co-financed by the European Regional Development Fund (ERDF); SEV- 2013-0317; GRISOLIA/2015/034.