Endogenous Mas-related G-protein-coupled receptor X1 activates and sensitizes TRPA1 in a human model of peripheral nerves

Abstract

Mas-related G-protein-coupled receptor X1 (MrgprX1) is a human-specific Mrgpr and its expression is restricted to primary sensory neurons. However, its role in nociception and pain signaling pathways is largely unknown. This study aims to investigate a role for MrgprX1 in nociception via interaction with the pain receptor, Transient Receptor Potential Ankyrin 1 (TRPA1), using in-vitro and in-vivo human neuronal models. MrgprX1 protein expression in human trigeminal nociceptors was investigated by the immunolabeling of the dental pulp and cultured peripheral neuronal equivalent (PNE) cells. MrgprX1 receptor signaling was monitored by Fura-2-based Ca2+ imaging using PNEs and membrane potential responses were measured using FluoVoltTM. Immunofluorescent staining revealed MrgprX1 expression in-vivo in dental afferents, which was more intense in inflamed compared to healthy dental pulps. Endogenous MrgprX1 protein expression was confirmed in the in-vitro human PNE model. MrgprX1 receptor signaling and the mechanisms through which it couples to TRPA1 were studied by Ca2+ imaging. Results showed that MrgprX1 activates TRPA1 and induces membrane depolarization in a TRPA1 dependent manner. In addition, MrgprX1 sensitizes TRPA1 to agonist stimulation via Protein Kinase C (PKC). The activation and sensitization of TRPA1 by MrgprX1 in a model of human nerves suggests an important role for this receptor in the modulation of nociception.

KEYWORDS
dental pulp, human, MrgprX1, nociception, peripheral neurons

Presenter: Hye In Kim

TMEM120A/TACAN inhibits mechanically activated Piezo2 channels

John Smith Del Rosario*, Matthew Gabrielle*, Yevgen Yudin and Tibor Rohacs# 

Department of Pharmacology, Physiology and Neuroscience, Rutgers, New Jersey Medical School, Newark, NJ 08540 

ABSTRACT

Mechanically activated Piezo2 channels are key mediators of light touch and proprioception in mice and humans. Relatively little is known about what other proteins regulate Piezo2 activity in a cellular context. TACAN (TMEM120A) was proposed to act as a high threshold mechanically activated ion channel in nociceptive dorsal root ganglion (DRG) neurons. Here we find that TACAN co-expression robustly reduced mechanically activated Piezo2 currents, but did not inhibit mechanically activated Piezo1 and TREK1 currents. TACAN co-expression did not affect cell surface expression of either Piezo1 or Piezo2 and did not have major effects on the cortical actin or tubulin cytoskeleton. TACAN expression alone did not result in the appearance of mechanically activated currents above background. In addition, TACAN and Piezo2 expression in DRG neurons overlapped, and siRNA mediated knockdown of TACAN did not decrease the proportion of slowly adapting mechanically activated currents, but resulted in an increased proportion of rapidly adapting currents. Our data do not support TACAN being a mechanically activated ion channel, and identify it as a negative modulator of Piezo2 channel activity.

Blockade of TRPC Channels Limits Cholinergic-Driven Hyperexcitability and Seizure Susceptibility After Traumatic Brain Injury

Chase M. Carver, Haley R. DeWitt, Aiola P. Stoja and Mark S. Shapiro

Abstract

We investigated the contribution of excitatory transient receptor potential canonical (TRPC) cation channels to posttraumatic hyperexcitability in the brain 7 days following controlled cortical impact model of traumatic brain injury (TBI) to the parietal cortex in male adult mice. We investigated if TRPC1/TRPC4/TRPC5 channel expression is upregulated in excitatory neurons after TBI in contribution to epileptogenic hyperexcitability in key hippocampal and cortical circuits that have substantial cholinergic innervation. This was tested by measuring TRPC1/TRPC4/TRPC5 protein and messenger RNA (mRNA) expression, assays of cholinergic function, neuronal Ca2+ imaging in brain slices, and seizure susceptibility after TBI. We found region-specific increases in expression of TRPC1, TRPC4, and TRPC5 subunits in the hippocampus and cortex following TBI. The dentate gyrus, CA3 region, and cortex all exhibited robust upregulation of TRPC4 mRNA and protein. TBI increased cFos activity in dentate gyrus granule cells (DGGCs) and layer 5 pyramidal neurons both at the time of TBI and 7 days post-TBI. DGGCs displayed greater magnitude and duration of acetylcholineinduced rises in intracellular Ca2+ in brain slices from mice subjected to TBI. The TBI mice also exhibited greater seizure susceptibility in response to pentylenetetrazolinduced kindling. Blockade of TRPC4/TRPC5 channels with M084 reduced neuronal hyperexcitation and impeded epileptogenic progression of kindling. We observed that the time-dependent upregulation of TRPC4/TRPC5-containing channels alters cholinergic responses and activity of principal neurons acting to increase proexcitatory sensitivity. The underlying mechanism includes acutely decreased acetylcholinesterase function, resulting in greater Gq/11-coupled muscarinic receptor activation of TRPC channels. Overall, our evidence suggests that TBI-induced plasticity of TRPC channels strongly contributes to overt hyperexcitability and primes the hippocampus and cortex for seizures.

Keywords: ion channels, TRPC channels, hippocampus, epilepsy, seizure, traumatic brain injury, epileptogenesis,
hyperexcitability

Inhibition of itch by neurokinin 1 receptor (Tacr1) -expressing ON cells in the rostral ventromedial medulla in mice

Taylor Follansbee1,2*, Dan Domocos3, Eileen Nguyen4, Amanda Nguyen1, Aristea Bountouvas1, Lauren Velasquez1, Mirela Iodi Carstens1, Keiko Takanami5, Sarah E Ross4, Earl Carstens1

Abstract

The rostral ventromedial medulla (RVM) is important in descending modulation of spinal nociceptive transmission, but it is unclear if the RVM also modulates spinal pruriceptive transmission. RVM ON cells are activated by noxious algesic and pruritic stimuli and are pronociceptive. Many RVM-spinal projection neurons express the neurokinin-1 receptor (Tacr1), and ON-cells are excited by local administration of substance P (SP). We hypothesized that Tacr1-expressing RVM ON cells exert an inhibitory effect on itch opposite to their pronociceptive action. Intramedullary microinjection of SP significantly potentiated RVM ON cells and reduced pruritogen-evoked scratching while producing mild mechanical sensitization. Chemogenetic activation of RVM Tacr1-expressing RVM neurons also reduced acute pruritogen-evoked scratching. Optotagging experiments confirmed RVM Tacr1-expressing neurons to be ON cells. We conclude that Tacr1-expressing ON cells in RVM play a significant role in the modulation of pruriceptive transmission.

Presenter: Gi Baek Lee